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Biomedical subjects

D D Carson

Publications and source records attributed to D D Carson.

At least 91 records · Page 5Linked to original sources
PubMed1987

Lactosaminoglycans are involved in uterine epithelial cell adhesion in vitro.

The cell type specificity of glycoconjugate synthesis between the epithelial and stromal cells of the uterus is described. Lactosaminoglycans (LAGs) constituted a major fraction of the cell-associated glycoconjugates synthesized by epithelial, but not stromal, cells. Furthermore, LAGs comprised the bulk (greater than 90%) of glycoconjugates that could be released from epithelial cell surfaces by proteases. Several lines of evidence indicate that the epithelial cell-specific lactosaminoglycans appear to interact directly with a cell surface galactosyltransferase (GalTase). This includes the observation that agents that perturb galactosyltransferase function also interrupt epithelial cell adhesion and cause LAG release from the cell layer. In addition, LAGs are galactosylated when UDP-[3H]galactose is added to intact epithelial cell layers. Interference with cell surface GalTase activity with alpha-lactalbumin or UDP-galactose, but not other agents, specifically interrupted epithelial cell adhesion; however, the same agents had absolutely no effect on stromal cells. Collectively, these studies describe the novel occurrence of lactosaminoglycans on cell surfaces in an adult tissue other than hematopoietic cells and provide evidence for cell type-specific involvement of lactosaminoglycans in uterine cell adhesion processes.

Amino Sugars↗
PubMed1987

Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro.

The involvement of embryonic cell surface proteoglycans in the attachment and outgrowth of cultured mouse embryos has been investigated. Several lines of evidence indicate that periimplantation stage blastocysts express heparin/heparan sulfate proteoglycans on their cell surfaces that can mediate embryo attachment and trophoblast outgrowth on a variety of matrices. First, in the presence of soluble heparin, the rate at which embryos attach and outgrow on laminin, fibronectin, or monolayers of uterine epithelial cells is reduced considerably. In the case of fibronectin, the rate of outgrowth in the presence of the heparin is slower than in the presence of the Arg-Gly-Asp-Ser-containing peptide that is recognized by a fibronectin receptor. Embryos also attach and exhibit a limited ability to outgrow on platelet factor IV, a heparin binding protein that does not possess the additional binding domains of laminin or fibronectin. Attachment on platelet factor IV is inhibited by heparin. Second, cell surface digestion of attachment-component embryos with heparinase, but not chondroitinase ABC, slows the rate of outgrowth on tissue culture plates in the presence of serum. Third, selective staining for sulfated molecules on the trophectoderm surface of periimplantation stage embryos indicates that such molecules are abundant and uniformly distributed on these cell surfaces. Last, heparin/heparan sulfate proteoglycans are detected as major cell surface components of embryos using vectorial labeling with lactoperoxidase and Na125I. Collectively, these data indicate that heparin/heparan sulfate-bearing molecules have a direct role in attachment and outgrowth of implantation stage blastocysts.

Animals↗
PubMed1986

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata.

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.

Acid Phosphatase↗
PubMed1986

Regulation of N-linked glycoprotein assembly in uteri by steroid hormones.

