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Biomedical subjects

M Gilbert

Publications and source records attributed to M Gilbert.

At least 37 records · Page 2Linked to original sources
PubMed2001

Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution.

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-linked galactose from a P(k) epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic P(k)-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha-2,3 and alpha-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha-2,6-sialyltransferase activity in vitro as well as in vivo.

Amino Acid Substitution↗
PubMed2001

Neuroligin 3 is a vertebrate gliotactin expressed in the olfactory ensheathing glia, a growth-promoting class of macroglia.

The molecular mechanisms that drive glia-glial interactions and glia-neuronal interactions during the development of the nervous system are poorly understood. A number of membrane-bound cell adhesion molecules have been shown to play a role, although the precise nature of their involvement is unknown. One class of molecules with cell adhesive properties used in the nervous system is the serine-esterase-like family of transmembrane proteins. A member of this class, a glia-specific protein called gliotactin, has been shown to be necessary for the development of the glial sheath in the peripheral nervous system of Drosophila melanogaster. Gliotactin is essential for the development of septate junctions in the glial sheath of individual and neighboring glia. Mutations that remove this protein result in paralysis and eventually death due to a breakdown in the glial-based blood-nerve barrier. To study the role of gliotactin during vertebrate nervous system development, we have isolated a potential vertebrate gliotactin homologue from mice and rat and found that it corresponds to neuroligin 3. Using a combination of RT-PCR and immunohistochemistry, we have found that neuroligin 3 is expressed during the development of the nervous system in many classes of glia. In particular neuroligin 3 is expressed in the olfactory ensheathing glia, retinal astrocytes, Schwann cells, and spinal cord astrocytes in the developing embryo. This expression is developmentally controlled such that in postnatal and adult stages, neuroligin 3 continues to be expressed at high levels in the olfactory ensheathing glia, a highly plastic class of glia that retain many of their developmental characteristics throughout life.

Animals↗
PubMed2001

Benefits of collaborative learning for environmental management: applying the integrated systems for knowledge management approach to support animal pest control.

Resource management issues continually change over time in response to coevolving social, economic, and ecological systems. Under these conditions adaptive management, or "learning by doing," offers an opportunity for more proactive and collaborative approaches to resolving environmental problems. In turn, this will require the implementation of learning-based extension approaches alongside more traditional linear technology transfer approaches within the area of environmental extension. In this paper the Integrated Systems for Knowledge Management (ISKM) approach is presented to illustrate how such learning-based approaches can be used to help communities develop, apply, and refine technical information within a larger context of shared understanding. To outline how this works in practice, we use a case study involving pest management. Particular attention is paid to the issues that emerge as a result of multiple stakeholder involvement within environmental problem situations. Finally, the potential role of the Internet in supporting and disseminating the experience gained through ongoing adaptive management processes is examined.

Animals↗
PubMed2001

Yolk protein endocytosis by oocytes in Drosophila melanogaster: immunofluorescent localization of clathrin, adaptin and the yolk protein receptor.

The process of yolk protein (YP) uptake by developing oocytes in Drosophila melanogaster has been investigated by immunofluorescent localization of the endocytosis proteins, clathrin, alpha-adaptin and the putative yolk protein receptor (YP receptor). Data suggests that YPs from the follicle cells are trafficked into the oocyte during early stages of vitellogenesis, and that hemolymph YPs are sequestered by nurse cells adjacent to the developing oocyte during late stages of vitellogenesis. Yolk proteins were immunolocalized to both follicle cells and nurse cells during these processes. Diapausing female Drosophila melanogaster undergo a pre-vitellogenic arrest of ovarian development associated with the absence of ovarian alpha-adaptin, clathrin and putative YP receptor. Diapause termination by transfer of whole animals from 11 degrees C to 25 degrees C, or by 20-hydroxyecdysone injection, results in the appearance of immunopositive material in the nurse cells for all three proteins between 12 h and 16 h post upshift and within four days of injection. Immunopositive material was not noted in the follicle cells during diapause termination. In vitro warming of diapausing ovaries, or incubation in the presence of 1 &mgr;M 20-hydroxyecdysone failed to initiate early vitellogenic development suggesting that diapause termination requires factor(s) external to the ovary. Western blotting analysis of extracts of 24 h post-eclosion wild type and ap(56f) females identified putative yolk protein receptor with a molecular weight of 208 kDa and clathrin with a molecular weight of 178 kDa.

