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Annetta North, Texas
Updated on Jun 22, 2018
County Parker
FIPS code 48-03340
Local time Saturday 6:48 PM
Time zone CST (UTC-6)
GNIS feature ID 1388247
Area 8.5 km²
Population 555 (2013)
Weather 9°C, Wind E at 8 km/h, 33% Humidity
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Annetta North is a town in eastern Parker County, Texas, United States, on FM 5. The population was 518 at the 2010 census.
Map of Annetta North, TX, USA
The Town of Annetta North was incorporated on August 11, 1979, and Phyllis M. Studer was elected as its first mayor. The current mayor is Rob Watson.
Annetta North is a small town that is mostly residential and farmland. There is only one church building, and the only other public places in the town are a car repair shop and riding arena. Both the Clear Fork and the South Fork of the Trinity River pass through the town, as well as Burgess Creek and the Dixon Branch. O'Neal Cemetery is located within the town limits as well.
Annetta North is located at 32°43′13″N 97°40′52″W (32.720231, -97.680992).
According to the United States Census Bureau, the town has a total area of 3.3 square miles (8.5 km2), all of it land.
At the 2000 census, there were 467 people, 164 households and 140 families residing in the town. The population density was 140.1 per square mile (54.1/km²). There were 172 housing units at an average density of 51.6 per square mile (19.9/km²). The racial makeup of the town was 98.93% White, 0.43% Native American, 0.21% from other races, and 0.43% from two or more races. Hispanic or Latino of any race were 1.93% of the population.
There were 164 households of which 41.5% had children under the age of 18 living with them, 78.7% were married couples living together, 4.9% had a female householder with no husband present, and 14.6% were non-families. 12.2% of all households were made up of individuals and 2.4% had someone living alone who was 65 years of age or older. The average household size was 2.85 and the average family size was 3.11.
27.6% of the population were under the age of 18, 4.9% from 18 to 24, 25.5% from 25 to 44, 29.6% from 45 to 64, and 12.4% who were 65 years of age or older. The median age was 41 years. For every 100 females there were 100.4 males. For every 100 females age 18 and over, there were 94.3 males.
The median household income was $68,750 and the median family income was $76,604. Males had a median income of $57,500 compared with $22,500 for females. The per capita income for the town was $36,091. About 1.6% of families and 0.9% of the population were below the poverty line, including none of those under age 18 and 2.7% of those age 65 or over.
Annetta North is served by the Aledo and Weatherford Independent School Districts.
Annetta North, Texas Wikipedia
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Article Free access | 10.1172/JCI19852
Acidic sphingomyelinase downregulates the liver-specific methionine adenosyltransferase 1A, contributing to tumor necrosis factor–induced lethal hepatitis
Montserrat Marí,1 Anna Colell,1 Albert Morales,1 Covadonga Pañeda,2 Isabel Varela-Nieto,2 Carmen García-Ruiz,1,3 and José C. Fernández-Checa1,3
1Liver Unit, Institut de Malalties Digestives, Hospital Clínic i Provincial, Instituto Investigaciones Biomédicas August Pi i Sunyer, Barcelona, Spain. 2Instituto Investigaciones Biomédicas Alberto Sols, Consejo Superior Investigaciones Científicas, Madrid, Spain. 3Department of Experimental Pathology, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.
Address correspondence to: José C. Fernández-Checa, Liver Unit, Institut de Malalties Digestives, Hospital Clínic i Provincial, C/ Villarroel 170, 08036-Barcelona, Spain. Phone: 34-93-2275709; Fax: 34-93-451 5272; E-mail: checa229@yahoo.com.
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Published in Volume 113, Issue 6 on March 15, 2004
Received: August 20, 2003; Accepted: January 13, 2004
S-adenosyl-L-methionine (SAM) is synthesized by methionine adenosyltransferases (MATs). Ablation of the liver-specific MAT1A gene results in liver neoplasia and sensitivity to oxidant injury. Here we show that acidic sphingomyelinase (ASMase) mediates the downregulation of MAT1A by TNF-α. The levels of MAT1A mRNA as well as MAT I/III protein decreased in cultured rat hepatocytes by in situ generation of ceramide from exogenous human placenta ASMase. Hepatocytes lacking the ASMase gene (ASMase–/–) were insensitive to TNF-α but were responsive to exogenous ASMase-induced downregulation of MAT1A. In an in vivo model of lethal hepatitis by TNF-α, depletion of SAM preceded activation of caspases 8 and 3, massive liver damage, and death of the mice. In contrast, minimal hepatic SAM depletion, caspase activation, and liver damage were seen in ASMase–/– mice. Moreover, therapeutic treatment with SAM abrogated caspase activation and liver injury, thus rescuing ASMase+/+ mice from TNF-α–induced lethality. Thus, we have demonstrated a new role for ASMase in TNF-α–induced liver failure through downregulation of MAT1A, and maintenance of SAM may be useful in the treatment of acute and chronic liver diseases.
TNF-α plays a central role in many forms of liver injury (1–3) and hence information on signaling of hepatocellular cell death by TNF-α may be of therapeutic relevance. The signaling mechanism mediating the intracellular effects of TNF-α is complex and can lead to multiple responses, including proliferation, inflammation, or cell death. The cytotoxic effects of TNF-α are the result of the generation of death signals that originate from the binding of TNF-α to the TNF-α–R1 receptor, a typical death receptor with a 60– to 80–amino acid cytoplasmic sequence known as the death domain. Research in this area has unveiled complex protein-protein interactions, recruitment of signaling intermediates, and activation of proteins that mediate the apoptotic effect of TNF-α (4, 5). Ceramide has emerged as a putative signaling lipid mediator that plays a role in the stress response, cell death, and TNF-α signaling (6–9). Ceramide levels can increase by de novo synthesis through activation of serine-palmitoyl transferase, the rate-limiting enzyme in ceramide synthesis, or ceramide synthetase (10, 11). However, the hydrolysis of sphingomyelin upon activation of sphingomyelinases (SMases) constitutes an effective means for the rapid generation of ceramide (8, 9). The binding of TNF-α to its plasma membrane receptor TNF-α–R1 results in the activation of two major types of SMases: a neutral SMase (NSMase), with an optimum pH around 7.5, which is membrane bound and Mg2+ dependent; and an acidic SMase (ASMase), with an optimum pH of about 4.8, which is further subclassified into two isoforms, an endosomal/lysosomal ASMase and a secretory Zn2+-dependent ASMase (8, 9, 12, 13).
While activation of these enzymes may generate ceramide at different cellular sites that mediate the diverse effects of TNF-α, their role in apoptosis is not well established. Thus, NSMase has been reported to be involved in the stress response mediating the cytotoxic effects exerted by chemotherapeutic agents (14), whereas ASMase has been shown to play an essential role in ionizing radiation–mediated cell death (15, 16) as well as in the developmental death of oocytes (17). In addition, the factor associated with NSMase activation, an adaptor protein involved in NSMase activation, has been shown to contribute to TNF-α–induced fibroblast apoptosis (18), although the role of NSMase ablation itself in TNF-α–induced apoptosis remains unknown. On the other hand, the role of ASMase in Fas-induced cell death appears to be restricted to hepatocytes, as the absence of ASMase in hepatocytes has been shown to confer resistance to Fas-mediated cell death (19, 20), while other cell types from ASMase knockout mice are sensitive to Fas (19). Moreover, we have recently demonstrated that ASMase plays a significant role in hepatocellular apoptosis and liver damage induced by TNF-α (21).
The liver plays a prominent role in the synthesis and consumption of S-adenosyl-L-methionine (SAM, also abbreviated as "AdoMet"), which functions as the one-carbon donor for methylation of DNA, RNA, and proteins and in the synthesis of biogenic amines and glutathione (GSH) (22–24). Furthermore, SAM is also required for DNA and RNA methylation in mitochondria and as an intermediate in the biosynthesis of ubiquinone and for sterol methylation (25, 26). SAM is synthesized from methionine and ATP by methionine adenosyltransferases (MATs). In mammals there are three known MAT isoforms (MAT I, MAT II, and MAT III) (23, 27, 28); MAT I and MAT III are a tetramer and a dimmer, respectively, of the same α1 subunit, a 43.7-kDa polypeptide encoded by the gene MAT1A, which is exclusively expressed in adult liver (23). MAT2A, on the other hand, is ubiquitously expressed and encodes a catalytic subunit (α2) found in a native MAT isoenzyme (MAT II) in association with a regulatory β subunit (29). In the liver, impairment of MAT I/III results in lowered SAM levels, which contributes to the progression of liver injury in experimental models and in patients with alcohol- or virus-induced cirrhosis (23, 30–33). Indeed, the most compelling evidence for the relevance of hepatic SAM has been recently documented with the development of MAT1A knockout mice (34). This paradigm illustrates the devastating consequences of chronic hepatocellular SAM depletion, which enhances the susceptibility of the liver to oxidant-induced cell death, metabolic disturbances, and spontaneous hepatocellular carcinoma (34, 35).
A common strategy for investigating the cellular effects of ceramide has been the incubation of cells with exogenous SMases, cell-permeable analogs, or natural ceramide, which has revealed a wide spectrum of responses ranging from cell cycle arrest to proliferation, oxidative stress, apoptosis, and gene regulation (6, 8, 21, 36, 37). As for the effect on gene regulation, membrane-permeant ceramide analogs (C2 and C6) as well as exogenous NSMase have been shown to modulate the expression of specific genes such as cytochrome P450 2C11, α1-acid glycoprotein, or γ-glutamylcysteine synthetase (γ-GCS) in rat hepatocytes, mimicking the effects of interleukin-1 or TNF-α (36, 37). In addition, we have recently reported that the cell-permeable ceramide C2 downregulated MAT1A expression in H35 rat hepatoma cells (38). Thus, the present study was undertaken to examine the regulation of MAT1A by individual SMases, namely Bacillus cereus NSMase (bNSMase) and human placenta ASMase (hASMase), previously characterized in rat hepatocytes (37), with the ultimate goal of assessing the contribution of ASMase to the regulation of MAT1A expression and to test the therapeutic role of SAM in TNF-α–induced fulminant liver failure.
