Biomedical subjects

D D Carson

Publications and source records attributed to D D Carson.

At least 73 records · Page 4Linked to original sources

PubMed1990

Selective activation of the N-glycosylation apparatus in uteri by estrogen.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional level.

Animals↗

PubMed1990

Lactosaminoglycan assembly, cell surface expression, and release by mouse uterine epithelial cells.

The kinetics of assembly, cell surface expression, secretion, and degradation of the major lactosaminoglycan (LAG)-bearing glycoproteins in mouse uterine epithelial cells have been studied. LAGs have been shown previously to be synthesized preferentially by these cells in the uterus and are expressed at the cell surface, where they participate in cell adhesion processes (Dutt, A., Tang., J.-P., and Carson, D. D. (1987) Dev. Biol. 119, 27-37). We utilized selection on pokeweed mitogen-Sepharose, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent electroelution to isolate the major LAG-bearing glycoproteins. The intact LAG-bearing glycoproteins exhibited very high apparent Mr (greater than 500,000) both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography under dissociative conditions. The subset of LAGs at the cell surface exhibited a half-life of approximately 11 h, whereas total cell-associated LAGs had a half-life of 6 +/- 1 h. Pulse-chase experiments indicated that the transit time of LAG core proteins from the rough endoplasmic reticulum to the site of LAG addition in the Golgi was 30-45 min. LAG glycoprotein transit from the Golgi to the cell surface required at least an additional 30-45 min. The major metabolic fate of the cell-associated LAGs was secretion to the medium with no evidence of lysosomal degradation. Some (30%) of the LAGs appeared to be released to the medium via the action of cell surface proteases. Epithelial cell surfaces bound fluoresceinated pokeweed mitogen, indicating the constitutive presence of LAG-bearing molecules at the cell surface; pokeweed mitogen binding to the cell surface was completely blocked by 10 mM chitotriose. These observations provide the first comprehensive description of the intracellular transport and metabolism of this interesting class of glycoproteins of the uterine epithelial cell surface.

Amino Sugars↗

PubMed1990

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells.

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.

Animals↗

PubMed1990

Vectorial secretion of prostaglandins by polarized rodent uterine epithelial cells.

Uterine epithelial cells (UEC) isolated from mature mice as well as immature mice and rats were cultured on EHS matrix-coated nitrocellulose filters in order to determine their ability to secrete prostaglandin (PG) F2 alpha and PGE2 in a polarized manner. Ultrastructural analyses were performed to validate the polar nature of mouse UEC and demonstrate the presence of separate apical and basolateral plasma membrane domains. These properties included the presence of tightly juxtaposed lateral membranes, apical microvilli, and a relatively flat basal surface. Biochemical indices of polarity included the preferential (approximately 5:1) basal uptake of [35S]methionine as well as a preferential (approximately 9:1) apical secretion of protein. UEC isolated from mice during the estrous and diestrous stages of the estrous cycle did not differ in their degree of polarity, as measured by these morphological and biochemical indices. UEC of estrous and diestrous mice as well as immature mice and rats preferentially secreted PGF2 alpha to the basal medium to an approximately 4-fold greater extent than to the apical medium. PGE2 was secreted at least 10-fold less than PGF2 alpha, and a preferential basal secretion could not be demonstrated. Polarized UEC accumulated relatively large cellular pools of PGF2 alpha, while nonpolarized cells grown on matrix-coated plastic did not. This difference was reflected by the inability of an inhibitor of PG biosynthesis, indomethacin, to inhibit PGF2 alpha secretion by polarized cells during short (4-h) incubations. In contrast, this drug effectively inhibited secretion in nonpolarized cells or polarized cells incubated with indomethacin for longer (24-h) intervals. Therefore, cellular PGF2 alpha pools apparently support continued secretion of this lipid even when de novo synthesis is transiently inhibited. Preferential basal secretion of PGF2 alpha was due to the polar nature of UEC, since disruption of tight junctions with EGTA modified the basal to apical ratio of PGF2 alpha secretion to near unity. Sodium azide inhibited the secretion of PGF2 alpha, indicating that PGF2 alpha secretion was energy dependent. PGF2 alpha secretion was not coupled to protein synthesis or secretion, since cycloheximide did not inhibit this process in polarized or nonpolarized cells. These studies describe the first evidence for polarized secretion of lipid-derived hormones by epithelial cells. The preferential basal secretion of PGF2 alpha may play an important role in regulating UEC interactions with the underlying stroma.

