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Biomedical subjects

G R Stewart

Publications and source records attributed to G R Stewart.

At least 19 recordsLinked to original sources

Mitochondrial DNA-Based phylogeography of North American rubber boas, Charina bottae (Serpentes: Boidae).

We used 783 bp of mitochondrial DNA sequences to study the phylogeography of Charina bottae (rubber boa) in western North America, with an emphasis on populations from California (U.S.A.). Maximum-parsimony and maximum-likelihood methods identified a basal divergence within C. bottae that corresponds to southern and northern segments of its current distribution. These clades coincide with the ranges of the two recognized subspecies, C. b. umbratica in the south and C. b. bottae to the north. A subsequent cladogenetic event in the C. b. bottae clade resulted in two groupings, which we refer to as the Sierra Nevada and the Northwestern subclades, based on the geographic distribution of their constituent populations. The two subclades have completely allopatric distributions, with a genetic break in the vicinity of Lassen Volcanic National Park in northeastern California, an area that was subjected to glaciation during the Pleistocene and that has been volcanically active in the past 100 years. An earlier genetic study documented fixed differences between populations of bottae and umbratica in four of seven allozymes surveyed, and despite noticeable variation and overlap in the characters that define C. b. bottae and C. b. umbratica, the two forms still can be separated in most cases using a suite of morphological traits. All available evidence thus indicates that C. b. umbratica is a genetically cohesive, allopatric taxon that is morphologically diagnosable, and we conclude that it is an independent evolutionary unit that should be recognized as a distinct species, Charina umbratica.

Animals↗

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection.

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Animals↗

Deletion of the putative antioxidant noxR1 does not alter the virulence of Mycobacterium tuberculosis H37Rv.

SETTING: The cloned M. tuberculosis noxR1 gene has been shown to confer resistance to reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) upon Escherichia coli and Mycobacterium smegmatis. OBJECTIVE: To investigate the role of noxR1 in resistance to RNI and virulence of M. tuberculosis. DESIGN: The noxR1 gene was deleted from M. bovis BCG and M. tuberculosis H37Rv by allelic exchange. The mutants were compared to wild type strains with respect to resistance to chemically generated RNI. The virulence of the M. tuberculosis mutant was investigated in a murine model of infection. RESULTS: The NoxR1 mutants grew normally in Sautons and 7H9 broths. The BCG mutant demonstrated decreased resistance to in vitro generated RNI compared to the wild type. Resistance to RNI could be restored to the mutant by reintroduction of the noxR1 locus on a replicating plasmid. However, deletion of noxR1 from M. tuberculosis H37Rv did not result in decreased resistance to RNI nor a difference in growth and survival of the bacterium during murine infection. CONCLUSION: The noxR1 gene locus in M. bovis BCG bestows ability to resist RNI generated in vitro. In M. tuberculosis H37Rv, however, noxR1 is either not involved in RNI resistance and virulence or is better compensated for by other mechanisms.

Animals↗

Deletion of the 19kDa antigen does not alter the protective efficacy of BCG.

Expression of the Mycobacterium tuberculosis 19kDa lipoprotein in saprophytic mycobacteria has been found to reduce their ability to prime a protective response to subsequent virulent challenge in the mouse model. The present study was designed to test whether 19kDa expression has an analogous detrimental effect on the efficacy of BCG vaccination. In contrast to the results in saprophytes, neither overexpression of the 19kDa antigen, nor deletion of the endogenous 19kDa gene altered the ability of BCG to protect against M. tuberculosis challenge in a mouse model.

Animals↗

Apparent contradiction: psychrotolerant bacteria from hydrocarbon-contaminated arctic tundra soils that degrade diterpenoids synthesized by trees.

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82 degrees N, 62 degrees W). According to most-probable-number assays, resin acid degraders were abundant (10(3) to 10(4) propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10(6) to 10(7) propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4 degrees C to 30 degrees C (DhA-91 and DhA-95) or 4 degrees C to 22 degrees C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22 degrees C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.

Arctic Regions↗

Onchocerciasis modulates the immune response to mycobacterial antigens.