Journal Article↗
PubMed2001

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.

Blood Bactericidal Activity↗
PubMed2001

Intrathecal treatment of neoplastic meningitis due to breast cancer with a slow-release formulation of cytarabine.

DepoCyte is a slow-release formulation of cytarabine designed for intrathecal administration. The goal of this multi-centre cohort study was to determine the safety and efficacy of DepoCyte for the intrathecal treatment of neoplastic meningitis due to breast cancer. DepoCyte 50 mg was injected once every 2 weeks for one month of induction therapy; responding patients were treated with an additional 3 months of consolidation therapy. All patients had metastatic breast cancer and a positive CSF cytology or neurologic findings characteristic of neoplastic meningitis. The median number of DepoCyte doses was 3, and 85% of patients completed the planned 1 month induction. Median follow up is currently 19 months. The primary endpoint was response, defined as conversion of the CSF cytology from positive to negative at all sites known to be positive, and the absence of neurologic progression at the time the cytologic conversion was documented. The response rate among the 43 evaluable patients was 28% (CI 95%: 14-41%); the intent-to-treat response rate was 21% (CI 95%: 12-34%). Median time to neurologic progression was 49 days (range 1-515(+)); median survival was 88 days (range 1-515(+)), and 1 year survival is projected to be 19%. The major adverse events were headache and arachnoiditis. When drug-related, these were largely of low grade, transient and reversible. Headache occurred on 11% of cycles; 90% were grade 1 or 2. Arachnoiditis occurred on 19% of cycles; 88% were grade 1 or 2. DepoCyte demonstrated activity in neoplastic meningitis due to breast cancer that is comparable to results reported with conventional intrathecal agents. However, this activity was achieved with one fourth as many intrathecal injections as typically required in conventional therapy. The every 2 week dose schedule is a major advantage for both patients and physicians.

Adult↗
PubMed2001

Effect of a low-glycaemic index--low-fat--high protein diet on the atherogenic metabolic risk profile of abdominally obese men.

It has been suggested that the current dietary recommendations (low-fat-high-carbohydrate diet) may promote the intake of sugar and highly refined starches which could have adverse effects on the metabolic risk profile. We have investigated the short-term (6-d) nutritional and metabolic effects of an ad libitum low-glycaemic index-low-fat-high-protein diet (prepared according to the Montignac method) compared with the American Heart Association (AHA) phase I diet consumed ad libitum as well as with a pair-fed session consisting of the same daily energy intake as the former but with the same macronutrient composition as the AHA phase I diet. Twelve overweight men (BMI 33.0 (sd 3.5) kg/m2) without other diseases were involved in three experimental conditions with a minimal washout period of 2 weeks separating each intervention. By protocol design, the first two conditions were administered randomly whereas the pair-fed session had to be administered last. During the ad libitum version of the AHA diet, subjects consumed 11695.0 (sd 1163.0) kJ/d and this diet induced a 28 % increase in plasma triacylglycerol levels (1.77 (sd 0.79) v. 2.27 (sd 0.92) mmol/l, P<0.05) and a 10 % reduction in plasma HDL-cholesterol concentrations (0.92 (sd 0.16) v. 0.83 (sd 0.09) mmol/l, P<0.01) which contributed to a significant increase in cholesterol:HDL-cholesterol ratio (P<0.05), this lipid index being commonly used to assess the risk of coronary heart disease. In contrast, the low-glycaemic index-low-fat-high-protein diet consumed ad libitum resulted in a spontaneous 25 % decrease (P<0.001) in total energy intake which averaged 8815.0 (sd 738.0) kJ/d. As opposed to the AHA diet, the low-glycaemic index-low-fat-high-protein diet produced a substantial decrease (-35 %) in plasma triacylglycerol levels (2.00 (sd 0.83) v. 1.31 (sd 0.38) mmol/l, P<0.0005), a significant increase (+1.6 %) in LDL peak particle diameter (251 (sd 5) v. 255 (sd 5) A, P<0.02) and marked decreases in plasma insulin levels measured either in the fasting state, over daytime and following a 75 g oral glucose load. During the pair-fed session, in which subjects were exposed to a diet with the same macronutrient composition as the AHA diet but restricted to the same energy intake as during the low-glycaemic index-low-fat-high-protein diet, there was a trend for a decrease in plasma HDL-cholesterol levels which contributed to the significant increase in cholesterol:HDL-cholesterol ratio noted with this condition. Furthermore, a marked increase in hunger (P<0.0002) and a significant decrease in satiety (P<0.007) were also noted with this energy-restricted diet. Finally, favourable changes in the metabolic risk profile noted with the ad libitum consumption of the low-glycaemic index-low-fat-high-protein diet (decreases in triacyglycerols, lack of increase in cholesterol:HDL-cholesterol ratio, increase in LDL particle size) were significantly different from the response of these variables to the AHA phase I diet. Thus, a low-glycaemic index-low-fat-high-protein content diet may have unique beneficial effects compared with the conventional AHA diet for the treatment of the atherogenic metabolic risk profile of abdominally obese patients. However, the present study was a short-term intervention and additional trials are clearly needed to document the long-term efficacy of this dietary approach with regard to compliance and effects on the metabolic risk profile.

