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3896757
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Progesterone (P4) regulation of LH secretion in sheep was studied in vitro using dispersed pituitary cell cultures. Neither cell content nor basal secretion of LH were normally altered by P4, but maximal stimulation of LH secretion by LHRH was suppressed up to 70% with 10(-7) M P4, without changing the ED50 of LHRH. The inhibitory effect of P4 appeared within 6 h and became maximal by 24 h but was partially reversed during continued treatment with P4. The inhibitory effect was completely reversed by 24 h after P4 withdrawal. The ED50 of P4 was 10(-9) M. P4 also decreased the sensitizing effect of 17 beta-estradiol (E2) on LHRH-induced LH secretion in ovine pituitary culture. It partially inhibited the E2-induced increase in maximal response to LHRH and decreased the ability of E2 to lower the ED50 of LHRH. Our results clearly show that P4 can act directly on the ovine pituitary to inhibit both normal and E2-sensitized LH responsiveness to LHRH.
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3896758
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Our study compares the properties of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from a human metastatic virilizing carcinoma and that from normal adrenal glands obtained from kidney donors. Optimal conditions for enzyme assay were obtained when a 50-mM imidazole buffer (pH 7.2) containing 5 mM EDTA, 250 mM NaCl, 1 mM phenyl-methylsulfonylfluoride, 0.1 mM leupeptin, and 5.5 mM dithiothreitol (DTT) was used. A 30-min preincubation period preceding addition of substrates enhanced reductase activity by 1.75-fold. In crude microsomal preparations, Km values were similar for both tumor and normal tissues and varied between 4 and 5 microM (S)HMG-CoA. The presence of NaF in homogenization and incubation media decreased the maximum velocity, but not the Km. A partially purified rat liver phosphorylase phosphatase preparation or a similar preparation from the carcinoma restored to maximal levels the reductase activity of microsomes prepared in the presence of NaF. A Km of 96 microM NADP was found for the carcinoma microsomal preparation. Preincubation of microsomes in the presence of monothioglycerol or DTT resulted in an increased reductase activity, suggesting a possible inactive enzyme precursor(s) consisting of disulfide-linked units. Reactions of the DTT-activated enzyme incubated in the presence of increasing amounts of NADPH showed sigmoidal kinetics. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a 92.5K mol wt protein band that reacted with a rat antireductase antibody. Reductase activity in different regions of the carcinoma varied from 679-1763 pmol/mg protein, with an average of 1146 pmol. In three normal adrenal glands we found values of 23.4, 48.1, and 36 pmol. We concluded that the expression of HMG-CoA reductase activity was elevated in human adrenal carcinoma.
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3896759
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GnRH has been shown to induce premature meiotic maturation in preantral follicles of the immature estrogen-primed hypophysectomized rat. As these animals are free of circulating gonadotropins and contain large numbers of full-grown oocytes in preantral follicles, we have investigated this model to determine its usefulness in studying meiotic maturation. We show that a maximum dose of the agonist D-Trp6,Pro9,Net-LRF (GnRH-a) induces approximately 25% of full grown oocytes to resume meiosis within a 12-h period. This response is dose dependent (ED50 = 0.24 microgram/rat) and specific for GnRH-a. GnRH-a stimulates germinal vesicle breakdown and first polar body formation within 2 and 8 h, respectively. More than 75% of those oocytes that initiate meiotic maturation reach metaphase II by 15 h. This effect of GnRH parallels the time course of physiological meiotic maturation triggered by LH as well as that of oocytes maturing spontaneously in vitro. Oocytes in primordial and primary follicles do not respond to GnRH. The majority of affected follicles are small tertiary follicles (200-400 micron in diameter) and show signs of atresia. This atresia is not caused by GnRH-a and does not, in itself, result in meiotic maturation, but appears to confer susceptibility to GnRH-a-induced meiotic maturation. Our studies indicate that this animal model will be useful to elucidate further the mechanisms and requirements for meiotic maturation. It will also facilitate investigation of the role of atresia in the GnRH response of tertiary follicles and the issue of follicle heterogeneity within these animals.
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3896760
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A transient increase in serum progesterone concentrations (to 1 ng/ml for 1-2 days) is observed in the majority of ewes before the first estrous cycle of the breeding season. To determine whether such a brief antecedent rise in progesterone ensures initiation of a full-length cycle by the next LH surge, synthetic GnRH was administered for 3 days to 24 anestrous ewes in a pulsatile fashion designed to mimic the pattern of LH secretion during the preovulatory period of the breeding season. Six ewes received no further treatment, and 6 ewes were treated sc with Silastic implants containing progesterone for 3 days before injecting GnRH. The remaining 12 ewes were treated with additional injections of GnRH every 4 h for the next 5 days. Four of these ewes received a second increase in GnRH pulse frequency, every 2 h and hourly on the subsequent 2 days. An LH surge was stimulated by each regimen of increasing GnRH pulse frequency in all ewes; progesterone pretreatment had no effect on its time of onset, duration, or amplitude. The LH surges induced full-length luteal phases in 10 of 10 ewes when preceded by either an exogenous (n = 6) or an endogenous (n = 4) progesterone increment, but in only 8 of 18 ewes not pretreated with progesterone. These results indicate that a transient increase in progesterone ensures that an ensuing LH surge will initiate an estrous cycle and suggest that progesterone may play an important role in the endocrine mechanisms governing transitions from acyclic to cyclic states.
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3896761
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Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.
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3896755
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Infantile autism is a behaviorally defined syndrome. Many investigators suggest that it is probably related to a brain dysfunctioning in which catecholamine metabolism is involved. The aim of the following paper is to study the validity of different clinical and biological markers of this syndrome: 1. clinical scores using a behavioral rating scale, 2. amplitude and variability of conditioned auditory evoked potentials, 3. urinary levels of homovanillic acid. The response of these markers in autistic children treated with vitamin B6 and magnesium have been assessed. The relationships found between clinical and biological data are discussed. It can be expected that such an approach would contribute to a better understanding of the underlying mechanism of autism.
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3896763
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Cold-exposed rats exhibit hypermetabolism, hyperphagia, and increased glucose oxidation. Their counterregulatory hormone secretion is markedly elevated, while insulin levels fall acutely, gradually returning to basal during acclimation. We assessed both hepatic and peripheral sensitivity to insulin in rats in the basal state and after 5 days of cold (5 C) exposure. The contribution of gluconeogenesis to total glucose turnover was measured and compared to daily urinary corticosterone excretion. Hepatic glucose production was equally suppressed by the infusion of insulin at 1.2 mU/kg X min in both control and cold-acclimated rats, but enhanced hepatic sensitivity to low dose (0.6 mU/kg X min) insulin infusion was only observed after cold exposure. The metabolic clearance of glucose was elevated with cold stress and was insensitive to the infusion of insulin at either level. Insulin resistance was not observed. Urinary excretion of corticosterone and urea nitrogen were markedly increased, but creatinine excretion was unchanged, suggesting that the concurrent increase in gluconeogenesis resulted from increased protein intake rather than increased catabolism of muscle protein.
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3896762
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Morphological evidence suggests that GnRH may be released into cerebrospinal fluid (CSF) of the third ventricle. Therefore, a method of cannulating the third ventricle of monkey brains was developed for the purpose of examining GnRH secretion in primates. A stainless steel guide cannula was stereotaxically implanted into the third ventricle of 14 ovariectomized rhesus monkeys. A Silastic cannula for collecting CSF was inserted via the guide cannula into the ventral portion of the ventricle, permitting repeated CSF sampling for long time periods from the same animal. One week to 6 months after cannulation, CSF was collected continuously for periods of 5-10 h at 2 different rates (480 and 120 microliter/h) from conscious monkeys seated in chairs. Samples were divided into 15-min fractions, and the GnRH concentration in each was determined by RIA. In contrast to most previous studies, third ventricular CSF was found to contain significant concentrations of GnRH. GnRH was detected in 40 of 50 collections. Concentrations ranged from less than 8 to greater than 800 pg/ml, a range similar to that observed in hypophyseal portal blood. Furthermore, fluctuations within individual collections indicated that GnRH was released in pulses. The mean GnRH pulse frequency during the higher rate of CSF withdrawal was 0.43 +/- 0.06 pulses/h (n = 31), while the mean pulse amplitude was 91 +/- 7 pg/ml (n = 64). Neither parameter was influenced by the rate of CSF removal, as frequency was 0.52 +/- 0.08 pulses/h (n = 19) and amplitude was 94 +/- 11 pg/ml (n = 82) during the lower collection rate. However, the CSF withdrawal rate had a profound influence on LH secretion. In 12 of 17 collections at the higher rate, LH levels plummeted to undetectable concentrations during the first 2 h of CSF exfusion and remained low throughout the collection period. Pituitary responsiveness was not reduced, as a GnRH bolus (0.25 or 2.5 micrograms) after 6 h of CSF removal elicited a dose-dependent stimulation of LH secretion. In contrast, a higher incidence of normal pulsatile LH secretion (12 of 19 collections) was observed when the CSF withdrawal rate was reduced. During these 12 collections, LH and GnRH pulses occurred at regular intervals and exhibited similar pulse frequencies (mean +/- SE, 0.76 +/- 0.07 and 0.67 +/- 0.09 pulses/h for LH and GnRH, respectively). Most GnRH and LH pulses were synchronized, as 86% of all GnRH pulses (43 of 50) were accompanied by a LH pulse.(ABSTRACT TRUNCATED AT 400 WORDS)
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3896764
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We compared the role of Ca2+ in regulating GnRH-induced LH synthesis and release from cultured rat pituitary cells. LH synthesis and release were measured after a 4-h treatment of cells with gallopamil hydrochloride (D600; 1 and 100 microM), a Ca2+ channel blocker, or pimozide (0.5 and 5.0 microM), a calmodulin inhibitor, with or without 1 nM GnRH. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. GnRH significantly (P less than 0.01) increased total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation). D600 significantly (P less than 0.01) depressed (1 microM) and completely blocked (100 microM) GnRH-induced LH glycosylation and release of [3H]LH, [14C]LH, and immunoreactive LH. D600 (100 microM) also reduced (P less than 0.05) total basal synthesis of [14C]LH. Neither dose of D600 altered uptake of [3H]glucosamine, but 100 microM D600 significantly (P less than 0.01) depressed its incorporation into total protein. D600 (100 microM) significantly (P less than 0.01) depressed [14C]alanine uptake and incorporation into total protein. Pimozide significantly (P less than 0.01) reduced, in a dose-related manner, GnRH-induced LH glycosylation, and release of immunoreactive LH, [3H]LH, and [14C]LH. Pimozide did not alter LH translation or uptake of radiolabeled precursors or their incorporation into total protein. These results demonstrate that D600 and pimozide inhibit both GnRH-induced LH glycosylation and release. Thus, the actions of GnRH on LH glycosylation and release are both mediated by similar Ca2+-dependent pathways.