Animals↗

PubMed1989

Estrogen induces N-linked glycoprotein expression by immature mouse uterine epithelial cells.

Characterization of complex glycoconjugates and the effects of estrogen on their expression in immature mouse uterine epithelial cells are reported. The secreted fraction contained nonanionic, O-linked lactosaminoglycan (LAG)-bearing proteins of Mr 30,000-40,000 as well as anionic, O-linked, LAG-bearing glycoproteins with very high apparent molecular weight (greater than 670K). Heparan sulfate (HS) proteoglycans and HS linked to little or no protein were found in the secreted fraction as well. A very similar array of glycoconjugates was found in the nonhydrophobic fraction of cell-associated macromolecules. In addition, the hydrophobic cell-associated fraction contained nonanionic, LAG-bearing glycoproteins of approximately 250K, anionic LAG-bearing glycoproteins distributing over a wide range of molecular weights, and HS proteoglycans with median molecular weights of approximately 250K. In contrast to the glycoproteins produced by their mature counterparts, virtually all glycoproteins produced by immature cells were O-linked. Estrogen treatment of immature mice caused uterine epithelial cells to secrete anionic, high molecular weight (greater than 670K) N-linked glycoproteins as a major product. These estrogen-responsive glycoproteins did not appear to contain LAGs. Estrogen treatment also markedly decreased the proportion of all hydrophobic glycoconjugates in the cell-associated fraction. Collectively, these observations indicate that one aspect of the estrogen-induced maturation of uterine epithelial cells is the stimulation of N-linked glycoprotein synthesis and secretion. Furthermore, stimulation of N-linked glycoprotein synthesis by itself is insufficient to support N-linked LAG glycoprotein production.

Animals↗

PubMed1989

A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.

Animals↗

PubMed1989

Extraction and isolation of glycoproteins and proteoglycans.

A number of diverse approaches have been devised to bring about the efficient extraction and isolation of glycoproteins and proteoglycans from biological tissues. Many of these approachs are general procedures that can also be used for the extraction of nonglycoproteins from cells or tissues. Others, such as lectin affinity chromatography, take advantage of specialized structures found only on discrete sub-classes of glycoproteins. Unfortunately, the development of a protocol suitable for the purification of a given glycoprotein of interest remains largely empirical. This article presents a general overview of some of the potential strategies that can be utilized in the development of new isolation procedures and attempts to point out some of the possible pitfalls that may be encountered.

Animals↗

PubMed1988

Estrogen preferentially stimulates lactosaminoglycan-containing oligosaccharide synthesis in mouse uteri.

The effects of steroid hormones on the synthesis of lactosaminoglycan (LAG)-containing oligosaccharides by mouse uteri are reported. The uterine LAG-containing oligosaccharides were degraded partially by Pseudomonas endo-beta-galactosidase, releasing an oligosaccharide of the apparent structure: Gal beta----N-acetylglucosaminyl(----N-acetylgalactosaminyl)beta 1,3----galactose. A larger fraction of the LAG-containing oligosaccharides bound to pokeweed mitogen than to Datura stramonium lectin, suggesting the presence of highly branched structures. LAG-containing oligosaccharides were resistant to sequential digestion with Pronase, nitrous acid, hyaluronidase, and chondroitinase ABC. These polysaccharides exhibited a Gal:GlcNAc:GalNAc ratio of approximately 1.0:1.0:0.3 and were not fucosylated. The ion-exchange behavior of the LAG-containing oligosaccharides before and after mild acid hydrolysis indicated the presence of sialic acid residues. The LAG-containing glycopeptides were highly resistant to beta-elimination but were released quantitatively by hydrazinolysis, demonstrating an N-linkage to protein. Binding to pokeweed mitogen was markedly enhanced following release of these oligosaccharides from peptides by hydrazinolysis, suggesting that peptide-bound oligosaccharides were partially inaccessible to the lectin. Molecular exclusion chromatography of the oligosaccharides released by hydrazinolysis revealed a broad distribution ranging from Mr 4,000 to 15,000 with a median Mr of approximately 8,000. We extended the above observations by determining how the steroid hormones 17-beta-estradiol (E2) and progesterone affected synthesis of the LAG-containing oligosaccharides in ovariectomized mice. Generally, E2 and a number of E2 agonists stimulated glycoconjugate synthesis; however, chronic E2 treatment or combined treatment with E2 plus progesterone caused the synthesis of most glycosaminoglycans to return to basal levels. In contrast, E2 either alone or in combination with progesterone stimulated synthesis of LAG-containing oligosaccharides in preference not only to glycosaminoglycans but also to other classes of N-linked oligosaccharides. This effect was apparent during both priming and nidatory E2 treatments. Collectively, these data provide the first demonstration of LAG-containing oligosaccharides in uteri and for the hormonally regulated synthesis of lactosaminoglycans. In addition, this is the first demonstration of the ability of steroid hormones to induce the synthesis of certain types of N-linked oligosaccharides in preference to others in the same tissue.