Hepatocyte isolation and incubation with SMases and glycosphingolipids. ASMase knockout mice (C57BL/6 strain) were propagated using heterozygous breeding pairs (a generous gift from R. Kolesnick, Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, USA; and E. Gulbins, University of Duisburg-Essen, Essen, Germany) and were genotyped as described previously (21). Hepatocytes from male Sprague-Dawley rats (200–300 grams) or male mice (8–12 weeks old) were isolated by collagenase perfusion with flow rates of 16–18 ml/min and 7–9 ml/min, respectively, and were cultured at a density of 2 × 106 cells on 60-mm dishes coated with rat tail collagen. Rat or mouse hepatocytes were incubated with recombinant human TNF-α (15–280 ng/ml; Preprotech EC, London, United Kingdom) or exogenous SMases (Sigma-Aldrich, St. Louis, Missouri, USA) by dilution of concentrated hASMase (250 U/0.55 ml in 50% glycerol, 25 mM potassium phosphate, pH 4.5, 0.1% Triton X-100, and 0.05 mM PMSF) and bNSMase (100 U/ml in 50% glycerol and 50 mM Tris-HCl, pH 7.5) with culture medium to a concentration of 0.1–1.0 U/ml as described previously (37). Some cultures were pretreated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol HCl (D-threo-PDMP), N-butyldeoxynojirimycin, bafilomycin A1, chloroquine, or mannose 6-phosphate (M 6-P) before the addition of TNF-α or hASMase. Alternatively, cells were incubated with cell-permeable ceramide (C2 or C6), ganglioside GD3 (GD3), ganglioside GM3 (GM3), glucosylceramide, lactosylceramide, or lyso-glucosylceramide (1–5 μM) (Matreya, Pleasant Gap, Pennsylvania, USA) in ethanol (0.1%). In addition, cells were incubated with the natural ceramide C16 in dodecane/ethanol (2:98 [volume/volume]; 0.05% final concentration) or solvent only as a control. The experimental protocols met the guidelines of and were approved by the Animal Care Committee of the Hospital Clinic-Universidad de Barcelona.
Real-time PCR. MAT1A and MAT2A mRNA levels were determined by real-time RT-PCR analyses using a LightCycler and a Faststart SYBR Green I DNA quantification kit (Roche, Mannheim, Germany) following the manufacturer's protocol. cDNA was prepared from total RNA (2 μg) at 50°C for 50 minutes using random hexamers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) following the manufacturer's protocol. The primers used were: MAT1A, forward, 5′-ATGGACCTGTGGATGGC-3′, and reverse, 5′-TGGCCTCCAGTGTTATGT-3′; MAT2A, forward, 5′CCATGAGGCGTTCATCG-3′, and reverse, 5′-GCGTAACCAAGGCAATG-3′; 18S, forward, 5′-GGGGAATCAGGGTTCGATT-3′, and reverse, 5′-GCGCCGGTCCAAGAATTTCA-3′. The second-derivative maximum method was taken to determine the crossing points automatically for individual samples, and relative amounts of target genes were calculated based on the crossing-point analysis. The result was expressed as a ratio of product copies to copies of housekeeping gene 18S from the same RNA.
Northern blot. MAT1A and MAT2A mRNA levels were also determined by Northern blot analyses of total RNA isolated using the Trizol reagent (Gibco/BRL, Grand Island, New York, USA). Ten micrograms of RNA were separated by electrophoresis under denaturing conditions and were transferred onto Hybond-XL nylon membranes (Amersham Biosciences, Uppsala, Sweden) and hybridized to random-primed 32P-labeled cDNA probes.
MAT I/III protein levels. Samples were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose membrane, and incubated with rabbit anti–MAT I/III (1:10,000 dilution), a generous gift from M. Pajares (Instituto Investigaciones Biomédicas Alberto Sols, Consejo Superior Investigaciones Científicas, Madrid, Spain), characterized previously (39). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:5,000 dilution; Amersham, Arlington Heights, Illinois, USA), and bound antibody was visualized with ECL detection (ECL; Amersham) on Kodak X-OMAT film (Eastman Kodak, Rochester, New York, USA).
SAM/S-adenosyl-l-homocysteine (SAH), mitochondrial isolation, and GSH determination. SAM or SAH levels were determined in liver homogenates (or in mitochondrial fractions isolated from liver homogenates) after homogenization with 0.5 M HClO4 followed by neutralization of the sample with 3 M KOH. SAM/SAH levels were determined by HPLC using a Partisil SCx 10-μm column by isocratic elution at a flow rate of 1 ml/min with 0.03 mol/l NH4H2PO4 containing 2% acetonitrile, pH 2.6, as described before (33). SAM and SAH peaks were identified at 254 nm by sample spiking with authentic standards. The amount of either metabolite was calculated from standard curves relating the peak area to calibration curves with standards. In some cases, mitochondria were isolated by Percoll gradient centrifugation as described (40). GSH levels in homogenates or mitochondria were determined using HPLC (21, 37).
Fulminant hepatitis model and therapeutic treatment with SAM. The combination of D-(+)-galactosamine (Gal) plus TNF-α (Gal/TNF) or Gal plus LPS (Gal/LPS) is a known model of lethal hepatitis (41, 42). C57Bl/6 mice were used at an age of 8–10 weeks. A single dose of recombinant murine TNF-α (10 mg/kg intravenously; Preprotech EC, London, United Kingdom) or LPS (50 μg/kg intraperitoneally; Escherichia coli serotype 0128:B12; Sigma-Aldrich) was administered after Gal treatment (700 mg/kg intraperitoneally), resulting in the death of mice (90–95%) within 6–8 hours. In some cases, SAM (p-toluensulfonate form; Europharma, Madrid, Spain) was given hourly for 8 hours after TNF-α or LPS injection (5 mg/mouse intraperitoneally). For estimation of the effect of prolonged SAM treatment, SAM was administered to Gal/TNF- or Gal/LPS-treated mice for 24 hours (hourly for the first 8 hours and then every 8 hours). For blocking of GSH synthesis, L-buthionine sulfoximine (BSO) was administered (4 mmol/kg) 30 minutes before and 3 hours after TNF-α or LPS administration. Alternatively, N-acetylcysteine (NAC) (30 mg/mouse intraperitoneally) was given to mice before treatment with Gal/TNF or Gal/LPS to assess the efficacy of antioxidants on the course of liver injury. For histology, liver specimens were washed with PBS and embedded in paraffin. Sections 7 μm in thickness were stained with hematoxylin and eosin using standard methods. Serum alanine aminotransferase (ALT) and aspartate aminotransferase levels were determined by a colorimetric test (Sigma-Aldrich).
TUNEL assay. Paraffin-embedded liver tissue was cut into slices 4 μm in thickness using a microtome (Leica RM2155; Leica Instruments, Bensheim, Germany) and was fixed to glass slides. The TUNEL assay was performed by incubation of the slices with 2 μg/ml proteinase K (Sigma-Aldrich) in 10 mM Tris-HCl buffer containing 5 mM EDTA at a pH of 7.4. Sections were then labeled following the instructions of a commercial Kit (In situ Cell Death Detection Kit; Roche Diagnostics), stained with 3,3′-diaminobenzidine (Roche Diagnostics), and counterstained with hematoxylin and eosin. The slides were mounted and examined using a Nikon Eclipse E1000 microscope.
Caspase 3 and caspase 8 activation. Liver was homogenized in a buffer containing 120 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0,5 % Igepal, 2 mM ethylene glycol-bis-(β-aminoethyl ether) tetraacetic acid, and 50 μM PMSF, incubated at 4°C for 15 minutes with shaking, and spun down at 12,000 g at 4°C for 15 minutes. Caspase activity was assayed with 200 μg of cell lysate and 1.25 ml of assay buffer containing 100 mM NaCl, 10% sucrose, 0.1% (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 10 mM DTT, pH 7.4, by the release of 7-aminomethyl coumarin from 40 nmol of Ac-DEVD-AMC (caspase 3) or Ac-IETD-AMC (caspase 8) (Calbiochem, San Diego, California, USA). Fluorescence was continuously recorded with emission at 460 nm and excitation at 380 nm as described (43).
In addition, caspase activation was also monitored by immunoblotting using rabbit anti–caspase 3 (1:1,000 dilution; Pharmingen, San Diego, California, USA) and anti–caspase 8 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, California, USA) as primary antibodies. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Amersham) and bound antibodies were visualized as described above for MAT I/III.
Measurement of gangliosides and ceramide levels. Gangliosides levels (including GM1, GM3, and GD3) were measured by high-performance thin-layer chromatography as described previously (43). Ceramide levels were quantified by HPLC (37).
TNF-α levels. Total TNF-α (bound and free) was measured in serum after Gal/LPS treatment using a mouse-specific competitive enzyme immunoassay kit (Chemicon, Temecula, California, USA).
Statistical analyses. Statistical analyses of mean values ± SD for multiple comparison were made by one-way analyses of variance followed by Fisher's exact test. Survival curves were compared using a log-rank χ2 test.