The effects of the steroid hormones 17 beta-estradiol (E2) and progesterone on N-linked glycoprotein assembly in ovariectomized mice have been examined. Both priming and nidatory E2 markedly stimulate [3H]mannose incorporation (3- to 6-fold) into uterine glycoproteins, whereas uterine bulk protein synthesis is not stimulated under the same conditions. Progesterone alone stimulates glycoprotein synthesis modestly (1.5-fold) over that in oil-injected controls, but antagonizes the action of E2 when coinjected with the estrogen. The E2 effect is not systemic, because livers from these same animals do not display an increase in glycoprotein synthesis. When mice were injected with tamoxifen or clomiphene, two drugs that mimic E2 actions in uteri without inducing the full extent of cell proliferation that normally accompanies E2 treatment, a similar enhancement of uterine glycoprotein synthesis was observed. Although mannosylphosphoryldolichol synthase activity rose in parallel with glycoprotein synthesis during E2 priming, the apparent activities of two other enzymes involved in the assembly of N-linked glycoproteins, namely chitobiosylpyrophosphoryldolichol synthase and oligosaccharyltransferase, remained relatively unchanged. Furthermore, the apparent in vivo rate of dolichol phosphorylation was not altered during E2 priming. Supplementation of uterine tissue slices with dolichylphosphate failed to enhance the rate of protein glycosylation in vivo. In addition, changes in the pool sizes of GDP-mannose did not correlate with changes in the in vivo rate of glycoprotein synthesis. Collectively, these observations indicate that the E2-dependent increase in glycoprotein synthesis is not likely to be due to increased enzyme activities for oligosaccharide assembly or transfer to protein, increased dolichylphosphate availability, or increased sugar nucleotide availability. To study the effects of E2 on the production of specific glycoproteins, the pattern of [3H]mannose-labeled glycoproteins produced as a function of days of E2 priming was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estrogen priming induced the secretion of 9-11 [3H]mannose-labeled glycoproteins by uteri; however, the pattern of tissue-associated glycoproteins remained constant throughout this interval. It appears, therefore, that estrogen priming induces the secretion of a few specific glycoproteins while generally enhancing the production of most tissue-associated glycoproteins. Most (70%) of the [3H]mannose-labeled oligosaccharide chains of these glycoproteins were of the polymannose type.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

A single enzyme catalyzes the synthesis of the mannosylphosphoryl derivative of dolichol and retinol in rat liver and Chinese hamster ovary cells.

It is well established that mannosylphosphoryldolichol participates in the synthesis of N-linked glycoproteins by donating mannosyl residues to oligosaccharide-lipid intermediates. It has been suggested that mannosylphosphorylretinol also is involved in glycoprotein biosynthesis. We conclude that one synthase catalyzes the synthesis of both mannosylphosphoryldolichol and mannosylphosphorylretinol in rat liver tissue and Chinese hamster ovary cells, based on the following results. 1) The enzyme in rat liver microsomes that synthesizes mannosylphosphoryldolichol and mannosylphosphorylretinol is inactivated at the same rate at 55 degrees C. 2) In membranes of both rat liver and Chinese hamster ovary cells, exogenous dolichyl phosphate and retinyl phosphate compete with each other for mannosyl-lipid synthesis. However, in both systems adding exogenous retinyl phosphate has no effect on the synthesis of mannosylphosphoryldolichol from endogenous dolichyl phosphate in the membranes. 3) Membranes prepared from a mutant of Chinese hamster ovary cells which is devoid of mannosylphosphoryldolichol synthase lack the ability to synthesize mannosylphosphorylretinol.

Animals↗
PubMed1985

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin.

The assembly of the spicules (primitive skeleton) of the sea urchin embryo is being studied in primary mesenchyme cells cultured in vitro. A monoclonal antibody (1223) has been prepared that inhibits the deposition of CaCO3 into the spicules. This antibody reacts with a 130,000 Mr cell-surface protein that is concentrated on the surface of approximately 5% of the cells of dissociated gastrula stage embryos. When primary mesenchyme cells in the embryo or cells cultured in vitro are examined, the 1223 antigen is detected on the surface of the cells and on the extracellular material associated with the spicule. We conclude that the 1223 antibody recognizes a cell-surface protein that plays an essential role in spicule formation.

Animals↗
PubMed1984

Synthesis and secretion of a novel binding protein for retinol by a cell line derived from Sertoli cells.

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.

Animals↗
PubMed1983

Vitamin A deprivation selectively lowers uridine nucleotide pools in cultured sertoli cells.

The effects of retinoid addition of vitamin A-depleted (UV-irradiated) culture medium on uridine metabolism in cultured Sertoli cells have been studied. After vitamin A depletion, a consistent 2- to 4-fold enhancement of [3H]uridine incorporation into RNA was observed. Several lines of evidence indicate that this enhancement is the result of an increase in the specific activity of the uridine-labeled precursors of RNA. Although vitamin A depletion did not affect either uridine uptake or alter cellular RNA content, a 5-fold increase in the specific activity of UMP was found in vitamin A-depleted cells. This increase results because the cellular content of uracil nucleosides plus nucleotides is selectively lowered in vitamin A-depleted cells. The decreased content of uridine derivatives could be accounted for by a 45-57% decrease in the activity of glutamine-dependent carbamylphosphate synthetase in vitamin A-depleted cells. The effects of vitamin A deprivation on uridine incorporation, as well as carbamylphosphate synthetase activity, could be completely restored to or above control values by supplementing vitamin A-depleted cell culture medium with either retinol or retinoic acid. This effect of vitamin A depletion appears to be highly specific. Under the same conditions, no gross alteration in either the pattern or extent of synthesis of cellular or secreted proteins, glycoproteins, glycosaminoglycans, and lipids was observed. In addition, vitamin A depletion/repletion had no effect on the growth rate or morphology of the cells.