Amino Sugars↗

PubMed1988

Collagens support embryo attachment and outgrowth in vitro: effects of the Arg-Gly-Asp sequence.

Collagen types I through VI support attachment and outgrowth of mouse blastocysts in vitro. We found that embryos acquire the ability to attach to collagens type II and VI relatively early in their developmental program. The time at which half of the embryos displayed outgrowth formation and the morphology of outgrowths formed on these two collagen types are similar to those observed for laminin, fibronectin, and hyaluronate. Embryos acquire the ability to outgrow on the other collagen types at a later time in culture. Both "native" and denatured collagens support embryo attachment and outgrowth, indicating that this activity is intrinsic to the primary collagens' structure. A synthetic peptide containing the sequence Arg-Gly-Asp inhibits embryo outgrowth on collagen type II and denatured collagen type IV, whereas a peptide containing the related sequence, Arg-Gly-Glu, has relatively little effect on embryo outgrowth. In contrast, embryo attachment to collagen types I, V, and VI was not inhibited specifically by the Arg-Gly-Asp peptide sequence. Consequently, it appears that embryos use multiple adhesion systems to attach to collagens. Among these are adhesion systems that have a peptide recognition specificity similar to that of fibronectin receptors. These studies indicate that embryo interactions with collagens may be one aspect of the tissue invasion processes that take place during implantation.

Amino Acid Sequence↗

PubMed1988

Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene.

An early gene which augments the expression of the delayed early/late 39K gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was identified by functional mapping. Transient expression of the plasmid p39CAT, containing the bacterial chloramphenicol acetyltransferase coding sequences under the control of the promoter of the 39K protein, was observed in cells cotransfected with AcNPV DNA digested with several restriction endonucleases. However, when p39CAT was cotransfected with viral DNA digested with Bg/II restriction endonuclease, no CAT activity could be detected. To map the location of the Bg/II-sensitive sequences required for efficient expression of 39CAT, p39CAT and Bg/II-digested viral DNA were cotransfected with a PstI library of AcNPV DNA. The PstI-N fragment restored 39CAT activity. A major early 1.3-kb transcript from this fragment was mapped by S1 nuclease analysis. Transient assay experiments indicated that this major transcript of the PstI-N fragment was produced by an immediate early gene, named IE-N. The PstI-N fragment alone did not activate expression of p39CAT but was required when IE-1 was present in limiting quantities.

Chromosome Mapping↗

PubMed1988

Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells.

The present study describes a culture environment in which luminal epithelial cells isolated from immature rat uteri and cultured on a matrix-coated permeable surface, with separate apical and basal secretory compartments, proliferate to confluence. Subsequently the cells undergo a process of differentiation accompanied by progressive development of functional polarity. Ultrastructural and immunocytochemical evidence verifies the ability of these primary cultures to regain polar organization, separate membrane domains, and form functional tight junctions as demonstrated by the development of transepithelial resistance. The appearance of uvomorulin is restricted to the lateral cell surface. Coordinated indices of functional polarity that develop progressively in post-confluent cultures include the preferential uptake of [35S]methionine from the basal surface and a rise in uterine epithelial cell secretory activity characterized by a progressive preference for apical secretion. The time dependent development of polarity was characterized by differences in the protein profiles of the apical and basolateral secretory compartments. The maintenance of hormone responsiveness by the cultured cells was validated by the secretion of two proteins identified as secretory markers of estrogen response in the intact uterus. The technique of culturing the cells on a matrix-coated permeable surface with separate secretory compartments produces a uterine epithelial cell that morphologically and functionally resembles its in situ equivalent. The culture method and analytical approach used in this present study may be applied to primary cultures of a variety of natural epithelia, which have hitherto proven resistant to more conventional culture methodologies.

Animals↗

PubMed1988

Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells.

