Performing JCAHO dose calculations with the aid of a microcomputer spreadsheet program.
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Humans↗
Biomedical subjects
G David
Publications and source records attributed to G David.
Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts.
Heparitinase digestion of the hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) of fetal human lung fibroblasts yields core proteins of various sizes: i.e. monomeric core proteins of 125, 90, 64, 48, and 35 kDa and a disulfide-linked dimeric core protein composed of approximately 35-kDa subunits. By immunizing BALB/c mice with liposome-incorporated HSPG, we have obtained a total of five anti-HSPG monoclonal antibodies (Mabs, i.e. Mabs S1, 1C7, 2E9, 6G12, and 10H4) with different specificities. Polyacrylamide gel electrophoresis of 125I-labeled membrane HSPG immunoprecipitated with these Mabs revealed that Mabs 1C7 and 2E9 bind only membrane HSPG which yield a 125-kDa core protein after heparitinase digestion, whereas Mab S1-bound HSPG yield a 64-kDa core protein, and Mabs 6G12 and 10H4 retain membrane HSPG with a 48-kDa core protein. Western blotting of the heparitinase-digested proteoglycans and immunostaining with the Mabs confirmed this pattern of reactivity. However, in this assay, Mabs 6G12 and 10H4 also detected a minor approximately 90-kDa core protein in addition to the 48-kDa core protein. Except perhaps for the 10H4 epitope, the epitopes recognized by these Mabs appear to be part of the peptide moieties as they resisted complete deglycosylation of the HSPG with trifluoromethanesulfonic acid. Since these data were inconsistent with a direct relationship between the major core proteins, the 48-, 64-, and 125-kDa core proteins were immunopurified and further compared by peptide mapping with Staphylococcus aureus protease V8, trypsin, and CNBr cleavage. Clearly distinct peptide patterns were obtained for the three different core proteins. These results imply that the 48-, 64-, and the 125-kDa membrane HSPG core proteins of human lung fibroblasts are derived from distinct proteoglycans.
Aggrecans↗
Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associated heparan sulfate proteoglycans of lung fibroblasts. Prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts.
Heparitinase treatment of cell surface-associated heparan sulfate proteoglycans (HSPG) of human lung fibroblasts reveals core proteins with apparent Mr values of 125,000, 90,000, 64,000, 48,000 and 35,000 (Lories, V., De Boeck, H., David, G., Cassiman, J.-J., and Van den Berghe, H. (1987) J. Biol. Chem. 262, 854-859). The 90- and 48-kDa core proteins share the epitope of the monoclonal antibody 6G12 which was used to screen a human lung fibroblast expression cDNA library. Rescreening of the libraries yielded clone 48K5 with an insert of 3439 base pairs. Polyclonal antibodies were raised in rabbits against a fragment of the protein encoded by the 48K5 cDNA different from the part carrying the 6G12 epitope. These antibodies specifically recognize the 90- and 48-kDa core proteins on Western blots of total cellular extracts of human lung fibroblast HSPG. The specific reactivity of the polyclonal antiserum confirms the identity of the 48K5 clone and further distinguishes the 48- and the 90-kDa core proteins, which do share the 6G12-defined epitope and at least one additional antigenic determinant with the 48K5 cDNA-encoded protein, from the 125-, 64-, and 35-kDa core proteins of cell surface HSPG of human lung fibroblasts which do not react with either antibody preparation. The protein encoded by the 48K5 clone contains a stop-transfer sequence indicative of an integral membrane protein and three potential glycosaminoglycan attachment sites. The 48K5 clone detects two major poly(A)+ RNA species in human lung fibroblasts presumably generated by the use of alternative polyadenylation signals. The 48K5 gene was mapped to chromosome 8q23 by in situ hybridization and hybridization to DNA of somatic cell hybrids.
Aggrecans↗
Cell-surface heparan sulfate and heparan-sulfate/chondroitin-sulfate hybrid proteoglycans of mouse mammary epithelial cells.
The hydrophobic cell-surface proteoglycans of mouse mammary epithelial cells were purified by gel filtration, ion-exchange chromatography, and liposome incorporation. The size of the proteoglycans appeared to be directly proportional to the size of their heparan-sulfate chains, larger proteoglycans yielding larger chains. The chondroitin sulfate chains, in contrast, showed no size heterogeneity. Digestion of 125I-labeled proteoglycans with heparitin-sulfate lyase and chondroitin ABC lyase yielded core proteins of approximately 93 kDa, approximately 85 kDa and approximately 38 kDa. Comparison with single enzyme digestions identified the 93-kDa and 85-kDa cores as components of hybrid proteoglycans that carried both heparan-sulfate and chondroitin-sulfate chains. Immunoblotting indicated that the 93-kDa and 85-kDa cores shared the epitope defined by monoclonal antibody 281-2. The 38-kDa core, in contrast, carried only heparan-sulfate chains and lacked the 281-2 epitope. Preparations enriched in heparan sulfate or in heparan-sulfate/chondroitin-sulfate hybrid proteoglycans were obtained by N-desulfation and ion-exchange chromatography. Hybrid proteoglycans accounting for the bulk of the chondroitin-sulfate and nearly half of the heparan-sulfate residues of the proteoglycans showed a similar polydispersity of heparan-sulfate chain sizes as found in proteoglycans that carried only, or predominantly, heparan-sulfate chains. These hybrids contained heparan-sulfate and chondroitin-sulfate chains in similar molar amounts. Analysis of 125I-labeled proteoglycans suggested that typical hybrid proteoglycans were composed of a 85-kDa core protein that carries a single chondroitin-sulfate chain and a single heparan-sulfate chain of variable length. A minority of hybrids seemed characterized by the variant, but possibly structurally related, 93-kDa core protein. The other half of the hydrophobic proteoglycans were composed of the 38-kDa core and carried only heparan-sulfate chains. The significance of the co-existence of hybrid and heparan-sulfate proteoglycans at the cell surface and possible relationships between the proteoglycans need to be further clarified.