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3896765
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Purified hog thyroid lysosomes, prepared by a procedure previously developed in this laboratory, were used to study lysosomal digestion of [131I]thyroglobulin [131I]Tg). The lysosomal proteases were solubilized with 0.1% Triton X-100. Rates of proteolytic digestion, measured by the release of ethanol-ammonium acetate-extractable 131I, were greatly stimulated by thiol reagents. The pH optimum was also affected by the presence of thiols. In the absence of a thiol reagent, a broad pH optimum was observed, ranging from 3.5-4.5. However, in the presence of 1 mM mercaptoethanol, the maximum rate of digestion occurred at pH 5.0, very close to reported values for the internal pH of lysosomes. Pepstatin, an inhibitor of cathepsin D, markedly inhibited lysosomal digestion of [131I]Tg at concentrations as low as 0.01 micrograms/ml. Its inhibitory effect was greater at pH 3.5 (pH optimum of cathepsin D) than at pH 5.0. Leupeptin, an inhibitor of thiol proteases, was not as potent as pepstatin, but it was significantly inhibitory at a concentration of 1 microgram/ml. In contrast to pepstatin, leupeptin displayed a greater inhibitory effect at pH 5.0 than at pH 3.5. The pH optimum of hog thiol proteases has been reported to range from 5.5-6.5. The effects of the two inhibitors were additive at pH 5.0. We conclude from these results that both cathepsin D and thiol proteases play a role in lysosomal digestion of Tg. Cathepsin D appears to be quantitatively more important than thiol protease in the initial phase of the digestion. The stimulatory effect of thiols on lysosomal digestion of [131I]Tg probably involves two separate effects: 1) stimulation of thiol proteases, and 2) reduction of S-S bonds in Tg, making the protein more susceptible to attack by proteolytic enzymes. Poorly iodinated [131I]Tg was more rapidly hydrolyzed than well iodinated [131I]Tg, based on the release of ethanol-ammonium acetate-extractable 131I. However, there was little or no difference in the rate of total peptide bond cleavage between poorly iodinated and well iodinated Tg. These results suggest that the first sites of iodination of Tg are preferentially attacked by lysosomal proteases. Long term (24-h) digestion of [131I]Tg with solubilized thyroid lysosomes at pH 5.0 in the presence of thiol compounds was just as effective as digestion with pronase at pH 8.0 in liberating free 131I-labeled iodothyronines and 131I-labeled iodotyrosines. Thus, thyroid lysosomes contain the full complement of proteases and peptidases required for cleaving free iodoamino acids from Tg.
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3896771
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In summary, it has been difficult to assess the permeability properties of the pulmonary capillary membrane. However, new mathematical and experimental techniques have recently been developed which are of sufficient sensitivity and specificity to begin to evaluate the complex mechanisms responsible for many forms of lung pathology. While future work will undoubtedly need to address problems associated with heterogeneity of pulmonary blood flow, and with an equally heterogeneous population of vascular permeability and fluid formation sites, we currently need to focus on using the correct experimental approaches for assessing vascular permeability. The appropriate techniques are described in the text and indicate that the measurements of reflection coefficients using lymph obtained at high vascular pressures, filtration coefficients obtained from both isolated and intact lungs, and two-pore models are useful in assessing vascular permeability.
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3896773
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Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.
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3896774
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A plasmid designated pNF101 was isolated by transforming rad10 mutants with a yeast genomic library and screening transformed cells for enhanced resistance to killing by u.v. radiation. Plasmid pNF101 fully complements the u.v. sensitivity of rad10 mutant strains and was shown to contain the RAD10 gene by genetic analysis of integrant strains. The nucleotide sequence of the RAD10 gene was determined. The coding region consists of 195 codons and could encode a polypeptide of calculated mol. wt. 22 616 daltons. RAD10 protein expressed in Escherichia coli maxicells has a mol. wt of approximately 30 kd measured by gel electrophoresis. The RAD10 gene was localized to chromosome XIII of Saccharomyces cerevisiae by hybridization of the cloned gene to yeast chromosomes resolved by electrophoresis, and by genetic analysis.
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3896775
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Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.
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3896772
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This report summarizes the potential impact of the acid precipitation phenomenon on human health. There are two major components to this phenomenon: the predepositional phase, during which there is direct human exposure to acidic substances from ambient air, and the post-depositional phase, in which the deposition of acid materials on water and soil results in the mobilization, transport, and even chemical transformation of toxic metals. Acidification increases bioconversion of mercury to methylmercury, which accumulates in fish, increasing the risk to toxicity in people who eat fish. Increase in water and soil content of lead and cadmium increases human exposure to these metals which become additive to other sources presently under regulatory control. The potential adverse health effects of increased human exposure to aluminum is not known at the present time.
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3896776
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We have used site specific mutagenesis in vitro to construct a set of deletion mutations within the 5' region of a cloned 23S rRNA gene. In contrast to previously studied mutations in this gene, some of these deletions prevent the incorporation of 23S rRNA into ribosomal particles. This result is discussed in terms of a model in which interaction with the assembly initiator protein, L24, is perturbed.
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3896777
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To investigate the differentiation-promoting potential of c-fos in embryonal carcinoma cells (EC cells) we have designed various human metallothionein promoter-mouse-c-fos gene constructs containing also the selectable SV40 promoter-driven neo gene. Upon transfection into F9 EC cells and selection for neo resistance, the following results were obtained. (i) With each of the constructs, colonies of morphologically altered and differentiated (i.e., TROMA-1 and TROMA-3 expressing) cells were identified. (ii) Expression of c-fos was required to affect the differentiation state of F9 cells to a significant extent, but a low level was sufficient; no enhancement of differentiation was noticeable even after 100-fold induction of c-fos expression by cadmium. (iii) F9 cell clones were isolated which, in spite of very high levels of exogenous c-fos expression, had stem cell morphology. These cells, however, continuously generated morphologically altered and differentiated cells upon subculturing. (iv) In other EC cell lines, which resemble stem cells more closely than the 'partially differentiated' F9 cells, c-fos expression showed either a less pronounced (P19 cells) or no differentiation-promoting effect at all (PC13 cells). Our results suggest that the c-fos gene product acts in concert with other, probably 'spontaneously' occurring events to promote differentiation of certain EC cell lines.
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3896782
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The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X-100. The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties. These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.
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3896783
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The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.
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3896785
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The mechanisms of insulin stimulation of protein synthesis in adipocytes are presently unknown. Addition of 10 nM insulin to isolated rat adipocytes caused a 1.5-2.5-fold increase in the protein synthetic rate and a corresponding increase in nascent chain level, indicating that the effect of insulin on protein synthesis in adipocytes is mediated by a stimulation of ribosomal initiation. The effect on protein synthesis exhibited a lag time of 6-8 min after insulin addition. A similar time dependence was also observed for the insulin-induced phosphorylation of ribosomal protein S6. This supports the proposal that these two phenomena are causally linked.
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3896784
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Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.
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3896787
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A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.
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3896786
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The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.
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3896789
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Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.
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3896788
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The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.
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3896790
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Aspergillus nidulans asparaginase activity may be assayed conductimetrically. The method is based on the increase of conductivity which is due to the production of ammonia and/or aspartate in a reaction mixture containing A. nidulans cell-free extract and asparagine or aspartate hydroxamate. This conductivity is linear with time and enzyme concentration and it follows Michaelis kinetics. Conductimetric activity was not detectable in mutants lacking asparaginase activity.
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3896791
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Escherichia coli like most gram-negative bacteria with walls maintains a cytoplasmic osmolarity exceeding that of the medium; the resulting hydrostatic pressure (turgor pressure) pushes the cytoplasmic membrane against the peptidoglycan and creates a tension in the two envelopes. Potassium is the only cation which takes part in the regulation of cellular osmolarity. The adaptation of intracellular K+ concentration to external osmolarity involves K+ turgor-controlled fluxes. When the medium osmolarity is raised an osmodependent influx of K+ can be observed; this is carried out by the K+ transport system TrkA which can also taken up rubidium. A specific and unidirectional pathway allows K+ ions to flow out of the cell when the medium osmolarity is decreased; this pathway reveals two characteristics: it has no affinity for rubidium and it can be blocked by the blockers of eukaryotic K+ channels. Osmodependent fluxes are turned on immediately after the medium osmolarity is disturbed; in contrast, they are turned off gradually as the rate of K+ fluxes approach zero. The rate of K+ influx seems to depend on the level of internal osmolarity and not on the extent of the increase in medium osmolarity. The rate of the efflux is directly proportional to the decrease in medium osmolarity and is independent on the level of internal osmolarity.
