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Biomedical subjects

G David

Publications and source records attributed to G David.

At least 19 recordsLinked to original sources
PubMed1992

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.

Antithrombin III↗
PubMed1992

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells.

We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.

Amino Acid Sequence↗
PubMed1992

Developmental changes in heparan sulfate expression: in situ detection with mAbs.

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

Animals↗
PubMed1992

Unexplained in-vitro fertilization failure: implication of acrosomes with a small reacting region, as revealed by a monoclonal antibody.

To determine the acrosomal characteristics related to in-vitro fertilization (IVF) outcome, spermatozoa from 50 men whose wives had resorted to IVF have been studied by indirect immunofluorescence microscopy with anti-human pro-acrosin monoclonal antibody 4D4 (mAb 4D4), prior to and after incubation in a capacitating medium. The antibody labelled only the acrosomal principal region (APR), revealing its shape (i.e. normal, small or amorphous) and its status (i.e. unreacted, partially or totally reacted). The IVF outcome distinguished: (i) spermatozoa which were able to fertilize at least one oocyte in vitro (group I; n = 25) and (ii) spermatozoa which failed to fertilize any oocyte in vitro (group II; n = 25). The semen characteristics of the two sperm groups, including the acrosome morphology, were similar according to conventional analysis. The mAb 4D4 detected in both the whole and the swim-up sperm cell fractions a lower percentage of normal APR in group II (< 50% for 10 patients in group II versus one patient from group I), which was related to a higher percentage of small APR. Moreover, after 21 h incubation, group II had a lower acrosomal loss index. The spermatozoa of five patients of this infertile group II did not undergo acrosomal modification whereas spermatozoa of all group I patients underwent the acrosomal reaction. The data showed that the relationship between acrosomal anomalies and IVF failure is mainly due to an increased incidence of acrosomes with a reduced size of the region involved in the acrosome reaction. Immunodiagnosis of this acrosomal region by means of mAb 4D4 is informative for IVF outcome.

Acrosin↗
PubMed1992

Using a computer to manage the radiation safety and quality control activities in diagnostic radiology.

Software has been developed to aid in the management of a radiation safety quality control program in diagnostic radiology. The core of the system is a data base of radiation safety activities. Software design goals were the prioritization, scheduling, and reporting of these activities. Computerization has helped organize the timely performance of surveys necessary for compliance with regulatory and accreditation agencies. Clear, concise program documentation and reporting are also provided.

Georgia↗
PubMed1992

Evidence that action potentials activate an internodal potassium conductance in lizard myelinated axons.

1. We have studied action potentials and after-potentials evoked in the internodal region of visualized lizard intramuscular nerve fibres by stimulation of the proximal nerve trunk. Voltage recordings were obtained using microelectrodes inserted into the axon (intra-axonal) or into the layers of myelin (peri-internodal), with the goal of studying conditions required to activate internodal K+ currents. 2. Peri-internodal recordings made using K2SO4-, KCl- or NaCl-filled electrodes exhibited a negligible resting potential (less than 2 mV), but showed action potentials with peak amplitudes of up to 78 mV and a duration less than or equal to that of the intra-axonally recorded action potential. 3. Following ionophoretic application of potassium from a peri-internodal microelectrode, the peri-internodal action potential was followed by a prolonged (hundreds of milliseconds) negative plateau. This plateau was not seen following peri-internodal ionophoresis of sodium. The prolonged negative potential (PNP) was confined to the K(+)-injected internode: it could be recorded by a second peri-internodal microelectrode inserted into the same internode, but not into an adjacent internode. 4. The peri-internodally recorded PNP was accompanied by an equally prolonged intra-axonal depolarizing after-potential, and by an increase in the conductance of the internodal axolemma. However, the K+ ionophoresis that produced the PNP had little or no detectable effect on the intra-axonally or peri-internodally recorded resting potential or action potential. These findings suggest that the PNP is generated by an inward current across the axolemma of the K(+)-injected internode, through channels opened following the action potential. 5. Following peri-internodal K+ ionophoresis a PNP could also be evoked by passage of depolarizing current pulses through an intra-axonal electrode or by passage of negative current pulses through an electrode in the K(+)-filled peri-internodal region. The threshold for evoking a PNP was less than the threshold for evoking an action potential, and the PNP persisted in 10 microM-tetrodotoxin. Thus the PNP is evoked by depolarization of the axolemma rather than by Na+ influx. 6. The PNP was reversibly blocked by tetraethylammonium (TEA, 2-10 mM), but was not blocked by 100 microM-3,4-diaminopyridine or 5 mM-4-aminopyridine.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1992

Dietary supplementation with xanthophyll as an effective way of identifying low-producing broiler breeder hens.