Regulation of MAT1A by hASMase. Preservation of high levels of MAT1A is critical for a differentiated and functional competent liver. Upon isolation from the intact organ, cultured rat hepatocytes undergo a gradual decline in MAT1A mRNA as it is replaced by MAT2A mRNA (23, 44). In vitro stabilization of MAT1A, however, can be achieved by the incubation of cultured hepatocytes with SAM (44). Because TNF-α downregulates MAT1A expression in cultured rat hepatocytes (38) and SMases mediate TNF-α signaling (8, 9), we first examined the role of individual SMases on MAT1A expression. Cultured rat hepatocytes were incubated with exogenous bNSMase and hASMase, and the levels of MAT1A and MAT2A mRNA were examined over time. Although the intracellular levels of ceramide generated by exposure of cultured hepatocytes to exogenous bNSMase or hASMase were equivalent (2–4 nmol ceramide/mg cellular protein), these enzymes regulated the expression of MAT1A and MAT2A in a divergent fashion. In the presence of exogenous SAM (3 mM), the level of MAT1A mRNA in control hepatocytes remained unchanged for 14–16 hours (not shown) in agreement with previous results (44). Next, we examined the effect of exogenous SMases on MAT1A mRNA by Northern blot and real-time PCR analyses. Either approach showed equivalent results, indicating a decrease in the levels of MAT1A mRNA by hASMase while those of MAT2A mRNA increased, thus mimicking the effect observed with TNF-α (Figure 1, A and B). In contrast, exogenous bNSMase did not affect the expression of MAT1A or MAT2A (Figure 1, A and B). The repressive effect of hASMase was observed within 3 hours of incubation, with a significant reduction in MAT1A mRNA levels to 39–45% of control values. To determine if the downregulation of MAT1A mRNA was reflected at the protein level, we analyzed the MAT I/III protein content by Western blot. As seen, both TNF-α and hASMase but not bNSMase reduced the levels of MAT I/III (Figure 1C). Consistent with these findings, we confirmed a severe reduction in MAT I/III enzymatic activity (8.7 ± 1.2 vs. 3.2 ± 0.8 pmol/min/mg protein in control or hASMase-treated cells, respectively; P < 0.05). Moreover, as TNF-α increases the cellular levels of glycosphingolipids synthesized in the Golgi from ceramide, and as glycosphingolipids play an important role in cell death (21, 43), we next examined the contribution of glycosphingolipids in the regulation of MAT1A by TNF-α. In agreement with previous results (21), preincubation of cells with D-threo-PDMP inhibited glucosylceramide synthase (GCS), the first enzyme in glycosphingolipid synthesis, decreasing the stimulation of GD3 and GM1 levels by TNF-α (325% ± 56% or 298% ± 76% vs. 8% ± 5% or 3% ± 4% for GD3 and GM1 in the absence or presence of D-threo-PDMP, respectively; P < 0.05). As expected, D-threo-PDMP enhanced TNF-α–generated ceramide levels (2.3 ± 0.3–fold; P < 0.05). However, under these conditions the downregulating effect of TNF-α on MAT1A expression was not affected by D-threo-PDMP (Figure 1A). Similar findings were observed using N-butyldeoxynojirimycin, another GCS inhibitor (not shown). Thus, these findings show the ability of ceramide generated specifically from hASMase to downregulate the expression of MAT1A and exclude the possibility of a role for glycosphingolipids in this effect.
Regulation of MAT1A by SMases. (A) Cultured rat hepatocytes were treated with TNF-α, with or without D-threo-PDMP (PDMP), or with hASMase or bNSMase alone for 16 hours. MAT1A and MAT2A mRNA levels were analyzed by Northern blot and were normalized to 18S mRNA content, and the corresponding ratio was expressed as percentage of control. The plots in the graph (bottom) are the mean ± SD of six individual experiments. *P < 0.05 versus control; denotes significance for MAT1A and MAT2A changes. (B) These experiments were similar to those shown in A except that MAT1A mRNA levels were determined by real-time PCR analyses as described in Methods. Results are expressed as the mean ± SD of three individual experiments. *P = 0.05 versus control. (C) MAT I/III protein content was determined by Western blot after 18 hours of incubation with TNF-α, hASMase, or bNSMase at the dose shown. Data are the mean ± SD of three individual experiments. *P = 0.05 versus control. C, control.
Abolishment of intracellular vesicle acidification prevents downregulation of MAT1A mRNA by hASMase. We have previously shown that exogenous hASMase enters hepatocytes by endocytosis being delivered to intracellular acidic compartments (37). To assess whether hASMase requires acidic vesicles for its effect on MAT1A expression, we next determined the effect of lysosomotropic agents that abolish acidification of intracellular vesicles. As shown (Figure 2), bafilomycin A1, an inhibitor of the H+-ATPase pump responsible for acidifying lysosomes and late endosomes (45), and chloroquine, known to disrupt acidic compartments (46), each prevented the downregulation of MAT1A induced by hASMase (Figure 2). Moreover, M 6-P, which blocks the delivery to endosomes/lysosomes of enzymes via the M 6-P receptor (47), abolished the reduction in MAT1A mRNA by hASMase (Figure 2). A similar effect for bafilomycin A1 and chloroquine was observed in the TNF-α–mediated downregulation of MAT1A mRNA levels (not shown). Thus, these findings indicate that the internalization of hASMase into acidic vesicles is required for the downregulation of MAT1A mRNA.
Regulation of the effect of hASMase on MAT1A expression. Cultured rat hepatocytes were incubated with hASMase (0.5 U/ml) for 16 hours in the presence of bafilomycin A1 (40 nM), chloroquine (50 μM), or M 6-P (3 mM), and the levels of MAT1A, MAT2A, and 18S mRNA were analyzed by Northern blot. The plot showing the change in the corresponding mRNA (bottom) is the mean ± SD of three individual experiments. *P = 0.05 versus control; denotes significance for MAT1A and MAT2A changes.
Structural requirement of ceramide in the downregulation of MAT1A. To assess the structural requirement of ceramide, we tested the role of sphingolipid analogs on MAT1A expression. As seen, membrane-permeant ceramide C2, as well as glycosphingolipids GD3, GM3, lactosylceramide, and glucosylceramide, reduced MAT1A mRNA levels to a similar extent while increasing those of MAT2A (Figure 3). Similar findings were observed for MAT1A regulation using the natural ceramide C16 (not shown). In contrast, incubation of cells with sphingomyelin, sphingosine, or dihydroceramide C2 did not change the relative levels of MAT1A or of MAT2A mRNA. In addition, incubation of cells with lyso-lactosylceramide (Matreya) did not affect the level of MAT1A or MAT2A mRNA (not shown). Thus, taking into account that carbohydrates are present only in glycosphingolipids but not in sphingolipids and that the N-fatty acyl-sphingosine moiety and the trans-4,5 double bond are structural features common to both lipid classes, it appears that the carbohydrate groups are not required for the regulation of MAT1A expression.
Regulation of MAT1A by sphingolipids. Cultured rat hepatocytes were incubated for 16 hours with various sphingolipids at the concentrations shown and the levels of MAT1A mRNA, MAT2A mRNA, and 18S mRNA were determined by Northern blot analyses. The plot showing the change in mRNA (bottom) is the mean ± SD of six individual experiments. *P < 0.05 versus control; denotes significance for MAT1A and MAT2A changes.
TNF-α fails to downregulate MAT1A in hepatocytes from ASMase-deficient mice. To assess the specific role of ASMase in the regulation of MAT1A by TNF-α, we used hepatocytes from ASMase–/– mice, as early signaling events in response to TNF-α are preserved in ASMase knockout mice (21). Compared with those in ASMase+/+ cells, the levels of MAT1A mRNA in hepatocytes lacking the ASMase gene were unaffected upon exposure to TNF-α (Figure 4, A and B). However, despite the unresponsiveness of ASMase–/– hepatocytes to TNF-α exposure, the addition of exogenous hASMase markedly decreased the levels of MAT1A mRNA while increasing those of MAT2A (Figure 4B). This effect was specific for ceramide derived from hASMase, as incubation of cells with exogenous bNSMase did not affect MAT1A or MAT2A mRNA content (Figure 4). These results show that the repressive effect of TNF-α on MAT1A expression is mediated through the generation of ceramide from ASMase.
Regulation of MAT1A in hepatocytes from ASMase knockout mice. ASMase+/+ (A) or ASMase–/– (B) hepatocytes were cultured for 16 hours with TNF-α, bNSMase, or hASMase at the dose shown, and the levels of MAT1A and MAT2A mRNA were determined by Northern blot. The plots (bottom) show the mean ± SD of six to seven individual experiments. *P < 0.05 versus control; denotes significance for MAT1A and MAT2A changes.
Hepatic SAM levels in ASMase–/– mice during lethal hepatitis. To address whether the contribution of ASMase to TNF-α–induced liver injury is through MAT1A downregulation and subsequent SAM decrease, we examined the changes over time of hepatic SAM produced by Gal and TNF-α or LPS in a well-established model of lethal hepatitis (41, 42). In ASMase+/+ mice (n = 25), Gal/TNF caused a time-dependent decrease of the hepatocellular SAM levels before the occurrence of liver damage, as monitored by the rise in ALT in serum (Figure 5A). Histological examination of liver samples from Gal/TNF- or Gal/LPS-treated mice indicated extensive hemorrhagic lesions with clusters of damaged cells (Figure 5B). TUNEL assay showed the presence of apoptotic hepatocytes in liver specimens from Gal/LPS-treated wild-type mice (Figure 6A). In contrast, the hepatic levels of SAM in ASMase–/– mice treated with Gal/TNF remained significantly higher than those in wild-type mice, and this outcome was accompanied by minimal liver damage, as monitored by serum transaminases (Figure 5A), liver histology (Figure 5B), and TUNEL assay (Figure 6A). Northern blot analyses indicated a decrease in MAT1A mRNA levels in ASMase+/+ mice by 3 hours after Gal/TNF treatment (not shown). The hepatocellular levels of SAM in the ASMase knockout mice after Gal/TNF treatment were similar to those in ASMase+/+ mice treated with Gal alone (63 ± 5 nmol/g liver and 53 ± 6 nmol/g liver 3 hours and 6 hours after, respectively). As this model of liver injury is caspase dependent, we examined the activation of caspases 8 and 3 in liver samples after Gal/TNF administration by a fluorescence assay using appropriate substrates and by immunoblot. Although each approach showed activation of caspases 3 and 8 by Gal/TNF in hepatocytes from wild-type mice (Figures 5C and 6, B and C), interestingly, caspase activation was much lower in ASMase–/– mice treated with Gal/TNF than in ASMase+/+ mice (Figures 5C and 6, B and C), suggesting that ASMase is necessary for optimal caspase activation in this model. Furthermore, as hepatic SAM regulates GSH homeostasis, which in turn can modulate caspase activation (48, 49), we next determined the time course of hepatic GSH levels in this model of hepatocellular cell death. As seen, hepatic GSH levels decreased in ASMase+/+ mice over time, lagging behind the reduction in SAM content in ASMase+/+ mice but not in ASMase–/– mice (Figure 5, A and D). Similar results were observed with Gal/LPS (not shown). Finally, the levels of mitochondrial GSH, an essential pool of GSH in the control of mitochondrial function and cell death (21, 40), were reduced to 51 ± 7% of control levels in ASMase+/+ mice (Figure 5E).