Chronic helminth infection induces a type-2 cellular immune response. In contrast to this, mycobacterial infections commonly induce a type-1 immune response which is considered protective. Type-2 responses and diminished type-1 responses to mycobacteria have been previously correlated with active infection states such as pulmonary tuberculosis and lepromatous leprosy. The present study examines the immune responses of children exposed to both the helminth parasite Onchocerca volvulus and the mycobacterial infections, Mycobacterium tuberculosis and M. leprae. Proliferation of peripheral blood mononuclear cells (PBMC) and production of IL-4 in response to both helminth and mycobacterial antigen (PPD) decreased dramatically with increasing microfilarial (MF) density. Although interferon-gamma (IFN-gamma) production strongly correlated with cellular proliferation, it was surprisingly not related to MF density for either antigen. IL-4 production in response to helminth antigen and PPD increased with ascending children's age. IFN-gamma and cellular proliferation to PPD were not related to age, but in response to helminth antigen were significantly higher in children of age 9-12 years than children of either the younger age group (5-8 years) or the older group (13-16 years). Thus, there was a MF density-related down-regulation of cellular responsiveness and age-related skewing toward type 2 which was paralleled in response to both the helminth antigen and PPD. This parasite-induced immunomodulation of the response to mycobacteria correlates with a previous report of doubled incidence of lepromatous leprosy in onchocerciasis hyperendemic regions. Moreover, this demonstration that helminth infection in humans can modulate the immune response to a concurrent infection or immunological challenge is of critical importance to future vaccination strategies.

Adolescent↗

Reduction in visceral adipose tissue is associated with improvement in apolipoprotein B-100 metabolism in obese men.

We investigated the effect of reduction in visceral obesity on the kinetics of apolipoprotein B-100 (apoB) metabolism in a controlled dietary intervention study in 26 obese men. Hepatic secretion of very low density lipoprotein (VLDL) apoB was measured using a primed, constant, infusion of 1-[13C]leucine. In seven men receiving the reduction diet, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) apoB kinetics were also determined. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry, and SAAM-II was used to estimate the fractional turnover rates. Subcutaneous and visceral adipose tissues at the L3 vertebra were quantified by magnetic resonance imaging. With weight reduction there was a significant decrease (P < 0.05) in body mass index, waist circumference, and visceral adipose tissue. The plasma concentrations of total cholesterol, triglyceride, insulin, and lathosterol also significantly decreased (P < 0.05). Compared with weight maintenance, weight reduction significantly decreased the VLDL apoB concentration, pool size, and hepatic secretion of VLDL apoB (delta+2.5+/-4.6 vs. delta-14.7+/-4.0 mg/kg fat free mass-day; P = 0.010), but did not significantly alter its fractional catabolism. Weight reduction was also associated with an increased fractional catabolic rate of LDL apoB (0.24+/-0.07 vs. 0.54+/-0.10 pools/day; P = 0.002) and conversion of VLDL to LDL apoB (11.7+/-2.5% vs. 56.3+/-11.4%; P = 0.008). A change in hepatic VLDL apoB secretion was significantly correlated with a change in visceral adipose tissue area (r = 0.59; P = 0.043), but not plasma concentrations of insulin, free fatty acids, or lathosterol. The data support the hypothesis that a reduction in visceral adipose tissue is associated with a decrease in the hepatic secretion of VLDL apoB, and this may be due to a decrease in portal lipid substrate supply. Weight reduction may also increase the fractional catabolism of LDL apoB, but this requires further evaluation.

Adipose Tissue↗

A cocktail of recombinant Onchocerca volvulus antigens for serologic diagnosis with the potential to predict the endemicity of onchocerciasis infection.

We report here the evaluation of the potential of a serologic test to determine the endemicity of onchocercal infection in hyper, meso, and hypoendemic communities by the detection of antibodies to a cocktail of recombinant antigens. Parasitologic parameters of infection prevalence and intensity were compared with serologic results. Infection prevalence by serology was consistently but not significantly higher than that defined by parasitology. Differences between the communities defined by microfilarial load (CMFL) and a measurement of Onchocerca volvulus-specific antibody levels (serologic index [SI]) were similar. When stratified by age, differences were more significant in the younger age groups. If a sentinel population of 5-15-year-old individuals was used to compare communities, all could be equally ranked by serologic and parasitologic parameters. The SI of the sentinel population gave a better distinction between each community than the SI of the whole and would be sufficiently sensitive to measure the changes in endemicity that would be required for onchocerciasis control programs.

Adolescent↗

A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia.

Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 >/= PN3 >>> rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 +/- 0.92 nM. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the beta-subunits.

Amino Acid Sequence↗

Cloning and characterisation of a glutamate dehydrogenase cDNA from tomato (Lycopersicon esculentum L.).

