Search PubMed⌕ Search

Biomedical subjects

S Strickland

Publications and source records attributed to S Strickland.

At least 73 records · Page 4Linked to original sources
PubMed1989

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator.

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.

Animals↗
PubMed1989

Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin.

Heparin has been shown recently to stimulate the activity of human tissue-type plasminogen activator (t-PA). To investigate this effect further, mutant proteins lacking various domains of t-PA were screened for the ability to be stimulated by heparin. Those mutants harboring either the finger domain or the 2nd kringle were found to have enhanced enzymatic activity in the presence of heparin. Only mutants containing these structures would bind to heparin-agarose beads; monoclonal antibodies directed against these domains blocked binding. The stimulatory effect of heparin was more pronounced in finger-containing mutants than kringle-2 proteins. Earlier results had localized the fibrin-binding domains to the same two structures. Unlike heparin, the 2nd kringle was shown to be more important than the finger for fibrin stimulation. Our results have implications for producing recombinant t-PA variants for use in thrombolytic therapy.

Antibodies, Monoclonal↗
PubMed1989

Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes.

The translational activation of dormant tissue-type plasminogen activator mRNA during meiotic maturation of mouse oocytes is accompanied by elongation of its 3'-poly(A) tract. Injected RNA fragments that correspond to part of the 3'-untranslated region (3'UTR) of this mRNA are also subject to regulated polyadenylation. Chimeric mRNAs containing part of this 3'UTR are polyadenylated and translated following resumption of meiosis. Polyadenylation and translation of chimeric mRNAs require both specific sequences in the 3'UTR and the canonical 3'-processing signal AAUAAA. Injection of 3'-blocked mRNAs and in vitro polyadenylated mRNAs shows that the presence of a long poly(A) tract is necessary and sufficient for translation. These results establish a role for regulated polyadenylation in the post-transcriptional control of gene expression.

Animals↗
PubMed1989

Differentiation-responsive elements in the 5' region of the mouse tissue plasminogen activator gene confer two-stage regulation by retinoic acid and cyclic AMP in teratocarcinoma cells.

F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.

Animals↗
PubMed1988

Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes.

Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.

Animals↗
PubMed1988

Tissue plasminogen activator mRNA in murine tissues.

The urokinase-type and tissue-type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue-type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase-type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non-protease finger, growth-factor and kringle domains.

Animals↗
PubMed1988

Molecular cloning of complementary DNA to mouse tissue plasminogen activator mRNA and its expression during F9 teratocarcinoma cell differentiation.

Two tissue plasminogen activator (tPA) cDNA clones were isolated from mouse cDNA libraries and characterized. The sequence coding for mature mouse tPA protein including its 29-residue signal peptide, as well as that for the 3' and part of the 5'-nontranslated regions of the mRNA was determined. The mature protein consists of 530 amino acids and is encoded by a transcript of approximately 2800 nucleotides. The mouse tPA protein has 81% homology with its human counterpart, and amino acid sequence conservations suggest that the multidomain-like structure found for human tPA is maintained in the mouse enzyme. The tPA cDNA has been used as a probe to analyze tPA gene expression during F9 cell differentiation. tPA mRNA is not detected in F9 stem cells. When F9 cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP, tPA mRNA accumulates. We demonstrate that this induction occurs at least in part because of an increase in tPA gene transcription.

Animals↗
PubMed1988

Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells.

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.

Animals↗
PubMed1988

Expression of SPARC/osteonectin transcript in murine embryos and gonads.

