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Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC. The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.
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[Possible structural-functional organization of the system of local cerebral blood flow regulation]. rCBF under normal conditions in the rabbit, cat, and monkey brain was found to have a spontaneous periodicity while rCBF responses to afferent flicker stimulation usually revealed a double-phasic fluctuative pattern. This suggests that the rCBF regulatory system consists of not less than two regulatory chains with different time constants, and a feedback. The data on cerebral vascular responses to microapplication of mCSF solutions with various pH, potassium and catecholamines concentrations, suggest that rapid regulatory chains may be conditioned by potassium and neurogenic vascular effects, while slow ones could be mediated by CO2 and related pH changes.
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[Differences in the responses of taste receptors to organic and inorganic acids with changes in the concentration of bicarbonate in the solution]. The thresholds of the pH for citric acid (pH=4.9) were found to exceed by 1.4 pH the thresholds for HC1 (3.5) at 1.2 mmol/1 bicarbonate in the solution. The reaction to citric acid was higher than to HC1 at equal pH. Decreasing of bicarbonate from 1.2 mmol/1 to 0 reduced pH threshold only for sitric acid from 4.90 to 3.15. pH threshold for HC1 remained 3.5 The chorda tympani response to stimulation with solutions containing bicarbonate (1.2 mmol/1) was higher than in absence of bicarbonate. The data obtained suggest two ranges of the acids in action.
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[Regulation of local tissue PO2 in the cerebral cortex of the cat]. Local PO2 was measured in the cat cortex on adjacent sites with a platinum multiwire surface electrode both during steady state conditions and with varying arterial oxygen supply. Concomitantly, PO2 in the sinus sagittalis was recorded continuously through the vascular wall. Under normoxia and steady state conditions local tissue PO2 values varied between O Torr and almost arterial levels of 85 Torr in accordance with theoretical calculations. With increased arterial oxygen supply local tissue PO2 as measured on agjacent sites was found to react fairly differently. Linear increases in local tissue PO2 as compared with arterial PO2, as well as constant levels, or only very small increases, were recorded. The constancy of local PO2 (="local PO2 autoregulation") was caused by local vasoconstriction. With reduced supply of arterial oxygen, however, tissue PO2 dropped in all studied sites down to hypoxia and anoxia. PO2 autoregulation during a decrease in arterial PO2, as described by Bicher (1973) could not be found.
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Analysis of skin grafts across the MSA-barrier in mice pretreated with sera from specifically or syngeeically grafted donors. Prolonged survival of weakly incompatible skin allografts in mice (across the barrier presented by the MSA) can be induced by pretreating the recipients not only with a specific anti-MSA serum (obtained on day 5 after a single MSA-incompatible skin graft) but also be means of control serum obtained in a similar way from the recipients of fully compatible (syngeneic) skin grafts. Administration of serum from non-grafted mice had no effect on graft survival. The similar biological effect of both sera had a counterpart in their similar content and spectrum of glycosaminoglycans. Also in the skin grafts themselves, the course of both qualitative and quantitative changes of GAG in the early postgrafting period was in the allogeneic and syngeneic situation similar. The possible role of these substances in the serum and at the site of grafting and their effect on the outcome of the allograft response are discussed.
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Effect of normal and ulcer-type diet on the acidity of gastric contents in patients with duodenal ulcer. Patients with uncomplicated duodenal ulcer were given two types of diet -a normal and a ulcer-type diet. The data obtained did not show any statistically significant difference between the action of the two diets. No evidence was then found to be in support of the still widely used restricted diet in the treatment of peptic ulcer.
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Impaired Sertoli cell function in experimental cryptorchidism in the rat. The production of testicular androgen-binding protein (ABP), as a measure of Sertoli cell function, was studied after unilateral or bilateral experimental cryptorchidism in adult rats. Two or 4 weeks after the testis had been translocated to the abdomen, no major changes were found in the concentration of ABP per mg protein, although there was a marked and progressive decrease in ABP content per testis. However, the rate of ABP production was greatly decreased, as measured by the accumulation of ABP during 16-h ligation of the efferent ducts or by the production of ABP by testis mince in an in vitro system. This indicates that the Sertoli cell function is severly impaired by the intra-abdominal position.
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The oxygen transport system of red blood cells during diabetic ketoacidosis and recovery. Daily evaluations of 8 newly detected ketoacidotic diabetics showed the Bohr-effect of haemoglobin to be decreased by 50% while erythrocyte 2,3-DPG was decreased below 10 mumoles/g Hb. 2,3-DPG correlated strongly with pH during acidosis and with plasma inorganic phosphate (Pi) subsequently to the first insulin administration. Oxygen affinity of haemoglobin, measured as P50 act pH, was unchanged in ketoacidosis compared to the time, however, P50 act pH fell striking (p less than 0.001) and remained decreased up to 7 days depending upon the resynthesis of 2,3-DPG in relation to Pi. The Hill-coefeficient in reflecting the slope of the oxygen dissociation curve was diminished in ketoacidosis (p less than 0.005), and decreased further after pH-normalization (p less than 0.005). There was a close association of n with 2,3-DPG (p less than 0.001) and additionally with Pi at 2,3-DPG-levels below 10 mumoles/g Hb. Based on these findings a decreased erythrocyte oxygen release of one fifth during acidosis and more than one third after pH-correction can be hypothesised. In view of the intimate relation of Pi to the oxygen transport system it is suggesed that treatment of ketoacidosis should include Pi-sugstitution.
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Fluorescence of oxidized flavoproteins from perifused isolated pancreatic islets. In perifused pancreatic islets, the fluorescence of oxidized flavoproteins (FAD) was recorded continuously. Elevation of glucose concentration in the medium form 0 or 5 mM to 20 mM led to decrease in FAD-fluorescence beginning 10 sec after change of medium. L-leucine (10 mM), (+/-)-B-BCH (20 mM) and alpha-ketoisocaproic acid (10 mM) caused typical kinetics of FAD-fluorescence decrease. The results are interpreted to indicate rapid changes of the functional state of B-cell mitochondria induced by the above-mentioned stimulators of insulin release.
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Comparison of amino acids bathing the oxyntic gland area in the stimulation of gastric secretion. This study was undertaken to compare the ability of L- and D-isomers of amino acids bathing the oxyntic gland area to stimulate acid secretion in conscious dogs with Heidenhain pouch (HP), gastric fistula (GF) and pancreatic fistula (PF). Acid outputs from HP were determined by an intragastric titration method when amino acid solutions were perfused into HP at various concentrations, pH values, and distention pressures. Only L-isomers of all natural amino acids were found to stimulate acid secretion, whereas D-isomers of amino acids tested were completely inert in this respect. The comparison of the secretagogue activity of amino acids shows that L-histidine among essential amino acids and glycine among nonessential amino acids exhibited the strongest stimulation of acid outputs, reaching, respectively, 52 and 40% of the maximal response to histamine. Decreasing the pH of L-histidine solution perfused into HP in sequential order from 5.0 to 1.0 resulted in a stepwise reduction of acid output, falling at pH 1.0 to about 40% of the peak response achieved at pH 5.0. Local irrigation of HP by 2% xylocaine and intravenous infusion of atropine (100 mug per kg per hr) or metiamide (2.9 mg per kg per hr) reduced but did not abolish HP response to chemical stimulation and the pH dependency of this response. We conclude that only L- and not D-isomers of amino acids bathing the oxyntic gland area stimulate acid secretion by a local, gastrin-independent mechanism sensitive to distention pressure and pH.
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Evidence of tandem duplication of genes in a merodiploid region of Pneumococcal mutants resistant to sulfonamide. A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d+ resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four strains showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d+) were quantitatively measured. From those heteroduplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's.
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A study of Pneumococcal merodiploids at the molecular level. The DNA of a sulfonamide-resistant Pneumococcal strain (heterozygous for sulr-c) and that of three highly resistant and persistently heterozygous cd transformants, derived by introducing sulr-c marker into a stable sulfonamide resistant strain (sulr-d), were studied to analyze the genetic basis of their merodiploidy. The physical properties of the native and denatured DNA from the heterozygotes and the nonheterozygous strains were not distinguishable. The denaturability and the renaturability of biological activity for the heterozygous markers were essentially identical to those of the normal markers. The heterozygosity extends to the closely linked locus giving rise to four different configurations of cd and cd+ transformants, characterized by their frequencies of segregation and donor-marker activities. The marker-activity ratios and the frequency of co-transfer of heterozygous markers were found to remain the same in each when the donor DNA was native, denatured or reannealed without fractionation or reannealed after remixing of resolved strands. Possible models were weighed against these observations and these considerations led to the suggestion that tandem duplication of a gene region may be responsible for the heterozygosity and instability of this region. A more detailed examination of this model will be presented in an accompanying paper.
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Genetic hybridization at the unlinked thy and str loci of Streptococcus. The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci.
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Fibrin plate method with reagents purified by affinity chromatography and its use for determination of fibrinolytic and other proteolytic activity in saliva, bile and plasma. A modification of the fibrin plate method is presented. Plasminogen-free human fibrinogen and plasminogen purified by affinity chromatography have been used. Fibrin plates without and with a constant amount of plasminogen and with agarose as stabilizing medium were used for the estimation of plasmin and plasminogen activator activity. Activator activity could be demonstrated in sterile bile and saliva. When plasmin activity was present, estimations of plasminogen activator were approximate. The method is sensitive, small volumes of reagents and samples are needed. The error of the method is comparatively low and the reproducibility is good.