A full-length cDNA (legdh1) has been cloned encoding glutamate dehydrogenase (GDH) from tomato (Lycopersicon esculentum L.). legdh1 is 1568 bp long and contains an open reading frame encoding a 44.8 kDa polypeptide with a putative mitochondrial-matrix-targeting pre-sequence at its N-terminus. Southern analysis indicates the existence of one copy of legdh1 per haploid genome, and no closely related genes were detected by Southern analysis at low stringency. We hypothesise that in tomato, the two GDH subunits may arise from post-transcriptional modifications of a single gene. Northern analysis reveals high expression of legdh1 in roots, lower levels of expression in stems, flowers and leaves, and no detectable expression in fruits. In general, there was no correlation between steady-state mRNA level and protein activity in the tissues analysed, again suggesting the importance of post-transcriptional events in the regulation of GDH. Comparison of cloned plant GDH proteins reveals a high degree of homology throughout the sequence except for a very specific, highly divergent region.

Amino Acid Sequence↗

Reversible Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor.

Substantial dysfunction and loss of cholinergic neurons occur in Alzheimer's disease (AD). Nerve growth factor (NGF) is a potent neurotrophic factor for cholinergic basal forebrain neurons, and the use of NGF to stimulate residual dysfunctional cells in AD is being considered. To define the effects of NGF on other cell populations in the brain, NGF was continuously infused into the lateral ventricle of rats for 7 weeks. At the end of treatment, Schwann cell hyperplasia and abundant sensory and sympathetic neurite sprouting were observed in the subpial region of the medulla oblongata and the spinal cord. Following withdrawal of NGF, the Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites disappeared completely. These findings suggest that better temporal and spatial delivery systems for NGF must be explored to limit potential undesirable side effects while maintaining the survival and function of diseased basal forebrain cholinergic neurons.

Animals↗

Hyperplastic changes within the leptomeninges of the rat and monkey in response to chronic intracerebroventricular infusion of nerve growth factor.

Recombinant human nerve growth factor (rhNGF) was delivered for up to 6 months by continuous intracerebroventricular (i.c.v.) infusion to CD (Sprague-Dawley derived) rats and cynomolgus monkeys. Rats (n = 15/sex/group) received doses of 0 (vehicle), 6, 60, or 300 ng/day; monkeys (n = 5/sex/group) received 0, 0.6, 6, or 60 microg/day of rhNGF. Animals tolerated i.c.v. infusion with no behavioral signs attributable to rhNGF. Body weight was transiently decreased in female rats at the highest dose. At the completion of dosing, histological examination in both species revealed an increase in the thickness of the leptomeninges along the ventral and lateral surfaces of the hindbrain and extending over the dorsal aspect of the spinal cord. The change was present to varying degrees at all doses of rhNGF and tended to be more severe at higher doses. At the light microscopic level, the leptomeninges contained layers of well-differentiated, spindle-shaped cells and a plexus of axonal fibers. Cells were immunoreactive for S-100 protein and were associated with an accumulation of Type IV collagen, suggesting Schwann cell origin. Electron microscopy revealed numerous fine caliber axons ensheathed by the presumptive Schwann cells, with myelination of individual axonal segments. These findings suggest that chronic i.c.v. delivery of rhNGF has stimulated axonal sprouting and secondary hyperplasia of Schwann or Schwann-like support cells within the pia-arachnoid.

Animals↗

The novel cuticular collagen Ovcol-1 of Onchocerca volvulus is preferentially recognized by immunoglobulin G3 from putatively immune individuals.

The cDNA sequence encoding an Onchocerca volvulus collagen, Ovcol-1, has been isolated and the corresponding native antigen has been identified. The cDNA encodes an open reading frame of 96 amino acid residues containing an uninterrupted 66-residue Gly-X-Y repeat triple-helical (TH) domain (where X and Y may be any amino acids) flanked by a 26-residue amino non-TH domain and a 4-residue carboxyl non-TH domain. The size (9.7 kDa) and structure of the deduced molecule are unique among previously identified collagen chains. This novel collagen type has been designated "mini-chain collagen." Native Ovcol-1 is aqueous soluble and resolves by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 14.2 kDa under reducing conditions. Immunoelectron microscopy of adult female O. volvulus localized Ovcol-1 to the cuticles of both the adult worm and uterine microfilaria. A group of individuals from an area in Ecuador where O. volvulus is hyperendemic have been classified as putatively immune (PI) to O. volvulus infection. Analysis of the humoral immune responses to Ovcol-1 demonstrated that immunoglobulin G3 (IgG3) of PI individuals preferentially recognized this antigen in comparison to IgG3 of infected individuals.

Amino Acid Sequence↗

Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons.

Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.

Amino Acid Sequence↗

Distribution of radioiodinated recombinant human nerve growth factor in primate brain following intracerebroventricular infusion.

