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Biomedical subjects

M Gilbert

Publications and source records attributed to M Gilbert.

At least 19 recordsLinked to original sources
PubMed2001

Azithromycin prophylaxis during a hospital outbreak of Mycoplasma pneumoniae pneumonia.

Outbreaks of Mycoplasma pneumoniae (MP) in closed communities can have a high attack rate and can last several months. Azithromycin chemoprophylaxis has not been evaluated as a means of limiting transmission. This randomized, double-blinded placebo-controlled trial of azithromycin was conducted among asymptomatic hospital employees during an MP outbreak. Oropharyngeal swabs were obtained for detection of MP by polymerase chain reaction, and questionnaires were administered to assess clinical illness. Of the 147 employees who were enrolled, 73 received azithromycin and 74 received placebo. Carriage was similar within and between groups at weeks 1 and 6 (9.6% vs. 6.7% and 10.3% vs. 13.2%, respectively). Four episodes of clinically significant respiratory illness occurred in the azithromycin group versus 16 episodes in the placebo group (protective efficacy, 75%; 95% confidence interval, 28%-91%). Use of azithromycin prophylaxis in asymptomatic persons during an MP outbreak in a closed setting may be of value in reducing clinical illness.

Adult↗
PubMed2001

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology.

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Bacterial Typing Techniques↗
PubMed2001

Skeletal muscle and insulin sensitivity: pathophysiological alterations.

Resistance to the normal action of insulin contributes to the pathogenesis of a number of common human disorders, Type II (non-insulin-dependent) diabetes mellitus. This review is focused on current understanding of the molecular mechanisms regulating insulin action and the factors contributing to insulin resistance in skeletal muscle. Since skeletal muscle is considered the major organ responsible for glucose uptake under insulin-stimulated conditions, defects in this target tissue are likely to contribute to metabolic disregulation in Type II diabetes mellitus. Defects in insulin signal transduction through the insulin-receptor substrate-1/phosphatidylinositol 3-kinase pathway is associated with reduced insulin-stimulated glucose transport activity in skeletal muscle from Type II diabetic patients. Glucose transport, the rate limiting step in glucose metabolism, is mediated by glucose transporter 4 (GLUT4) translocation and can be activated in skeletal muscle by two separate and distinct signaling pathways; one stimulated by insulin and the second by muscle contractions. Level of physical exercise has been linked to improved glucose homeostasis and enhanced insulin sensitivity. Understanding the molecular mechanism for the activation of signal transduction pathways by which insulin and muscle contraction increase glucose transport will provide a link to defining new strategies to enhance glucose metabolism in the diabetic patient.

Animals↗
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

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

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↗
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↗
PubMed2000

Ready access to sialylated oligosaccharide donors.

[formula: see text] Numerous glycoconjugates contain the disaccharide Neu5Ac alpha (2-->3)DGalp. An efficient way to incorporate this disaccharide into synthetic glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These disaccharide donors were used to prepare synthetic trisaccharides.

Carbohydrate Conformation↗
PubMed2000

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

Amino Acid Sequence↗
PubMed2000

Enhanced antisense efficacy of oligonucleotides adsorbed to monomethylaminoethylmethacrylate methylmethacrylate copolymer nanoparticles.

The purpose of this study was the investigation of cationic nanoparticles as drug delivery systems for antisense oligonucleotides. Cationic monomethylaminoethylmethacrylate (MMAEMA) copolymer nanoparticles were prepared from N-monomethylaminoethylmethacrylate hydrochloride and methylmethacrylate. Oligonucleotides were adsorbed onto MMAEMA nanoparticles. Cell penetration was investigated in vitro with fluorescently labeled oligonucleotides and nanoparticles. Antisense effects of oligonucleotides adsorbed to MMAEMA nanoparticles were evaluated by sequence specific inhibition of ecto-5'-nucleotidase expression. The amount of enzyme expressed in PC12 cells was detected and quantified by immunocytochemistry using fluorescein isothiocyanate-labeled antibodies. Oligonucleotides were adsorbed to MMAEMA nanoparticles by the formation of ion-pairs between the positively charged secondary amino groups located on the particle surface and the anionic phosphodiester or phosphorothioate backbones of the oligonucleotides. Adsorption to nanoparticles led to an increased cellular uptake of oligonucleotides and to a significantly enhanced antisense efficacy of unmodified phosphodiester oligonucleotides as well as phosphorothioates. The results of the cell penetration and the antisense assay demonstrated that MMAEMA nanoparticles are promising carriers for oligonucleotide administration.

5'-Nucleotidase↗
PubMed2000

A phase II study of preradiation chemotherapy followed by external beam radiotherapy for the treatment of patients with newly diagnosed glioblastoma multiforme: an Eastern Cooperative Oncology Group study (E2393).

Recent publications support the use of preradiation chemotherapy in the treatment of selected primary brain tumors. In the pediatric population, this treatment strategy often delays radiotherapy and may improve the outcome in patients. This manuscript describes the use of a preradiation chemotherapy approach for adult patients with newly diagnosed glioblastoma multiforme. The main objective of this trial was to determine the feasibility of delivering up to 3 monthly cycles of a 72 h continuous simultaneous intravenous infusion of BCNU (40 mg/m2/day) and cisplatin (40 mg/m2/day). Patients were evaluated for tumor response or progression after each cycle. Following the completion of the chemotherapy treatments or evidence of tumor progression, patients underwent external beam radiotherapy. A dose of 45 Gy was delivered to the pretreatment tumor volume plus surrounding edema and a margin of 3.0 cm. An additional 14.4 Gy was delivered to the preoperative volume plus a 2 cm margin. Fifty patients were enrolled, 47 were eligible and analyzable. Overall, 79% of patients were able to complete at least 2 cycles of treatment, exceeding the predefined measure of feasibility. One patient achieved a complete response, 10 patients a partial response and 18 patients had stable disease at completion of the chemotherapy treatments. Twenty-four patients experienced grade 4 toxicity, mostly hematologic. All patients were able to undergo radiotherapy following chemotherapy. These results indicate that a preradiation strategy is feasible. Although responses to the chemotherapy were seen, a phase III trial is needed to determine whether this approach provides an advantage over standard treatment; such a phase III trial has been undertaken by ECOG.

Adult↗