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[Thefts without motive of gain as a psychopathologic syndrome (author's transl)]. Thefts without motive of pain have been known since the early 19th century. But the problem has not been solved. While they were formerly considered a mental disease, today they are not seen as something special. But they still happen. Only a small percentage of common shop-lifting can be called a psycholopathologic syndrome. Many explanations and analyses have been published which are discussed in detail. In a group described here comprehensively difficult marital situations full of conflict, marital sexual frustration, depression, physical and mental exhaustion and aggressive and suicidal tendencies are found. Theft appears to be closely connected with these. But the pattern of motivation and causation is by no means stereo-typed. In order to clear up such actions one will have to consider as exactly as possible the biographic connection and what happens during the act - quite apart from somatic conditions. Present assessment in reports is totally unsatisfactory. To clear up the controversial questions is urgently necessary.
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The structures of the phytochrome chromophore in both photoreversible forms. Spectral measurements of phytochrome are performed after unfolding of the peptide chain. By comparison with bile pigments of known structure, structure 1a, containing a hydrogenated ring A, is deduced for the PR chromophore. Its spectral properties indicate that the chromophore of the physiologically active PFR form has lost the double bond of the bridge joining rings A and B.
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A micro-method for quantitative determination of acylneuraminic acids from erythrocyte membranes. A micro-method is presented which enables the fast and exact determination of acid-hydrolyzed acylneuraminic acids in erythrocyte membranes. Erythrocytes from 1 ml of human and rabbit blood containing ACD buffer are, washed and hemolyzed on Millipore filters of pore size 1.2 mu. Acylneuraminic acids are released from the erythrocyte membranes still on the filters under the optimal conditions of 0.1 N HCl at 80 degrees C for 50 min. A prerequisite for the determination of the true amount of acylneuraminic acids using the periodic acid/thiobarbituric acid assay is the small-scale extraction of lipids from the hydrolysate and anion-exchange chromatography of acylneuraminic acids. The values thus obtained must be corrected, as 20% of acylneuraminic acids are destroyed during acid hydrolysis. In samples of human blood from 10 healthy individuals, on an average 223 nmol acylneuraminic acids per ml of packed erythrocytes were found, and in the same amount of rabbit erythrocytes, 1e method for a screening of the acylneuraminic acid content of erythrocyte membranes in hemolytic diseases or of other cell membranes is discussed.
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Human liver acid phosphatases. Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively. Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.
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Isolation and characterization of pepsin-treated type III collagen from calf skin. Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.
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High-dosage and versatile drug therapy with treatment-resistant psychotic patients. The author believes that many of the chronic patients in psychiatric institutions and mental health facilities could be helped if physicians were more willing to try different combinations and higher dosages of psychotropic drugs than are commonly used. He presents case studies of two chronic patients who were helped by innovative use of drugs and discusses factors to be considered in implementing high-dosage and versatile drug therapy.
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A new variant of glucosephosphate isomerase deficiency. A new variant of glucose-6-phosphate isomerase deficiency is described. The enzyme kinetics and properties were studied. Genetic and electrophoretic data pointed to a double heterozygous state in the patient. These data are compared to the other variants described in the literature until now.
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[Infusion treatment in shock]. Nowadays, above all dextran, gelatin and starch solutions are available for the infusion theraphy of the various forms of shock. The application of these volume substitutes must be strictly controlled to avoid in particular cardial and pulmonal commplications. Blood transfusion combined with a volume substitute should only be applied in cases of heavy loss of blood. The treatment of metabolic acidose which usually occurs simultaneously is carried out with an alkaline solution.
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Suppressor T cells and host resistance to tye 111 pneumococcus after treatment with antilymphocyte serum. The antibody response to type III pneumococcal polysaccharide (SS-II) was significantly increased in mice treated with antilymphocyte serum (ALS). BALG/c mice given 0.25 ml of ALS on days -1, 0, and 1 relative to the days of immunization with 0.5 mug of SSS-II had a 20-fold increment (11,383 increased to 199,917) in the number of splenic plaque-forming cells enumerated on day 5 compared with untreated, immunized controls. This effect has been attributed to the elimination of subpopulation of thymus-derived lymphocytes (T cells) that has suppressor function. The present series of experiments relate the augmented antibody response to SSS-II in mice treated with ALS to increased host resistance after infection with Streptococcus pneumoniae, type III (Pn-II). The 50% lethal dose of Pn-III in niminnunized mice was 102 and the 100% lethal dose was 103 organisms. Mice immunized with 0.5 mug of SSS-III and challenged 5 days later with Pn-III were completely protected against a dose of up to 108 organisms. Mice treated with 0.25 ml of ALS on days -1, 0, and 1, immunized with SSS-III on day 0, and challenged with 2.5 X 10(9) Pn-III on day 5 had a mean survival time of greater than 100 h compared with 16 h for immunized non-serum-treated controls. Animals given a single injection of ALS before immunization showed no increase in resistance, whereas mice treated after immunization had significant prolongation of survival times. Untreated, immunized mice challenged with 5 X 10(9), 1 X 5 X 10(8) Pn-II survived 14 to 19 h, whereas ALS-treated animals had mean survival times of 48, 174, and 222 h, respectively. These findings suggest that immunoregulatory T cells may have a biologically significant effect in a narrow zone in which the normal host immune response is insufficient but still potentially capable of providing some additional degree of protection if suppressor cells are elimated.
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[Past and present aspects of diarrheal disease in childhood. Clinical study and treatment (author's transl)]. The etiologic and pathophysiologic findings described in the first part of this paper have important consequences: The recognition of the specific etiology of diarrhea requires new laboratory methods: most of these, however, are technically easy to perform and do not require a large laboratory. A long-ranging consequence of this changed concept is a well-founded modification of therapy. The most important discovery was, that in a well balanced glucose electrolyte solution sodium and glucose enhance their absorption mutually and increase the absorption of water by solvent drag. Since in most acute diarrheas the mechanisms of absorption of glucose and electrolytes are retained this mechanism can be utilized for fast oral rehydration and reinstitution of normal intestinal homeostasis. Prompt institution of a diet consisting of the previously mentioned glucose-electrolyte solution usually prevents severe dehydration and the need for stationary treatment. The elimination of lactose and long chain fatty acids from the diet prevents continuation of the pathologic osmotic and chemical conditions in the intestine. Antibiotics are not indicated in acute diarrhea with the exception of diarrhea caused by enteroinvasive E. Coli or Shigella, in the case of Salmonella-gastroenteritis even contraindicated. Further research concentrates on the development of drugs for neutralisation of E. Coli enterotoxin and the prevention of diarrheas by development of effective vaccines.
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Population growth-a menace to what? Originally, many of the initiators of the World Population Conference, which took place in Bucharest in 1974, had hoped that the Conference would imply a final breakthrough for the view that family planning measures should be given top priority in all less-developed countries. In fact, however, the Plan of Action passed by the Conference contains very little relating to population and family planning. Instead, the document is dominated by wordy phrases about the necessity of attaining social and economic development in those countries. Will the insight that family planning programs work efficiently only if they are an integral part of programs for the social and economic development of a country lead to such programs being realized? There is every reason to doubt that the plan of Action will have any such effect. The reasons for the underdevelopment of Third World countries cannot be removed through such United Nations resolutions. In the People's Republic of China, family planning is widely accepted, especially in the towns, and now also among the rural population. Limiting the number of children is considered part of China's development effort. China is a less-developed country that is in the process of rapid social and economic development. The issue at stake in other Third World countries is how to achieve a similar development. As soon as this goal is achieved, family planning efforts are meaningful and have a chance of success. The experience of China demonstrates that even there it took time before the efforts succeeded. There are many Third World countries that could, without much difficulty, support a population considerably larger than the present one. But there are no doubt also a number of countries where the population is already so large that a continued population increase would be harmful. The need to achieve rapid development becomes increasingly urgent, not in the least to make it possible to attain a reduced population growth. The sad truth is that so little development takes place in those countries. Without social and economic development, the present rapid population increase will continue in those countries where there is already an overly dense population.
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Training of the barefoot doctor in the People's Republic of China: from prevention to curative service. Among the changes that have been brought about in health delivery in the People's Republic of China, the introduction of the barefoot doctor has been one of the most important and effective ways that the government has devised to radically alter the concept of health care. Through close identification with the community in terms of recruitment, training, and practice, the barefoot doctor is a concrete manifestation of the ideological principles of following the mass line and being self-reliant. The paper focuses on the building of rural health services, with special reference to the training of the barefoot doctor as the first-level contact person in primary care in the communes. It describes the training programs in a school of public health and the career mobility possible to the barefoot doctor in joining the ranks of medical practitioners.
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Effect of the beta-receptor blocking agent Visken on the action of coumarin. In a double-blind study, the influence of Visken on the effect of anticoagulant therapy with Marcoumar was examined. In comparison to a placebo group, neither any influence on the Quick time, nor any increased tendency to haemorrhage bleeding could be detected.
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Investigations of the excretion of gamma-glutamyl-transpeptidase into the urine. The excretion of the enzyme gamma-glutamyl-transpeptidase and its isoenzymes into the urine was investigated in patients with renal diseases and compared with the excretion of the enzymes leucine-aminopeptidase and lactate-dehydrogenase. In animal experiments an increased excretion of these enzymes was found after autotransplantation. Increased excretion of gamma-glutamyl-transpeptidase was also found in patients with glomerulonephritis and in the polyuric phase of acute tubular necrosis, but not in cases of pyelonephritis and in the oliguric phase of acute tubular necrosis. The alterations of the isoenzyme pattern during diseases with increased enzyme excretion are in accordance with the hypothesis that the enzymes are liberated from the kidney tissue into the urine, and only a minority stems from the blood. Investigation of the excretion of gamma-glutamyl-transpeptidase and its isoenzymes into the urine seems to be of both scientific and clinical interest.