Fulminant hepatitis in ASMase+/+ and ASMase–/– mice. (A) ASMase+/+ and ASMase–/– mice (n = 25 each) were treated with Gal/TNF, and the levels of hepatic SAM were determined over time in relation to the appearance of liver damage, as monitored by serum ALT activity in serum. *P < 0.05 versus values at time 0; #P < 0.05 versus corresponding time points from ASMase+/+ mice. (B) Representative hematoxylin and eosin staining of liver sections from ASMase+/+ or ASMase–/– mice collected 6 hours after treatment with Gal/TNF or Gal/LPS. Scale bars: 5 μm. (C) Caspase activity in liver samples taken from ASMase+/+ or ASMase–/– 6 hours after treatment with Gal/TNF. *P < 0.05 versus control mice; #P < 0.05 versus ASMase+/+ mice. (D) Hepatic GSH levels were determined in liver samples from ASMase+/+ and ASMase–/– mice at various times after treatment with Gal/TNF. *P < 0.05 versus values at time 0. (E) Mitochondrial fractions were prepared from the livers of ASMase+/+ mice 6 hours after challenge with saline (C) or Gal/LPS with or without SAM administration given hourly as described in Methods. MitGSH, mitochondrial GSH. *P < 0.05 versus control; #P < 0.05 versus Gal/LPS.
TUNEL assay and immunoblot analyses of caspase activation. (A) Liver samples from ASMase+/+ or ASMase–/– mice treated with Gal/TNF or ASMase+/+ mice treated with SAM for 8 hours were subjected to TUNEL assay for the detection of apoptotic hepatocytes (magnification, ×200). (B and C) Cellular extracts from these groups of mice were incubated with anti–caspase 3 (B) or anti–caspase 8 (C) to monitor the processing of the corresponding caspase. Representative immunoblots of three individual experiments showing similar results are shown.
Lethal liver failure in ASMase+/+ mice is prevented by SAM administration. Because the fall in SAM levels was a primary event in this model of liver failure, we next examined the therapeutic role of SAM in ASMase+/+ mice. Initial experiments indicated that a single intraperitoneal injection of 10 mg/kg of SAM, a dose previously used in experimental models of liver injury and fibrogenesis (33, 50), before Gal/TNF or Gal/LPS administration was ineffective in preserving hepatic SAM levels and in preventing liver damage (not shown). However, continuous SAM treatment, hourly after Gal/LPS exposure, prevented liver injury through restoration of hepatic SAM (Figure 7, A–C). Furthermore, SAM treatment decreased caspase activation, as assessed by immunoblot (Figure 6, B and C) or stimulated fluorescence (Figure 7D), and these effects were accompanied by restoration of intracellular GSH stores in cytosol (Figure 7E) and mitochondria (Figure 5E). Indeed, the specific contribution of GSH to the protective effect of SAM was further assessed by treatment of mice with BSO, an inhibitor of γ-GCS. Pretreatment of ASMase+/+ mice with BSO after Gal/LPS administration abolished the protective effect of SAM treatment (Figure 7, A and B). BSO treatment, however, did not affect the levels of SAM but prevented the recovery of GSH (not shown). In addition, to ensure that the protective effect of SAM was not due to reduced TNF-α secretion caused by LPS, we monitored plasma TNF-α levels over time. As seen, the time course of TNF-α appearance in plasma was similar in Gal/LPS-treated mice with or without SAM therapy (Figure 7F). Moreover, we checked the safety of chronic SAM therapy in Gal/LPS-treated mice. As seen, sustained administration of SAM was safe and devoid of side effects, as mice were protected from Gal/LPS-induced liver damage (Figure 8A). In addition, NAC, an antioxidant, reduced the liver injury caused by Gal/LPS (Figure 8B).
SAM therapy in fulminant hepatitis. Experimental ASMase+/+ mice (n = 40) were treated with Gal/LPS; control mice (Saline, in B; n = 40) were treated with saline. Mice were divided in three groups: one group received SAM (5 mg/mouse intraperitoneally) or saline every hour for 8 hours after treatment with Gal/LPS; in addition, half of the mice receiving SAM therapy were pretreated with BSO (4 mmol/kg). (A) Serum ALT or AST levels. *P < 0.05 versus Gal/LPS; #P < 0.05 versus ASMase+/+ mice treated with Gal/LPS + SAM. AST, aspartate aminotransferase. (B) Hematoxylin and eosin staining of livers with or without SAM administration or BSO treatment. Scale bars: 5 μm. (C) Hepatocellular SAM levels of ASMase+/+ mice challenged with Gal/LPS with or without SAM therapy. *P < 0.05 versus values at time 0; #P < 0.05 versus Gal/LPS group. (D) Caspase activity in livers from ASMase+/+ mice with or without SAM treatment. *P < 0.05 versus untreated mice; #P < 0.05 versus Gal/LPS group. (E) Hepatic GSH levels in ASMase+/+ mice with or without SAM treatment. *P < 0.05 versus values at time 0; #P < 0.05 versus Gal/LPS group. (F) Serum TNF-α levels in ASMase+/+ mice at various time points after Gal/LPS treatment with or without SAM therapy.
Protection against liver failure by chronic SAM treatment or antioxidants. (A) ASMase+/+ mice treated with Gal/LPS were administered SAM for 16 or 24 hours, and liver damage was assessed by serum transaminase analyses. Data are the mean ± SD of ten individual experiments. *P = 0.05 versus Gal/LPS. (B) Alternatively, ASMase+/+ mice were treated with NAC instead of SAM to analyze its effect on liver injury induced by Gal/LPS, as assessed by serum transaminases. Data are the mean ± SD of four individual experiments. *P = 0.05 versus Gal/LPS.
Finally, the protective role of SAM "translated" to an improved survival rate (Figure 9). As seen, mice (n = 15) treated with SAM were significantly protected from lethality, compared with the control group treated with saline. Surviving mice were still alive for at least 5 days after the Gal/LPS treatment. Treatment of mice with SAM for 24 hours resulted in a similar survival rate (not shown). Similar results in terms of survival were observed in mice treated with Gal/TNF instead of LPS. In addition, 80–90% of ASMase–/– mice survived at least 5 days after Gal/TNF treatment.
Effect of SAM therapy on survival of mice treated with Gal/LPS. ASMase+/+ mice (n = 30) were treated with Gal/LPS; half were treated with SAM (Gal/LPS + SAM) and the other half were treated with saline (Gal/LPS) every hour for 8 hours. The percentage of dying mice was plotted as a function of time after induction of hepatitis. *P < 0.05 vs. Gal/LPS group.
The present study reveals a novel mechanism of action of ASMase whereby this signaling enzyme promotes TNF-α–induced hepatocellular damage by decreasing the expression of MAT1A. Our first approach was the use of exogenous hASMase. Certain structural features of this enzyme have been characterized in comparison with those of recombinant human ASMase (51). Those studies demonstrated that recombinant human ASMase and the native protein purified from human placenta showed a similar glycosylation pattern and the presence of a critical disulfide bond in the sphingolipid activator N-terminal domain. In addition, prior studies demonstrated that hASMase enters cells including hepatocytes via M 6-P receptor–mediated endocytosis and is delivered into acidic compartments (37, 47, 52). Consistent with the pH optima of hASMase, we verified that agents that block intracellular vesicle acidification abolished the effect of hASMase on MAT1A expression. In addition, the action of hASMase on MAT1A expression was prevented by an excess of M 6-P, which would be expected to compete with hASMase for endocytosis without dissipating the pH gradient of acidic intracellular compartments. Thus, based on these findings it appears that endocytosis of hASMase and its delivery into acidic vesicles where the enzyme becomes active are required for the downregulation of MAT1A expression. However, the precise intracellular site where hASMase becomes activated remains to be identified. In addition to this approach, we also used hepatocytes deficient in ASMase–/–. The data we obtained show the resistance of hepatocytes from ASMase knockout mice to TNF-α–induced downregulation of MAT1A. However, the levels of MAT1A mRNA were reduced by hASMase, indicating that the response of hepatocytes from ASMase knockout mice to TNF-α in the regulation of MAT1A is dependent on the presence of ASMase.
An important implication from these results is the divergent functions of NSMase versus ASMase in the regulation of MAT1A expression. Not only did bNSMase, a Mg2+-dependent exogenous SMase at neutral pH, characterized previously (8, 9, 37), fail to affect the expression of MAT1A, but also, more importantly, the activation of NSMase by TNF-α remains intact in hepatocytes from ASMase knockout mice (21) and yet in ASMase–/– hepatocytes, TNF-α failed to downregulate MAT1A. Thus, our findings clearly indicate that the ceramide pool signaling the downregulation of MAT1A originates from an acidic intracellular compartment, whereas the ceramide pool derived from NSMase is inactive, illustrating that the role of ceramide in cell signaling depends on the specific SMase activated. This outcome regarding the effect of ASMase on gene regulation contrasts with previous reports discarding a role for ceramide produced in lysosomes in apoptosis signaling. For instance, stress-induced apoptosis was observed in Epstein-Barr virus–transformed B lymphocytes from control individuals as well as from patients affected with Niemann-Pick disease, an autosomal recessive condition characterized by deficient ASMase in lysosomes (14). In contrast, DeMaria et al. (52), who also used Niemann-Pick lymphoblast lines, concluded that ASMase was required for Fas-mediated apoptosis. On the other hand, hepatocytes deficient in ASMase have been shown to be resistant to ionizing radiation–, Fas- or TNF-α–mediated cell death (15, 19–21). Furthermore, consistent with our findings regarding MAT1A regulation, Monney et al. (53) reported that agents that alkalinize acidic vesicles attenuate TNF-α–induced apoptosis in U937 human monocytes and L292 mouse fibroblasts. Thus, it appears that the role of individual SMases in signaling pathways (apoptosis or gene regulation) depends on several conditions, such as the stimuli used and type of cell studied. Hence, taken collectively, our data establish that MAT1A is selectively downregulated by ceramide generated from the activation of ASMase in acidic intracellular compartments, which mediates the effects observed with TNF-α.
