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L Wells

Publications and source records attributed to L Wells.

At least 19 recordsLinked to original sources
PubMed2003

A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.

The ability to regulate energy balance at both the cellular and whole body level is an essential process of life. As western society has shifted to a higher caloric diet and more sedentary lifestyle, the incidence of type 2 diabetes (non-insulin-dependent diabetes mellitus) has increased to epidemic proportions. Thus, type 2 diabetes has been described as a disease of 'chronic overnutrition'. There are abundant data to support the relationship between nutrient availability and insulin action. However, there have been multiple hypotheses and debates as to the mechanism by which nutrient availability modulates insulin signaling and how excess nutrients lead to insulin resistance. One well-established pathway for nutrient sensing is the hexosamine biosynthetic pathway (HSP), which produces the acetylated aminosugar nucleotide uridine 5'-diphospho-N-acetylglucosamine (UDP-Glc-NAc) as its end product. Since UDP-GlcNAc is the donor substrate for modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (O-GlcNAc), the possibility of this posttranslational modification serving as the nutrient sensor has been proposed. We have recently directly tested this model in adipocytes by examining the effect of elevated levels of O-GlcNAc on insulin-stimulated glucose uptake. In this review, we summarize the existing work that implicates the HSP and O-GlcNAc modification as nutrient sensors and regulators of insulin signaling.

Acetylglucosamine↗
PubMed2001

Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.

Acetylglucosamine↗
PubMed2001

Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc.

The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, O-GlcNAc and O-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. The diversity of proteins modified by O-GlcNAc implies its importance in many basic cellular and disease processes. Here we systematically examine the current data implicating O-GlcNAc as a regulatory modification important to signal transduction cascades.

Acetylglucosamine↗
PubMed2001

Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain.

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser/Thr residues is ubiquitous in higher eukaryotes and is analogous to protein phosphorylation. The enzyme for the addition of this modification, O-GlcNAc transferase, has been cloned from several species. Here, we have cloned a human brain O-GlcNAcase that cleaves O-GlcNAc off proteins. The cloned cDNA encodes a polypeptide of 916 amino acids with a predicted molecular mass of 103 kDa and a pI value of 4.63, but the protein migrates as a 130-kDa band on SDS-polyacrylamide gel electrophoresis. The cloned O-GlcNAcase has a pH optimum of 5.5-7.0 and is inhibited by GlcNAc but not by GalNAc. p-Nitrophenyl (pNP)-beta-GlcNAc, but not pNP-beta-GalNAc or pNP-alpha-GlcNAc, is a substrate. The cloned enzyme cleaves GlcNAc, but not GalNAc, from glycopeptides. Cell fractionation suggests that the overexpressed protein is mostly localized in the cytoplasm. It therefore has all the expected characteristics of O-GlcNAcase and is distinct from lysosomal hexosaminidases. Northern blots show that the transcript is expressed in every human tissue examined but is the highest in the brain, placenta, and pancreas. An understanding of O-GlcNAc dynamics and O-GlcNAcase may be key to elucidating the relationships between O-phosphate and O-GlcNAc and to the understanding of the molecular mechanisms of diseases such as diabetes, cancer, and neurodegeneration.

Acetylglucosaminidase↗
PubMed2001

Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics.

The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell's proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation, and acetylation, provide additional levels of functional complexity to the cell's proteome. The reversible post-translational modification O-linked beta-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states, including diabetes and Alzheimer's disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal (O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification.

Acetylglucosamine↗
PubMed2001

Older immigrant Tamil women and their doctors: attitudes toward breast cancer screening.

Cultural beliefs have been hypothesized to be powerful barriers to breast cancer screening in minority women and physician recommendation is consistently reported to be the strongest incentive. This study investigated (1) beliefs regarding breast cancer and (2) the perception of barriers to mammography and clinical breast examination in a sample of immigrant Tamil women, as well as in a sample of primary care physicians. Three focus groups, each consisting of 10 immigrant Tamil women from Sri Lanka aged 50 years or over were conducted and 52 primary care physicians who serve this population completed mailed surveys. The most common barriers to screening reported by the women were (1) lack of understanding of the role of early detection in medical care, (2) religious beliefs and, (3) fear of social stigmatization. Physicians reported the most common barriers to their screening recommendations for this group of women to be (1) women's episodic care, (2) unrelated presenting problems and, (3) women refusing to be screened. Interventions to increase screening in this and other minority groups requires an elaborated understanding of utilization barriers for both women and their doctors.