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Interaction of adrenergic antagonists with prostaglandin E2 and tetrahydrocannabinol in the eye. Both alpha- and beta-adrenergic antagonists have been utilized in an atempt to discern the site of action of prostaglandin (PG) and tetrahydrocannabinol (THC) in the eye. Both alpha- and beta-adrenergic antagonists (alpha-antagonists, phentolamine and phenoxybenzamine; beta-antagonists, propranolol and sotalol) cuased a dose-dependent reduction in intraocular pressure and blood pressure and increased total outflow facility. The results are consistent with the concept that both alpha- and beta-adrenergic receptors are present in the anterior uvea and that vasomotor tone is essential to the maintenance of normal intraocular pressure. No antagonist reduced the PG-induced elevation of intraocular pressure unless the blood pressure was severely lowered. All antagonists inhibit the normal PG-induced increase in total outflow facility, indicating that these agents protect the blood-aqueous barrier from breakdown without altering the vasodilatory response to PG. All antagonists reduced the fall in intraocular pressure produced by THC by approximately 50 per cent, except for sotalol which completely abolished the intraocular pressure fall. Only the alpha-adrenergic antagonists prevented the THC-induced increase in total outflow facility. The results indicate that true outflow facility may well be regulated exclusively by alpha-receptors. The data are consistent with the effect of THC being primarily a vasodilation of the efferent blood vessels of the anterior uvea. The partial inhibition by alpha-adrenergic antagonists may also suggest a lesser role of THC on the afferent vessels.
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Induction of corneal graft rejection by passive cell transfer. An experimental model is presented demonstrating that penetrating corneal grafts in the rabbit may be rejected by passive transfer into the anterior chamber of specifically sensitized lymphoid cells. Destruction of histo-incompatible corneal endothelium is always marked by the formation of focal pock-like areas of damage in this system, rather than by the typical moving line of rejecting endothelium usually seen in spontaneous graft rejection. Where the transferred lymphoid cells are compatible with the tissues of the graft recipient, the picture is one of a severely affected graft on a field of uninvolved recipient corneal endothelium. Where the lymphoid cells are compatible with the graft and not with the tissues of the recipient, one sees a clear corneal graft surviving on a field of endothelial destruction on the recipient bed. The specificity of these reactions is illustrated in terms of the histocompatibility relationships between corneal donor, graft recipient, and the donor of the sensitized lymphoid cells.
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Acute tubular necrosis. An experimental model detailing the biochemical events accompanying renal injury and recovery. Male Charles River mice, divided into control or experimental groups, received on Day 0 either sterile 0.3 MNaHCO3 in 0.9 per cent saline (pH7.4) intraperitoneal injection or pteroylglutamic acid (200 mug per body weight), similarly buffered to pH7.6, and were sacrificed on Days 0, 1/4, 1/2, 1,2,3,4,7, and 14. The experimental kidneys demonstrated intratubular deposits of pteroylglutamic acid with edema between Days 1 and 4 with cortical scarring by Day 14. The experimental kidneys reached maximal increases in weight (+90 per cent) on Day 2, RNA (+61 per cent, protein (+67 per cent) on Day 3, and DNA (+25 per cent) on Day 4 before falling to below control levels on Day 14. The control kidneys demonstrated the gradual incremental increases of normal renal growth throughout this period. No change in renal size, protein, RNA, or DNA could be detected in those animals who failed to demonstrate renal tubular damage. It is postulated that the response of the kidney to folic acid administration is a reparative response and not a response directed toward accelerated renal growth.
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Properties of the 3-o-methyl-D-glucose transport system in Acholeplasma laidlawii. Transport of 3-O-methyl-D-glucose (3-O-MG) by Acholeplasma laidlawii cells was studied. The 3-O-MG transport system appeared to be constitutive in cells grown on 3-O-MG and glucose; the transport process depended on the concentration of substrate used and exhibited typical saturation kinetics, with an apparent Km of 4.6 muM. 3-O-MG was transported as a free carbohydrate and was not metabolized further in the cell. Dependence on pH and temperature and the results of efflux and "counterflow" experiments demonstrated the carrier nature of the transport system. 6-Deoxyglucose and glucose competitively inhibited 3-O-MG transport, whereas maltose inhibited in non-competitively. p-Chloromercuribenzoate, p-chloromercuribenzene sulfonate, N-ethylmaleimide, and iodoacetate inhibited transport of 3-O-MG. Cells were able to accumulate 3-O-MG against a concentration gradient. Some electron transfer inhibitors (rotenone and amytal), arsenate, dicyclohexylcarbodiimide, and proton conductors such as 2,4-dinitrophenol, carbonylcyanide, m-chlorophenylhydrazone, pentachlorophenol, and tetrachlorotrifluoromethylbenzimidazole inhibited this process.
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Starvation survival of Salmonella enteritidis. Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.
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Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency. A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.
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Microcalorimetric study of the anaerobic growth of Escherichia coli: growth thermograms in a synthetic medium. A microcalorimetric technique was used for studying the growth of Escherichia coli during anaerobiosis. The growth thermograms obtained are complex and the shape of curves is dependent on the hydrogen lyase activity of the cells. Fermentation balances are given for different culture conditions, and simple growth thermograms are obtained when the hydrogen lyase activity is inhibitied.
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Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level. The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.
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Microcalorimetric study of the anaerobic growth of Escherichia coli: measurements of the affinity of whole cells for various energy substrates. Microcalorimetry has been used to determine the affinity of whole cells of Escherichia coli for glucose, galactose, fructose, and lactose. Anaerobic growth thermograms were analyzed, and the Km and Vmax values for these energy substrates were measured at pH 7.8. Results obtained with this technique using various organisms growing anaerobically on different sugars are compared. This comparison shows that in practically all cases the cellular rate of catabolic activity is a hyperbolic function of the energy substrate concentrations at low sugar concentrations. In some cases this technique also allows determination of kinetics at high sugar concentrations.
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Purification and properties of polyol dehydrogenase from Cephalosporium chrysogenus. A polyol dehydrogenase of broad specificity was purified 178-fold from extracts of the filamentous fungus Cephalosporium chrysogenum. The enzyme was found to act as an oxido-reductase in two substrate-coenzyme systems: D-sorbitol (or xylitol)-nicotinamide-adenine dinucleotide (NAD) and D-mannitol-nicotinamide adenine dinucleotide phosphate (NADP). The dehydrogenase was composed of five isozymes, which, as a mixture, exhibited these properties: Km to D-sorbitol and D-mannitol, 7.15 to 10(-2) M; PH optimum, 9 to 10; molecular weight, 300,000 subunit weight, 29,000; PI, 5.8 to 7.5. The NADP-linked activity was labile to treatment with heat or ethylenediaminetetraacetic acid. Mixed substrate assays support the hypothesis that both NAD-, and NADP-linked activities are associated with isozymes of a single dehydrogenase.
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Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies. Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles.
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Protein-carbohydrate-lipid complex isolated from the cell envelopes of Chlamydia psittaci in alkaline buffer and ethylenediaminetetraacetate. Exposure of isolated cell envelopes from purified infectious elementary (EB) of Chlamydia psittaci to sodium carbonate-bicarbonate buffer at pH 10 plus ethylenediaminetetraacetate (EDTA) results in partial solubilization of the total protein. The released materials represent 20% of the dry weight, 16% of the total protein, 40% of the total carbohydrate, and 9% of the total lipid of the cell envelopes. Sucrose density gradient centrifugation, and Sephadex G-200, Sepharose 4B, or diethylaminoethyl-cellulose column chromatography, reveal a protein-carbohydrate-lipid complex of several hundred thousand molecular weight that contains 50% protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated EB cell envelopes reveals two major protein bands, A and B, with estimated molecular masses of approximately 85,000 and 53,000, respectively, both of which also stain for the presence of carbohydrate and lipid. Gel electrophoresis of the protein-carbohydrate-lipid complex reveals two protein bands, C and D, with estimated molecular weights of approximately 17,000 and 13,000, respectively, which contain lipid and a small amount of carbohydrate; bands A and B are not present in the complex. Gel electrophoresis of the cell envelope residues after extraction of the complex with alkali and EDTA shows a single main band, corresponding to the position of band B, which contains protein, carbohydrate, and lipid; band A is completely missing. B and A is believed to be a component of the complex, which is split into two subunits on alkali solubilization.
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Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli. beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.
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Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum. Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study.
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Altered nutritional requirements associated with mutations affecting the structures of ribonucleic acid polymerase in Lactobacillus casei. Rifampin-resistant mutants were isolated from Lactobacillus casei S1 and examined for possible simultaneous alteration in nutritional properties. Among the 36 mutants obtained either spontaneously or after mutagenesis with 2-aminopurine, 22 were found to be altered with respect to the specific growth requirements. The majority (20 of 22) of the latter mutants were shown to require L-glutamine in addition to the nutrients required by the parental strain for maximal growth, whereas the remaining mutants had apparently lost the requirement for L-aspartate. Further studies with one of the glutamine-requiring mutants revealed that the rifampin resistance of this strain is due to the resistance of ribonucleic acid polymerase itself and that a single mutation is responsible for both rifampin resistance and the glutamine requirement. These results strongly indicate that a structural alteration of the ribonucleic acid polymerase caused by the rifampin resistance mutation somehow affected glutamine metabolism, possibly through change in selective transcription of the genes involved.