The relationship between dietary levels of xanthophyll, the degree of pigmentation in the hen, and egg production rate was studied in commercial broiler breeders (Anak 2000). In the first study, the degree of shank and beak coloration, measured with a 15-grade Roche yolk color fan, was determined in broiler breeder pullets until 34 wk of age. Although overall body coloration decreased with age following initiation of egg production, the degree of shank coloration was two- to threefold higher than that of the beak. In the second study, Roche Carophyll-red (canthaxanthin, 10%) was supplemented at levels of 0, 10, and 20 mg/kg in a corn and soybean diet containing xanthophyll, and given to hens for a period of 4 wk beginning at 55 wk of age. Increasing dietary canthaxanthin levels increased pigmentation of beak and egg yolk in a quadratic manner, whereas pigmentation increased linearly in the shank. The production rate was inversely related to the degree of shank and beak pigmentation. In a third study, the relationship between hen pigmentation (using Carophyll-red, 30 mg/kg of diet) and production rate was examined in a commercial flock of broiler breeder hens. Hens were sampled according to shank coloration (Grades 1 to 3) and egg production was monitored. The production rate of hens with high coloration was significantly lower (by approximately 33%) than the flock average. At peak production, shank pigmentation was too low to differentiate visually between degrees of coloration. Dietary supplementation of 5% corn gluten meal increased shank pigmentation and enabled efficient identification of the nonlaying hens.

Animal Feed↗
PubMed1991

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.

Acrosin↗
PubMed1991

Human acrosome biogenesis: immunodetection of proacrosin in primary spermatocytes and of its partitioning pattern during meiosis.

Proacrosin biosynthesis timing during human spermatogenesis has been studied using the monoclonal antibody 4D4 (mAb 4D4). Frozen and paraffin-embedded sections of testicular biopsies were labelled by standard indirect immunofluorescence and avidin-biotin immunoperoxidase procedures. The labelling specificity was checked by immunochemistry assays on unrelated tissues and by western blotting of testis extracts showing that only the 50-55 x 10(3) Mr proacrosin was recognized by mAb 4D4. Proacrosin was first observed in the Golgi region of midpachytene primary spermatocytes. In late pachytene primary spermatocytes, proacrosin was observed in two regions located at opposite nuclear poles. During the subsequent steps of the first meiotic division, the two bodies containing proacrosin were located: (i) on opposite sides of the equatorial plate during metaphase; (ii) along the microtubular spindle during anaphase; and (iii) close to each chromosomal aggregate during telophase. Two bodies containing proacrosin were still observed in interphasic secondary spermatocytes. The single labelled area observed in early spermatids was found to increase considerably in size during spermiogenesis. Anomalies of proacrosin scattering were observed in patients with Golgi complex partitioning failure. These data reveal proacrosin biosynthesis during diploid and haploid phases of human spermatogenesis and the proacrosin partitioning pattern during meiosis.

Acrosin↗
PubMed1990

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin.

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.

Cells, Cultured↗
PubMed1990

Growth properties and in vitro life span of Alzheimer disease and Down syndrome fibroblasts. A blind study.

A blind study was set up to examine the in vitro growth characteristics of skin fibroblasts from 2 individuals with and 9 at risk for familial Alzheimer disease, 4 individuals with sporadic Alzheimer disease, 18 with Down syndrome as well as 5 younger and 6 older controls. Several variables (biopsy size, number of explants, medium, passage procedure) were standardized. Two growth characteristics were examined quantitatively: (i) the actual in vitro replicating life span was determined by counting the number of cells plated the previous week at 500,000 cells/flask (cumulative population doublings); and (ii) the growth potential was examined by a colony size distribution assay. A difference from the age-matched controls in the growth characteristics of skin fibroblasts was only observed for two patients with and one older individual at risk for familial Alzheimer disease. The growth properties of skin fibroblast cultures from patients with sporadic Alzheimer disease or Down syndrome were not at variance with their age-matched controls. The decrease in the growth potential observed in the familial Alzheimer disease fibroblasts is however modest and needs confirmation. It is clear that the growth properties of skin fibroblasts, as examined in this study, do not provide a good marker for any form of Alzheimer disease, nor do they provide an appropriate in vitro system to study factors which may contribute to the etiopathogenesis of Alzheimer disease or Down syndrome.

Adult↗
PubMed1990

Adding a microcomputer bar-code network to a minicomputer-based radiology information system.

A bar-code terminal network under software control of a microcomputer was added to the minicomputer-based radiology information system at the Medical College of Georgia in Augusta. The bar-code network was specifically installed to address the inherent inaccuracies occurring when procedure information was entered at the time of registration before procedures were actually performed. Technologists now enter procedure data into bar-code terminals after procedures are performed, substantially reducing database errors. This approach allowed us to take advantage of a microcomputer product without the necessity of completely converting our highly customized information system software from mini-to microcomputer.

Electronic Data Processing↗
PubMed1990

Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts.

Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell-associated forms act as precursors for the nonhydrophobic medium-released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell-associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.

Amino Acid Sequence↗