Journal Article↗
PubMed2001

Kingella kingae endocarditis in a sixteen-month-old-child.

We report the case of a child with acute neurologic symptoms who was found to have bacterial endocarditis caused by Kingella kingae. The case alerts microbiologists and pediatricians to an organism that has rarely been reported to cause endocarditis in children.

Endocarditis, Bacterial↗
PubMed2000

Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase.

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.

Catalytic Domain↗
PubMed2000

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.

Animals↗
PubMed2000

Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase.

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (hGALT) results in the potentially lethal disorder classic galactosemia. Although a variety of naturally occurring mutations have been identified in patient alleles, few have been well characterized. We have explored the functional significance of a common patient mutation, F171S, using a strategy of conservative substitution at the defined residue followed by expression of the wild-type and, alternatively, substituted proteins in a null-background strain of yeast. As expected from patient studies, the F171S-hGALT protein demonstrated <0.1% wild-type levels of activity, although two of three conservatively substituted moieties, F171L- and F171Y-hGALT, demonstrated approximately 10% and approximately 4% activity, respectively. The third protein, F171W, demonstrated severely reduced abundance, precluding further study. Detailed kinetic analyses of purified wild-type, F171L- and F171Y-hGALT enzymes, coupled with homology modeling of these proteins, enabled us to suggest that the effects of these substitutions resulted largely from altering the position of a catalytically important residue, Gln-188, and secondarily, by altering the subunit interface and perturbing hexose binding to the uridylylated enzyme. These results not only provide insight into the functional impact of a single common patient allele and offer a paradigm for similar studies of other clinically or biochemically important residues, but they further help to elucidate activity of the wild-type human GALT enzyme.

Amino Acid Substitution↗
PubMed2000

Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae.

The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.

Base Sequence↗
PubMed2000

The continuing fall in incidence of hypoxic-ischaemic encephalopathy in term infants.

OBJECTIVE: To examine trends in incidence of hypoxic-ischaemic encephalopathy in term infants over a twenty-one year period. DESIGN: A retrospective analysis of medical records of all term infants admitted to a neonatal unit with hypoxic-ischaemic encephalopathy during the years 1992-1996 (period C) and a comparison with data from the years 1976-1980 (period A) and 1984-1988 (period B) from the same unit (previously published). SETTING: A District Health Authority in Central England serving a population of about 450,000. SAMPLE: All term infants admitted with clinical features of hypoxic-ischaemic encephalopathy. MAIN OUTCOME MEASURES: Incidence of three grades of hypoxic-ischaemic encephalopathy, disability and mortality. RESULTS: In each five year period there were similar numbers of births. Over the time-span of this study the stillbirth rate and neonatal mortality rate has consistently fallen. The overall incidence of hypoxic-ischaemic encephalopathy in term infants was significantly lower (P < 0.001; OR 0.42 CI 0.29-0.59) in the present study period (C) compared with the earlier study period B (1.9 vs 4.6 per 1,000 total live births). The fall in moderately and severely affected infants between the present and the first study period was significant (1.2 vs 2.6 per 1,000 total live births, P < 0.001: OR 0.46 CI 0.29-0.72). The number of deaths and the incidence of cerebral palsy in survivors fell progressively over the 21 years spanned by this study. CONCLUSION: This study shows that the incidence of hypoxic-ischaemic encephalopathy and its sequelae in term infants has fallen significantly. The use of cardiotocography and caesarean section rates have risen but the relative contributions of changes in clinical practice are uncertain.

England↗
PubMed1999

Ondansetron/promethazine combination or promethazine alone reduces nausea and vomiting after middle ear surgery.