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3896792
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An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.
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3896794
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Percutaneous nephrostomy (PCN) was performed 70 times in 46 patients. In the majority of cases the procedure was carried out as an emergency to relieve urinary obstruction. 20 of the patients had subsequent elective surgery, in others PCN allowed time for other forms of treatment to become effective. The technique carries a low mortality and morbidity, can be performed using either fluoroscopic or ultrasound imaging and should be available in all radiology departments.
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3896795
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Two cases are described in which a tortuous pulmonary density, suspicious of vascular anomaly, was studied using venous digital subtraction angiography. One proved to be a pulmonary varix, the other an A-V fistula. The establishment of the diagnosis of such lesions is discussed and the value of D.S.A. is emphasized in comparison with conventional pulmonary angiography by right heart catheterization.
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3896793
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An enzyme catalyzing the reduction of methylglyoxal was isolated from Saccharomyces cerevisiae and its enzymatic properties were analyzed. The enzyme, specifically eluted from a blue-dextran--Sepharose CL-6B column by the substrate, methylglyoxal, was homogeneous on polyacrylamide gel electrophoresis. The enzyme consisted of single polypeptide chain with a relative molecular mass of 43 000. The enzyme was glycoprotein and contained 6.6% carbohydrate. NADPH was specifically required for activity and the Km for NADPH was 2.0 X 10(-7) M. The enzyme was active on various glyoxals such as glyoxal, methylglyoxal (Km = 5.88 mM) and phenylglyoxal (Km = 1.54 mM). The reaction catalyzed by the enzyme was virtually irreversible. The activity was inhibited by sulfhydryl agents and activated by reducing agents such as glutathione. Intermediates in glycolysis, nucleosides, nucleotides, polyamines and various metal ions showed little inhibitory or activating effects on enzyme activity. Tricarboxylic acids showed a slight inhibitory effect. The activity of the enzyme was strongly inhibited by anionic detergents. The enzyme was rapidly inactivated by incubating with the substrates probably because of the non-enzymatic interaction between glyoxals and NH2 groups in arginine residues in the enzyme. NADP, one of the reaction products, also inhibited the enzyme activity and the Ki for NADP was about 0.07 mM. We tentatively designated the enzyme methylglyoxal reductase.
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3896796
|
A series of 82 consecutive patients scheduled for operation, with pre-operatively obtained P.O. cholecystography and in some cases also I.V. cholangiography, is presented. All patients had cholecystosonography performed "blindly" the day prior to the operation (76 had a cholecystectomy and six had a vagotomy). Based upon the operative findings, the diagnostic value of ultrasonic examination for gallstones can be calculated to predictive value of positive test--1.00; predictive value of negative test--0.71. The diagnostic failures are discussed but it is not possible to predict which patients will benefit more from peroral cholecystography rather than from ultrasound. It is concluded that cholecystosonography is a safe alternative to peroral cholecystography.
|
3896798
|
One case of pyogenic splenic abscess, diagnosed and treated by several ultrasonically guided fine needle punctures, is presented. This noninvasive procedure provided a definitive therapy preserving the spleen. Evacuating fine needle puncture seems to be an alternative therapy of splenic abscess, especially in patients with reduced operability.
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3896797
|
The sonographic aspect of the gallbladder in seven cases of haemobilia is analysed. The changes include a diffuse echogenic gallbladder content during the initial stage. Later, irregular shaped inhomogeneous non shadowing masses are seen in the dependent parts of the lumen. This variable aspect was studied in an experimental model by percutaneous injection of blood in the gallbladder of Guinea pigs. The hyperechoic gallbladder content, seen early after injection is a transitory phenomenon which seems related to red cell aggregation before coagulation occurs. During the later stage the intraluminal masses were shown by histology to represent bloodclots. In a patient the observation of such a rapid evolution from diffuse hyper-reflectivity to less reflective masses is strongly suggestive of haemobilia.
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3896799
|
Two rare diseases of the thyroid gland are described. One, an intracystic carcinoma, has apparently only been described once before. The other, a mousetyphoid induced abscess, is extremely rare. Without ultrasonography both conditions would have been misdiagnosed in the first instance. It is advocated that all uncertain thyroid conditions and all "cold" areas on scintigraphy should be examined with ultrasonography.
|
3896800
|
The appearance of a renal abscess on realtime-ultrasound, excretion urography and computed tomography is presented of an operatively and histologically proven case. An unusually hyperdense rim of the lesion is observed on native CT-scan. Knowledge of clinical symptoms is most important for correct interpretation of diagnostic imaging techniques.
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3896801
|
Omnipaque (iohexol) 350 mg I/ml has been compared with Telebrix (ioxithalamate) 380 mg I/ml in 48 patients undergoing intravenous urography. The contrast medium dose corresponded to 400 mg I/kg body weight. No cardiovascular reactions (BP and pulse rate) were observed. Subjective reactions occurred somewhat more frequently after Telebrix than after Omnipaque. Sensation of warmth was significantly less with Omnipaque (p less than or equal to 0.05). The overall radiological quality was equally good for the two contrast media.
|
3896803
|
We present a detailed immunofluorescence study of the distribution of microtubules in Lytechinus pictus from fertilization until first cleavage, using an improved technique for extraction and fixation of sea urchin eggs. Eggs were prepared for fixation by brief treatment with a buffer selected for its ability to maintain mitotic spindle birefringence while extracting opaque cytoplasm. Subsequent glutaraldehyde fixation and borohydride treatment provided reliable preservation of microtubule arrays with very low background fluorescence. 4'-6-Diamino-2-phenylindole (DAPI) staining of the chromosomes allowed events of the chromosomal cycle to be related to those of the microtubule cycle. By sampling cells frequently between fertilization and first cleavage, we obtained good images of transitional stages between monaster, interphase asters, pause asters, and the mitotic spindle, as well as the changes in spindle structure during mitosis. These showed that: During the growth of the sperm aster, microtubules are present elsewhere in the cell. The monaster does not persist into interphase and divide, but rather breaks down simultaneously with the formation of the bipolar interphase array. During mitotic spindle formation fibers from the pause asters extend around the nuclear envelope, on the surface of which chromosomes occupy discrete sites. Upon nuclear envelope breakdown, these fibers penetrate the nuclear region as the chromosomes move to the metaphase plate, consistent with chromosomal capture by the forming spindle. During anaphase, the mitotic poles become hollow and elongated perpendicular to the long axis of the spindle, consistent with recent studies on centrosomal shape changes during mitosis.
|
3896802
|
Meglumine/sodium diatrizoate was administered as a one minute bolus of 60 ml. (60%) and subsequent 5 minute infusion of 250 ml. (24%) into antecubital veins during pelvic CT Scanning of 15 patients. Blood samples were taken from the opposite arm before and at 1 and 5 minutes after starting the contrast medium injection. The average plasma concentrations of diatrizoate were 2.2 +/- 0.5 and 3.8 +/- 0.8 mg./ml. in the 1 and 5 minute blood samples, respectively. The systemic levels of thromboxane A2 (TXA2) increased in 10 patient's plasma during contrast medium infusion while the level of prostacyclin (PGI2) remained unchanged. One patient had immediate adverse reaction due to contrast without any changes in PGI2 of TXA2 levels. Contrast medium infusion increased the systemic ratio of TXA2/PGI2 which may predispose to hemostatic disorders.
|
3896804
|
The intermediate filament (IF) system of the various cells of human, pig and rat ovaries was studied by electron microscopy, by immunolocalization using antibodies to cytokeratins, vimentin, desmin and desmoplakin, and by two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissue samples. In human ovaries, surface epithelial cells (mesothelium) were stained by antibodies against cytokeratins, desmoplakins and vimentin. Biochemical analysis revealed cytokeratins Nos. 8, 18 and 19, together with variable amounts of No. 7. Granulosa cells of follicles of all stages were also positive for cytokeratins, desmoplakins and vimentin, in agreement with the electron microscopic finding of desmosomes in these cells. As the follicle matured, the cytokeratin content usually appeared to decrease, whereas vimentin remained unchanged. On gel electrophoresis, granulosa cells presented cytokeratins Nos. 8 and 18 and vimentin. Rete ovarii cells were also positive for both cytokeratins, desmoplakins and vimentin, and the electron microscopy revealed numerous desmosome-tonofilament complexes. Oocytes appeared to be devoid of IFs. Corpus luteum cells were rich in vimentin but biochemical analysis also revealed small amounts of cytokeratins Nos. 8 and 18. In contrast, cells of the ovarian stroma and luteinized stromal nodules were positive for vimentin only. A certain type of scattered stromal cells, especially around tertiary follicles and corpora lutea, and also desmin-positive. Pig and rat ovaries differed from human ones in that vimentin was not detected in ovarian mesothelium and cytokeratins were not seen in granulosa cells. The latter, however, contained significant amounts of vimentin. These results indicate that three cell types of human ovary, i.e. surface epithelial, granulosa and rete ovarii cells, can be regarded as true epithelial cells which, however, simultaneously express vimentin, a phenomenon frequently seen in cultured epithelial cells but uncommon in epithelial tissues. The presence of cytokeratins in granulosa cells in all types of human follicles is discussed with regard to the development of these cells. In contrast, granulosa cells of the other two mammalian species only display vimentin IF. Such differences between different mammalian species in IF composition of ovarian components present an example which precludes extrapolation of data from one species to another. The results are discussed in relation to current views of the histogenesis of various ovarian tumors.
|
3896805
|
In this study we show that harmine treatment (10 mg/l for 2 or 24 h) of PtK2 cells had a marked effect on the localization of the nucleolar phosphoproteins C23 and B23. C23 was localized with silver staining in the fibrillar areas of completely segregated nucleoli. B23 was localized mainly on the periphery of the nucleoli with the aid of immunofluorescence.
|
3896806
|
The endosomal compartment of giant HeLa cells was labelled with a transferrin-horse radish peroxidase (HRP) conjugate. Serial thin sections from the leading lamella of a cell are presented; they show that the endosomal compartment contains a tubular system connected to vesicular structures. In addition, small (approximately 50 nm) coated vesicles are seen in the leading lamella.