Animals↗
PubMed1981

Enhancement of protein glycosylation in tissue slices by dolichylphosphate.

Glycosylation of N-linked glycoproteins has been stimulated in hen oviduct and bovine pancreas tissue slices by supplementing the tissue culture media with concentrations of dolichylphosphate from 1-100 micrograms/ml. In oviduct, overall incorporation of radioactive sugars into alkali-stable, hot trichloroacetic acid-precipitable material (N-linked glycoproteins) is stimulated approximately 2-fold in dolichylphosphate-supplemented tissues although no stimulation in protein synthesis is observed. Rather, the elevation in glycosylation seems to be a general one involving many protein acceptors. In vitro analysis of microsomal preparations derived from control or dolichylphosphate-supplemented oviduct tissue slices demonstrated a similar enhancement in glycosylation activities that appears to be attributable to an enhanced level of endogenous dolichylphosphate in the microsomes from supplemented tissues. Additionally, when bovine pancreas tissue slices are preincubated with dolichylphosphate and then doubly labeled with [3H]mannose and 14C-labeled amino acids, a 4-fold increase in the ratio of [3H-mannose to 14C-amino acids in secreted ribonuclease is observed relative to the nonsupplemented control. Furthermore, while only 12% of the ribonuclease secreted from control tissue slices specifically binds to concanavalin A-Sepharose, more than 90% of that secreted by dolichylphosphate-supplemented tissue slices binds to the lectin. These data support the notion that dolichylphosphate availability is a limiting factor in the in vivo glycosylation of proteins in these systems.

Animals↗
PubMed1981

Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis.

Cellular autolytic activity as well as lipid and lipoteichoic acid metabolism have been studied in cultures of Streptococcus faecalis receiving various combinations of the following treatments: chloramphenicol addition, starvation for an essential amino acid (valine), and cerulenin treatment. Lipoteichoic acid initially accumulated in chloramphenicol-treated and amino acid-starved cells and decreased relative to the cellular mass in cerulenin-treated cells. The relative phosphatidylglycerol content of amino acid-starved cultures or of cultures treated with either antibiotic rapidly decreased upon initiation of each treatment. In all cases, cerulenin initially stimulated diphosphatidylglycerol synthesis. Pretreatment of cultures with cerulenin prevented the inhibition of cellular synthesis autolysis normally observed during chloramphenicol treatment, but did not affect amino acid starvation-induced inhibition of autolytic activity. Variations in the levels of the nonionic lipid fraction, predominantly diglycerides, correlated best with the patterns of autolytic activity observed during chloramphenicol treatment, whereas variations in the levels of diphosphatidylglycerol and lipoteichoic acid correlated best with the patterns of autolytic activity observed during amino acid starvation. Components of the nonionic lipid fraction were demonstrated to inhibit autolytic activity 50% in whole cell and in cell wall assays at 60 and 120 nmol/mg (dry weight), respectively.

Amidohydrolases↗
PubMed1980

Effects of fatty acids on lysis of Streptococcus faecalis.

Palmitic, stearic, oleic, and linoleic acids at concentrations of 200 nmol/ml all inhibited autolysin activity 80% or more in whole cells or cell-free extracts. This concentration of the saturated fatty acids palmitic acid and stearic acid had little or no effect on the growth of whole cells or protoplasts. However, the unsaturated fatty acids oleic acid and linoleic acid induced lysis in both situations. This lytic effect is apparently not related to any uncoupling activity or inhibition of energy catabolism by unsaturated fatty acids. It is concluded that unsaturated fatty acids induce cell and protoplast lysis by acting as more potent membrane destabilizers than saturated fatty acids.

Amidohydrolases↗
PubMed1980

Morphological effect of cerulenin treatment on Streptococcus faecalis as studied by ultrastructure reconstruction.

