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Stephen Marshall

Publications and source records attributed to Stephen Marshall.

16 recordsLinked to original sources
PubMed2006

Role of insulin, adipocyte hormones, and nutrient-sensing pathways in regulating fuel metabolism and energy homeostasis: a nutritional perspective of diabetes, obesity, and cancer.

Traditionally, nutrients such as glucose and amino acids have been viewed as substrates for the generation of high-energy molecules and as precursors for the biosynthesis of macromolecules. However, it is now apparent that nutrients also function as signaling molecules in functionally diverse signal transduction pathways. Glucose and amino acids trigger signaling cascades that regulate various aspects of fuel and energy metabolism and control the growth, proliferation, and survival of cells. Here, we provide a functional and regulatory overview of three well-established nutrient signaling pathways-the hexosamine signaling pathway, the mTOR (mammalian target of rapamycin) signaling pathway, and the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Nutrient signaling pathways are interconnected, coupled to insulin signaling, and linked to the release of metabolic hormones from adipose tissue. Thus, nutrient signaling pathways do not function in isolation. Rather, they appear to serve as components of a larger "metabolic regulatory network" that controls fuel and energy metabolism (at the cell, tissue, and whole-body levels) and links nutrient availability with cell growth and proliferation. Understanding the diverse roles of nutrients and delineating nutrient signaling pathways should facilitate drug discovery research and the search for novel therapeutic compounds to prevent and treat various human diseases such as diabetes, obesity, and cancer.

AMP-Activated Protein Kinase Kinases↗
PubMed2006

Genetic research and genetic information: a health information professional's perspective on the benefits and risks.

BACKGROUND: The completion of the Human Genome Project, and recent breakthroughs in identifying the genes both directly responsible for and contributing to the severity of specific diseases and disabilities in humans offer great potential for future health care. AIM: The aim of the paper is to inform the health information community of these developments in order to foster debate over their wide-reaching implications. METHODS: The article outlines recent research in this area and explains how our growing understanding opens the way for improved diagnosis of disease, detection of possible predisposition to specific diseases, the interaction between genetics and environmental aspects such as diet, as well as enabling gene therapy and preventative actions. This new knowledge also opens the way for the use of pharmacogenetic information to enhance the efficacy of pharmaceuticals and to avoid toxic reactions. RESULTS: These advances have significant social, legal and ethical implications. Most countries have limited legislative control over the use and protection of genetic information about individuals. CONCLUSION: There is a role for health information professionals to play in promoting wider public knowledge of genetic research and its implications, in fostering public debate on key issues, and in seeking clearer policies in the health sector concerning the use of genetic information about individuals.

Genetic Counseling↗
PubMed2005

Turnover and characterization of UDP-N-acetylglucosaminyl transferase in a stably transfected HeLa cell line.

To estimate the turnover of UDP-N-acetylglucosaminyl transferase (OGT), we exposed stably transfected HeLa cells to tetracycline for 16h to induce OGT gene expression and increase cytosolic enzyme levels. Removal of tetracycline led to a progressive decrease in OGT activity (after a 6h lag period), yielding an estimated OGT half-life of 13h. A similar half-life (12h) was obtained by measuring the loss of biosynthetically labeled OGT ([35S]methionine pulse-chase experiments). Since OGT turnover was relatively slow, it is unlikely that changes in OGT gene expression or protein expression play a role in the short-term regulatory actions mediated by the hexosamine signaling pathway. We also found that the overexpressed 110kDa murine OGT subunit (recombinant enzyme) was enzymatically similar to the endogenous holoenzyme derived from rat brain tissue. Thus, stably transfected HeLa cells provide an abundant source of enzyme that can be used to study the structure, function, and regulation of OGT.

Gene Expression Regulation, Enzymologic↗
PubMed2005

Glucosamine induces rapid desensitization of glucose transport in isolated adipocytes by increasing GlcN-6-P levels.