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3896807
|
Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.
|
3896809
|
The cellular localization of the human intestinal disaccharidase, sucrase-isomaltase, was visualized in ultrathin cryosections by the use of specific monoclonal antibodies [25] followed by protein A-gold. The principle site of immunoreaction concerned the microvillus membrane, which supports current concepts of the localization of these hydrolases. One antibody against sucrase-isomaltase also showed labeling of the Golgi apparatus, apical vesicles, and lysosomes, but not of the basolateral membrane. The labeling of the Golgi complex was uniform, suggesting the absence of accumulation of sucrase-isomaltase in cisternae during its passage through this organelle. Absence of labeling of the basolateral membrane appears to support the view that newly synthesized sucrase-isomaltase is transferred directly from the Golgi complex to the microvillus membrane, bypassing the basolateral membrane. However, the results do not exclude the possibility of a very rapid passage through the basolateral membrane. A substantial fraction of the sucrase-isomaltase occurred in lysosomes, which indicates that this organelle plays a major role in the catabolism of microvillar hydrolases. Transport of sucrase-isomaltase to lysosomes might occur by endocytosis or via the crinophagic pathway. The latter was previously postulated to reflect a regulatory mechanism at the post-Golgi level for the surface expression of microvillar membrane proteins.
|
3896810
|
Rabbit antisera against homogeneous rat liver thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) were prepared and used for immunohistochemical analysis in adult rats. Immunoreactive thioredoxin and thioredoxin reductase were widely distributed in tissues and organs, but varied a lot between cell types. Generally, epithelial cells, neuronal cells and secretory cells, both exocrine and endocrine, showed high immunoreactivity whereas mesenchymal cells with exceptions showed low activity. Surface lining epithelial and keratinizing cells showed high activity. The immunofluorescence was localized in the cytoplasm of cells with enrichments at secretory granules, at the plasma membrane or in the subplasma membrane zone. Variations in secretory cells were seen related to feeding and starvation and to metabolic activity. The distribution of thioredoxin and thioredoxin reductase is compatible with function in thiol-disulfide interchange reaction related to protein synthesis, intracellular transport and different forms of secretion.
|
3896811
|
A double blind randomized study of 800 patients was carried out to determine if very early intervention with metoprolol (15 mg I.V. followed by oral administration) in suspected acute myocardial infarction affected overall mortality in selected subgroups, (age, site of infarct, delay to intervention). Sudden death occurred less frequently in patients allocated to metoprolol but there was no significant difference in total mortality on discharge, at three months and at twelve months. Ventricular fibrillation after intervention was not significantly reduced. Adverse reactions did not occur significantly more frequently in patients assigned to metoprolol.
|
3896812
|
The usefulness of ultrasound diagnosis during polychemotherapy for ovarian cancer is valued. Between September 1980 and March 1983, 32 patients with ovarian cancer underwent polychemotherapy, had an ultrasound follow-up. Ultrasounds are very important for selecting the patients who must undergo second look.
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3896814
|
A variety of methods have been proposed to estimate the glomerular filtration rate (GFR) from the renal uptake of technetium Tc 99m-DTPA using a gamma camera. To compare alternative methods, we calculated the GFR in several different ways from measurements in 33 patients and compared the results with an independent GFR measurement based on eight-point plasma clearance of ytterbium Yb 169-DTPA. The best agreement was obtained using an algorithm that has not been described previously, in which correction was made for overlap of the kidneys by the liver and spleen. The correlation coefficient was 0.958, and the residual standard deviation was 12.1 ml/min. This method required a single 20-min blood sample as well as the camera data. The best method not requiring a blood sample was significantly less accurate, with a correlation coefficient of 0.837 and a residual standard deviation of 23.1 ml/min. The accuracy of these methods was comparable to that reported for creatinine clearance, the most commonly used estimate of the GFR in current clinical practice.
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3896815
|
Lymphoscintigraphy has been used as a noninvasive means of evaluating lymph node involvement. The potential exists for its wider application to other malignancies. Additionally, it is useful in determining the etiology of edema of the extremities. We report the use of 99mTc-antimony sulfide colloid lymphoscintigraphy to determine the etiology of edema of an extremity in a lymphoma patient.
|
3896816
|
An investigation of the staff of a car assembly plant (3,351 persons) revealed a similarity between the change in relative body weight and diastolic blood pressure with age. There is a good temporal correlation between the course of alcohol consumption during life and the change of the relative body weight. German women had significantly less blood pressure for the same relative body weight than German men, and foreign employees had lower blood pressure than Germans. In both cases the main cause is the difference in alcohol consumption. Besides obesity and hereditary factors, alcohol is the main cause of "essential" hypertension today. Epidemiological and experimental data indicate that there are two ways from alcohol to high blood pressure, a more direct one and an indirect one via obesity. Alcohol causes obesity via a change in metabolism (hyperinsulinism) rather than by higher caloric intake. In both ways alcohol is an important cause of stroke. To reduce body weight and blood pressure, a reduction of alcohol consumption should be recommended in addition to reduced caloric intake and increased physical activity as means of preventive neurology.
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3896819
|
Using the Mishell and Dutton culture system, we have developed an assay for eliciting and quantifying parasite-specific immune responses in vitro. The ability of spleen cells from noninfected and Trypanosoma cruzi-infected mice to respond to parasite-associated antigens was assessed by examining the primary plaque-forming cell response to trinitrophenylated T. cruzi (TNP-TC). The response to TNP-TC of both normal, noninfected, unprimed mice and mice infected with T. cruzi is T cell dependent and appeared to involve recognition of parasite antigens by T. cruzi-specific T cells. In most experiments, spleen cells from infected mice respond to TNP-TC at levels equal to, or below, that of spleen cells from normal mice. This near "normal" response is in apparent contrast to the suppressed response of spleen cells from infected mice to another antigen (sheep red blood cells) or TNP on a different carrier (TNP-chicken erythrocytes). Demonstration that the response of spleen cells from infected mice to TNP-TC can be potentiated by addition of interleukin 2-containing supernatants or by depletion of plastic and Sephadex G-10-adherent cells suggests that the mechanisms which control the response of infected mice to nonparasite antigens may also limit parasite-specific immune responses.
|
3896820
|
Previous studies have shown that the fecal flora of patients with Crohn's disease (CD) differed from the flora of healthy subjects by a higher number of anaerobic gram-positive coccoid rods. Sera from patients with CD agglutinated four strains of coccoid rods (Me44, C18, Me46 and Me47) more frequently and stronger than sera from healthy subjects and patients with other diseases. One of these bacteria, Coprococcus comes strain Me46, was not ingested by neutrophils after coating with specific IgG. In the present study, therefore, the binding of IgG and of Fab and Fc fragments to the four coccoid rods was investigated using immunofluorescence and absorption techniques. Results were compared with those obtained with Staphylococcus aureus strain Cowan I and showed that Me46, like S. aureus, bound (nonspecifically) IgG through its Fc portion whereas strain Me47 bound IgG through the Fab portion. Possible implications of the findings for CD are discussed.
|
3896821
|
LG 82-4-00 (5-(2-(1-imidazolyl)-ethoxy)-thiophene-2-carboxylate) and LG 82-4-01 (4-chloro-thiophenic-substituted derivative) were examined for specific inhibition of thromboxane (TX) synthetase. Thromboxane formation was measured by a radioimmunoassay specific for TXB2. In thrombin (0.6 IU/ml)-stimulated, washed human platelet suspensions (WPS) the IC50 (microM) for inhibition of TX formation were 1.1 (LG 82-4-00), 1.3 (LG 82-4-01) and 0.7 (dazoxiben). LG 82-4-00, LG 82-4-01 and dazoxiben also inhibited collagen (0.6-2.5 micrograms/ml)-induced TXB2 formation and platelet aggregation in human platelet-rich plasma. Neither LG 82-4-00 nor LG 82-4-01 had vasoconstrictor, proaggregatory or TX antagonistic activity or affected primary wave ADP aggregation. There was less than 10% inhibition of PGI2 formation from bovine coronary artery slices with concentrations up to 100 microM. At 100 microM, dazoxiben inhibited thrombin-induced 12-HPETE formation in WPS by 81 +/- 10% whereas LG 82-4-00 and LG 82-4-01 were much less active. These data indicate that LG 82-4-00 and LG 82-4-01 are specific inhibitors of thromboxane synthetase in human platelets.