Of note, we would like to emphasize that the downregulating effect of TNF-α or hASMase on MAT1A mRNA levels was observed despite the presence of exogenous SAM, which stimulates the transcription of MAT1A (44). Thus, in further examining the putative mechanism involved in the downregulation of MAT1A by ASMase, we determined the stability of MAT1A mRNA in the presence of actinomycin D. Our data show that the presence of hASMase shortened the half-life of MATA1 mRNA from 5.4 ± 0.3 hours to 3.3 ± 0.2 hours (P = 0.005). In an attempt to characterize this process further, we observed that cycloheximide pretreatment abolished the effect of hASMase on MAT1A mRNA decay, suggesting the involvement of specific proteins. Thus, although at present we do not understand the mechanism involved in the posttranscriptional decay of MAT1A mRNA by ASMase, an important pathway in the control of mRNA includes the recruitment of the exosome by AU-binding proteins to affect 3′-to-5′ mRNA degradation (54). Although clearly more work will be required to fully characterize this novel mechanism in the regulation of MAT1A expression by ASMase-induced ceramide generation, including the identification of the specific proteins involved and the role of the AU site in the 3′ untranslated region of MAT1A, similar effects on the modulation of mRNA stability have been described recently for cell-permeable ceramides in other cell types. For instance, in Jurkat cells, ceramide C2 shortened the half-life of Bcl-2 mRNA through a conserved AU-rich element in the 3′ untranslated region of Bcl-2 mRNA (55). In addition, other remaining questions include how ceramide recruits or activates this regulated pathway (54). Whether ceramide generated within acidic vesicles undergoes a transbilayer movement similar to what has been recently reported for bNSMase (56) in erythrocyte ghosts remains to be established. In hepatocytes we have shown that the ceramide pool generated from bNSMase must undergo a "flip-flop" movement across the bilayer to signal the transcriptional regulation of genes such as γ-GCS (37).
To examine the in vivo consequences of the downregulation of MAT1A by ASMase, we analyzed the regulation of hepatic SAM during the progression of liver failure in a model of lethal hepatitis. This approach revealed that the depletion of hepatocellular SAM levels precedes the activation of caspases induced by TNF-α, resulting in liver damage and lethality. The outcome of ASMase–/– mice challenged with Gal/TNF was similar to that of ASMase+/+ mice treated with Gal alone. In both cases, the mild decrease in hepatic SAM reflects the effect of Gal, a hepatotoxin that blocks transcription in hepatocytes. MAT I/III is sensitive to oxidative stress due to the targeting of a critical cysteine residue (30, 32). Hence the downregulation of MAT1A and the inactivation of MAT I/III accelerate the depletion of SAM by TNF-α in ASMase+/+ mice, initiating a cascade of events that culminate in the death of mice. As shown by TUNEL assay, the fulminant outcome of this model reflects a prominent hepatocellular apoptosis. In comparing caspase activation in ASMase–/– versus ASMase+/+ mice, we unexpectedly observed a defective activation of caspase 8 by TNF-α in ASMase knockout mice. Previous findings reported the involvement of upstream caspases, such as caspase 8, in the activation of ASMase (57–59); however, our data indicate that ASMase is needed for optimal caspase 8 activation by TNF-α (Figure 5C) and suggest an inverse hierarchical ordering in caspase 8/ASMase activation, as reported for Fas (58, 59). This effect seems to be dependent on SAM through replenishment of GSH levels, as SAM therapy attenuated caspase 8 activation in ASMase+/+ mice (Figure 7D). Furthermore, our findings also indicate the regulation of caspase 3 by ASMase. In the absence of ASMase, we observed a lowered activation of caspase 3 by TNF-α (Figures 5C and 6B), and this ASMase-independent caspase 3 activation may be due to the participation of other signaling proteins, such as cathepsin B (5). In addition, SAM seems to regulate caspase 3 activation. This outcome may reflect the ability of SAM to regulate the mitochondrial commitment to cell death (60, 61). Depletion of SAM in the livers of MAT1A–/– knockout mice has been shown to modulate mitochondrial integrity through regulation of the expression of prohibitin, a protein encoded in the nucleus that participates in the functional organization of mitochondria (60). However, in addition to this potential mechanism, it is likely that the protective effect of SAM in lethal hepatitis is dependent on the replenishment of GSH levels, particularly in mitochondria (39, 62), as BSO pretreatment abrogated the therapeutic role of SAM. Indeed, mitochondrial GSH has been shown to play an essential role in the survival of hepatocytes in the face of TNF-α–mediated cell death (21, 37, 40, 62).
The present study complements recent observations on the mechanisms of TNF-α–induced hepatocellular damage (21) (Figure 10). Through MAT1A downregulation and subsequent SAM decrease, mitochondrial GSH levels become limited, leaving hepatocytes unprotected from death signals generated by TNF-α–induced ASMase activation, such as GD3 (21). As GD3 can target mitochondria (63), the consequences of this interaction, which leads to apoptosome assembly and caspase 3 activation, depend largely on the levels of mitochondrial GSH (21, 43, 63). Thus, the combination of mitochondrial GSH depletion and the trafficking of GD3 to mitochondria mediates the hepatocellular damage of TNF-α. An implication of the current and previous findings (21) is that in addition to the therapeutic effect of SAM, interference with ASMase signaling may be beneficial, as it would spare SAM/GSH levels and avoid the production of harmful lipids such as GD3. The effects of ASMase inhibitors in vivo on liver function and protection against liver failure remain to be established, however.
Scheme depicting the mechanisms used by ASMase in mediating the cytotoxic effects of TNF-α. Our present findings illustrate the consequences of the downregulation of MAT1A by ASMase resulting in GSH depletion, particularly in the mitochondria, subsequent to the decrease in SAM levels. Although this mechanism leaves hepatocytes unprotected, glycosphingolipids, especially GD3, synthesized from the pool of ceramide generated by ASMase activation, increase and traffic to the mitochondria, initiating the apoptosome activation responsible for hepatocellular apoptosis and liver failure. Both processes promote the damaging effects induced by TNF-α. SAM treatment would replenish GSH stores, including those in mitochondria (Mit), producing favorable conditions in which hepatocytes withstand the effects of TNF-α.
Taken together, our findings characterize a novel function of ASMase in the cytotoxicity of TNF-α. Given that TNF-α is essential in several forms of liver damage, such as alcohol-induced liver injury (31, 40) and lethal hepatitis (41, 42), our findings may be of therapeutic relevance for the prevention of fulminant liver failure and the treatment of liver diseases through replenishment of SAM and subsequent restoration of mitochondrial GSH.
We want to express our gratitude to R. Kolesnick and E. Gulbins for the gift of ASMase knockout mice, and M. Pajares for anti–MAT I/III. The work presented was supported in part by the Research Center for Liver and Pancreatic Diseases, P50 AA 11999; and grant 1R21 AA014135-01, funded by the US National Institute on Alcohol Abuse and Alcoholism; Plan Nacional de I+D grants SAF01-2118, SAF2002-3564, and SAF2003-04974; and Red Temática de Investigación Cooperativa G03/015 and Red de Centros C03/02, supported by Instituto de Salud Carlos III. We want to thank Susana Nuñez for expert technical assistance and genotyping of ASMase–/– mice. M. Marí and A. Morales are Ramón y Cajal investigators.
Nonstandard abbreviations used: acidic sphingomyelinase (ASMase); alanine aminotransferase (ALT); Bacillus cereus sphingomyelinase (bNSMase); d-(+)-galactosamine (Gal); d-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol HCl (d-threo-PDMP); ganglioside GD3 (GD3); ganglioside GM3 (GM3); glucosylceramide synthase (GCS); γ-glutamylcysteine synthetase (γ-GCS); glutathione (GSH); human placenta sphingomyelinase (hASMase); l-buthionine sulfoximine (BSO); liver-specific methionine adenosyltransferase (MAT1A); mannose 6-phosphate (M 6-P); methionine adenosyltransferase (MAT); N-acetylcysteine (NAC); neutral sphingomyelinase (NSMase); S-adenosyl-l-homocysteine (SAH); S-adenosyl-l-methionine (SAM); sphingomyelinase (SMase).
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Contreras, FX, Villar, AV, Alonso, A, Kolesnick, RN, Goñi, FM. Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes. J. Biol. Chem. 2003. 278:37169-37174.
Schwander, R, Wiegmann, K, Bernardo, K, Kreder, D, Kronke, M. TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem. 1998. 273:5916-5922.
Brenner, B, et al. Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases. Cell Death Differ. 1998. 5:29-37.
Sawada, M, et al. Acid sphingomyelinase activation requires caspase 8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells. Exp. Cell Res. 2002. 273:157-168.
Santamaria, E, et al. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc. Natl. Acad. Sci. U. S. A. 2003. 100:3065-3070.
Ansorena, E, et al. S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells. Hepatology. 2002. 35:274-280.
García-Ruiz, C, et al. Feeding S-adenosyl-L-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes. Hepatology. 1995. 21:207-214.
García-Ruiz, C, et al. Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-α. J. Biol. Chem. 2002. 277:36443-36448.