Exponential-phase cells of Streptococcus faecalis ATCC 9790 were treated with a concentration of cerulenin (5 micrograms/ml) that has been shown to block both lipoteichoic acid and lipid synthesis and cell division within 10 min. The morphological effect of this treatment was studied by making three-dimensional reconstructions of cells based on measurements taken from axial thin sections. This analysis indicated that cerulenin interferes with cell division by inhibiting normal constriction of the division furrow and centripetal growth of the cross wall in envelope growth sites. Rather than dividing, many of the sites in treated cells apparently continue to elongate and produce abnormally large amounts of peripheral wall surface. These observations were interpreted in terms of a previously proposed model in which cerulenin would prevent the synthesis of a lipid-containing inhibitor of autolytic enzyme activity needed for division. In addition, measurements showed that the average number of envelope growth sites per cell increased during treatment, suggesting that although cerulenin treatment blocks division, it does not interfere with the formation of new envelope growth sites. It was also observed that the size and frequency of mesosomes did not decline during the 60-min period of drug treatment. This tends to decrease the likelihood that mesosomes are formed from a pool of intracellular membrane precursors that would be depleted during a period of restricted lipid biosynthesis.

Antifungal Agents↗
PubMed1979

Inhibition of polyisoprenoid and glycoprotein biosynthesis causes abnormal embryonic development.

Compactin, a potent inhibitor of polyisoprenoid biosynthesis, induces abnormal gastrulation during sea urchin development at concentrations that have no effect on earlier embryonic development or on macromolecular synthesis. Three lines of evidence suggest that the developmental lesion caused by compactin results from inhibition of dolichol biosynthesis and a concomitant inhibition in the biosynthesis of the oligosaccharide chains of N-linked glycoproteins. (i) Embryos cultured in the presence of compactin gastrulate normally when supplemented with dolichol alone, whereas supplementation with cholesterol or coenzyme Q or both does not prevent the compactin-induced developmental lesion. (ii) Exogenously supplemented [3H]dolichol is incorporated into a compound with the chromatographic properties of oligosaccharide-pyrophosphoryldolichol. (iii) Embryos cultured in the presence of compactin exhibit a decreased capacity to synthesize mannose-labeled glycolipids and N-linked glycoproteins. This decrease in synthesis is abolished if the embryos are cultured in the presence of dolichol along with compactin.

Animals↗
PubMedDate not supplied

Cell surface glycoconjugates as modulators of embryo attachment to uterine epithelial cells.

Attachment of mammalian embryos to the uterine wall involves the coordinated development of both the embryo and the uterine epithelium to an attachment-competent state. This coordination is achieved directly or indirectly through the actions of ovarian steroids. Acquisition of attachment competence is proposed to reflect two processes. The first is the loss of non-adhesive glycoproteins at the cell surface of embryos, e.g. zona pellucida subunits, as well as uterine epithelial cells, e.g. mucin glycoproteins. The second process is the functional expression of complementary adhesion-promoting molecules at these cell surfaces. A series of studies indicates that heparan sulfate proteoglycans and their corresponding binding sites can play an important role in the initial stage of embryo attachment to the uterine surface.

Animals↗
PubMedDate not supplied

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds.

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

Bone Marrow Cells↗
PubMedDate not supplied

Mucin expression and function in the female reproductive tract.

Reproductive tract epithelia are characterized by the presence of a thick, apical glycocalyx. This glycoprotein coat is drastically reduced in the uterus of many species during the time of embryo implantation. Recent studies indicate that mucin glycoproteins constitute a large proportion of the apical glycocalyx. One of these mucins, Muc-1, has particularly important functions at the luminal surface of the uterus and other female reproductive tract tissues. Muc-1 appears to play a dominant role in maintaining a functionally non-receptive uterine surface with regard to blastocyst attachment. Conversion to a receptive uterine state is brought about by the concerted actions of ovarian steroid hormones that in several species also strongly modulate Muc-1 protein and mRNA expression. Muc-1 also appears to serve a general function in protecting reproductive tract mucosa since Muc-1 null mice are particularly prone to bacterial infection. Collectively, these studies indicate that mucins, including Muc-1, play important barrier roles in reproductive processes and protection from bacterial pathogenesis in the female reproductive tract.

Animals↗