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3896822
|
Electron microscopic and cytochemical studies indicate that microtubules play an important role in the organization of the Golgi complex in mammalian cells. During interphase microtubules form a radiating pattern in the cytoplasm, originating from the pericentriolar region (microtubule-organizing centre). The stacks of Golgi cisternae and the associated secretory vesicles and lysosomes are arranged in a circumscribed juxtanuclear area, usually centered around the centrioles, and show a defined orientation in relation to the rough endoplasmic reticulum. Exposure of cells to drugs such as colchicine, vinblastine and nocodazole leads to disassembly of microtubules and disorganization of the Golgi complex, most typically a dispersion of its stacks of cisternae throughout the cytoplasm. These alterations are accompanied by disturbances in the intracellular transport, processing and release of secretory products as well as inhibition of endocytosis. The observations suggest that microtubules are partly responsible for the maintenance and functioning of the Golgi complex, possibly by arranging its stacks of cisternae three-dimensionally within the cell and in relation to other organelles and ensuring a normal flow of material into and away from them. During mitosis, microtubules disassemble (prophase) and a mitotic spindle is built up (metaphase) to take care of the subsequent separation of the chromosomes (anaphase). The breaking up of the microtubular cytoskeleton is followed by vesiculation of the rough endoplasmic reticulum and partial atrophy, as well as dispersion of the stacks of Golgi cisternae. After completion of the nuclear division (telophase), the radiating microtubule pattern is re-established and the rough endoplasmic reticulum and the Golgi complex resume their normal interphase structure. This sequence of events is believed to fulfil the double function to provide tubulin units and space for construction of the mitotic spindle and to guarantee an approximately equal distribution of the rough endoplasmic reticulum and the Golgi complex on the two daughter cells.
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3896823
|
When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.
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3896824
|
To examine the hypothesis that p53 protein may play a central role in regulating reproduction of mammalian cells, we compared the absolute amounts and relative rates of synthesis of p53 protein in two pseudonormal cell lines, 3T3 and C3H 10T1/2, during quiescence, during log proliferation, and in quiescent cells stimulated with serum. The absolute amount of p53 protein per cell was found to be severalfold lower in quiescent cells than in log-phase cells. The ratio of the rate of synthesis of p53 protein to the rate of synthesis of total protein was slightly higher in quiescent cells than the same ratio in log-phase cells. Thus, entry into quiescence is not accompanied by a differential switch-off of synthesis of p53 protein. In quiescent cells stimulated with serum the amount of p53 protein per cell and its rate of synthesis increase, but only in proportion to the increase in total protein per cell and the increase in rate of total protein synthesis. Similarly, 12-14 h after serum stimulation, the time of the G1 to S transition, the accumulated increase in p53 protein per cell is about what would be expected for a short-lived protein whose rate of synthesis has increased in proportion to the increase in rate of synthesis of total protein. The results are not those expected for a protein that functions specifically in release from quiescence or in transition from G1 to S.
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3896826
|
In Saccharomyces cerevisiae both the induction of heat shock proteins (98, 85, 70 kD) and the intracellular pH, determined by means of 31P-NMR spectroscopy, show a similar dose response to increasing temperature or concentrations of 2,4-dinitrophenol (DNP). Temperature increases from 23 degrees to 32 degrees C or more, or concentrations of DNP higher than 1 mM cause a significant increase in the synthesis rate of heat shock proteins and a significant decrease of the intracellular pH. A similar correlation is found in a mitochondrial mutant (Q) defective in oxidative phosphorylation. Intracellular signal transduction may thus involve H+-concentration changes independent of intact oxidative phosphorylation.
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3896825
|
A sensitive radioisotope dilution method was used to measure the S-adenosyl-L-methionine (AdoMet) content in macroplasmodia of the slime mold Physarum polycephalum during the mitotic cycle. The AdoMet pool had two maxima, one during mitosis, the other in the middle of G2 phase.
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3896827
|
Adult frog (Xenopus laevis) hepatocytes were found to proliferate in a culture medium containing adult homologous serum. Insulin and dexamethasone were required for a net proliferation of hepatocytes. Dose-response analysis showed that a low concentration of serum (greater than or equal to 0.5%) was enough to induce DNA synthesis and mitosis, but a higher concentration (5%) caused certain necrotic changes. Under optimal conditions, there was a two- to threefold increase in nuclei per culture 10 days after serum treatment. Heterologous sera (fetal bovine, calf and chick) showed less proliferative activity. Based on our results, hepatocyte-proliferating activity in adult frog serum is considered to be heat-unstable and acidic protein(s). Thus, adult frog serum may contain hepatopoietin possibly different from well-known growth factors.
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3896828
|
We have obtained several hybridoma clones producing antibodies to microtubule-associated proteins (MAPs) from bovine brain. Interaction of one of these antibodies, named RN 17, with cultured cells was studied by indirect immunofluorescence and immunoelectron microscopy. RN 17 antibody recognized both high molecular weight (HMW) MAPs, MAP 1 and MAP 2, in immunoblotting reaction with brain microtubules. In lysates of cultured cells, it bound to a protein doublet with a molecular weight of 100 kD. By immunofluorescence microscopy we showed that RN 17 antibody stained cytoplasmic fibrils, mitotic spindles and small particles in the cytoplasm of various cultured cells. The cytoplasmic fibrils were identified as both microtubules and intermediate filaments by double fluorescence microscopy and by their response to colcemid and 0.6 M KCl. This identification was confirmed by immunoelectron microscopy which also showed that the particles stained by RN 17 antibody are coated vesicles. Thus, cultured non-neural cells may contain a novel protein that binds to microtubules, intermediate filaments, and coated vesicles.
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3896829
|
In studies on the specific migration of macromolecules across the nuclear envelope, a karyophilic protein was injected into the cytoplasm of cultured cells and its subsequent location in the cell was examined. Nucleoplasmin of frog nuclear protein was used for this experiment. When [125I]nucleoplasmin was introduced into the cytoplasm of mammalian cells (human and mouse) by red blood cell-mediated microinjection, it rapidly accumulated in the nucleus. When nucleoplasmin conjugated with [125I]IgG against chromosomal protein was introduced similarly, it also accumulated rapidly in the nucleus, and reacted with its antigen inside the nucleus. On the contrary, when IgG alone or IgG conjugated with BSA were introduced, they did not migrate from the cytoplasm into the nucleus. These findings imply that the migration of macromolecules from the cytoplasm to the nucleus does not depend only on their molecular size but also on a specific transport mechanism, and that karyophilic proteins may act as useful carriers in the transfer of exogenous proteins into the nucleus.
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3896830
|
The cellular targets for the Kirsten murine sarcoma virus (KiMSV)-transforming protein, p21ras, are unknown. Other studies have indicated that the mature form of p21 is distributed diffusely on the cytoplasmic face of the plasma membrane. However, after fixation without buffer washes, indirect immunofluorescent staining of sparse cultures revealed a particularly well preserved cellular architecture and a strikingly heterogeneous subcellular distribution of p21 in transformed normal rat kidney (NRK) cells but not in their untransformed counterparts. The transformed cells included A KiMSV-transformed NRK line. NRK cells newly transformed with KiMSV. A temperature-sensitive (ts) KiMSV-transformed NRK line. An uninfected, spontaneously transformed NRK line in which p21 was neither phosphorylated nor overproduced. In the tsKNRK line p21 was abundant at both permissive and non-permissive temperatures; however, its distribution was heterogeneous at the permissive temperature only. Observation of this array of cells indicates that the transformation-associated p21 distribution does not require overexpression of the gene, nor phosphorylation of the protein, nor the viral oncogene. Furthermore, it is reversible in the tsKNRK cells, and so appears to be highly correlated with acquisition of a transformed morphology. Accumulations of p21 occurred preferentially in subcellular locations similar to those where ruffles were observed by phase contrast microscopy and lamellar and villous extensions were observed by scanning electron microscopy (SEM). Since enhanced ruffling is a morphological correlate of transformation in a variety of cells, the distribution of p21 observed here may relate to its function as a transforming molecule.
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3896831
|
This article represents the second in a series of invited annual reports from the International Bone Marrow Transplant Registry (IBMTR) published in Experimental Hematology. In the first report, the results of IBMTR analyses for the two-year period 1 January 1982 through 31 December 1983 were described. In this report, data are presented reflecting the phenomenal recent growth in the field of bone marrow transplantation. Also presented are summaries of the results of IBMTR analyses during the period 1 January through 31 December 1984.
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3896833
|
Rat litters were treated daily from birth with the antithyroid drug propylthiouracil for 2 or 4 weeks, respectively. Transverse cryostat sections of cerebellar cortex with underlying white matter were incubated with antiserum against glial fibrillary acidic protein. Semiquantitative microscopic evaluations showed a marked decrease in width of the molecular layer in thyroid deficient animals 2 weeks postnatally, as compared to controls. Also, the well delineated GFA-positive glial cell layer extending along the border between white matter and the internal granular layer was absent in thyroid deficient animals. The density of GFA immunoreactivity was increased in white matter in the experimental group. No effects of thyroid deficiency on GFA immunoreactivity could be found after 4 weeks of treatment, indicating a transient disturbance of glial development in the thyroid deficient cerebellum.
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3896836
|
Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.
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3896835
|
The biochemical characterization of dipeptidyl aminopeptidase II activity was investigated in the supernatant of centrifuged homogenates of adult Schistosoma japonicum using a lysine-alanine oligopeptide derivative of 4-methoxy-2-naphthylamide as a substrate. It was observed that the pH optimum of the enzyme is in the acid range, with an optimum at pH 6.3. Time and enzyme concentration studies, along with temperature studies, support the premise that the reaction is enzymatic. The Km was 3.3 X 10(-3) M, at pH 5.5 and 37 C. Tris and diisopropyl phosphofluoridate, when incorporated into the assay system at final concentrations of 500 and 2 mM, respectively, significantly inhibited the reaction by 70.9 and 75%, respectively. Leupeptin (5 X 10(-4) mM) had no effect. The results indicate that the enzyme under study in the present investigation strongly resembles mammalian dipeptidyl aminopeptidase II due to its affinity for substrate, sensitivity to Tris and diisopropyl phosphofluoridate inhibition, and pH optimum. Its inhibition by diisopropyl phosphofluoridate indicates that it may belong to the serine class of proteases. Cytochemical studies revealed reaction product in the lipid-like globules in the gastrodermis, adding further credence that these globules are lysosomal.