We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate-containing molecules (HS[PG]) were the major sulfated products synthesized and secreted by these cells. The ability of epithelial cells to secrete KSPG greatly increased in parallel with the development of cell polarity. Furthermore, KSPG secretion occurred preferentially to the apical medium in highly polarized cultures. In contrast, HS(PG) secretion did not increase along with development of polarity, although most HS(PG) (85%) were secreted apically as well. Pulse-chase studies indicated that highly polarized cultures secreted 80-90% of the sulfated macromolecules they synthesized, predominantly to the apical secretory compartment. The half-lives for KSPG and HS(PG) secretion were approximately 3 and 4 h, respectively. Parallel studies of cells cultured on tissue culture plastic-coated with biomatrix indicated that neither the state of confluency nor the biomatrix was primarily responsible for inducing the KSPG secretion observed in polarizing cultures. Experiments with uterine strips indicated that the steroid hormone, 17-beta-estradiol, markedly stimulated synthesis and secretion of sulfated macromolecules, but had no preferential effect on KSPG production. The ratio of KSPG to HS(PG) secretion from uterine strips was similar to that found in the apical medium of highly polarized cell cultures. Thus, the pattern of proteoglycan secretion observed in polarized cell cultures mimicked that observed for uterine cells, although the preferential increase in KSPG production by polarized cells could not be attributed to an estrogen response. Collectively, these studies describe the major sulfated molecules secreted by rat uterine epithelial cells under varying conditions and provide evidence for a novel influence of cell polarity on the cell's ability to secrete sulfated glycoconjugates.

Animals↗

PubMed1988

Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells.

A number of studies have implicated glycoconjugates in cell recognition events associated with implantation of mammalian blastocysts into the uterus. We have found that p-nitrophenyl-D-xylosides inhibit mouse embryo attachment and outgrowth on monolayers of uterine epithelial cells when cocultured in vitro. Inhibition of attachment and trophoblast formation by alpha- and beta-xylosides was observed in embryos cultured on tissue culture plastic in serum containing medium or on monolayers of epithelial cells. The biochemical basis for this inhibition has been investigated. Consistent with their accepted mode of action, beta- but not alpha-D-xylosides greatly stimulated glycosaminoglycan chain production by uterine epithelial cells and likewise reduced proteoglycan assembly. In contrast, both alpha- and beta-anomers selectively inhibited embryo attachment and outgrowth without stimulating glycosaminoglycan chain production by embryos. The inhibitory effect of the xylosides on embryos was reversible and did not require concentrations that reduced the rate of protein synthesis. Both alpha- and beta-D-xylosides inhibited the synthesis of proteoglycans including heparan sulfate as well as certain other glycoconjugates by embryos. Collectively, these data indicate that proper assembly of glycoconjugates, including proteoglycans, is required for implantation-related processes, although the inhibition of embryo outgrowth by xylosides may be by an as yet uncharacterized mechanism.

Animals↗

PubMed1988

Inhibition of T-lymphocyte proliferation by the cyclic polypeptide didemnin B: no inhibition of lymphokine stimulation.

We investigated possible mechanisms by which the cyclic depsipeptide didemnin B (DB) inhibits lymphocyte proliferation. DB inhibited the proliferation of Con-A stimulated murine splenocytes, the interleukin-2 (IL-2) dependent proliferation of the CTLL-2 cell line, and the interleukin 4 (IL 4) dependent growth of both CTLL-2 and D10.G.4.1 cell lines at approximately equimolar concentrations (SD50 = 3 to 10 X 10(-9)M). Inhibition of CTLL-2 growth by 10(-8)M DB was partially reversible, and significantly blocked the incorporation of [3H]-thymidine even when added 24 hr after initial IL 2 stimulation. Concentrations of DB (10(-8)M) that completely blocked mitogen-driven spleen cell blastogenesis only partially inhibited the synthesis and secretion of IL-2. Although DB blocked the growth of T lymphocyte clones in response to both recombinant human IL 2 and recombinant murine IL 4, the suppression was not due to an uncoupling of lymphokinetic signaling but was closely correlated with an inhibition of protein and RNA synthesis. Addition of DB to an in vitro translation system did not inhibit protein synthesis. Thus, we conclude that DB functions as an antiproliferative, and not as a specifically immunosuppressive, compound.

Animals↗

PubMed1987

Heparan sulfate proteoglycan synthesis and metabolism by mouse uterine epithelial cells cultured in vitro.