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Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus. Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.
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Urea-hydrolyzing activity of a T-strain mycoplasma: Ureaplasma urealyticum. The urea-hydrolyzing activity of a T-strain mycoplasma was studied in experiments using whole cells and cell-free enzyme preparations by measuring the release of 14CO2 from [14C]urea. Under the conditions used, the urea concentration optimum is approximately 5.6 X 10(-3) M urea. The activity is soluble and not membrane bound. It is stable at -70 C for several weeks but is more labile at higher temperatures. The pH optimum is between 5.0 and 6.0. The effect of several inhibitors on the activity was tested and revealed similarities, as well as differences, between T-strain mycoplasma urease activity and the urease activity of other organisms and plants.
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Arginine decarboxylase from a Pseudomonas species. An arginine decarboxylase has been isolated from a Pseudomonas species. The enzyme is constitutive and did not appear to be repressed by a variety of carbon sources. After an approximately 40-fold purification, the enzyme appeared more similar in its properties to the Escherichia coli biosynthetic arginine decarboxylase than to the E. coli inducible (biodegradative) enzyme. The Pseudomonas arginine decarboxylase exhibited a pH optimum of 8.1 and an absolute requirement of Mg2+ and pyridoxal phosphate, and was inhibited significantly at lower Mg2+ concentrations by the polyamines putrescine, spermidine, and cadaverine. The Km for L-arginine was about 0.25 mM at pH 8.1 AND 7.2. The enzyme was completely inhibited by p-chloromercuribenzoate. The inhibition was prevented by dithiothreitol, a feature that suggests the involvement of an -SH group. Of a variety of labeled amino acids tested, only L-arginine, but not D-arginine was decarboxylated. D-Arginine was a potent inhibitor of arginine decarboxylase with a Ki of 3.2 muM.
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Derepression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli K-12 by growth in Fe3+-deficient medium. 3-Deoxy-arabino-heptulosonic acid 7-phosphate synthase, prephenate dehydratase, tryptophan synthase, and 2,3-dihydroxybenzoylserine synthase enzyme activities are derepressed in wild-type Escherichia coli K-12 cells grown on Fe3+-deficient medium. This derepression is reversed when FeSO4 is added to the growth medium. Addition of shikimic acid to the Fe3+-deficient growth medium caused repression of the first three enzyme activities but not of 2,3-dihydroxybenzoylserine synthase activity. Addition of 2,3-dihydroxybenzoic acid to the Fe3+-deficient growth medium has no effect on any of the above-mentioned enzyme activities. The Fe3+ deficiency-mediated derepression of 3-deoxyarabino-heptulosonic acid 7-phosphate synthase activity is due to an elevation of the tyrosine-sensitive isoenzyme; the phenylalanine-sensitive isoenzyme is not derepressed under these conditions.
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Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development. When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5.
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Serum stimulation of phosphate uptake into 3T3 cells. The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguised from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system.
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Dinoflagellate bioluminescence: a comparative study of invitro components. In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.
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Effect of lumbar sympathetic blockade and chlorpromazine-induced adrenergic alpha-receptor blockade on skin temperature in peripheral arterial diseases. The post-cooling toe temperature changes after lumbar sympathetic blockade and after intramuscular administration of an adrenergic alpha-receptor blocking substance (chlorpromazine) were studied in 14 patients with impending gangrene because of peripheral arterial insufficiency. The post-cooling temperature rise was similar after sympathetic blockage and chlorpromazine administration and significantly different from the basal toe temperature changes after cooling. It is concluded that administration of an adrenergic alpha-receptor blocking substance is as good as the lumbar sympathetic blockage for evaluation of a remaining sympathetic vasomotor tone in arterial disease patients.
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Control of phenylalanine hydroxylase synthesis in tissue culture by serum and insulin. Stationary-phase, minimal deviation hepatoma H4-II-E-C3 cell cultures that are serum-deprived respond with a biphasic time course of phenylalanine hydroxylase induction when dialyzed fetal calf serum or insulin is added. These two agents induce phenylalanine hydroxylase additively, during both the initial 3-hour and the delayed 24-hour phases. The initial phase of induction by insulin is inhibited by cycloheximide but not by actinomycin D. The delayed induction by both dialyzed fetal calf serum and insulin is inhibited by 10(-6) M cycloheximide and 0.20 mug/ml actinomycin D. H4-II-E-C3 cells in culture do not synthesize the factor(s) in serum that induce phenylalanine hydroxylase.
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High-speed ion-pair partition chromatography in pharmaceutical analysis. Ion-pair chromatography offers attractive possibilities in pharmaceutical analysis. The specificity of the separation systems can be varied over a wide range by appropriate selection of the stationary phase. The choice of a suitable counter-ion can also drastically improve the detection limit, permitting the determination of drug substances in low dosage and possibly of by-products or breakdown products. Ion-pair chromatography of tropane and ergot alkaloids has been investigated using picrate as counter-ion. Alumina, Kieselguhr and various grades of silica gel have been tested as supports. Partition properties studied in a batch procedure have been compared with the actual chromatographic conditions. Columns (10 cm) filled with silical gel (particle size, 5 mum; pore size, 1000 A) show the best performance in the separation of hyoscyamine, scopolamine and ergotamine as picrate ion-pairs. Close control of pH and temperature is essential for reproducible separations. Improvements in detection limits between 100 and 300 times have been observed with these systems. Ion-pair extractions of these alkaloids from dosage forms can be used for sample preparation prior to injection on the the column. This provides an added degree of selectivity and sensitivity.
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Analytical separation of amino acids on a cation-exchange resin cross-linked with m-divinylbenzene. The elution bands of acidic and neutral amino acids of protein hydrolysates, emerging from the column of a cation-exchange resin cross-linked with pure m-divinylbenzene, are narrower than those from a resin prepared from styrene and technical divinylbenzene. As a result of these narrower bands, a more complete resolution of the critical pairs threonine-serine, glycine-alanine and tyrosine-phenylalanine is obtained. The most probable reason for the narrower elution peaks is the more rapid diffusion of the exchanged components through the bulk of the resin as a result of a more regular arrangement of cross-linkages in the cation-exchange resin prepared from m-divinylbenzene.
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[Template chromatography on immobilized oligonucleotides. Synthesis and application of oligodeoxyadenosine-5'-phosphate--DEAE-cellulose (author's transl)]. Oligomers of deoxyadenylic acid, obtained by polycondensation, were covalently attached to polyvinyl alcohol. These polymer-bound oligonucleotides undergo very strong adsorption on DEAE-cellulose such that at neutral pH and with 1 M NaCl, partial desorbtion occurs only above 60 degrees. The use of this PV(pA)n-DEAE-cellulose for the column chromatographic separation of deoxythymidylic acid oligomers obtained by polycondensation, according to the principle of base-pairing, is discussed. Linear oligomers and also pyrophosphate derivatives of thymidylic acid which contain more than five monomer units undergo strong retardation under the conditions of base-pairing. Cyclic oligonucleotides do not show any noticeable interaction with the stationary phase. Thus, through the use of a temperature gradient, it is possible to fractionally separate the polycondensate, giving an average degree of polymerisation of 6--10.
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A rapid and comprehensive system for the routine identification of drugs in biological material based on microphase extraction and drug colour profiles. The separation of basic, acidic and neutral drugs from propanol-2 extracts of serum, urine and tissue homogenates at different pH values using a micro-phase extraction technique is described. Following preliminary screening, the various drug-containing fractions obtained are further examined by two-dimensional thin-layer chromatography. The drugs present are identified with reference to documented standards with the aid of a drug colour profile system and RF values relative to three different reference standards. By means of gas chromatographic analysis of the same extracts, semi-quantitative estimates of the amounts of drugs present, which are sufficiently accurate for clinical emergency purposes, can be made in many instances. The main advantages of the system are "clean" extracts with a minimum of background interference, rapidity (4-6 h for a complete analysis) and systematically documented and visually presented behaviour of drugs after spraying with various chromogenic and fluorogenic reagents, allowing the systematic identification of unknown substances.
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Chromatographic behaviour of alkaloids on thin layers of anion and cation exhangers. I. AG 1-X4 and cellex D. The chromatographic behaviour of 48 alkaloids has been studied on Bio-Rad AG 1-X4, Cellex D and microcrystalline cellulose, eluting with solutions of different pH but constant ionic strength (0.5). Many interesting separations were effected on both AG 1-X4 and Cellex D layers. The influence of pH on the chromatographic behaviour of alkaloids has been quantitatively studied and an equation was used that expresses the behaviour of the alkaloids on both AG 1-X4 (AcO-) and microcrystalling cellulose layers. The nonapplicability of this equation to Cellex D layers is discussed.
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Determination of trimethylsilyl methylated nucleic acid bases in urine by gas-liquid chromatography. A method for the determination of the urinary excretion level of methylated nucleic acid bases by gas-liquid chromatography (GLC) has been developed. A clean-up procedure prior to GLC analysis consisted of hydrolysis, filtration, charcoal adsorption, and anion exchange. Studies to determine optimum derivatization conditions for conversion of the methylated bases to their trimethylsilyl derivatives and to evaluate GLC parameters and columns to obtain the best separation were conducted. Application of the method was shown by determining the excretion levels of methylated bases in urine of normal controls and of patients with various types of malignancy.