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3896834
|
Immunocytochemical procedures employing the unlabeled antibody enzyme (PAP) method were used to visualize those neuronal elements in the basilar pontine nuclei (BPN) and nucleus reticularis tegmenti pontis (NRTP) of the rat which contain glutamic acid decarboxylase (GAD), a key enzyme involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). Neuronal somata, axons, and axon terminals in the BPN and NRTP exhibited GAD-immunoreactivity. Both thick and thin varieties of labeled axons and terminals were distributed in varying densities throughout the BPN and NRTP. The greatest accumulation of labeled terminals was noted in the ventrolateral and lateral border regions of the BPN while a slightly less dense aggregation was observed along the ventral, ventromedial and midline regions of the pontine gray. Labeled fibers and terminals were also observed in the dorsal and ventral peduncular regions as well as central portions of the lateral, ventral, and medial pontine areas. Axonal and terminal labeling was present throughout the NRTP but no focal increases in density similar to those in the BPN were apparent. No obvious GAD-labeled fiber bundles could be observed to enter the BPN or NRTP. However, small fascicles of labeled axons were seen to course ventrally around the dorsolateral aspect of the cerebral peduncle to reach lateral pontine areas while other labeled axons descended through clefts in the mid-portion of the cerebral peduncle or passed through the medial lemniscus and around the medial portion of the cerebral peduncle to enter the pontine gray.
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3896837
|
Chicken and human plasma and some of their proteins, partially isolated by means of isoelectrofocusing and ion-exchange chromatography, induce changes in the epimastigotes of Trypanosoma (Schizotrypanum) cruzi into forms resembling extracellular amastigotes in synthetic media.
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3896838
|
Two neutrophil chemotactic factors were identified in soluble egg antigen preparations of Schistosoma japonicum. The higher-molecular-weight neutrophil chemotactic factor was not separable from eosinophil chemotactic factor by means of gel filtration, anion-exchange chromatography, isoelectric focusing, or affinity chromatography; this neutrophil chemotactic factor is apparently identical to the higher-molecular-weight eosinophil chemotactic factor which we purified previously from the soluble egg antigen. The chemotactic activity of the eosinophil chemotactic factor for neutrophils was stable to periodate oxidation but was notably affected by heating or Pronase digestion, suggesting that the determinant for neutrophil chemotaxis exists on the peptide moiety of the eosinophil chemotactic factor. The lower-molecular-weight neutrophil chemotactic factor was separable from the higher-molecular-weight eosinophil chemotactic factor by gel filtration or anion-exchange chromatography. This neutrophil chemotactic factor was rather hydrophobic and heat-stable, but was sensitive to Pronase or carboxypeptidase A digestion. These results suggest that the receptors on the surfaces of neutrophils and eosinophils for those chemoattractants would be different from each other. We suppose that neutrophil chemotactic factors and eosinophil chemotactic factors from the eggs are responsible for neutrophil and eosinophil accumulation around the eggs in schistosomiasis japonica.
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3896840
|
The term photopollution is proposed for artificial light having adverse effects on wildlife. The differences between natural and artificial light are discussed in relation to the concepts of orientation, disorientation, misorientation and abnormal orientation. The ways in which optic orientation systems are attuned to natural illumination conditions are analysed, and it is shown why they therefore may fail to cope with artificial light. It is concluded that for many nocturnally active animals a natural light-field between sunset and sunrise is a requirement for survival. A review is given of data on a) bird kills at man-made lighted obstacles, and b) the interference of artificial light with nest site selection by female sea turtles and water-finding by hatchlings at nesting beaches. Conventional remedies against the hazards of photopollution are critically reviewed and new ones are suggested. It is emphasized that measures should aim not only at reducing threats to a species or population but also at preventing suffering in individual animals.
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3896839
|
In the fasting state, totally gastrectomized rats had higher glycemia, higher glucagonemia and lower insulinemia than normal rats. However, they are not prediabetic or diabetic, because after a glucose tolerance test the response of glucagon secretion, though accentuated at the beginning, decreased sharply when insulin secretion was stimulated. The evolution of glucagonemia and insulinemia indicated that the positive-negative feedback of the couple alpha-beta cells of islets of Langerhans still regulated glucose homeostasis. Hypoglycemia seen after the glucose test in operated rats disappeared when soluble starch replaced glucose. With soluble starch, hyperglycemia, hyperglucagonemia and hyperinsulinemia were less marked than with glucose at 30 min. Therefore, we conclude that 1) Gastrectomy, in removing the stomach with all its physiological role, has conferred to the operated rat in the fasting state a new level of hormonal glucose counterregulation close to the diabetic or prediabetic situation; but once the animal is fed, its endocrine pancreas responses quite normally, and 2) In the sugar tolerance test, soluble starch induces less release of glucagon and insulin than glucose.
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3896846
|
The synthesis and antifungal activities of 1-aryl-4-p-nitrophenylimidazoles and their 2-mercaptoderivatives is reported. Antimicrobial data in comparison with antifungal antibiotic pyrrolnitrin attributed the best activity to 1-p-methoxyphenyl-4-p-nitrophenylimidazole. The tested compounds were prepared by reacting p-nitrophenacylanilines with potassium thiocyanate in acidic medium to afford 1-aryl-2-mercapto-4-p-nitrophenylimidazoles, which were then transformed into the title compound by treatment with nitric acid.
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3896847
|
The uptake of 2-[1-14C]deoxyglucose in vivo by brown adipose tissue was greater than that of brain or heart in control mice on a whole tissue basis. When mice were treated with noradrenaline the rate of uptake of 2-[1-14C]deoxyglucose by brown adipose tissue was increased 6-fold with the only other change occurring in heart where a 5-fold increase was observed. After administration of insulin the uptake of 2-[1-14C]deoxyglucose in vivo was increased in heart, brown adipose tissue, white adipose tissue and muscle. The amount of 2-[1-14C]deoxyglucose taken up by brown adipose tissue compared to other tissues and the changes in this uptake after administration of noradrenaline or insulin suggest that brown adipose tissue is capable of playing a quantitatively important role in glucose removal from the blood.
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3896850
|
The astroglial growth factor (AGF), which induces a characteristic morphological change in cultured rat astroglial cells and stimulates their proliferation, was purified to homogeneity from bovine brain. Two different methods were used, the second one including heparin-Sepharose affinity chromatography. AGF is actually composed of two factors, AGF1 and AGF2, which both modify the morphology and stimulate the proliferation of the astroglial cells. Several data suggest that the AGFs are similar or possibly identical to the fibroblast growth factors (FGFs) isolated from brain [(1984) Proc. Natl. Acad. Sci. USA 81, 357-361; and 6963-6967]. A specific antiserum against AGFs was raised in mouse.
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3896848
|
Three closely related forms of a 21 kDa protein which is co-secreted with insulin have been purified and analysed. These differed in behaviour on ion-exchange chromatography but were indistinguishable by their susceptibility to staphylococcal V8 proteinase digestion, amino acid composition or N-terminal amino acid sequence. Their amino acid composition and N-terminal sequences were remarkably similar to adrenal medullary chromogranin A, a much larger protein (72 kDa). Antibodies to chromogranin A also reacted strongly with the 21 kDa protein in isolated insulin granules. It is concluded that the 21 kDa proteins either represent a repeated domain within the chromogranin molecule or a closely related gene product. The name beta-granin is proposed for these proteins.
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3896849
|
IgA proteases were estimated in a turbid aqueous two-phase system with 10% polyethylene glycol-Tris buffer, where IgA spontaneously concentrates in microscopic spherical particles (less than 1 micron). After enzymatic cleavage of IgA into Fab alpha and Fc alpha fragments, these fragments are soluble and decreasing turbidity is observed. The reaction may be followed by conventional spectrophotometry. In this manner, IgA proteases may be estimated in 10 min. Examples of the utility of the method are given with results from inhibitor studies, estimation of Km and purification of IgA protease from Haemophilus influenzae.
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3896851
|
An insect neurohormone, melanization and reddish coloration hormone (MRCH), is responsible for cuticular melanization and epidermal red pigmentation in the armyworm. The three molecular forms of MRCH were isolated from adult heads of the silkworm, Bombyx mori, and their N-terminal amino acid sequences revealed a sequence homology with the C-terminal region of human insulin-like growth factor-II as well as N-terminal heterogeneity of MRCHs.
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3896852
|
Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.
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3896845
|
Alcohol dehydrogenase activity (ADH; KP 1.1.1.1.) in blood serum of rats and rabbits is 1 and 2 orders of magnitude higher than in humans. In chronic alcoholics, blood ADN is activated with an increase in alcoholism standing. Twelve hours after acute alcoholic intoxication alcoholics and heavy drinkers manifest a significant reduction in blood ADH activity. Acute alcoholic intoxication does not influence blood ADH in men who do not abuse alcohol. Chronic exposure of rabbits to ethanol leads to a decrease in ADH activity in the liver and to its rise in the blood. ADH activation is observed only in those animals which demonstrate the signs of fatty and protein liver dystrophy. It is concluded that chronic exposure to ethanol does not induce ADH synthesis in the liver. The blood ADH content ascends as a results of an increase in ADH transport from hepatocytes to the bloodstream.
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3896854
|
dpG antibodies were fractionated on a cellulose-double-stranded DNA column. The flow-through fraction bound denatured DNA but not double-stranded DNA (dsDNA). The dpG-eluted fraction bound dsDNA preferentially. The results show that proteins can recognise dpG in DNA by different mechanisms, some involving DNA unwinding.