We have examined the hypothesis that glucosamine (GlcN) can rapidly induce insulin resistance through an allosteric mechanism. When insulin-treated adipocytes were exposed to 2mM GlcN, glucose uptake was rapidly reduced by approximately 60% with a T(1/2) of 2 min. We also observed an increase in intracellular GlcN-6-P (at 5 min) from undetectable levels to approximately 260 nmol/g. Continued GlcN treatment resulted in additional accumulation of GlcN-6-P (>1200 nmol/g at 2h), but caused no further decrease in glucose uptake. Although the acute inhibitory action of GlcN could be completely reversed by removing extracellular GlcN, a slow and progressive decrease in insulin-stimulated glucose transport was observed with longer treatment times (T(1/2) of 45 min, 62% loss by 5h). From these data, we conclude that: (1) GlcN elevates intracellular GlcN-6-P levels within minutes, resulting in desensitization of the glucose transport system through allosteric inhibition of hexokinase; (2) prolonged treatment elevates GlcN-6-P to levels that cannot be effectively lowered by cell washing; and (3) residual levels of GlcN-6-P continue to allosterically inhibit glucose uptake, resulting in a slower rate of desensitization that is temporally similar to glucose-induced desensitization, but mechanistically different.

Adipocytes↗
PubMed2005

Glucosamine-induced activation of glycogen biosynthesis in isolated adipocytes. Evidence for a rapid allosteric control mechanism within the hexosamine biosynthesis pathway.

Enhanced flux through the hexosamine biosynthesis pathway (HBP) induces insulin resistance and facilitates lipid storage through the up-regulation of enzyme mRNA levels. Both actions occur over several hours and require gene expression. We now identify a regulatory arm of the HBP that involves rapid allosteric activation of glycogen synthase (GS) and stimulation of glycogen biosynthesis (GBS). When insulin-pretreated adipocytes were exposed to 2 mM GlcN, incorporation of [14C]glucose into glycogen doubled by 10 min (t(1/2) of <5 min), whereas UDP-glucose levels were concomitantly decreased during this time (t(1/2) of 1.4 min; >90% depletion). Stimulation of GBS and depletion of UDP-glucose both correlated with an early and rapid rise in the levels of glucosamine-6-phosphate (GlcN-6-P), a known activator of GS. The lowering of GlcN-6-P levels by removing extracellular GlcN (>80% reduction by 45 min) was accompanied by the restoration of UDP-glucose levels. Prolonged GlcN treatment (20 min to 2 h) inhibited GBS, which corresponded to a massive intracellular accumulation of GlcN-6-P (t(1/2) of approximately 32 min; >1,400 nmol/g). From these data, we conclude the following. 1) GlcN treatment elevated intracellular GlcN-6-P levels within minutes, resulting in allosteric activation of GS, stimulation of GBS, and a reduction in steady-state levels of UDP-glucose due to increased precursor utilization. 2) Prolonged treatment with high concentrations of GlcN caused massive accumulation of GlcN-6-P that adversely affected cellular metabolism and reduced GBS. 3) The biphasic actions of GlcN on GBS may explain many of the discrepant reports on the role of the HBP in glycogen metabolism.

Adipocytes↗
PubMed2004

Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels.

Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-Glc-NAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t((1/2)) < 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED(50) = 4mm). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mm) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min ( approximately 400 nmol/g) and continued to rise over 2 h (t((1/2)) approximately 20 min) before reaching a plateau at >1,400 nmol/g (ED(50) = 900 microm). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDP-GlcNAc levels (t((1/2)) approximately 14-18 min), but greater levels were obtained with GlcN (4-5-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 microm, ED(50) = 80 microm) could markedly elevate UDP-GlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.

Adenosine Triphosphate↗
PubMed2004

Differential effects of vanadate on UDP-N-acetylglucosaminyl transferase activity derived from cytosol and nucleosol.

UDP-N-acetylglucosaminyl transferase (OGT) is a key enzyme of a novel signal transduction pathway that regulates protein function through O-linked glycosylation. In the current study, we found that sodium vanadate potently inhibits OGT activity in brain cytosol (IC50 = 55 microM) and nucleosol (IC50 = 150 microM), but fails to alter activity of a related enzyme (UDP-galactosyltransferase). Vanadate also inhibits OGT activity in cytosol (IC50 of 2.3 microM) and nucleosol (IC50 of 130) derived from a stable HeLa cell line that overexpresses OGT. When HeLa cytosol was immunopurified to separate OGT from other cellular proteins, vanadate still inhibited OGT activity (IC50 = 2 microM). We conclude that OGT derived from cytosol exhibits greater vanadate sensitivity than nucleosol OGT and that a large difference exists (25-fold) in vanadate sensitivity when comparing OGT activity in different cell types (IC50 of 55 microM for brain cytosol vs. 2.3 microM for HeLa cytosol). Understanding the mechanism(s) by which a tyrosine phosphatase inhibitor differentially reduces OGT activity should lead to new insights into OGT function and regulation.