Animals↗
Genetic aspects of artificial insemination with donor semen: the French CECOS Federation guidelines.
The genetic problems raised by assisted reproduction using donor gametes (AID) are numerous and often complex. They concern the legitimacy and the appropriate forms of genetic screening for both gamete donors and recipients; the identification of genetic indications justifying the use of this method of reproduction; and ascertainment of the state of health of the conceptus at birth. The experience and guidelines of the French CECOS Federation, which comprises 20 AID treatment centers, are described. The discussion emphasizes the need for an international exchange of view on this subject.
Eugenics↗
Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility.
The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondialdehyde determined by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10(8) spermatozoa after 1 hour of incubation) and 1) initial motility r = -0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.
Humans↗
Mini-micro-mainframe computer marriage: combining technologies in a radiology results reporting system.
The minicomputer-based information system in the Department of Radiology at the Medical College of Georgia Hospital and Clinics was placed in service in February, 1982. This system represents a sizable investment in minicomputer hardware in addition to more than 6 years of software customization. One serious deficiency in the original system was the lack of a radiology results reporting facility. Several options were considered to provide the department with this capability. The most obvious option was retiring the existing system and replacing it with one of a number of commercial products already offering results reporting. In-house development of a reporting facility lent itself more readily to microcomputers than to the existing minicomputer system. Due to system customization, economic and time constraints, it was decided to merge an in-house developed microcomputer-based report module into our existing minicomputer system. The minicomputer was able to communicate with and transfer files to and from both micro and mainframe systems. Combining technologies allowed us to continue taking advantage of our sizable investment in money, time, and customization while providing a microcomputer-based report module. Radiology reports are now typed on microcomputer word processors and bulk transferred to the minicomputer. The minicomputer provides access to both unapproved and approved reports on system terminals throughout the department. It also enhances reports by merging patient demographics and registration information. Using existing communications facilities to the hospital mainframe system, reports are provided throughout the institution.
Computer Communication Networks↗
Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts.
Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.
Aggrecans↗
Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.
Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.
Amino Acid Sequence↗
[Pretherapeutic conservation of sperm].
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Antineoplastic Agents↗
Post-translational modification of the beta-subunit of the human fibronectin receptor.
Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.
Antibodies, Monoclonal↗
Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization.
Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.
Antibodies, Monoclonal↗
Mapping of human fibronectin receptor beta subunit gene to chromosome 10.
Human-mouse hybrid cells were examined by indirect immunofluorescence with Mab DH12, a monoclonal antibody that recognizes the beta subunit of the human fibronectin receptor. Cells that expressed the antigen at their surface were sorted by FACS and karyotyped. Immunoaffinity chromatography on Mab DH12 was used to confirm the presence of the human antigen. The chromosome assignment was strengthened by isozyme analysis of markers for chromosomes 9 and 10. The results are suggestive for a 10p mapping of this beta subunit of the fibronectin receptor. Since the gene coding for the beta subunit of the VLA proteins was previously assigned to the same chromosome, our result could provide further evidence for the relationship between the beta subunit of the human fibronectin receptor and the VLA protein family.
Animals↗
[Present-day pathological anatomy and prospects for its development in the 21st century].
Scientific Societies of Pathologists from Socialist countries held three symposia to analyse recent developments and future trends in pathology. The author presents his state-of-the-art review at the third symposium (Berlin, 1987) based on international literature data and first-hand experience gained in the field of pathology for the last decades. Some negative tendencies are considered. It is stated, in particular, that the percentage of autopsies performed in those who died at hospitals and at home continues to decrease due to fading interest to autopsy results both from physicians and medical authorities. This can entail growing number of misdiagnoses, unassessed quality of medical aid in complicated cases, biased statistics, undetected infection during life, defects in medical training. Tendencies and tasks facing histopathology and cytological diagnosis are determined as follows: 1) purpose-specific introduction of new techniques and equipment 2) active promotion of interdisciplinary task-force groups of clinicians, pathologists, roentgenologists, etc. to verify diagnosis and develop new techniques 3) establishment of special reference centres 4) overall computerization 5) urgent need in additional skilled staff to satisfy growing technical requirements of molecular biology, genetics and tissue culture. Current research on pathology is believed unsatisfactory. The role of experimental pathology is underestimated. To improve the situation in fundamental pathological research, the author suggests large-scale perestroika involving additional investments and human resources, active participation of scientists, clinical pathologists and medical authorities.
Europe, Eastern↗
Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.
Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed.
Animals↗
Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody.
Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).
Antibodies, Monoclonal↗
Anomalous protein complement of sperm nuclei in some infertile men.
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Humans↗
Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms.
Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.
Cells, Cultured↗