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High-resolution liquid chromatographic analysis of methylated purine and pyrimidine bases in transfer RNA. Methylated and major purine and pyrimidine bases were separated and quantified by high-resolution liquid chromatography after hydrolyzing transfer ribonucleic acids (tRNAs). Separation was accomplished by eluting the hydrolyzed samples from an anion-exchange column with a concentration gradient of ammonium acetate at pH 9.2. Isolated sample of tRNA were hydrolyzed to the free bases with a trifluoroacetic acid-formic acid mixture of 200 degrees. Detection limits of 100-200 ng/ml were measured for the methylated bases; analytical data are reported for ten methylated bases plus the four major bases of calf liver and rat liver tRNA.
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A new gas chromatographic method for the estimation of reserpine and rescinnamine. Under the conditions described for alkaline hydrolysis of reserpine and rescinnamine in absolute and aqueous methanol, and after esterification (with diazomethane) of the resulting acid fraction, methyl 3,4,5-trimethoxybenzoate was quantitatively recovered, whereas methyl trans-3,4,5-trimethoxycinnamate, in normal lighting conditions, was either partly isomerized to methyl cis-trimethoxycinnamate or formed an adduct with a molecule of methanol, yielding methyl 3-methoxy-3-(3,4,5-trimethoxyphenyl)propionate. The structures of the products were established by synthesis, nuclear magnetic resonance studies and mass spectrometry. This investigation of the hydrolytic conditions allowed a reliable and rapid gas chromatographic determination of reserpine and/or rescinnamine in amounts down to 500 and 2000 mug, respectively, to be devised.
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Specific ion-exchange chromatography and fluorimetric assay for urinary 3-O-methyldopamine. A technique for the selective extraction of 3-O-methyldopamine, normetanephrine and metanephrine from a single urine sample has been investigated. After hydrolysis of the conjugates, the diluted mixture is passed through a Dowex 50W-X2 column and the methoxylated amines are eluted by means of concentrated ammonia. The eluate, containing metanephrine, normetanephrine and 3-O-methyldopamine is evaporated, and a solution of the residue in borate buffer is fractionated under strictly controlled conditions on an Amberlite CG-50 column. The three amines so separated are estimated by specific fluorimetric methods. The extraction recovery is 80 +/- 3% for pure solutions and 78 +/- 4% for 3-O-methyldopamine added to urine. The fluorimetric procedure, carried out under well-defined conditions, allows the estimation of 10 ng of 3-O-methethyldopamine. The spectral characteristics of the fluorescent derivative are similar to those obtained with dopamine, so that it can be assumed that iodine oxidation of 3-O-methyldopamine demethylates this compound and oxidises the resulting dopamine to the dopamine fluorophore (5,6-dihydroxy-indole). Of the compounds that might interfere in the fluorimetric procedure, dopamine, DOPA and alpha-methyl-DOPA are destroyed by the ammoniacal elution from the Dowex column and 3-O-methyl-DOPA is eliminated in the effluent from the Amberlite column. The elimination of interfering compounds and the improved separation on Amberlite ensure high specificity for this procedure. We have applied the method to normal urine and to pathological urines from patients with adrenergic tumours or untreated and treated parkinsonian subjects; vital information has been obtained on the prognosis of adrenergic tumours. The presence of large amounts of dopamine, normetanephrine and/or metanephrine does not affect the assay for 3-O-methyldopamine. The method is also applicable to rat and dog urine, and can be applied to tissue extracts with little modification.
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Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH. When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported.
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Counterimmunoelectrophoresis of pneumococcal antigens:improved sensitivity for the detection of types VII and XIV. Rapid identification of Streptococcus pneumoniae has been reported using counterimmunoelectrophoresis for the detection of specific capsular antigens in serum, cerebrospinal fluid, and urine. Previous clinical studies have failed to detect type VII or XIV pneumococcal antigen. These two types, however, account for a significant portion of pneumococcal disease. The incorporation of a sulfonated derivative of phenylboronic acid in the buffer system provides a method for the sensitive detection of these types in artificial mixtures without greatly reducing the sensitivity for the detection of other pneumococcal types. A problem with false positives encountered using human serum and barbitalbuffer was reduced by the use of buffer containing sulfonated phenylboronic acid.
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Isolation of streptococcal nuclease B by batch adsorption. A method has been developed for the preparation of streptococcal nuclease B by batch adsorpton to diethylaminoethyl-cellulose. The enzyme is homogeneous with respect to nuclease activity and is suitable for use as an antigen in measurement of anti-deoxyribonuclease B levels in sera.
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Rapid screening of Veillonella by ultraviolet fluorescence. Among 51 strains of anaerobic gram-negative cocci belonging to the family Veillonellaceae, all strains of Veillonella (V. parvula and V. alcalescens) displayed red fluorescence under long-wave (366 nm) ultraviolet light, whereas no Acidaminococcus or Megasphaera demonstrated fluorescence. In contrast to Bacteroides melaninogenicus, growth of Veillonella does not require hemin and menadione, and flourescence is rapidly lost upon exposure to air. The fluorescent component of a strain of V. parvula examined could not be extracted in solution with water, ether, methanol, or chloroform, but was readily extracted with 0.4 N NaOH. Spectrophotofluorometrically, the fluorescence maximum of this extract was 660 nm with an excitation maximum of 300 nm, when measured at pH 7.2 and 25 C. Coupled with the Gram stain, ultraviolet fluorescence may be a useful tool for rapid screening of Veillonella and is particularly helpful for detection and, isolation of this organism from mixed culture.
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Retention of mercurial preservatives in desiccated biological products. A variety of bacterins, vaccines, and antisera retained greater than 90% of their original level of mercurial preservative after lyophilization, and this might influence certain uses of these products.
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Distribution and removal of added mercury in milk. Distribution patterns of added mercury in raw whole milk after equilibration for 30 min and 2 h at 37 C showed a distribution among acid casein, whey proteins, fat globule membrane, and soluble fat globule membrane of 33, 28, 16, and 2%. On the basis of protein content, the fat globule membrane had the highest amount of mercury. Mercury added to milk as mercuric chloride was removed by treatment with thiolated aminoethyl celluloses and reduced human hair. In a 5 min treatment, 70, 43, and 41% of the mercury was removed by thiosuccinylated aminoethyl cellulose, thionitrocarboxyphenylated aminoethyl cellulose, and reduced human hair, respectively, from whole milk initially containing 1 ppm mercury and equilibrated for 2 h at 37 C prior to treatment. After treatment for 60 min, 82, 52, and 64% of the mercury was removed by thiosuccinilated aminoethyl cellulose, thionitrocarboxyphenylated aminoethyl cellulose, and reduced hair, respectively. However, increasing incubation temperature and time prior to treatment decreased the removal efficiencies. Thiosuccinilated aminoethyl cellulose and reduced human hair showed increasing efficiency directly with pH, while thionitrocarboxyphenylated aminoethyl cellulose showed the opposite effect and had higher affinity for mercury at pH 5.5 than at pH 7.5. Moreover, the rate of removal of mercury at 4 C compared to 37 C was much slower. The removal of mercury from soluble casein and soluble whey proteins was more efficient than from micellar casein. Protein, lactose content, and pH of milk were not changed by the polymer treatments.
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Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. A nonadrenergic inhibitory nervous system has been demonstrated in the guinea pig trachea. Electrical field stimulation of this system, in the presence of adrenergic and cholinergic blockade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was blocked by tetrodotoxin, which indicated that nerve stimulation was responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprung's disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, due to a lack of inhibition, may be either partially contracted or unable to relax, and thus appear hyperreactive to stimuli.
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Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes. Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes.
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The effect of neonatal rat graft-vs-host disease (GVHD) on Fc receptor lymphocytes. The level of Fc receptor rosette-forming lymphocytes (Fc-RFL) was examined in spleen and lymph node cell suspension from neonatal DA and BN rats inoculated within 24 hr of birth with either allogeneic L (experimental) or syngeneic (control) lymphoid cells. In addition, these levels were compared to fetal and neonatal animals that received no injection. The indicator cells (EA) were sheep erythrocytes sensitized with one-half concentration of the highest dilution of rabbit anti-sheep erythrocyte IgG(A) which agglutinated an equal amount of 1% suspension of E. Care was taken to exclude scoring macrophages by injecting colloidal carbon at least 1 hr before killing the test animals. The spleen of 19-day DA fetal rats exhibited a level of 19.3% Fc-RFL, similar to that of animals having received adult syngeneic cells at birth (40.0%) by day 7. Thereafter the level of Fc-RFL did not vary appreciably between these two groups. However, as early as 2 days after inoculation there was a significantly greater number of Fc-RFL in the spleen of experimental DA neonates compared to controls. The lymph nodes of experimental animals did not exhibit a significantly greater number of Fc-RFL until day 6 with both tissue compartments peaking at day 10 and remaining significantly higher than controls until death. In neonatal BN animals significantly higher levels of Fc-RFL in experimental animals were not evident as early and peaked later (day 12) in both tissue compartments but again these differences remained until death. Cytotoxic alloantisera demonstrated that on days 6, 10, and 12 most, if not all, of the Fc-RFL were host in origion in both DA and BN GVHD, with a very significant host plasma cell response in such GVHD animals. One-micron tissue section revealed the presence of a great number of plasma cell especially prominent in the medulla of lymph nodes with the cortex of lymph nodes and white pulp of the spleen markedly depleted of lymphocytes indicative of cytotoxicity.
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Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization. A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III.