STUDY OBJECTIVES: To determine the incidence of postoperative nausea and vomiting when a combination of ondansetron and promethazine is given prophylactically, and to ascertain the effect of postoperative nausea and vomiting on recovery room duration and patient satisfaction. DESIGN: Prospective, randomized, placebo-controlled, double-blind study. SETTING: University-affiliated tertiary-care hospital. PATIENTS: 87 ASA physical status I and II adult patients scheduled for middle ear surgery. INTERVENTIONS: Patients were randomly assigned to receive one of the following interventions intravenously: ondansetron 4 mg (Group 1), promethazine 25 mg (Group 2), ondansetron 2 mg plus promethazine 12.5 mg (Group 3, combination), or placebo (Group 4). MEASUREMENTS AND MAIN RESULTS: Independent, study blinded observers recorded complaints of nausea and number of episodes of vomiting for 24 hours following the patient's first response to commands. All patients were contacted the day after discharge to inquire about nausea and vomiting. The awakening time, postanesthesia care unit and day surgery unit durations, opioid use, and side effects were recorded. At the end of the 24-hour period, the study blinded observers asked patients for an overall assessment of their global anesthesia experience using an 11-point scale. During the 24-hour period, the incidence of postoperative nausea and vomiting was reduced from 74% (placebo) to 39% (promethazine; p = 0.03) and 29% (combination; p = 0.003). Compared with placebo, the severity of vomiting was significantly less in the combination group (p = 0.04). The number of very satisfied patients correlated negatively with the incidence of postoperative nausea and vomiting (p < 0.0001) and with the severity of vomiting (p = 0.003). CONCLUSION: The prophylactic use of an antiemetic with middle ear surgery may reduce postoperative nausea and vomiting over 24 hours, and the ondansetron/promethazine combination or promethazine alone are cost-effective choices. Finally, the combination reduced significantly the severity of vomiting.

Adolescent↗
PubMed1998

Effects of acute intravenous cocaine on cardiovascular function, human learning, and performance in cocaine addicts.

Continuous non-invasive cardiovascular monitoring in eight healthy cocaine addicts receiving intravenous cocaine (0.325 mg/kg or 0.650 mg/kg) or placebo in double-blind, randomized, cross-over fashion demonstrated significant dose-dependent increases in pulse and mean arterial pressure following cocaine. Pulse and mean arterial pressure peaked 5 min post-cocaine injection and maximal response was sustained for a further 15 min and 35 min afterwards, respectively. Cocaine administration had no significant effect on peripheral oxygen saturation, and no clinically significant abnormalities of rhythm or conduction were seen on the electrocardiogram. These doses and method of single-dose intravenous cocaine administration, and our procedures for cardiovascular monitoring, appear relatively safe for laboratory studies of healthy cocaine addicts with no pre-existing cardiovascular disease. In addition, cocaine-taking (0.325 mg/kg i.v. and 0.650 mg/kg i.v.) was associated with enhanced attention (i.e. increased numbers of correct responses on the Rapid Visual Information Processing Task), but the trend towards reduced reaction time did not achieve statistical significance. Cocaine-taking resulted in a small but statistically insignificant improvement in learning on the Digit Symbol Substitution Task. These results suggest that cocaine-taking in rested subjects is associated with some cognitive enhancement.

Adult↗
PubMed1998

Isradipine prevents global and regional cocaine-induced changes in brain blood flow: a preliminary study.

The L-type calcium channel antagonist, isradipine, reduces brain ischemia in animal models of ischemic stroke. These effects of isradipine appear more pronounced in dopamine (DA) rich brain regions. These same DA-rich brain regions have also been shown to be the areas most affected by cocaine-induced ischemic changes. Using a novel quantified approach to single photon emission computerized tomography, we demonstrated that isradipine pre-treatment prevented cocaine-induced ischemic changes, especially in these DA-rich brain regions. This is the first demonstration that any medication, including isradipine, can prevent the ischemic effects of cocaine on brain blood flow. Isradipine may, therefore, be a useful therapeutic agent for the prevention of brain ischemia in cocaine addicts.

Adult↗
PubMed1998

Demonstration of dose-dependent global and regional cocaine-induced reductions in brain blood flow using a novel approach to quantitative single photon emission computerized tomography.

Ischemic stroke is a common cause of morbidity and mortality in cocaine addicts. Because the previous semiquantitative single photon emission computerized tomography (SPECT) method for measuring brain blood flow does not quantify blood flow, the magnitude and specificity of cocaine's effects during drug taking has not been well established. Here, using a novel quantitative approach to SPECT, we established that intravenous cocaine administration to nine recently abstinent cocaine-dependent subjects was associated with significant decreases in global and regional brain blood flow to dopamine-rich areas such as the prefrontal, frontal temporal, and subcortical gray matter. Establishing the utility of this relatively new quantitative SPECT technique provides an important tool for the management of vascular disorders of the brain. Additionally, identifying the site-specific effects of cocaine provides targets for the development of putative therapeutic medications to attenuate or minimize ischemic stroke in cocaine addicts.

Adolescent↗
PubMed1997

Biochemical characterization of the S135L allele of galactose-1-phosphate uridylyltransferase associated with galactosaemia.

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia. The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems. Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems. In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity. Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold. Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold. Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo. We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.

Alleles↗