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3896853
|
Tissue-type plasminogen activator (t-PA) has a high affinity for fibrin and induces lysis of fibrin (fibrinolysis) on the surface of fibrin without degrading circulating fibrinogen. cDNA for t-PA which lacks the 'finger-domain' (the site for fibrin affinity) was isolated from Detroit 562 cells. Analysis of the nucleotide sequence revealed a lack of the sequences which code for the finger-domain. A plasmid (pDPAT 1) containing the Escherichia coli tac promoter/operator and the cDNA sequence coding for 'finger-domain lacking t-PA' was constructed for expression in E. Coli. The polypeptide so produced was a new type of t-PA lacking finger-domain, but revealed plasminogen activator activity with the function of fibrin affinity.
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3896861
|
Defined changes in the concentration of estrone glucuronide in daily samples of early morning urine have been used to locate the limits of the fertile period and the time of maximum conception probability during 118 cycles (106 menstrual, 12 conceptional) in 73 women. The peak day of urinary luteinizing hormone was used as an index of ovulation. Follicular growth was monitored daily by ultrasonography throughout 38 cycles, and the time of maximum follicular diameter was used as an alternative reference point to define the times of potential fertility according to the life spans of the gametes. With optimized algorithms and the best index of ovulation, the estrogen test delineated the limits of the fertile period in 89% of the tests (mean length, 10.8 days; range, 5 to 17 days) and the time of maximum conception probability in 82% of the cycles, with a mean time to the maximum follicular diameter of 0.42 days (range, -4 to +4 days).
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3896862
|
The present study was conducted to induce endometriosis in an experimental animal model in which the condition and its response to pharmacologic agents could be quantified. Endometriosis was induced in New Zealand White rabbits by transplanting endometrial sections into various sites throughout the peritoneum. After 7 weeks, the mean implant weight increased in concomitant controls from 10.3 to 89.0 mg. In the next 8 weeks, endometrial implant weight increased to 163.6 mg. Daily subcutaneous administration of a luteinizing hormone-releasing hormone agonist, histrelin, or oral administration of danazol, reduced the ectopic implant weight within 8 weeks to 21.7 and 46.0 mg, respectively. In a group of animals that were bilaterally ovariectomized, implant weight decreased significantly in the same 8-week period to 22.4 mg. Furthermore, histologic analysis of the endometriomas showed that ovariectomy, histrelin, or danazol treatment reduced the number of endometrial glands and atrophied the stroma. We conclude that this animal model represents an excellent method for quantitative evaluation of potential therapeutic agents for endometriosis.
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3896882
|
A significantly high prevalence (41%) of non-organ-specific autoantibodies, tested in immunofluorescence with sera diluted 1:40, is reported in sera from 86 alopecia patients. No correlation was found with extension and duration of the disease, topical treatment with contact agents or response to it. The relevance and significance of these findings, with particular reference to anti-smooth muscle and anti-basal cell layer antibodies, is discussed.
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3896883
|
Measurements of the ring size (circumference in millimeters) of the middle phalanx in 22 females suffering from generalized scleroderma (acrosclerosis) were carried out and compared to control measurements in 22 age-matched healthy females. The ring size was increased (p less than 0.01) in the patients (mean 52.8 mm; range 45-61) as compared to the controls (mean 49.4 mm; range 46-52). Ring size measurements correlated well (r = 0.71; p less than 0.001) with ultrasound measurement of soft tissue thickness over the middle phalanx. Ring size was larger (p less than 0.001) in a group of healthy men as compared to female controls. Ring size was not correlated to age in healthy females (r = 0.06; n.s.), whereas it was in 19 healthy men studied (r = 0.78; p less than 0.001). The normal range (mean +/- 2 SD) for ring size in females was 45.5-53.3 mm; in males less than or equal to 60 years of age, the range was 48.5-59.2 mm. It is concluded that ring size is suited to quantify soft tissue changes of the digits in acrosclerosis. This method has the advantage of being simple.
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3896884
|
Insulin binding on circulating red cells has been studied in hypertriglyceridemic (HTG) patients before and after normalization of plasma TG levels by a low fat and low CHO diet, followed for 2 months. Under basal condition HTG patients showed lower insulin binding on red cells (B/T) than control subjects. The reduction in binding was due to a lower receptor number (binding capacity). After the diet and normalization of TG levels, insulin binding was identical in HTG patients and always lower than in controls. The fasting values of blood glucose, IRI, FFA, were also unchanged after TG reduction, suggesting, together with the low insulin binding, a state of insulin resistance. We conclude that our lean patients, affected by HTG, present a state of insulin resistance. Despite normalization of plasma TG, obtained with diet alone, insulin receptor binding on red cells in unchanged.
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3896886
|
Plasma free insulin was measured repeatedly for 4 hours following a standardized breakfast in 20 C-peptide negative chronically pumped type I diabetic patients and 5 normal subjects. In the former group, insulin was given as a 1 u/h basal infusion and a 1h superimposed meal-dose of 6 u via a peritoneal (IP) catheter lying in the low (n = 10), or in the mid-abdomen (n = 10). The results of the IP patients were correlated with glycosylated haemoglobin and home capillary blood glucose. Fasting free insulin of IP patients was lower than those of normals (14.7 +/- 0.5 vs 21.0 +/- 1.3 mU/l, p less than 0.01). Dose-induced peak occurred similarly in IP patients and normals (70 +/- 6 vs 70 +/- 12 min.). Values tended to baseline after 165 +/- 15 and 185 +/- 22 min. in IP patients and normals (NS). Results of the mid--and low peritoneum subgroups differed only for peak values (31.5 +/- 2.9 vs 25.0 +/- 1.6 mU/l respectively) and did not correlate with diabetic control.
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3896885
|
The aim of this prospective, randomized, double-blind, placebo controlled study was to investigate the effect of nifedipine on carbohydrate metabolism in diabetic patients after a 3-day and a 3-month course of treatment. Sixteen non obese, well controlled non-insulin dependent diabetics, (HbA1 less than 10%), with moderate untreated hypertension were divided in two groups: nifedipine (group N, 8 patients) and placebo (group P, 8 patients). An oral glucose tolerance test (OGTT, 75 g glucose) and an arginine infusion were performed before, after a 3-day, and a 3-month course, either of nifedipine 30 mg/D or placebo. Blood samples obtained during OGTT were assayed for glucose and insulin, and during arginine infusion for insulin, glucagon and growth hormone. The differences between basal and peak values during tests were compared between both groups before and after treatment using Wilcoxon's rank sum test. Neither acute nor chronic administration of nifedipine or placebo modified the glucose tolerance. However, basal insulin levels were reduced by 3 month-administration of nifedipine (from 19 +/- 2 micromicrons/ml to 10 +/- 1 micromicrons/ml, p = 0,01). Otherwise the basal and peak hormonal values during tests were not significantly affected by nifedipine either at the start of after 3 months of treatment. These results suggest that nifedipine, when given in standard dosage for 3 months, has minor effects on carbohydrate metabolism in non-insulin dependent diabetic patients.
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3896888
|
The development of methods for fetal blood sampling in the second trimester of pregnancy offers the possibility of fetal diagnosis for couples at risk for having children with a haemoglobinopathy. We review the evolution of 10 years' experience of fetal blood sampling for 681 patients. The obstetric risk associated with the procedure has fallen from an initial 15% (in the first 87 pregnancies) to about 3%, in parallel with increased experience, improved ultrasound control, identification of the causes of complications and implementation of simple steps to avert them.
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3896889
|
Receptor mediated endocytosis has been proposed as the method of cellular iron uptake from transferrin (TF). However, the experimental evidence for endocytosis in every situation is found wanting. This is particularly true for the hepatocyte where an alternative mechanism of iron release at the cell surface can account for all iron uptake. It may be, that under appropriate physiological conditions (e.g. degree of iron saturation of TF) cells may take up iron by either an endocytotic or nonendocytotic mechanism.
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3896891
|
Insulin infusion through the portal vein immediately after a pulse of [3-14C]pyruvate in 24 hr starved rats enhanced the appearance of [14C]glucose at 2, 5 and 10 min and glucose specific activity at 1, 2 and 20 min in blood collected from the cava vein at the level of the suprahepatic veins. Insulin infusion for 5 min decreased liver pyruvate concentration and enhanced both liver and plasma lactate/pyruvate ratio, and it decreased the plasma concentration of all amino acids. When insulin was infused together with glucose, [14C]glucose levels and glucose specific activity decreased in blood but there was a marked increase in liver [14C]glycogen, glycogen specific activity and glycogen concentration, and an increase in liver lactate/pyruvate ratio. The effect of insulin plus glucose infusion on plasma amino acids concentration was smaller than that found with insulin alone. It is proposed that insulin effect enhancing liver gluconeogenesis is secondary to its effect either enhancing liver glycolysis which modifies the liver's cytoplasmic oxidoreduction state to its more reduced form, increasing liver amino acids consumption or both. In the presence of glucose, products of gluconeogenesis enhanced by insulin are diverted into glycogen synthesis rather than circulating glucose. This together with results of the preceding paper (Soley et al., 1985), indicates that glucose enhances liver glycogen synthesis from C3 units in the starved rat, the process being further enhanced in the presence of insulin.
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3896893
|
We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.
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3896894
|
The neural crest of early vertebrate embryos gives rise to a wide variety of cell types. One way in which phenotypic diversity may be generated in neural-crest-derived cells is by a series of partial developmental restrictions. In order to test the possibility that the crest-derived mesenchymal cells of the branchial arches (BAs) of avian embryos are partially restricted intermediates during this segregation of developmental fates, we examined some of their phenotypic and developmental properties. We found that the mesenchymal cells of the posterior BAs differ from those of the anterior BAs in that the posterior BA cells express the neuron-specific antigen NAPA-73, whereas the anterior BA cells do not. This phenotypic difference first appears in the different populations of migrating neural crest cells which populate the different BAs. Anterior and posterior BA cells also differ in their abilities to give rise to various crest derivatives in heterospecific grafting experiments. Whereas anterior BA cells only produce connective tissue derivatives, posterior BA cells give rise to neurons, glial cells, and glandular tissue, in addition to the connective tissues. However, neither anterior nor posterior BA grafts give rise to melanocytes--another neural crest derivative. This developmental restriction of melanogenic potential occurs either during crest migration, or shortly after colonization of the BAs. These results are consistent with the notion that the mesenchyme of both anterior and posterior BAs contain different partially restricted intermediate cell types derived from the neural crest.