Characterization and metabolism of heparan sulfate glycosaminoglycans and proteoglycans (HSPGs) synthesized by primary cultures of mouse uterine epithelial cells are reported. HSPGs were detected in both the medium and in the cell-associated fraction, whereas glycosaminoglycans containing little or no protein (free glycosaminoglycans) were found primarily in the cell-associated fraction. The cell-associated HSPGs were relatively large (Kav = 0.1 on Superose 12), had a buoyant density in cesium chloride gradients of 1.45-1.55 g/ml, and contained heparan sulfate chains that fell into two size classes, exhibiting Kav values on Superose 12 of 0.2-0.5 and 0.7-0.8, respectively. The free glycosaminoglycan chains displayed a Kav on Superose 12 of 0.6-0.7. The secreted HSPGs were smaller (median Kav on Superose 12 of 0.28) than the cell-associated HSPGs. More than 90% of the cell-associated HSPGs contained hydrophobic portions, as evidenced by their ability to bind to octyl-Sepharose. In contrast, only 10-15% of the secreted HSPGs bound to octyl-Sepharose. HSPGs were detected at both apical and basal cell surfaces/extracellular matrices by indirect immunofluorescence in vitro and in utero and by accessibility to external proteases in vitro. It was estimated that 60-70% of the total cell-associated HSPGs were exposed at the cell surface. The HSPGs released from the cell surface by proteases were slightly smaller than the intact HSPGs and lacked the hydrophobic properties of the latter. These observations suggested that the cell surface HSPGs contain a small, hydrophobic domain that functions in the attachment of HSPGs to cells. The free glycosaminoglycans appeared to be primarily intracellular and were not secreted. The cell-associated HSPGs turned over rapidly (t1/2 = 1.5 h) and appeared to be the precursors to the free glycosaminoglycans. Metabolic turnover of the free glycosaminoglycan pool was a relatively slow process (t1/2 = 10-12 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri.

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Polyadenylation at a cryptic site in the pBR322 portion of pSV2-neo: prevention of its utilization by the SV40 late poly(A) signal.

Transcripts originating from the SV40 late promoter of pSV2-neo or pSV2-cat contain pBR322 sequences and are polyadenylated at the SV40 late poly(A) site, resulting in an RNA of 3500 nt. If the SV40(L) poly(A) signal is destroyed, late orientation transcripts are polyadenylated at a site within pBR322 sequences, yielding in an RNA of 2500 nt. This cryptic poly(A) site is located 42-46 nucleotides downstream from an AAUAAA. Utilization of the pBR322 poly(A) signal is undetectable in late orientation transcripts from pSV2-neo or pSV2-cat, although it is located 966 nucleotides upstream from the SV40(L) poly(A) signal. The pBR322 site is not utilized when the spacing between the two poly(A) signals is varied from 209 to 1913 nucleotides. The pBR322 poly(A) site was utilized only in constructs in which all or portions of the SV40(L) poly(A) signal were deleted, such as in a construct with a 7 bp deletion into the SV40(L) AATAAA and adjacent sequences.

Animals↗

PubMed1987

Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function.

The synthesis of various glycoconjugate classes by mouse uteri during the pre- and peri-implantation period has been examined using [3H]glucosamine as a metabolic precursor. A unique and dramatic (five- to sixfold) increase was observed in the synthesis of hyaluronate on the day upon which embryo implantation normally occurs. Mated, but nonpregnant mice did not display increased hyaluronate biosynthesis. In contrast to hyaluronate, the synthesis of most other types of glycoconjugates remained fairly constant during the first 5 days of pregnancy. Low (1500-5000)-molecular-weight N-linked oligosaccharides constituted the major class of oligosaccharides synthesized under all conditions. High (greater than 10,000)-molecular-weight glycoconjugates constituted the second most abundant class of glycoconjugates synthesized (20-30%). Most (85%) of the newly synthesized hyaluronate was associated with the nonepithelial cell types of the uterus. Experiments using ovariectomized mice receiving steroid hormones demonstrated that uterine hyaluronate synthesis was induced preferentially by an artificial decidual stimulus and implicated stromal cells as the site of hyaluronate synthesis. In addition, it was demonstrated that tissue culture plates coated with hyaluronate, but not other polysaccharides, support attachment and spreading of a large fraction (60 to 70%) of embryos cultured in serum-free medium. Collectively, these studies indicate that increased hyaluronate biosynthesis accompanies decidual responses in the endometrium and may promote embryo implantation following initial penetration of the uterine epithelium.

Animals↗