Animals↗
PubMed2004

Musculoskeletal symptoms among commercial fishers in North Carolina.

Musculoskeletal symptoms were reported by 215 fishermen followed at 6-month intervals over 18 months. Exposure information was collected through field observation and in-depth ethnographic interviews allowing potential ergonomic stressors to be identified and catalogued by task and stage of work. Symptoms causing work interference in the last 12 months were reported by 38.5% of the cohort at baseline. Low back symptoms were the most common cause of work impairment (17.7%), followed equally by pain in the hands or wrists and shoulders (7%). Symptoms in any body region were more likely to have been reported among individuals who did not fish full-time and those who worked other jobs part or all year had significantly lower symptom prevalence; both likely reflect a healthy worker effect. A number of ergonomic stressors were identified in all stages of fishing with exposure variability dictated by some unpredictable factors such as weather; but also by type of boat, gear, crew size, and level of experience. Reducing ergonomic exposures associated with work among these traditional workers is important, regardless of whether they directly cause or contribute to their musculoskeletal symptoms, or aggravate existing pathology.

Adolescent↗
PubMed2004

Potential regulation of nuclear UDP-N-acetylglucosaminyl transferase (OGT) by substrate availability: ability of chromatin protein to bind UDP-N-acetylglucosamine and reduce OGT-mediated O-Linked glycosylation.

UDP-N-acetylglucosaminyl transferase (OGT) resides in both cytosolic and nuclear compartments and catalyzes O-linked glycosylation of various proteins. In the current study, we have extracted protein from nuclear DNA (chromatin protein) using 0.2% NP-40 detergent. Addition of chromatin protein to either cytosolic or nuclear preparations (containing abundant OGT) resulted in a dose-dependent loss of OGT activity. Since chromatin-mediated loss of OGT activity could be restored by immunopurification of OGT, we conclude that loss of enzyme activity is not due to direct inactivation of OGT. Addition of UDP-galactose (to saturate potential UDP binding proteins) effectively restored OGT activity in cytosol containing chromatin protein. This indicates that chromatin protein inhibits OGT activity by binding UDP-GlcNAc. These studies suggest that nuclear substrate availability may comprise one of the in vivo mechanisms regulating OGT activity and O-linked glycosylation of nuclear proteins. This is potentially significant, since most transcription factors are O-linked glycosylated and such post-translational modifications can alter gene expression.

Acetylglucosamine↗
PubMed2003

Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.

We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h. Enhanced OGT activity was due to overexpression of OGT protein as determined by Western analysis. Trichostatin A (TSA), a potent and specific histone deacetylase inhibitor (HDI), markedly enhanced tetracycline-induced OGT gene expression, resulting in a >10-fold increase in OGT activity (>50-fold compared to that of uninduced cells). Other HDIs such as butyrate (ED(50)=1.6mM) and propionate (ED(50)=8mM) were similarly effective, but less potent than TSA (ED(50)=120 nM). We next examined the appearance of recombinant OGT in cytosol and nucleosol at various times (10 min to 6h) after inducing OGT gene. Within 2h, recombinant OGT was detected by Western analysis in both cytosol and nucleosol. This indicates rapid biosynthesis and accumulation of recombinant OGT in the cytosol and subsequent nuclear translocation. Entry of OGT into the nucleus was closely correlated with enhanced O-linked glycosylation of nuclear proteins, indicating that recombinant OGT was enzymatically active. The ability to rapidly induce OGT expression in a stable cell line provides an excellent model system to study the mechanism(s) underlying OGT nuclear translocation and a useful system to elucidate the cascade of signaling events related to O-linked glycosylation.

Animals↗
PubMed2003

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose, glutamine, and glucosamine on glycerophosphate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase mRNA levels.

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.

Acetyl-CoA Carboxylase↗
PubMed2003

Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.

UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification). To study the enzymology of OGT, independent of potential regulatory proteins within cytosol, we also developed and characterized an alternate OGT assay that uses antibody-purified OGT as the enzyme source. The major advantage of this assay lies in the ability to measure OGT in the absence of other cytosolic proteins.

Animals↗
PubMed2003

UDP-N-acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme.

