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Biomedical subjects

K E Marshall

Publications and source records attributed to K E Marshall.

8 recordsLinked to original sources
PubMed2006

Alternative splicing of G6PC2, the gene coding for the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), results in differential expression in human thymus and spleen compared with pancreas.

AIMS/HYPOTHESIS: Autoimmunity to insulin, glutamic acid decarboxylase and the tyrosine-phosphatase-like protein IA-2 is associated with type 1 diabetes. The production of self-molecules in thymus and secondary lymphoid tissues is critical for self-tolerance; reduced levels may impair tolerance and predispose to autoimmunity, as shown for insulin. Alternative splicing causes differential expression of IA-2 gene (PTPRN) transcripts and IA-2 protein in human thymus and spleen compared with pancreas. IA-2 sequences not present in lymphoid tissues become autoimmune targets in type 1 diabetes. The beta cell molecule islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an autoantigen in the non-obese diabetic (NOD) mouse, a model of type 1 diabetes. IGRP is a candidate autoantigen in the human disease, but robust assays for IGRP autoantibodies and/or autoreactive T cells are not available. Both full-length and IGRP splice variants encoded by the G6PC2 gene are expressed in the pancreas. In this study we tested the hypothesis that IGRP splice variants could be differentially expressed in thymus and spleen compared with the pancreas. METHODS: We evaluated the expression of G6PC2 transcripts in matched human thymus, spleen and pancreas specimens by RT-PCR. RESULTS: Alternative splicing results in differential expression of G6PC2 transcripts in thymus and spleen compared with pancreas. The full-length transcript is expressed in human pancreas but not in thymus or spleen. Five alternative spliced forms are always expressed in pancreas but those lacking exons 2, 3 and 4, alone or in combination, were rarely detected in thymus or spleen. CONCLUSIONS/INTERPRETATION: Differential tissue expression might favour autoimmune responses to IGRP in humans; target epitopes may be encoded by exons 3 and 4, or at the junctions of the conserved exons in the spliced transcripts. This information may aid in designing synthetic peptides for the identification of IGRP-specific autoreactive T cells in patients with type 1 diabetes.

Alternative Splicing↗
PubMed2001

Pharmacokinetics study of a novel chimeric single-chain variable fragment antibody against western equine encephalitis virus.

A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.

Administration, Intranasal↗
PubMed1998

Inhibition of angiogenesis in liver metastases by carboxyamidotriazole (CAI).

Carboxyamidotriazole (CAI), an inhibitor of calcium-mediated signal transduction, is a promising new cytostatic anti-cancer drug which has entered Phase II clinical trials, and for which multiple modes of action have been proposed. We tested the hypothesis that CAI can inhibit tumor angiogenesis in vivo. The ability of orally administered CAI to inhibit experimental metastases of B16F1 melanoma cells in mouse liver was assessed. A computer-assisted stereological technique was then used to analyze images from histological sections of CAI-treated vs. control livers; the vascular volume percentage (percentage of tumor volume consisting of functional microvessels) was determined to assess the effect of CAI on tumor angiogenesis. CAI treatment significantly reduced the size (8 x reduction in volume; P = 0.02) but not the number of metastases. In association with this reduction in tumor size, CAI significantly decreased the vascular volume percentage within metastases by at least a factor of two (P = 0.001). A reduction in both number of microvessels/mm2 and microvessel size (cross-sectional area) was found to contribute to this decrease. CAI treatment did not affect the vascular volume percentage of normal liver tissue surrounding metastases (P = 0.8). This study documents for the first time that CAI can inhibit tumor angiogenesis within metastases in vivo.

Journal Article↗
PubMed1993

Identification of zinc-binding ligands in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

An expression and mutagenesis system for the E. coli Class II fructose-1,6-bisphosphate aldolase has been created by modification of the vector pKfda (Biochem. J. 257 (1989) 529-534). Large amounts of Class II aldolase (about 1 g/l in crude extracts), with properties consistent with those previously reported for the naturally occurring enzyme (Biochem. J. 169 (1978) 633-641) are obtained. The enzyme contains 2 zinc ions per enzyme dimer. We have investigated the nature of the zinc-binding site of the enzyme by site-directed mutagenesis. His-108, His-111, Cys-112 and His-142 were identified as possible zinc-binding ligands by sequence alignments and comparisons with other known zinc-containing enzymes. Mutation of these residues identified His-108 and His-111 as two of the ligands directly responsible for the tight binding of zinc. Mutation of the other two residues results in only a small effect on the amount of zinc bound per monomer and a corresponding change in specific activity. These residues are, therefore, unlikely to be directly involved in zinc binding, but may be indirectly involved in some manner in the zinc-binding environment.

Amino Acid Sequence↗
PubMed1980

Rapid identification of Enterobacteriaceae associated with bacteremia: a preliminary report.

A preliminary report of a method to rapidly identify Enterobacteriaceae associated with bacteremia is described. When both bottles of a blood culture set revealed gram-negative rods on the Gram stain, an aliquot of the positive culture sample was repeatedly washed in sterile deionized water to remove RBCs and culture media. The concentrated suspension of organisms was used to inoculate API 20ETM identification strips which were generally read within 6-8 hr. Samples were also processed using conventional techniques. Ten episodes of bacteremia associated with Enterobacteriaceae were correctly identified using this rapid system. One Klebsiella pneumoniae with growth only in the aerobic bottle was excluded from the study, but was identified by conventional methods. Further advantages of this protocol are described.

Enterobacter↗