Analysis of Variance↗
PubMed2001

Evaluation of viral resistance to reverse transcriptase inhibitors (RTI) in HIV-1-infected patients before and after 6 months of single or double antiretroviral therapy.

We evaluated samples of peripheral blood mononuclear (PBMC) cells from 46 AIDS patients, before starting therapy with HIV-1 reverse transcriptase inhibitors (RTI), and after 6 months of drug use. PBMC were stored and tested by a Line Probe Assay (LiPA), in order to assess the frequency of RT mutations in this population. Six patients were taking AZT before initial blood collection (1 to 16 weeks of drug use) and 40 patients had no prior therapy. After baseline evaluation, 19 patients received AZT, 23 AZT plus DDI, 3 started AZT only with DDI added after 3 months, and 3 received a combination of AZT plus 3TC. Detection of at least one mutation was found in 33% (15/46) of patients at baseline, and 83% (38/46) had at least 1 mutation after 6 months of therapy. In the majority of cases, samples presented with the wild type and variants of HIV, simultaneously. Patients receiving monotherapy had a higher frequency of mutations (L41 and F214, Y215) than did patients receiving double-drug therapy (19 vs. 10). No specific mutation associated with DDI was identified in 26 patients so treated. Despite the finding of a mean increase in CD4 count and a mild decrease in viral load, patients tended to have an inverse correlation between the CD4 variation and number of mutations detected after 6 months, suggesting potential loss of drug efficacy in the presence of these genotypic changes.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Endoscopic versus open surgical treatment of carpal tunnel syndrome.

The impact of CTS is significant as evidenced by the fact that only back injuries result in greater rates of employee absenteeism in the workplace. CTR is now the most commonly performed surgical procedure in the United States. Earlier efforts using open surgical techniques were associated with significant morbidity, which some would argue is greater than that associated with the disease itself. The addition of endoscopy to surgeon's armamentarium offers the promise of decreased morbidity associated with the surgical treatment of CTS. Evidence indicates that when compared with open CTR, endoscopic CTR results in earlier achievement of patient satisfaction and functional outcomes. As a result, it is becoming clear that endoscopic surgery is a safe and effective method of treating CTS.

Arthroscopy↗
PubMed2000

Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP.

The x-ray crystallographic structure of selenomethionyl cytosine-5'-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-A resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-A resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.

Amino Acid Sequence↗
PubMed2000

Polymer-supported and chemoenzymatic synthesis of the Neisseria meningitidis pentasaccharide: a methodological comparison.

Neisseria meningitidis trisaccharide [GlcNAc[(1-->3)Galbeta(1-->4)Glc-R], tetrasaccharide [Galbeta(1-->4)GlcNAcbeta(1--> 3)Galbeta(1-->4)Glc-R], and a pentasaccharide [Neu5Acalpha(2-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)G albeta(1-->4)Glc-SPh] were prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting from D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion, and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent stereo- and regioselectivities as well as high yield (> 90% from Gal(1-->4)Glc-SPh) of the pentasaccharide were obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides.

Antigens, Bacterial↗
PubMed2000

Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis.

Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.

Apoptosis↗
PubMed2000

Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion.

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Adsorption↗