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3896895
|
We describe a nonradioactive microcytotoxicity assay for ICSA using a cloned rat insulinoma cell line. This assay system had good reproducibility (r = 0.93) and was suitable for the study of large numbers of samples. The following results were obtained by testing the sera of 111 patients with IDDM (type I diabetes) and all of their first-degree relatives. (1) Thirty-five percent of IDDM patients had ICSA, as compared with only 2% of healthy controls. (2) ICSA was found more frequently in patients within 2 yr of onset (45%) than in those with disease for longer than 2 yr (27%) (P less than 0.05). (3) The prevalence of ICSA was associated with the presence of cytoplasmic islet cell antibodies (ICA) (P less than 0.05). (4) No association was found between the prevalence of ICSA and specific HLA-DR alleles. Association with the HLA haplotypes in families with ICSA-positive probands, on the other hand, is suggested although not proven by these data. (5) Among the nondiabetic relatives of IDDM patients, 5% of the parents and 14% of the sibs had ICSA. Increased prevalence of ICSA occurred in the unaffected sibs of ICSA-positive probands (31%) but not in those of ICSA-negative probands (4%) (P less than 0.001); in fact, the relatives of ICSA-negative probands had ICSA with a frequency not higher than in unrelated controls. (6) Female relatives of ICSA-positive probands were more often ICSA-positive than males, but no such difference was found among probands. (7) In multiplex sibships, ICSA were not associated with disease in the sibs.(ABSTRACT TRUNCATED AT 250 WORDS)
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3896897
|
The effects of experimental diabetes on cardiac function and ultrastructure were studied in rats that had been diabetic for 6-24 wk. Experimental diabetes was produced by the intravenous (i.v.) injection of 65 mg/kg streptozocin (STZ) into rats 42-43 days old. Diabetic rat hearts perfused at 15 cm H2O on the working heart apparatus demonstrated depressed cardiac function (i.e., lower left ventricular pressure and +/- dP/dt) at 6, 12, and 24 wk of diabetes. Electron microscopic analysis of ventricular myocardium revealed increased lipid deposition from 6 to 24 wk of diabetes and progressive deterioration of the myocardial cell integrity at 12 and 24 wk of diabetes. This deterioration was characterized by loss of contractile protein, vacuolization (swollen sarcoplasmic reticulum), myelin formations, myocytolysis, and contracture bands. These alterations paralleled the depression of cardiac function at 12 and 24 wk of diabetes. There was, however, depressed function at 6 wk of diabetes but no observable alterations in myocardial ultrastructure. Therefore, experimental diabetes produced ultrastructural alterations in the rat heart that manifested themselves only after a demonstrable depression in cardiac function.
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3896896
|
NIDDM is characterized by decreased insulin secretory responses to glucose and to nonglucose stimuli, hyperglucagonemia, and decreased tissue sensitivity to insulin. However, it has been unclear which of these abnormalities, if any, precedes the others. Since women with histories of gestational diabetes mellitus (GDM) are at high risk for eventual development of NIDDM, we measured B- and A-cell function and tissue sensitivity to insulin in eight normoglycemic, postpartum women with recent histories of GDM and in eight control subjects pair-matched for age and percent of ideal body weight. Fasting plasma glucose levels in subjects with former GDM tended to be slightly higher than in matched controls (98 +/- 3 versus 92 +/- 2 mg/dl, P = 0.07). Basal plasma insulin in subjects with former GDM was significantly higher than in controls (22 +/- 4 versus 14 +/- 2 microU/ml, P = 0.05). During an intravenous glucose tolerance test (IVGTT), relative first- and second-phase insulin responses to glucose were decreased in subjects with former GDM (2316 +/- 560 versus 7798 +/- 1036% of basal X min, P = 0.004; and 8340 +/- 946 versus 14,509 +/- 2556, P = 0.04). An index of sensitivity to insulin, SI, calculated from the IVGTT, was also lower in former GDM (1.23 +/- 0.69 X 10(-4) versus 3.58 +/- 0.78 X 10(-4) min-1/microU/ml, P = 0.001). Acute insulin responses to 5 g i.v. arginine were measured at plasma glucose levels of approximately 95, 215, and 600 mg/dl. The response at 600 mg/dl is termed the AIRmax and is used as an index of glucose-regulated insulin secretory capacity.(ABSTRACT TRUNCATED AT 250 WORDS)
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3896898
|
The effects of various forms of glucose administration on insulin action were investigated in isolated human fat cells. Subcutaneous (s.c.) adipose tissue was obtained before and (1) 30 min (eight subjects) and 60 min (seven subjects) after an intravenous (i.v.) glucose load, and (2) after a 60-min continuous i.v. glucose infusion (five subjects). In addition, five subjects were reinvestigated before and 60 min after oral glucose ingestion. Lipolysis (glycerol release) and insulin receptor binding were determined. After all forms of i.v. glucose administration, adipocyte insulin binding was significantly reduced by 20% owing to a decrease in the high-affinity binding, whereas the concentrations of insulin producing the half-maximum inhibitions of basal and isoprenaline-induced rates of glycerol release were unaltered. Sixty minutes after oral glucose ingestion, insulin sensitivity increased 7-30-fold (P less than 0.05-0.01) and high-affinity insulin binding increased by 25% (P less than 0.05). The maximum insulin-induced inhibitions of basal and isoprenaline-stimulated lipolysis were not altered after oral or i.v. glucose. The plasma level of glycerol was markedly and rapidly reduced after oral glucose, but the fall was slow and less pronounced after i.v. glucose. It is concluded that oral, but not i.v., glucose administration mediates a rapid increase in the antilipolytic potency of insulin in human fat cells in vitro. This may explain why antilipolysis in vivo is more pronounced after oral than after i.v. glucose challenge.(ABSTRACT TRUNCATED AT 250 WORDS)
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3896899
|
The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Cr-labeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring after the onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in the absence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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3896900
|
The reduction in blood glucose in non-insulin-dependent diabetes mellitus (NIDDM) brought about by the use of phenobarbital (PB), a hepatic microsomal enzyme inducer, suggests an improvement in insulin sensitivity. The effect of PB on insulin-mediated glucose metabolism was hence investigated using the euglycemic clamp technique in 10 women with NIDDM aged 56-75 yr. The addition of PB to sulfonylurea therapy, concurrently for 6 wk, reduced fasting blood glucose (BG, from 12.8 +/- 1.6 to 10.2 +/- 3.2 mmol/L, P less than 0.01) and immunoreactive insulin (IRI) levels (from 32.4 +/- 13.6 to 24.7 +/- 9.8 mU/L, P less than 0.01), whereas body weight remained unaltered. During the trial, there was a significant change in the glucose disposal rate (M, from 1.27 +/- 0.60 to 2.82 +/- 0.86 mg/kg/min, P less than 0.001), the metabolic clearance rate of glucose (from 0.89 +/- 0.41 to 2.24 +/- 1.27 ml/kg/min, P less than 0.01), the insulin sensitivity index (from 1.10 +/- 0.44 to 2.86 +/- 1.54 mg/kg/min: mU/L X 100, P less than 0.001), and the plasma antipyrine clearance rate (from 28.3 +/- 11.7 to 51.4 +/- 20.2 ml/min, P less than 0.001), an in vivo index of liver microsomal enzyme activity. The antipyrine clearance rate correlated with insulin-mediated glucose metabolism (r2 = 0.560, P less than 0.01). This correlation could be interpreted as indicating that, in NIDDM patients, peripheral glucose utilization and the liver microsomal enzyme system share common regulators. Our study suggests a new approach to the improvement of insulin sensitivity in NIDDM patients.
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3896901
|
Insulin antibodies, as measured by plasma radiolabeled insulin-binding capacity, were determined in 124 newly diagnosed insulin-dependent diabetic (IDDM) children before and after 1, 3, and 5 days of insulin therapy. Controls were 35 nondiabetic children with plasma insulin binding capacity of 1.0 +/- 0.7%. The patients were divided into three groups according to their plasma insulin-binding capacity. Group 1 (N = 79) had binding within two standard deviations (SD) of the control mean, group 2 (N = 20) had insulin binding 2-6 SD above controls, and group 3 (N = 25) showed insulin-binding capacity of more than 6 SD above the control mean. After exogenous insulin therapy, plasma 125I-insulin-binding capacity dropped significantly in both groups 2 and 3, concurrent with significant increases in plasma insulin levels. The three groups differed from each other in that patients in group 3 were significantly younger than in the other groups and clinically seemed to be more severely dehydrated, as reflected in their higher levels of serum urea nitrogen, plasma glucose, potassium, and elevated pulse rate. The three groups did not differ in respect to sex, HLA-DR antigens, Coxsackie-B antibody titers, islet cell cytoplasmic antibodies, immunoglobulin level, and C-peptide levels. Only two of 446 siblings of IDDM children showed elevated insulin binding, one of whom developed IDDM 6 wk later. The presence of an insulin-binding substance probably representing insulin antibodies in some cases of newly diagnosed IDDM suggests that autoimmunity in this disorder is not limited to the B-cell membrane and cytoplasm and lends further support to the heterogeneity of IDDM.
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