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Kinetics of the antibody response to type III pneumococcal polysaccharide. II. Factors influencing the serum antibody levels after immunization with an optimally immunogenic dose of antigen. When the number of PFC present in the spleen was measured at 24-hr intervals after immunizing with an optimally immunogenic dose of type III pneumococcal polysaccharide (SSS-III), maximal numbers of PFC were attained 4 days after immunization; thereafter, the number of PFC decreased rapidly. By contrast, serum antibody levels, which were measured in the same mice using a Farr test, reached peak values 5 days after immunization and then declined much more slowly than did the number of PFC. Two factors were found to contribute to this disparity. First, experiments conducted with splenectomized mice showed that extrasplenic antibody synthesis, which began between days 3 and 4 after immunization and peaked on days 6 to 7, accounted for nearly one-third of the total amount of serum antibody produced. Second, the average rate of antibody synthesis by PFC increased through day 6 after immunization and then declined. Antigen-antibody dissociation tests showed that the avidity of the serum antibody obtained 4 to 7 days after immunization was the same. Moreover, during the same interval, all the antibody detected by the Farr test was of the IgM class. Thus, a change in avidity or class of immunoglobulin after day 5 did not account for the disparity observed. The clearance rate of antibody injected i.v. into nonimmune and immunized mice was studied. The data obtained indicated that accelerated clearance of antibody was occurring prior to day 3 after immunization; however, after day 3 the antibody clearance rate was constant and was the same as that found when antibody was injected into nonimmune mice. These findings affirmed the results of previous studies showing that treadmill neutralization was not important in determining the serum antibody levels present after immunization with an optimally immunogenic dose of SSS-III.
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Mouse epidermal aryl hydrocarbon hydroxylase. Mouse skin contains a NADPH-dependent, aryl hydrocarbon hydroxylase (AHH), which is inducible by polycyclic aromatic hydrocarbons. In general, unsubstituted polycyclic hydrocarbons caused a greater induction of epidermal AHH than substituted one (1,2,3,4-dibenzanthracene greater than 1,2,5,6-dibenzanthracene greater than benz (a)anthracene equal 3-methylcholanthrene greater than 7,12-dimethlbenz (a)anthracene) which did not correlate with their ability to initiate tumors in mouse skin. Two different techniques were used to isolate epidermis and similar results were obtained with both. However, the technique of isolating epidermis using a mild heat treatment required that the temperature be maintained at 52 degrees C for 30 sec. If the temperature was raised to 54 degrees C or above, there was a large reduction in the AHH activity. Isolated epidermis has 4 to 5 times the AHH activity as dermis and about twice that of whole skin. This was true for control mice or mice in which AHH was induced by pretreatment with benz(a)anthracene.
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Defects in the biochemistry of collagen in diseases of connective tissue. The collagens are the major structural glycoproteins of connective tissues. A unique primary structure and a multiplicity of post-translational modification reactions are required for normal fibrillogenesis. The post-translational modifications include hydroxylation of prolyl and lysyl residues, glycosylation, folding of the molecule into triple-helical conformation, proteolytic conversion of precursor procollagen to collagen, and oxidative deamination of certain lysyl and hydroxylysyl residues. Any defect in the normal mechanisms responsible for the synthesis and secretion of collagen molecules or the deposition of these molecules into extracellular fibers could result in abnormal fibrillogenesis; such defects could result in a connective tissue disease. Recently, defects in the regulation of the types of collagen synthesized and in the enzymes involved in the post-translational modifications have been found in heritable diseases of connective tissue. Thus far, the primary heritable disorders of collagen metabolism in man include lysyl hydroxylase deficiency in Ehlers-Danlos syndrome type VI, p-collagen peptidase deficency in Ehlers-Danlos syndrome type VII, decreased synthesis of type III collagen in Ehlers-Danlos syndrome type IV, lysyl oxidase deficency in S-linked cutis laxa and Ehlers-Danlos syndrome type V, and decreased synthesis of type I collagen in osteogenesis imperfecta.
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Some properties of proteolysis by polymorphonuclear leukocyte-granule extracts. The extracts of granules of human polymorphonuclear leukocytes hydrolyzed a variety of proteins including human and bovine hemoglobin, human fibrinogen, human and bovine serum albumin, bovine elastin, and casein. The hydrolysis of all the proteins except fibrinogen and elastin was increased by addition of urea. Various inhibitors of trypsin, kallikrein, plasmin, Clr, Cls, and other proteolytic enzymes had no inhibitory effect. Slight inhibition was observed with polyanethol sulfonate and strong inhibition with normal human serum. Serum of patients with hereditary angioneurotic edema having no functional C1-esterase inhibitor was as effective in inhibiting the proteolysis as normal serum. The inhibitor was localized in 4S fractions of normal serum fractionated on Sephadex G-200. Fractionation of normal serum by ammonium sulfate precipitation, Sephadex G-200 filtration, and CM-Sephadex chromatography did not result in appearance of inhibitory activity in more than one protein peak, suggesting the possibility that only one inhibitor might be responsible. Since all fractions which contained the inhibitor of proteolysis also contained alpha1-antitrypsin, since sera of patients having low alpha1-antitrypsin levels contained less inhibitory activity, and since antibodies against alpha1-antitrypsin reversed the inhibition obtained from normal serum, the inhibition of proteolysis may be attributed to alpha1-antitrypsin.
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Inhibition of the antibacterial activity of gentamicin by urine. Urinary levels of antibiotics determine the outcome of treatment of most urinary tract infections. The antibacterial effect of gentamicin against Escherichia coli and Pseudomonas aeruginosa in urine was studied. With use of urinary constituents in concentrations normally found in human urine, it was shown that urine has an inhibitory effect that is dependent upon the acidity and total osmolality of the urine, as well as upon the presence of individual solutes. Up to 40 times as much gentamicin may be needed to prevent the growth of E. coli or P. aeruginosa in concentrated, acidic human urine as is required in broth. This inhibitory effect may be particularly important when urinary concentrations of gentamicin are reduced either because of a reduction in dosage or because of decreased excretion due to renal insufficiency.
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The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition. A large-molecular-weight capsular polysaccharide was isolated from strains of Bacteroides fragilis subspecies fragilis. By means of electron microscopy and staining with ruthenium red, the thick polysaccharide capsule was also visualized. With use of a radioactive antigen-binding assay, antibody to this capsular polysaccharide was demonstrated in antisera prepared in rabbits to each of eight strains of B. fragilis fragilis. Antibody of similar specificity was not found in antisera prepared to Bacteroides melaninogenicus or to strains of Bacteroides fragilis subspecies vulgatus and Bacteroides fragilis subspecies distasonis; such antibody was found in antisera to only one of two strains of Bacteroides fragilis subspecies thetaiotaomicron. The radioactive antigen-binding assay is a sensitive test for the detection of antibody to capsular polysaccharide. This polysaccharide antigen may form the basis of a serogrouping system for B. fragilis.
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Radioassay for serum and red cell folate. A simple, reliable assay for serum and red cell folate is described. It uses plain untreated liquid or powdered milk, requiring no special handling or purification, as binder. Such milk makes it possible to ignore endogenous serum folate binder, since crude (but not purified) milk contains a factor which releases folate from serum binder. It simplifies counting radioactivity by employing a gamma emitting isotope of pteroylglutamic acid (PGA), namely the 125I-tyramide of PGA. Like the 3H-PGA assay of Givas and Gutcho, it permits the use of stable PGA rather than unstable methyltetrahydrofolic acid (MeTHFA) standards, because it is carried out at pH 9.3, a pH at which milk folate binder is unable to distinguish PGA from MeTHFA, which is the predominat folate in human tissues. The equipment required to do the radioassay is present in most diagnostic chemistry laboratories. Results are essentially identical to the generally accepted Lactobacillus casel microbiologic method of folate assay, except that false low results are not produced in the radioassay by antibiotics, tranquilizers, and chemotherapeutic agents. Three caveats in its use are the relative instability of 125I-PGA as compared to 3H-PGA, the fact that various powdered milks differ widely in folate-binding capacity, and that only about 60 per cent of commercially obtained skim or powdered milk preparations appear to contain the substance which splits folate from serum binder.
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The effect of environmental pH on glycosaminoglycan metabolism by normal human chondrocytes. The synthesis and release of sulfated glycosaminoglycans by normal human chondrocytes in culture are markedly affected by environmental pH. The biosynthetic rate is increased threefold as the pH of the growth medium is raised from 7.0 to 8.0. This coincides with a corresponding elevation in total protein and cell growth. The rate of release of newly synthesized sulfated glycosaminoglycans from the cell layer as well as their distribution between intra- and extracellular localization in the cell layer is also modulated by environmental pH. At pH 8, 35 per cent is found within the cells, this value is reduced to 13 per cent at pH 7. Pulse-chase experiments showed that previously incorporated sulfated proteoglycans were released at a faster rate at pH 7 than at pH 8. The data suggest that proton concentrations affect the biosynthesis and the mode of distribution of newly synthesized sulfated glycosaminoglycans.
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Effect of PGA1, PGE2, diazoxide on myocardial contractile force. Experiments were conducted in anesthetized dogs comparing the effects of PGA1, PGE2, and diazoxide on myocardial contractile force (MC). The three agents were given in successive bolus injections intravenously in equidepressor doses and myocardial contractile force was measured by means of a strain-gauge arch sutured onto the right ventricle. The drugs were administered before and during ganglionic (hexamethonium) and beta-blockade (practolol). Both PGA1, and PGE2 caused a marked rise in MC, 24 and 20 per cent, respectively, before blockade and 10 and 11 per cent during blockade. Diazoxide caused only a minimal rise, 0.9 per cent, before blockade and a marked fall, 27 per cent, during blockade. Diazoxide administration during left ventricular bypass indicates that the decrease in MC is not a direct result of alterations in preload or after load. It is suggested that hypertensive patients treated with autonomic blocking agents may be more susceptible to heart failure in response to diazoxide therapy.
