Biomedical subjects

J A McCann

Publications and source records attributed to J A McCann.

14 recordsLinked to original sources

PubMed2001

Evidence against GRB10 as the gene responsible for Silver-Russell syndrome.

Recent evidence shows that Silver-Russell syndrome (SRS), the major functional deficit of which is limited growth, both intrauterine and postnatal, is due to a double dose of a gene within 7p11.2-p13 that is normally expressed exclusively from the maternal copy. Of the several growth-related genes in this chromosomal region, only GRB10 has been demonstrated to be imprinted; however, imprinting was limited to brain and muscle and was incomplete. Using reverse-transcript PCR, we now confirm GRB10 imprinting in these two tissues is isoform-specific and, more importantly, demonstrate absence of imprinting in growth plate cartilage, the tissue most directly involved in linear growth. Thus, it is unlikely that GRB10 is the gene responsible for SRS.

Aged↗

PubMed2001

In vitro assembly of novel cholera toxin-like complexes.

Cholera toxin (CT) is responsible for the major pathological features of cholera, but in addition to its cytotoxic properties, CT is a potent mucosal adjuvant when coadministered with antigens at mucosal sites. Discovery of CT adjuvanticity has prompted the generation of CT chimeras with reduced toxicity and improved efficiency for antigen presentation at mucosal sites. To date, chimeric forms of CT have been produced in bacterial strains by coexpressing the CT B subunit and a chimeric form of the CT A subunit consisting of a target protein antigen fused with the A2 polypeptide of CT. In this study, a chimeric protein consisting of green fluorescent protein (GFP) fused with polypeptide A2 was generated to investigate the feasibility of assembling CT holotoxin-like complexes in vitro. The assembly of such holotoxin-like complexes would expand the variety of antigenic compounds that could be incorporated into CT-based vaccines. In this study, GFP-A2/CTB complexes could be generated in vitro using a stepwise denaturation-renaturation process. These findings suggest that it is possible to generate novel mucosal vaccines consisting of macromolecules that are chemically coupled to polypeptide A2 and reconstituted into CT-like complexes in vitro.

Adjuvants, Immunologic↗

PubMed2001

Galphas transcripts are biallelically expressed in the human kidney cortex: implications for pseudohypoparathyroidism type 1b.

Pseudohypoparathyroid type 1b patients are characterized by renal resistance to PTH in the absence of Albright's hereditary osteodystrophy or other endocrine abnormalities. Kindred studies have suggested that the cause of this resistance is a specific decrease in Galphas activity in renal proximal tubules due to paternal imprinting of Galphas. To test this, allelic expression of Galphas was analyzed in human fetal kidney cortex samples by RT-PCR assays. The results showed that, in contrast to the parent-specific expression of exon 1A and XLalphas (paternal) or NESP (maternal) mRNAs, Galphas transcripts are biallelically expressed in human kidney cortex. These data implicate abnormal imprinting of alternative regions within the GNAS1 locus as a more likely cause of pseudohypoparathyroid type 1b.

GTP-Binding Protein alpha Subunits, Gs↗

PubMed2000

The heat shock protein Ssa2p is required for import of fructose-1, 6-bisphosphatase into Vid vesicles.

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Deltassa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Deltassa1, Deltassa3, or Deltassa4 strains. Likewise, in vitro import was impaired for the Deltassa2 strain, but not for the other Deltassa strains. The cytosol was identified as the site of the Deltassa2 defect; Deltassa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Deltassa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Deltassa2 Vid vesicles, providing Deltassa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

Adenosine Triphosphate↗

PubMed1999

Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis.

Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a consequence of specific binding to globotriaosylceramide (Gb3) receptors on endothelial cell surfaces. Molecular models based on the VT1 crystal structure were used previously to investigate the structural basis for receptor recognition by VT1 and other verotoxins. Interestingly, these model-based predictions of glycolipid binding to VT1 differ somewhat from recently published structural data from cocrystals of the VT1 B-subunit (VT1B) and an analogue of the sugar moiety of Gb3. In this study, fluorescence spectroscopy was used to test model-based predictions of the location of Gb3 binding on the B-subunit pentamer of VT1. Resonance energy transfer was used to calculate the distance from a coumarin probe used to replace the acyl tail of Gb3 and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggests that these two moieties are approximately 13.3 A apart when a single distance is assumed. This distance is consistent with proposed models for the fit of Gb3 within the "cleft site" of the VT1 B-subunit. When the distances from Trp34 to the other coumarinGb3 molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consideration, it appears likely that the coumarin-modified Gb3 analogue used in this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c for coumarinGb3. To provide additional information on the association of Gb3 with the VT1 B-subunit, the influence of Gb3 glycolipid binding on the accessibility of Trp34 to different quenching agents in solution was then examined. Taken together, the data suggest that coumarin-labeled Gb3 preferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.