redpajama_set_name: RedPajamaCommonCrawl


text:
U.S. dairy farmers praise the power of collaboration in Mexico
By Mark O'Keefe
On a week-long governance mission, DMI board members saw how USDEC facilitates business partnerships in Mexico that benefit farmers back home.
Trade agreements, as critical as they are, do not in themselves create trade.
Trade happens when people connect with people, a reality five Dairy Management Inc. board members, all dairy farmers, witnessed on an October 20-25 governance mission to Mexico, organized by the U.S. Dairy Export Council.
Simply put, one-to-one business connections matter. They always have. They always will.
"That was my biggest takeaway, my aha moment, realizing how valuable the relationships are," said Marilyn Hershey, a Pennsylvania dairy farmer who chairs the DMI board that oversees USDEC, a nonprofit subsidiary.
"It's not something that's accomplished in one year. It's something that happens over the years through developing relationships and increasing collaboration. That's when the payoff comes, when you see that collaboration."
Five Dairy Management Inc. board members, all dairy farmers, recently went on a USDEC-sponsored trip to Mexico to learn more about the No. 1 market for U.S. dairy exports. They are shown here at a dairy farm owned by Lala, the largest dairy processor in Mexico. Standing left to right are Kathleen Skiba, Marilyn Hershey, Chace Fullmer, Evan Hillan and Larry Hancock.
Other DMI board members seeing what nearly 25 years of USDEC work in Mexico has accomplished had the same takeaway.
"Relationships are vital," said Larry Hancock, a Texas dairy farmer with 4,200 cows who was recently elected USDEC's new chairman. "The thing I want to really get across to the DMI and USDEC boards after this trip is how important relationships are and how well USDEC facilitates those relationships. It was a really cool and amazing for me to see."
USDEC sells no products. Its mission is to see U.S. dairy exporting companies do the exporting, benefiting farmers and the entire industry. In 2018, sales to Mexico reached $1.4 billion, about one-fourth of all U.S. dairy exports around the world.
Because value is dependent on world prices, which go up and down, volume may be a more reliable indicator of year-after-year progress. The chart below shows the results of sustained effort -- a 450% increase in 25 years.
The United States has seen consistent, year-after-year growth in the volume of U.S. dairy exports to Mexico. Chart: Created by USDEC utilizing government data.
Consistent export volume growth means more and more U.S. milk has crossed the border to Mexico through dairy products and ingredients.
Two-way relationship
Twenty-five years ago, the North American Free Trade Agreement (NAFTA) opened a door of opportunity for tariff-free trade with Mexico. Processors and farmers alike were united in the belief that an organization was needed to facilitate U.S. dairy exports not just to Mexico, but the world.
DMI founded USDEC 24 years ago. Through the dairy checkoff, farmers continue to fund USDEC, accounting for about three-fourths of USDEC's budget.
Missions to Mexico and other U.S. dairy markets give DMI board members an opportunity to assess with their own eyes and ears USDEC programs and activities, the ROI for farmers and the potential for continued growth.
Click here to see more...
Alta. MPs commit to help farmers
U.S. ag faces propane shortage


Scroll down to explore all 20 images uploded under Resume Templates and Examples's gallery and find brilliant inspirations and ideas. Enjoy!
this post was published by revando under Resume on Thursday, October 19th, 2017 and tagged with resume templates tumblr, free resume templates zety, resume templates for burger king, resume templates best, resume templates job, resume templates overleaf, resume templates google docs free, resume templates jobstreet, resume templates canva, resume templates macbook.


Mayor Robert Garcia unveiled a new non-profit Thursday which has aims of fundraising to supplement educational programs in the city. The Mayor's Fund For Education will initially try to tackle the task of increasing access to early childhood education opportunities before addressing some of its broader goals.
Garcia, joined by nearly every top educator in the city, revealed the Mayor's Fund just one floor above the clamor of pint-sized students at Little Owl Preschool in North Long Beach. He said that the fund, of which is official position will be to solicit donations for, will first focus on the youngest learners in the city.
The mayor noted that while the city is not interested in running schools outright, he is committed to supporting the efforts made at every level, especially in the years leading up to elementary school.
The focus of the fund in the foreseeable future will be on exploring what an early childhood education plan looks like at the city level, supporting early literacy campaigns promoted by the city's libraries, supporting the preschool centers at CSULB and Long Beach City College and ensuring that the new Long Beach Educare Center is opened.
Long Beach Unified School District Superintendent Chris Steinhauser said that the the Educare Center, a state-of-the-art education school set to open at Barton Elementary as early as next year, is making progress with the help of an anonymous $8.5 million donation.
The center will serve at-risk children in North Long Beach from birth to age five providing year-round quality education programs.
In a study released last month by Nobel Prize winning educator James Heckman, investing in students with programs like Educare can have substantial payoffs in the long-run.
Heckman's study found that children enrolled in such zero-to-five programs show a greater likelihood to graduate from high school, had higher IQs and were healthier over the course of their lives. His work also found that these programs resulted in lower rate of incarceration than their peers who were enrolled in lower-quality programs or just stayed home.
His work also put a price on the investment, estimating that for every dollar invested into zero-to-five programs society gained over $6 in benefits. The savings, or "societal benefits", stem from a combination of less taxpayer dollars spent on public health, drops in crime and less money being poured into publicly funded jails.
The findings of Heckman's study were based off of students in North Carolina whom his group started tracking in the 1970s and followed until age 35.
The Mayor's fund could provide a unique opportunity for the city to enhance its efforts to combat the opportunity gap that exists in some portions of the city without having to dip into the general fund to do so. The fund already has some big name donors who have contributed including AT&T, The California Endowment, Intertrend and Renzei.
How much money would be needed to meet the fund's goals was not clear as the board has yet to outline the annual operating needs, said the fund's executive director, Karissa Selvester. She said that at this point the fund is more worried about assessing needs before it sets too specific of goals in terms of fundraising.
As far as what the fund will try to do specifically, when it branches out past its initial focus on early childhood education and incorporates efforts at the college level, Selvester said that those talks, too, are preliminary, but that the fund's web of education—early childhood, the Long Beach College Promise, and internship opportunities—could work hand in hand to ensure that the investments made in the coming years result in college degrees.
Garcia has long been an advocate of education, with his first policy address held at Cal State Long Beach, where he declared he would do everything he could to support the education institutions in the city. Since then, Garcia has expanded the the College Promise and fought for an expansion of internship opportunities in the city, working with Pacific Gateway and other entities in Long Beach to draw the city's student to internship ratio closer together.
Having graduated from and instructed at the city's two post-secondary institutions, Garcia said that education will always be a priority for him. At next week's State of the City, Garcia said he plans to keep education as one of the core areas of interest that the city will act on in the coming year.


This hymn about God's kingdom was written by Welsh Congregational minister Bryn Austin Rees (1911-1983). It was written in 1973 and first published in 1974.
Verse 2 originally started "The kingdom of God ss mercy and grace, the lepers are cleansed, the sinners find place" but most users and some publishers have changed this to "The kingdom of God Is mercy and grace, the captives are welcomed, the sinners find place,"
TETHERDOWN by organist and composer Gerald Barnes (b 1935) - this tune was written for the text, and is named for the church in Muswell Hill, London, where Rees served as minister from 1950 to 1962.
It is included in a number of hymn-books, including The Celebration Hymnal for Everyone, Mission Praise and Sing the Faith Plus, and these are the best source of sheet music for it today.
It is not the same as the Taizé chorus "The Kingdom of God is Justice and Peace" despite the similarity of first lines.
It does not appear to have been registered with any of the major church-copyright organisations: the copyright owner is named as Mrs. Olwen Scott in some on-line sources, while the 2012 edition of The Celebration Hynmal for Everyone names Mrs Morfydd E Rees, St Benedict's Abbey, Charlbury Grove, Ealing, London W52DY.


Making use of our pioneering spirit, we make the first line of organic certified seeds available on the market. Korin seeds were developed in conjunction with the Mokiti Okada Research Center (CPMO), a pioneer in the development of biological technologies. Koirn seed production follows the management of Natural Agriculture, being certified by audit, with organic compliance guarantee and free of chemical treatments.


RICHMOND, VA (WWBT) - Virginia Senate Majority Leader Tommy Norment was the managing editor of a Virginia Military Institute yearbook in the 1960s that included photos of people wearing blackface.
"With 114 editions of The Bomb available online dating back to 1885, I am not surprised that those wanting to engulf Republican leaders in the current situations involving the Governor, Lt. Governor, and Attorney General would highlight the yearbook from my graduation a half century ago," he said in a statement.
Norment says he "emphatically" condemns the use of blackface.
"Despite all of the distractions from the continuing controversies involving our statewide elected officials, I am intent on fulfilling the work of the people of Virginia by passing a fiscally responsible budget that provides tax relief for working families," he said.
Senator Norment has emphatically condemned the racist and abhorrent photos in the yearbook, as do I. None of the photos include him, he's spoken to his role as a member of the yearbook staff and shared his views on the admissions policy. It's unfair to compare assisting in the production of a yearbook to the other revelations from this week.
Norment, represents the 3rd District, which includes Gloucester, King and Queen, King William and New Kent counties and the City of Poquoson, and portions of Isle of Wight, James City, Surry, and York counties, and the cities of Hampton and Suffolk.


text:
.. title:: CMake Reference Documentation

Command-Line Tools
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.. toctree::
   :maxdepth: 1

   /manual/cmake.1
   /manual/ctest.1
   /manual/cpack.1

Interactive Dialogs
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.. toctree::
   :maxdepth: 1

   /manual/cmake-gui.1
   /manual/ccmake.1

Reference Manuals
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.. toctree::
   :maxdepth: 1

   /manual/cmake-buildsystem.7
   /manual/cmake-commands.7
   /manual/cmake-developer.7
   /manual/cmake-generator-expressions.7
   /manual/cmake-generators.7
   /manual/cmake-language.7
   /manual/cmake-modules.7
   /manual/cmake-packages.7
   /manual/cmake-policies.7
   /manual/cmake-properties.7
   /manual/cmake-qt.7
   /manual/cmake-toolchains.7
   /manual/cmake-variables.7