The distribution of radioiodinated recombinant human nerve growth factor ([125I]rhNGF) was evaluated in adult cynomolgus monkeys following unilateral intracerebroventricular (icv) administration. Animals were cannulated into the right ventricle and recovered for 7 days. Monkeys were infused with 1.2 micrograms of [125I]rhNGF or [125I]rhNGF with a 140-fold excess of rhNGF. Twenty-four hours after infusion, animals were anesthetized and transcardially perfused with an aldehyde fixative. Coronal brain sections were processed for quantitative film autoradiography or for choline-acetyltransferase immunohistochemistry and then emulsion dipped. Specific radiolabel was distributed bilaterally and, with equal density, throughout the basal forebrain and was colocalized with choline acetyltransferase-positive neurons. Specific labeling was also present in the superficial ventral cortex. Nonspecific binding was observed surrounding the ventricles and lining blood vessels. These results demonstrate that unilateral icv infusion is an effective approach for delivering NGF to basal forebrain cholinergic neurons in primates and represents a viable drug delivery strategy for the therapeutic use of NGF in Alzheimer's Disease.

Animals↗

Mineralization of the globus pallidus following excitotoxic lesions of the basal forebrain.

The excitotoxin N-methyl aspartic acid was injected into the rat nucleus basalis to destroy basal forebrain cholinergic (BFC) neurons. In long-term survival experiments (up to 11 months post-lesion), conspicuous mineralized deposits were found in the globus pallidus and to a lesser extent in the thalamus. Deposits stained in a manner consistent with a composition of calcium and iron. Typically, deposits were absent from the center of the injection site, where BFC cell loss was most severe, but were present within the ventral and lateral globus pallidus where there was substantial sparing of BFC neurons. The similarity of this pathology to basal ganglia calcification and its relationship to Alzheimer's Disease and Down's syndrome is discussed.

Alzheimer Disease↗

Dose-response comparison of recombinant human nerve growth factor and recombinant human basic fibroblast growth factor in the fimbria fornix model of acute cholinergic degeneration.

Both nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) have been proposed for the treatment of Alzheimer's disease. This study describes a comparative, dose-response analysis of recombinant human (rh)NGF and rhbFGF in a rat unilateral fimbria-fornix model of acute cholinergic neuronal degeneration. Doses for rhNGF were 0.6, 6, 60, 600 and 1,800 ng/rat/day and for rhbFGF were 600, 1,800, 3,000 and 6,000 ng/rat/day, delivered for 4 weeks. The number of surviving septal cholinergic neurons was evaluated using ChAT immunohistochemistry. In control animals, the number of ChAT-positive neurons remaining on the lesioned side was between 22 and 18% compared to the non-lesioned side. Infusion with either neurotrophic factor increased the number of ChAT-positive neurons on the lesioned side in a dose-dependent manner. The maximal response to rhbFGF peaked at 3,000 ng/rat/day with a cell savings of 47%. However, there was evidence of neuropathological changes associated with rhbFGF. In contrast, rhNGF produced a maximal response with an infusion of 600 ng rhNGF/rat/day and a cell savings of 70% and no evidence of neuropathology, indicating that rhNGF was better tolerated and more efficacious than rhbFGF.

Acetylcholinesterase↗

Isotype-specific characterization of antibody responses to Onchocerca volvulus in putatively immune individuals.

Isotype/subclass-specific antibody responses to adult Onchocerca volvulus extract (OvAg) were assessed by both ELISA and immunoblotting for a group of putatively immune individuals (PIs, n = 29) from a hyperendemic area in Ecuador and for a group of infected individuals (INFs, n = 470) from the same regions. As a group, the PIs have been previously shown to possess lower levels of OvAg specific IgG1, IgG2, IgG3 and IgG4 than INF's but semi-quantitative analysis revealed that the relative proportions of these subclasses differs between the two groups. The IgG of the PI group contained a higher proportion of IgG3 and a lower proportion of IgG4 than the INF group. The frequency distribution of IgG3 responses was similar for the PI and INF groups. The frequency distributions for IgG1, IgG4 and IgE were significantly different between the PI and INF groups. A subgroup of the PIs were identified from frequency distributions and multivariate plots of individual isotype responses as having antibody responses (mainly IgG4) possibly indicative of cryptic infection. High IgE responses were exclusive to INF individuals, and a rare response type of high IgG3 with negligible levels of other isotypes/subclasses was seen only in the PI group. However, the majority of the PIs had negligible responses for all antibody classes. Immunoblots demonstrated no obvious differences in qualitative recognition between the PIs and INFs.

Adult↗