In brain tissue, UDP-N-acetylglucosaminyl transferase (OGT) is known to catalyze the addition of a single N-acetylglucosamine moiety (GlcNAc) onto two proteins linked to the etiology of neurodegenerative disease--beta-amyloid associated protein and tau. Hyperphosphorylation of tau appears to cause neurofibrillary tangles and cell death, and a functional relationship appears to exist between phosphorylation and glycosylation. Since a greater understanding of brain OGT may provide new insights into the pathogenesis of Alzheimer's disease, we examined the characteristics and subcellular distribution of OGT protein and OGT activity and its relationship to O-linked glycosylation. We found that cytosolic OGT activity is 10 times more abundant in brain tissue compared with muscle, adipose, heart, and liver tissue. Temperature studies demonstrated that cytosolic OGT activity was stable at 24 degrees C but was rapidly inactivated at 37 degrees C (T1/2 = 20 min). Proteases were probably not involved because OGT immunopurified from cytosol retained temperature sensitivity. Subcellular distribution studies showed abundant OGT protein in the nucleus that was enzymatically active. Nuclear OGT activity exhibited a high affinity for UDP-GlcNAc and a salt sensitivity that was similar to cytosolic OGT; however, nuclear OGT was not inactivated at 37 degrees C, as was the cytosolic enzyme. Two methods were used to measure O-linked glycoproteins in brain cytosol and nucleosol -[3H]galactose labeling and western blotting using antibodies against O-linked glycoproteins. Both methods revealed a greater abundance of O-linked glycoproteins in the nucleus compared to cytosol.

Adipose Tissue↗
PubMed2003

Implementing a community-based oral health care program: lessons learned.

OBJECTIVES: The objective of this paper is to report key findings of a process evaluation that may be useful to other institutions seeking to implement a community-based oral health care program primarily targeting children in dentally underserved communities. By partnering with community-based organizations, public schools, and community health care providers, the Columbia University School of Oral and Dental Surgery (SDOS) established the Community DentCare Network (DentCare) in the Harlem and Washington Heights/Inwood neighborhoods of northern Manhattan. These low-income neighborhoods are characterized by poor oral health and have been designated by the federal government as health professions shortage areas. METHODS: The method used in the process evaluation was open-ended qualitative interviewing by a sociologist with extensive experience in this methodology aided by a participant-observer within the DentCare program. RESULTS: The heterogeneity of the two communities required different strategies and resources to gain trust and acceptance. Fundamental changes were required of SDOS over a 10-year period, beginning with prioritizing community service into a primary mission. Collaborating with medical clinics facilitated the implementation of the network when the partners shared the same philosophical goals. Faculty and staff with different skills were needed during the start-up and the sustained development phases of the program.

Black People↗
PubMedDate not supplied

Recovery of postural control after cerebral concussion: new insights using approximate entropy.

CONTEXT: The return-to-play decision after sport-related cerebral concussion depends in part on knowing when an athlete has fully recovered postural control after injury. OBJECTIVE: To describe the postconcussion recovery of postural control using approximate entropy (ApEn), a regularity statistic from nonlinear dynamics. DESIGN: Retrospective case series analysis. SETTING: Sports medicine research laboratory. PATIENTS OR OTHER PARTICIPANTS: Collegiate athletes from whom center-of-pressure and symptom data were collected at preseason, less than 48 hours after injury, and 48 to 96 hours after injury. MAIN OUTCOME MEASURE(S): Approximate entropy values reflecting the amount of randomness contained in center-of-pressure oscillations were calculated for anterior-posterior (AP) and medial-lateral (ML) time series. Equilibrium scores reflecting the amplitude of center-of-pressure AP oscillations were used to indicate postural stability. The number and severity of symptoms were described. RESULTS: Compared with the healthy preseason state, ApEn values for the AP and ML time series generally declined immediately after injury in both steady and unsteady injured athletes. At 48 to 96 hours after injury, ApEn values for the ML time series remained significantly depressed (mean difference compared with preseason = -0.268, standard error = 0.072), even among athletes whose initial postural instability had resolved. We found few significant relationships between changes in ApEn values and changes in symptoms before and after injury. CONCLUSIONS: The effects of cerebral concussion on postural control appear to persist for longer than 3 to 4 days, even among athletes with no signs of unsteadiness. Our results may reflect changes in neurophysiologic or mechanical constraints on postural control. Approximate entropy provides a theoretically distinct, valuable measurement alternative that may prove useful for reducing uncertainty in the return-to-play decision.

Journal Article↗