.. only:: html or text

 Release Notes
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 .. toctree::
    :maxdepth: 1

    /release/index

.. only:: html

 Index and Search
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 * :ref:`genindex`
 * :ref:`search`

redpajama_set_name: RedPajamaGithub


"He that overcometh, and keepeth my works unto the end, to him will I give power over the nations. " — Rev. 2:26 Here we find a convert, who has now developed into a powerful saint. He is the same one who was formerly a baby on a pillow, in a little crib, but now he has grown until he can be trusted with power. Power is a glorious, yet dangerous thing. Put a child, or even an untrained man at the head of a long passenger train, or in charge of a great ocean liner, and he would wreck and ruin things. But after careful and severe discipline, he comes to the place where it is easy and natural to handle complicated machinery.
We all like to look at powerful things. I like to get out of a train and walk up to the great iron horse pulsating with energy, and say to it: "You certainly are a monster! You pulled fifteen large Pullman cars over the steep grade, and down the other side of the mountain in safety." When I was a boy, I worked in the iron mills where we had a great hammer which could be operated so accurately that it would crack a hazel nut without smashing it. The very next moment enough power could be turned on so it would strike sixty tons! It had a cement foundation thirty feet deep, and jarred the earth for some distance every time it came down.
How sad that we poor mortals put a premium on wrong kinds of power — for instance, money power. We think it a great thing to make and possess money. If one has enough of it he can very nearly control city councils, state officials, and even Congress itself. One young schoolma'am in London received a prize when she gave the best and shortest definition of "Money". Here it is: "Money is that commodity in life which will purchase everything but happiness, and give a passport to every place but Heaven." Yes, if you have enough money, you can buy yourself out of nearly all kinds of trouble, and buy yourself into nearly every office in the United States!
There is another kind of power — political power. A man who is a shrewd politician can pull wires and get special privileges that few others enjoy.
Then, there is brute power, or physical force. A man who is a great athlete or pugilist can knock another man "stiff", can draw immense crowds and make a million dollars in an hour. But this is not the power we want.
Again, there is intellectual power. If a man has a great mind, he can sway audiences, write books and become a great factor in the molding of men, but this is simply mind power.
Then, there is a higher grade of power, namely, that of a good name. "A good name is rather to be chosen than great riches …" He may be a very poor man, a very feeble man; but yet if he has lived in a community a long time and is recognized as a good citizen, his influence is worth a great deal.
But the ideal power is Divine power — holy unction. Mr. Finney has a great sermon on "Power from on High." Where he got the idea, I do not know, but I have stood at the Niagara Falls many times and watched with awe that great phenomenon of nature. Here both Canada and the United States have large dynamos where they generate power that runs machinery, lights the cities, and blesses the people — also sends men into eternity in the electric chair! How do they make it? Lake Erie is one hundred sixty-nine (169) feet higher than Lake Ontario. Were these lakes on a level we could not make power from "on high".
In like manner, if we would have Divine unction and anointing we must humble ourselves and go down, down, down in self-abnegation and humility until the gates of heaven are opened and the mighty power of the Holy Ghost is poured upon us. Mark the language! — To him "that overcometh will I give power over the nations." Not power over simply one man or a neighborhood, or even a city, but over the "nations"! Is this too strong? Yes, if your faith does not claim it, but "all things are possible to him that believeth!" That is, if you can get on believing ground you are almost omnipotent.
Has not God Almighty said: "Concerning the work of my hands command ye me"? Think of it! God is waiting for a feeble man who will believe him, then stand in the gap and in the name of Jesus demand supernatural things! We get feeble answers because we have feeble faith and a small vision. George Muller said that perhaps there never lived a man who fully proved all the possibilities of prayer. He might have demanded greater things and seen greater victories had he only insisted upon it.
Yes, "according to your faith," or lack of faith, "be it unto you." Perhaps we will not know until the books are opened, to what heights and lengths we might have attained had we not "limited the Holy One of Israel." Who would reject such power? Who would not have it for the asking?
But such power cannot be had in a moment. You remember the young, enthusiastic prophet, Elisha, requested of his master, Elijah, that "he might have a double portion of his spirit." And do you remember the answer? "Thou hast asked a hard thing!" Yes, it meant a hard thing to the flesh, a hard thing to finery, a hard thing to popularity. And, dear friend, if you have ever gotten a sight of your carnal heart and died the death to the "old man" of sin, you know by experience that it was no child's play. When I see preachers and instructors talk and sing seekers of heart purity through in a hurry; then giggle and laugh if they can provoke a smile on that distressed face, I say to myself: "You know little about spiritual crucifixion." We all want resurrection power, but, remember, it must be preceded by crucifixion suffering!
How may we obtain power over the nations? First, by getting power over ourselves. No one is fit to rule over others until he can rule over old self. No one is fit to teach others until he can sit and patiently be taught. Friend, have you ever received a stinging letter? If so, did you have power to hold still and not answer sarcastically? Were you ever crossed or contradicted, and did you have power to hold still and answer never a word? This is the power that the Holiness people need.
Yes, this power from on high will affect kings and rulers. They may not be aware of it, they may not acknowledge it; but, friend, you can so prevail with God that sooner or later things will come to pass that never would have happened had it not been for your prevailing prayer. I remember hearing wife tell how that when she and her sister were but fifteen or seventeen years of age, while assorting clothes in the attic in the wicked city of Chicago, all at once they were drawn out in intercessory prayer for President McKinley. They stopped their work and both knelt together and wrestled with God in behalf of this great man, who had never heard of them. The very next day he was assassinated. Perhaps God could not find great preachers to pray at that crucial time, but these two, pure, clean channels could be used by the Holy Ghost to prepare a great President for the sudden change that so soon awaited him!
Let us, then, resort to this mighty arm that moves the world; but remember that purity and power are inseparable. Do not try to seek for power until you are purified, for if God granted it upon an unsanctified heart, you would be ruined. This is why so many great and good men have crippled or killed their usefulness. They had the power of eloquence; they had the power to understand, and unravel prophecies; they had power to lay hands upon the sick and they recovered. Sad to say, as a result they became lifted up and later were ruined by their own gifts! They had power without purity. Again I repeat — purity first, then holy unction!
But, oh, I need my soul well blest!
Please daily keep my soul well blest!
If Thou wilt only keep me blest!
But, Spirit, kindly keep me blest!


Ludos Flamma, sounds flammable! This is an awesome gaming mouse, with programmable buttons and RGB LEDs. It uses nice smooth silkish rubber coating and anti-slip scroll wheel.
Let's take a look at this simple digital alarm clock.
Here we have a cool gaming combo by AcGam.
Fully Programmable Compact Mechanical Keyboard with blue switches, Fully Programmable Gaming mouse and perfectly long mouse + keyboard mat!
AcGam seem to be stepping up with really cheap, yet good quality gaming gear. This keyboard is fully programmable, even tho it has only white backlight, it has everything else that a gamer needs. You can re-program any button and it has detachable cable so you can easily store it in a bag, making it perfect for travelling.
Mouse is also fully programmable and has 10000 dpi sensitivity. Software allows you to program 5 profiles and each profile can have 5 dpi settings and colors are also fully programmable to your liking. Each profile can have just the color you like.
Mouse mat is very simple, but that too, has some good feature – the stitched edges, making it durable around the edges.
Here we have a mini wireless smart switch. It's rated for 240 volts @ 5 amps max. It requires eWeLink mobile app, but can also be turned on / off manually by pressing the tiny button on top (which is real nightmare to press).
This switch is excellent for lights, but can also be used for other stuff. Just remember that 5 amps @ 240 volts = 1200 watts, so anything that takes more than 1200 watts of power would probably fry the thing.
It also works with Alexa, so you can have voice commands 🙂 For any further details, check out the video below. Video contains unboxing, first look, installation guide + app setup / pairing. In other words, it's a full review of the product, everything you need to know.
It's battery powered and splash proof, so you can use it with wet hands. For more details, check out the review video below.
Here we have a color changing LED light bulb that can be controlled remotely via app. It's quite heavy on it's own, but apart from that, it can change colors and brightness and saturation via app. When unconfigured, it's just bright white.
It's not quite enough to properly light up the room, but will do just fine for ambient light when watching TV or gaming. Untested, but it says it also works with Alexa and google assistant.


This article is about the mineral variety, for the English term for the helmet worn in the 16th century see Morion (helmet).
Morion or morion quartz is a dark-brown to black opaque variety of smoky quartz resulting from the natural or artificial irradiation of aluminium-containing milk quartz.
This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Morion". A list of authors is available in Wikipedia.


Each client is important — even previous ones. To help generate new business, maintaining relationships with previous clients may be one of your keys to success. Here are some reasons to maintain these connections and suggestions on how to make them stronger.
In the frequently crowded real estate market, maintaining a thriving client base is essential to the growth of your business. And while there's no denying that winning new clients is crucial, strengthening your connections with past clients can be just as important.
Why maintain relationships with past clients?
Converting leads into actual clients can take a great deal of time and dedication. So once you've invested so much effort into building a relationship with a buyer or seller, why not keep in contact to see how you can help meet their future housing needs?
They are moving due to important life developments like career changes, family considerations or shifting priorities.
They are looking to purchase a second home or vacation property.
They are seeking future investment opportunities.
In addition to helping previous clients directly, you can also receive valuable referrals from past buyers or sellers. A successful transaction can mean a boost in your business, especially when previous clients offer their trusted opinions and references to friends, family and associates.
Notify past clients of recent sales or listings.
Send personalized postcards letting them know you're still available to assist with their housing needs.
Mail cards for special occasions such as holidays or birthdays.
Follow up with thank you greetings.
You may also want to consider friendly reminders for other services you can assist with such as helping past clients protect their investments with an American Home Shield® Home Warranty Plan.