Bacterial Toxins↗

PubMed1997

Conformational changes in cholera toxin B subunit-ganglioside GM1 complexes are elicited by environmental pH and evoke changes in membrane structure.

Fluorescence resonance energy transfer (FRET) was used to monitor pH-dependent structural changes in the cholera toxin B subunit (CTB) and the membranes with which CTB associates. The distance separating the single tryptophan (Trp88) of each CTB monomer and a pyrene probe linked to the membrane-imbedded tail of ganglioside GM1 is not influenced by pH in a range from 3.5 to 7.5, consistent with the position of Trp88 in the GM1 binding site of CTB. In contrast, the distance between the pyrene probe on GM1 and coumarin, stilbene, or fluorescein probes covalently linked to specific sites on CTB appears to increase significantly as the pH is lowered to 5.0 or less. This conformational change is not accompanied by detectable changes in the distance between Trp88 and these extrinsic probe positions in the presence of nonfluorescent GM1. However, when the distance from Trp88 to the extrinsic probes is monitored as a function of pH in the absence of GM1, a conformational change is seen which indicates that receptor binding influences the character of pH-dependent conformational changes that occur within CTB. Interestingly, the observed change in CTB conformation is accompanied by a change in the relative position of GM1 within the membrane as judged by FRET from the pyrene probe on GM1 to a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe linked to the polar head group of phosphatidylethanolamine and positioned at the membrane surface. Taken together, the data imply that low endosomal pH is capable of inducing structural changes in CTB, which, in turn, exert effects on the structure of the membrane to which CTB is bound. These phenomena may have a role in (1) processing of cholera toxin within the endosomal compartments of some target cell types, (2) determining the lag time between cholera toxin binding and the target cell response to cholera intoxication, or (3) the efficiency of CTB and cholera toxin as mucosal adjuvants.

Biopolymers↗

PubMed1996

Fluorescence analysis of galactose, lactose, and fucose interaction with the cholera toxin B subunit.

The cholera toxin B subunit (CTB) recognizes ganglioside GM1 receptors on target cells to facilitate entry of the toxin's A1 polypeptide into the host cytoplasm. GM1 binding to the CTB homopentamer occurs cooperatively with the most prominent interactions involving the terminal galactose residue of the ganglioside. Here, it is shown that association of galactose, lactose, or fucose (6-deoxy-galactose) with CTB is readily monitored using fluorescence spectroscopy. In many respects, however, the formation of CTB complexes with these small sugar analogues of GM1 greatly differs from the formation of complexes with the ganglioside itself. Each of these monosaccharides has a much weaker affinity for CTB than does GM1 and none of the sugars appear to be bound cooperatively. Moreover, GM1 binding conveys a stabilizing effect to CTB which is not seen upon binding of galactose or lactose. These data indicate that CTB-GM1 interactions involving sites other than the terminal galactose of the ganglioside serve prominently in the proper placement of CT on the target cell surface.

Cholera Toxin↗

PubMed1990

Interaction of insulin and phorbol esters on the regulation of DNA synthesis in rat hepatoma cells.

Insulin and phorbol esters stimulated DNA synthesis in rat H4 hepatoma cells. Insulin and phorbol ester induction of thymidine incorporation was dose-dependent, with a maximal 4.2- and 3.0-fold increases at concentrations of 1 x 10(-9)M and 1 microM, respectively. Phorbol esters in combination with increasing concentrations of insulin resulted in additive effects, but only at submaximal insulin concentrations. The combination failed to increase thymidine incorporation above the maximal effects produced by insulin alone. When cells were pretreated with phorbol esters for 24h to produce protein kinase-C (PKC) deficiency, basal DNA synthesis was depressed. Pretreatment with phorbol esters abolished the effects of phorbol esters to induce DNA synthesis but did not impair the magnitude of insulin-induced DNA synthesis. Thus, although phorbol ester-activatable PKC-activity was necessary for basal DNA synthesis, it was not necessary for insulin-induction of DNA synthesis in H4 cells.

Animals↗

PubMed1987