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Peptide-binding macromolecules in the blood of seriously ill or mentally retarded patients. This report describes macromolecules that bind (des-aspartic acid1)-angiotensin II, the des aspartic acid1 derivative of angiotensin I, and several biologically active and inactive analogues of these polypeptides. The macromolecules were found in the plasma of approximately 2 per cent of ambulatory adults and hospitalized children and 32 per cent of the patients at two institutions for the mentally retarded. The binding properties of these macromolecules were studied by incubating with peptides labeled with 125iodine, and separating bound from free labeled peptide using small gel filtration columns. The peptide-binding macromolecules from several patients were compared. They showed very similar specificity for a group of arginyl peptides of the des-aspartyl1-angiotensin sequence. The plasma binders differed from one another in their optimum pH and their mobility in electrophoretic fields. Those with more acid pH optima displayed more rapid electrophoretic mobility. The binders fell into two classes based on apparent molecular weight, approximately 140,000 and 250,000. Those with the higher apparent molecular weight contained a large proportion of binder that could be precipitated with antiserum to human IgA. Kinetic measurements showed that the plasma binders were somewhat heterogeneous with respect to affinity for (des-asp1)-angiotensin, with apparent association constants ranging from 10(7) to 10(8) M-1. Binding activity was labile to heat, and to treatment with pepsin or trypsin. It was inhibited by calcium, protamine, streptomycin, and some other cationic compounds. The plasma peptide binder differed in specificity and molecular weight from soluble angiotensin-binding molecules extracted from tissues, and from properties expected of a receptor for angiotensin. These macromolecules may be useful reagents for measuring (des-asp1)-angiotensins. Their presence in plasma samples may interfere with angiotensin assays in some circumstances.
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The transmission of impulses in the ectodermal slow conduction system of the sea anemone Calliactis parasitica (Couch). 1. The SS 1 fatigues in response to repetitive electrical stimulation. This fatigue is manifested by an increased conduction delay and a decreased SS 1 pulse amplitude. 2. Continued repetitive stimulation leads to the failure of the system. Recovery may take many seconds. Narrow strips of column fail more rapidly than wide strips. 3. The increased conduction delay is explained in terms of a decrease in the population of spiking cells. 4. A computer model is described and analysed. It suggests that conduction between electrically coupled ectoderm cells could be the basis for the SS1. The SS 1 may have properties not so far experimentally demonstrated; for example, under certain conditions it could behave as a local system.
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Colonial conduction systems in the Anthozoa: Octocorallia. 1. The octocorals Alcyonium digitatum, Pennatula phosphorea and Virgularia mirabilis each have a through-conducting nerve net. The nerve net demonstrated electrophysiologically may well be the same as that previously shown by the use of histological techniques. 2. It exhibits both facilitation and defacilitation in the rate of conduction of pulses. 3. The distance of spread of nerve net activity is not limited by the number of stimuli applied. 4. The nerve net controls fast muscle contractions; the frequency of pulses is important in determining which muscles contract and in which sequence. 5. The nerve net is 'spontaneously' active. 6. A previously undescirbed slow system has been identified in Pennatula. It has many of the properties of slow systems in sea anemones and may well be ectodermal. It is suggested that multiple conduction systems are of common occurrence in the Anthozoa.
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Water regulation by a presumptive hormone contained in identified neurosecretory cell R15 of Aplysia. Injection of an homogenate of identified neuron R15 into the hemocele of Aplysia produced a weight increase of 3-10% within 90 min. Control injections of several other identified neurons or of seawater, were ineffective. The weight increase occurred even when the animals were maintained in 5% hyperosmotic seawater. The activity of the R15 homogenate was retained after acidification to pH 2 and heating to 100 degrees C; but activity was destroyed by proteolytic digestion with Pronase. Dialysis in cellulose dialysis tubing resulted in a significant loss of aion on Sephadex G-50 (nominal exclusion limits 1,500-30,000 daltons), activity was present in the partially included volumes, but was absent in the totally excluded or totally included volumes. The data support the notion that R15 contains one or more hormones involved in ionic regulation or water balance. The results of bioassays of R15 extracts subjected to different treatments are consistent with the hypothesis that activity is due to one or more stable polypeptides of relatively low molecular weight.
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Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. The intracellular pH (pHi) of squid giant axons has been measured using glass pH microelectrodes. Resting pHi in artificial seawater (ASW) (pH 7.6-7.8) at 23 degrees C was 7.32 +/- 0.02 (7.28 if corrected for liquid junction potential). Exposure of the axon to 5% CO2 at constant external pH caused a sharp decrease in pHi, while the subsequent removal of the gas caused pHi to overshoot its initial value. If the exposure to CO2 was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial fall in pHi was followed by a slow rise, and (b) after the exposure, the overshoot was greatly exaggerated. Application of external NH4Cl caused pHi to rise sharply; return to normal ASW caused pHi to return to a value below its initial one. If the exposure to NH4Cl was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial rise in pHi was followed by a slow fall, and (b) after the exposure, the undershoot was greatly exaggerated. Exposure to several weak acid metabolic inhibitors caused a fall in pHi whose reversibility depended upon length of exposure. Inverting the electrochemical gradient for H+ with 100 mM K-ASW had no effect on pHi changes resulting from short-term exposure to azide. A mathematical model explains the pHi changes caused by NH4Cl on the basis of passive movements of both NH3 and NH4+. The simultaneous passive movements of CO2 and HCO3-cannot explain the results of the CO2 experiments; these data require the postulation of an active proton extrusion and/or sequestration mechanism.
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Analysis of an L-histidinol-utilizing mutant of Pseudomonas aeruginosa. Transductional analysis was applied to the Pseudomonas aeruginosa mutant PAO14 (hnc-1). This mutant can utilize L-histidinol as sole source of carbon and nitrogen and has a 60-fold increased histidinol dehydrogenase (HDH) content (Dhawale, Creaser & Loper, 1972). Transductional analysis was carried out using 18 histidine-requiring mutants to see where the hnc-1 locus maps in relation to the structural genes of histidine biosynthesis. The hnc-1 marker cotransduced with group IV genes at 97 to 100 % and not at all with group I, which is known to be the structural gene for HDH. The data obtained in the studies of Km (histidinol) and Km (NAD), and the effect of pH and temperature on the HDH activity from PAO1 and PAO14 are in full agreement with the genetic data that the hnc-1 mutation is not in the structural gene for HDH. It is suggested that hnc-1 may be a mutation in a regulatory gene affecting HDH synthesis in PAO14 and may map close to his-IV whose function in histidine biosynthesis is not known.
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Studies on the rumen flagellate Neocallimastix frontalis. The vast increase in the population density of the rumen flagellate Neocallimastix frontalis shortly after the host animal has commenced eating is caused by stimulation of a reproductive body on a vegetative phase of the organism to differentiate and liberate the flagellates. The stimulant is a component of the host's diet. The vegetative stage of N. frontalis bears a strong morphological resemblance to that of certain species of aquatic phycomycete fungi, and consists of a reproductive body borne on a single, much branched rhizoid. The flagellates liberated in vivo within 15 to 45 min of feeding lose their motility within I h and develop into the vegetative phase, thus producing a rapid decrease in population density of the flagellates. Conditions for maximum flagellate production are similar to those occurring in the rumen: pH 6-5, 39 degrees C, absence of O2, presence of CO2. Differentiation of the reproductive body is inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The organism was cultured in vitro in an undefined medium in the absence of bacteria or other flagellates.
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Properties of some bacteriocins produced by Rhizobium trifolii. Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1-8 X 105 to 2-0 X 105. All were of protein composition, as indicated by buoyant density (1-32 to 1-34 g/cm3) in CsC1 and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production occurred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture.
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Production and purification of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'. The gamma haemolysin of Staphylococcus aureus 'Smith 5R' was produced on Dolman-Wilson agar overlain with cellophane. Maximal yields of crude lysin with titres of 2000 to 4000 haemolytic units/ml were obtained within 24 h at 37 degrees C in 10% (v/v) CO2 in air, on medium adjusted to pH 7-0. The crude lysin was purified 2700-fold (with 75% recovery) by ultrafiltration, gel filtration and ammonium sulphate fractionation. The specific activity of the lysin was 10(5) haemolytic units/mg protein after the dialysed active precipitate was extracted with NaCl and reprecipitated with ammonium sulphate. Purified gamma lysin was homogeneous by disc electrophoresis and immunoelectrophoresis.
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Basic and neutral amino acid transport in Aspergillus nidulans. Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.