Choose from our range of pet decorations for Christmas and for all year round.
These lovely pet decorations are ideal for Christmas but also great as year round memorials to your pet.
So many dog owners love having personalised dog decorations that have their dogs name engraved onto them and as they are part of the family why not have something to show their importance.


Agile Transformation Consulting is agile expertise to achieve an agile transformation for any organisation. As a focused package, Agile Transformation Kick-Start sets you off in the right direction and gain momentum. Agile Transformation Consulting maintains this momentum, hence keeping the organisation, leaders, teams and individuals focused on the outcomes required for business agility.
Communications across the organisation keeps people aligned with outcomes.
Addressing blockers to progress helps people to make change.
Evolving the roadmap for change, so that people respond to change.
Building confidence across the organisation means that people take ownership of future change.
Achieving an agile transformation requires investment and initial decisions are therefore key to future success. Agile consulting ensures key stakeholders remain engaged because people are guided and challenged to ensure good alignment between parts of the organisation.
We help to address and remove blockers.
People take ownership of continuous improvement.
There is access to expert agile transformation advice from an experienced agile consulting team.
We assure success by avoiding typical issues and problems.
Progress is achieved and maintained, so that those involved stay motivated.
Benefits are delivered early, therefore ensuring return on investment.
Agile Transformation Consulting is organised and completed quickly due to Agility in Mind's unique and highly flexible agile consulting and delivery model.


text:
QB3 Berkeley / News / An interview with Professional in Residence Mike Holmes: Pioneering genome editing tools in gene therapy
An interview with Professional in Residence Mike Holmes: Pioneering genome editing tools in gene therapy
By Diana Aguilar Gómez | April 06, 2021
Mike Holmes, PhD, is the Chief Scientific Officer at Ambys Medicines. Holmes is joining the QB3-Berkeley Professionals in Residence (PIR) program on April 16th and 23rd. He spoke with PhD student Diana Aguilar Gómez about his career from being a graduate student to industry. They also discussed his pioneering work in genome editing techniques in gene therapy and the application and importance it has to severe liver disease patients.
Register here to join the PIR events with Mike Holmes on April 16th and 23rd.
Diana Aguilar Gómez: Could you briefly describe your career trajectory?
Mike Holmes: As an undergraduate, I was doing research in a genetics laboratory. I worked very closely with a postdoc who taught me how to work in a lab and do science. I fell in love with doing research. From that point, I decided that was what I wanted to do. I saw myself doing academic research and decided to go to graduate school. I ended up going to UC Berkeley, my first choice. While I really loved doing research, I wanted to do something more applied. I liked the idea of going into industry and trying to build medicines or novel therapies for patients with significant, unmet needs. I became passionate about the idea of applying my research towards building gene therapies and medicines for patients.
DAG: Could you explain what gene therapy is and if genome editing is a type of gene therapy?
MH: Gene therapies very broadly revolve around the idea of delivering genetic payloads to cells. These payloads would change or provide missing factors, to down-regulate or reduce the expression of factors that might be responsible for driving disease. The general sense of gene therapy is the idea of delivering to cells new genetic payloads that would ameliorate the impact of disease. I consider genome editing to be a tool that can be used by gene or cell therapy to invoke a therapeutic response. For example, we might use gene therapy to deliver genome-editing reagents or we might use genome-edited cells to develop better cell therapy.
DAG: What did you do when you finished your PhD at UC Berkeley?
MH: I joined a small company called Sangamo Therapeutics. It was working on how to build engineered transcription factors as a potential gene therapy approach for patients with severe unmet medical needs. Three weeks into my new job, our boss was tragically killed in an accident. We were in a very young company that suddenly didn't have a clear mentor. Very quickly, many of us within the company found ourselves in leadership roles. With the opportunity I was given out of this tragedy, one of the projects I ended up leading was in genome editing — trying to figure out how to develop genome editing as a way to expand what we could do from a gene therapy standpoint.
DAG: Why did you leave Sangamo Therapeutics to go to Ambys Medicines?
MH: I was at Sangamo for 17 years. I started off as a "scientist one," and then I left as the Chief Technology Officer. I worked my way up to heading up the research group there. Working with a tremendous group of scientists to invent a field and figure out how to use genome editing in a variety of applications, both for advancing basic science research as well as perfecting it for use as a therapy — it was a dream job for me in terms of just how much success we had and how far we are taking the technology. After 17 years, I found myself wanting to do something kind of new and different. The company had grown to 300 people. Oftentimes, I was in a variety of meetings that had nothing to do with science; it had to do with running a larger company. I wasn't passionate about the nuts and bolts of running a large company, and I want to get back to my roots.
I moved to a startup company called Ambys Medicines in 2018. This company has the mission of trying to find novel therapies for patients with severe liver disease. For patients with both acute and chronic liver disease where the disease is so severe, the standard of care for them is one of liver transplant. There are probably more than 30,000 people in the United States who are in need of significant therapy for liver disease, but we only do about 8,500 transplants per year. The number of patients is expected to actually grow over the next decades, and therefore, there is a significant amount of unmet need.
DAG: How are you tackling this problem at Ambys Medicines?
MH: We are using cell and gene therapy to try and regenerate the liver in patients with severe liver disease. From a gene therapy perspective, the idea is in the context of cirrhosis, when the liver is no longer able to regenerate itself to maintain sufficient liver activity and function. In the context of gene therapy, can we deliver genes that can modulate the different pathways that are involved in driving severe liver disease and cirrhosis? We want to stop and potentially reverse the changes that have occurred in the liver. Our aim is to restore the natural regenerative processes in the liver. In the context of cell therapy, what we're doing is we're getting primary human hepatocytes, and we're expanding them outside the body. We can introduce them back into patients, such that we can augment liver function, ameliorate disease, and significantly improve the quality of patients' lives. Those are the two scientific pillars of Ambys. To replace the need for transplants is our big mission.
DAG: What does a typical day look like for you?
MH: Right now, in the context of COVID-19, I spend a lot of time in meetings over Zoom, staying in touch with people and having meetings to talk about the science that we're doing and to discuss next steps. We are essential. We do have many of the people here on site. However, it's one of the things where, in science, sometimes some of the best discoveries or best discussions are you just going down the hall, seeing the person, and working on the whiteboard. Zoom has helped us a lot in terms of staying in touch, but that's the one thing that we haven't found a technology for to replace those one-on-one informal discussions that a lot of times discoveries come out of.
DAG: Do you have any advice for PhD students who are considering going to industry?
MH: Think about the scientific problems facing society, and the topics that you're really passionate about. Find the job and the company that best matches up with that passion. Science is tough; it's challenging, many experiments just don't work. You need to work in a field that is exciting for you. One of the things that drew me to industry was the tangible applications like a project becoming a drug or treatment for disease.
DAG: Why did you decide to take part in this program with UC Berkeley?
MH: I greatly appreciate all the opportunities that UC Berkeley gave me, both in terms of the education and the connections I made. I certainly greatly appreciate the experience I had in getting me ready to build my career. I like the idea of giving back to Berkeley and do anything I can do to help the students and the people. In learning from my own experience, how can I better help them in making the right choice? I remember being at the same stage and trying to figure out what I wanted to do. I remember going to a variety of very similar presentations — people talking about going into consulting or industry. I appreciate these different perspectives. If I can participate in this and help even one person, then I think it's worthwhile and paying back to all those before me who helped me.
Mike Holmes, PhD, is a passionate scientist who is currently Chief Scientific Officer at Ambys Medicine. He gained his PhD in molecular and cell biology from UC Berkeley. After he graduated and did a short postdoctoral training, Mike transitioned to industry. He worked 17 years for Sangamo Therapeutics, where he and a group of talented scientists were pioneers in developing gene therapies involving genome editing tools. Mike received his BS in molecular biology from UC San Diego.
Diana Aguilar Gómez is a PhD Candidate in the Computational Biology program which is affiliated with QB3-Berkeley. She is working on her thesis at the Nielsen lab and her research involves population genetics and evolution of coloration in lizards and frogs.
Categories: News, Professional in Residence
Chan Zuckerberg Biohub awards $9 million to QB3-Berkeley faculty members
An Interview with Elise Kikis: Balancing Teaching and Research at a Liberal Arts College
New Berkeley pedagogy course introduces the science of teaching
QB3 workforce development programs receive $5 million state funding boost
UC Berkeley partners with new Arc Institute to tackle complex diseases


That's right, in a few weeks, I will be in charge of a holiday. My parents are flying in from California and Paul's parents are coming down from North Dakota to celebrate with a big ol' turkey.
As Thanksgivings go, it will be fairly small for me, only 6 adults in attendance.
What if the turkey doesn't thaw in time?
What if we don't have enough potatoes?
What appetizers should we serve?
One thing for certain, I will NOT be making ridiculous turkey gourd decorations. I don't have the time nor the talent. I bought some stuff at various stores to hang/place here and there to make our apartment festive, but I'm not going to try and create something I see on Pinterest and create a whole new (and unnecessary) ball of stress.
Speaking of Pinterest, I am using it to stay organized. When I see an article or recipe that might be useful, I pin it to my Thanksgiving board. Also, I've been keeping lists. Thanksgiving To Do & Thanksgiving to Buy are updated regularly….when I think of something I definitely want to have or should do before the parents see our apartment for the first time.
When it comes right down to it, I love holidays. The decorations, the gathering with family and friends, the good cheer, the food, it all makes me happy and I choose not to let it stress me out…too much.


Bauchi State Police Command has confirmed the kidnappings of some foreign nationals by unknown gunmen at a road construction company site in Jama'are Local Government Area of the State. Police spokesman, ASP Hassan said the incident which occurred last night left a security guard of the company dead. The Police gave the breakdown of the kidnapped Nationals as 4 Lebanese, 1 Briton, 1 Greek and 1 Italian. The Police Spokesman said, the gunmen had earlier attacked a Police station and a Prison in Jama'are but no casualty recorded and no one has yet claimed responsibility for the attack.