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Polyamino acid induced aphid transmission of plant viruses. Aphids transmitted poly-L-ornithine (PLO)-treated tobacco mosaic virus (TMV) when given acquistion and inoculation access periods as brief as 30 s and 2 min, respectively; the ability to transmit was lost within 90 min. Aphids without claws were able to transmit the virus. Transmission thus seems similar to that of nonpersistent viruses. The ratio of virus to polyamino acid, as well as the KCl concentration, markedly affected transmission. Transmission was best from mixtures which contained 250 mug/ml TMV, 2-5 MUG/ML PLO (mol. wt. 120000) and 0-6 M-KCl. A similar mixture favoured transmission when poly-L-lysine (mol. wt. 85000) was substituted for PLO, but with poly-L-lysine (mol. wt. 30 000) it was necessary to decrease the KCl to 0-3 M to obtain transmission. Less KCl (0-08 to 0-24 M) also favoured aphid transmission of PLO-treated potato virus X and tobacco rattle virus. PLO-treated TMV ultracentrifuged in the presence of, and resuspended in, 0-6 M-KCl remained aphid transmissible while PLO-treated virus in 2 M-DCl, which favours greater dissociation of the virus-PLO complex, was transmissible neither before nor after sedimentation by ultracentrifuging, and resuspension in 0-6 M-KCl. these results show that transmissibility is not due to a permanent alteration of the virus by PLO and indicate that the formation of a TMV-PLO complex is required for transmission. Sequential acquisition experiments suggest that PLO may act by binding TMV to receptor sites in aphids. However, the possibility that PLO affects the infection process was not ruled out.
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Disruption of Vi bacteriophage III and localization of its deacetylase activity. It has been shown that particles of Vi bacteriophage III catalyse deacetylation of O-acetyl pectic (polygalacturonic) acid, a structural analogue of Vi polysaccharide (Vi antigen). Using this substrate, and determining the acetic acid liberated by gas-liquid chromatogrphy, a method for the estimation of Vi phage deacetylase activity has been developed. Purified particles of Vi phage III were exposed to a variety of mildly dissociative reagents and conditions, and then tested for plaque-forming and for deacetylase activity. They have also been inspected under the electron microscope. Osmotic shock, and incubation in the presence of ethylenediamine tetraacetic acid (greater than or equil 0-01 M), or of L-arginine (0-25 M), were found to cause disintegration of the virions into empty head capsids, deoxyribonucleic acid, and base plates still carrying the spikes. The mixtures of viral fragments exhibited an increased deacetylase activity. Using zonal sedimentation and ion exchange chromatography, the phage fragments obtained by treatment with ethylenediaminetetraacetic acid have been fractionated and the base plates isolated. Amongst the viral components, these structures showed the highest specific deacetylase activity. They had the shape of six-pointed stars (about 9-5 nm inner, and 14-5 nm outer diam.) with a central hole or plug (approximately 3 nm), carrying six spikes, roughly cylindrical organelles of approx. 11 X 4 nm, one at each of the points. Of the polypeptides of six sizes (P.1, about 153,000 daltons; P.2, 91,000; P.3, 71,000; P.4 56,500; P.6, 22,000), detected in whole Vi phage III virions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, only two, P.2 and P.3 were found in the base plates.
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A model to replace psychiatric hospitals. A comprehensive system of community treatment in southwest Denver has reduced the need for adult psychiatric inpatient beds to less than 1/100,000 population. Six small, community-based therapeutic environments, crisis intervention, home treatment, social systems intervention, and rapid tranquilization comprise the essential components of this total community care system. The system operates within a framework of citizen participation and community control, the elimination of formal staff offices, and a focus on working in the real-life setting of the client and his family. To evaluate the effectiveness of community care, patients about to be hospitalized were randomly assigned to a psychiatric hospital or to community alternative treatment. Outcome measures at discharge and at follow-up completed by the client himself, treatment staff, and family members indicate that community treatment was more effective than psychiatric hospitalization.
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Dynamic changes in regional CBF, intraventricular pressure, CSF pH and lactate levels during the acute phase of head injury. The authors measured regional cerebral 133xenon (133Xe) blood flow (rCBF), intraventricular pressure (IVP), cerebrospinal fluid (CSF) pH and lactate, systemic arterial blood pressure (SAP), and arterial blood gases during the acute phase in 23 comatose patients with severe head injuries. The IVP was kept below 45 mm Hg. The rCBF was measured repeatedly, and the response to induced hypertension and hyperventilation was tested. Most patients had reduced rCBF. No correlation was found between average CBF and clinical condition, and neither global nor regional ischemia contributed significantly to the reduced brain function. No correlation was found between CBF and IVP or CBF and cerebral perfusion pressure (CPP). The CSF lactate was elevated significantly in patients with brain-stem lesions, but not in patients with "pure" cortical lesiosn. The 133Xe clearance curves from areas of severe cortical lesions had very fast initial components called tissue peaks. The tissue peak areas correlated with areas of early veins in the angiograms, indicating a state of relative hyperemia, referred to as tissue-peak hyperemia. Tissue-peak hyperemia was found in all patients with cortical laceration or severe contusion but not in patients with brain-stem lesions without such cortical lesions. The peaks increased in number during clinical deterioration and disappeared during improvement. They could be provoked by induced hypertension and disappeared during hyperventilation. The changes in the tissue-peak areas appeared to be related to the clinical course of the cortical lesion.
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Nursing deans as leaders in collegiate health professions. Deans of Nursing who choose to be administrators of the Health Sciences may very well enhance Nursing programs by facilitating the nursing faculty to become leaders through offering service courses in Health, may thereby help to cut the high cost of nursing education, and may make a contribution to other Health majors by sharing their faculty's special expertise in Health theory.
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Studies of the effect of dietary cholesterol on hepatic protein synthesis, reduced glutathione levels and serine dehydratase activity in the rat. A basal diet or a basal diet plus 1% of cholesterol and 0.33% cholic acid was fed to rats for varying lengths of time and (1) the activities of liver phosphoenolpyruvate-carboxykinase (PEP-CK), tyrosine transaminase (TT), and serine dehydratase (SD); (2) the rate of total hepatic protein synthesis and (3) the concentration of hepatic reduced glutathione (GSH) were quantitated. The specific activity of PEP-CK was significantly depressed by cholesterol plus cholic acid feeding, while the specific activity of TT was unchanged. No significant effect of dietary cholesterol plus cholic acid was found on the total liver activities. In contrast, SD specific activity was increased 3-fold. The rate of (U-14C)-L-leucine incorporation into total TCA precipitable protein following ingestion of cholesterol plus acid was significantly reduced when the data were expressed as dpm (U-14C)-L-leucine/mg protein. After correcting this expression for specific radioactivity of the liver tissue free leucine pool, no significant effect of dietary cholesterol plus cholic acid on hepatic protein synthesis existed. In fact, the amount of 14C-leucine incorporated into protein on a total liver basis was 50% greater for the cholesterol group. On a per gram of liver basis, the concentration of GSH in the liver of rats fed a cholesterol plus cholic acid diet was significantly decreased. Considering the liver enlargement in rats fed cholesterol plus cholic acid, total organ GSH was found to be significantly greater than for rats fed a basal diet.
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Thiamine triphosphatase activity of myosin and accelerating effect of thiamine di- and tri-phosphates on superprecipitation of actomyosin. TTP accelerated ATP-induced superprecipitation of actomyosin in as low a concentration as 30 muM and decreased light scattering by actomyosin. These effects could also be observed in the same way, but to a lesser degree, by addition to TDP. Myosin was able to hydrolyze TTP to TDP, but some important differences were confirmed between myosin TTPase and ATPase. Myosin TTPase was inhibited by actin and showed a much larger Km than that of ATPase. TTP significantly inhibited myosin B ATPase and ATP greatly inhibited myosin B TTPase. These findings suggest that the accelerating effect of TDP and TTP may be due to the binding of thiamine phosphate to the regulatory site of myosin followed by a change in its physical chemical property, rather than due to the competitive binding of thiamine phosphate to the catalytically activity site of myosin.
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The meaning of the Leydig cell in relation to the etiology of cryptorchidism: An experimental electron-microscopic study. In our electron-microscopic studies of testicular biopsies, both normal and cryptorchid, we found a simple atrophy of the Leydig cell in the cryptorchid testis. Based on experiments by Raynaud1,2 and Jean3 on pregnant mice, we tried to find the reason for changes in the Leydig cell relating to the etiology of cryptorchidism. We found on electron microscopic study of testes in the offspring of pregnant mice treated with estrogen the same atrophy of the Leydig cell as we see in human cryptorchidism. These changes are not evident when estrogen and HCG are given together. We can conclude from this experiment that lack of gonadotropin stimulation leads to the atrophy of Leydig cells. This atrophy then produces a lack of androgen which could be responsible for cryptorchidism.
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The in vitro adsorption of drugs from horse serum onto carbon coated with an acrylic hydrogel. In vitro studies have shown that uncoated carbon and carbon coated with an acrylic hydrogel are capable of adsorbing drugs from horse serum at 37 degrees. Increase in the coating weight from 2 to 4% decreased the rate of adsorption but not the total capacity. In vivo data supports the concept of carbon haemoperfusion for use in the treatment of drug overdose.
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The interaction of antihistamines with lecithin monolayers. The interaction of a series of antihistamines with monolayers of L-alpha-dipalmitoyl lecithin has been examined. An increase in the monolayer surface pressure was noted for monolayers spread on the antihistamine solutions, suggesting penetration of the film by drug molecules. At high surface pressures there was an apparent ejection of drug molecules from the film. The ability of the antihistamines to increase surface pressure was correlated with their surface activity at the air-solution interface. The effect of drug concentration on the magnitude of the surface pressure was examined for diphenhydramine hydrochloride. Application of the Gibbs adsorption equation at low surface compressions indicated an approximate area per molecule for diphenhydramine in the film which was in good agreement with the value previously obtained at the air-solution interface. Preliminary measurements showed that the surface pressure increase was larger in the presence of phosphate buffer at pH 6-8. It was not clear whether this effect was caused by the buffer components or was a pH effect.
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