A cDNA clone, p2-4, was isolated from mouse teratocarcinoma-derived parietal endoderm-like cells and used to analyze expression of the corresponding transcript during mouse embryogenesis. Nucleotide-sequence analysis revealed extensive homology between this clone and SPARC/osteonectin cDNA cloned from mouse parietal endoderm and bovine bone cells. The SPARC/osteonectin transcript became more abundant when embryonal carcinoma (EC) cells differentiated into parietal endoderm-like cells. In embryos, the transcript began to appear in the embryo proper on day 11 and continued to be expressed throughout the gestation period. The transcript was also present in extraembryonic membranes and placenta from days 9 and 11 onward, respectively. Thus, expression of the transcript was regulated during differentiation of EC cells and during embryogenesis. In adult mice, several non-bone tissues, including testis, also expressed the transcript. Analysis of germ-cell-deficient mice indicated that non-germ-cell components of the testis expressed the transcript. Analysis of mouse testicular cell lines further suggested that the transcript was abundant in Sertoli cells and Leydig cells. Cumulus oophorus cells that envelope the ovulated egg also expressed high levels of the transcript.

Animals↗
PubMed1987

Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells.

Granulosa cells from immature rats produce tissue plasminogen activator (tPA) in response to follicle stimulating hormone (FSH) or luteinizing hormone (LH) both in vitro and in vivo. We have used the in vitro system to investigate the level at which the hormonal induction of tPA is regulated. Within 12 h following FSH addition, a dramatic but transient increase in tPA secretion occurs for by 24 h secretion returns to basal levels. This pattern of enzyme induction is similar with LH, but the onset of the increase is delayed. When steady-state tPA mRNA levels are examined after hormone treatment, the results mirror those obtained if one measures enzyme activity; a large increase in tPA mRNA followed by a decrease to basal levels is observed with both hormones, and the lag in induction by LH is also apparent. These results demonstrate that the regulation of tPA activity by gonadotropins occurs at the level of the steady-state concentration of the mRNA. In the presence of cycloheximide, the induction of tPA mRNA by FSH or LH is not greatly affected, indicating that this phase of the response to gonadotropins does not require the synthesis of new protein. However, the decrease in tPA mRNA levels observed 24 h after FSH treatment is affected by cycloheximide, in that the drug delays the reduction in mRNA levels seen with hormone alone.

Animals↗
PubMed1987

Chromosomal assignments of genes for tissue plasminogen activator and urokinase in mouse.

The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.

Animals↗
PubMed1987

Expression of the murine apolipoprotein E gene is coupled to the differentiated state of F9 embryonal carcinoma cells.

Apolipoprotein E (apoE) expression was studied in F9 embryonal carcinoma cells that differentiate in response to retinoic acid into cells resembling either parietal endoderm or visceral endoderm of the parietal or visceral yolk sac. F9 cells secreted newly synthesized apoE when incubated with radiolabeled amino acid. Upon differentiation to parietal endoderm-like cells, apoE synthesis and secretion were markedly down-regulated. In contrast, apoE secretion was up-regulated upon differentiation to visceral endoderm-like cells. These changes in apoE expression were due, at least in part, to regulation of apoE mRNA abundance since visceral endoderm-like cells contained 5- to 7-fold more apoE mRNA than parietal endoderm-like cells. The apoE phenotype reflected the differentiated state of the F9 cell since either the up-regulated or down-regulated pattern was stable to the removal of the retinoic acid inducer. To determine how well apoE regulation in F9 cells reflects regulation in visceral and parietal yolk sacs, apoE mRNA was measured in yolk sac tissues of the 12-day mouse embryo. Visceral yolk sac contained 109 +/- 11 pg of apoE mRNA per micrograms of RNA while parietal yolk sac contained 14.3 +/- 3.1 pg/micrograms of RNA. This 7- to 8-fold difference is similar to the difference in the apoE mRNA contents of visceral endoderm-like and parietal endoderm-like F9 cells. RNA gel blot analysis showed that apoE mRNA is the same size in F9 cells as in yolk sac tissues and fetal or adult liver. In addition, primer-extension analysis showed that transcription is initiated at or near the same site on the apoE gene in F9 cells and mouse tissues. These data suggest that both quantitative and qualitative features of apoE gene expression in development are retained in the F9 cell. The F9 cell should provide a useful system to study the developmental activation of endogenous apolipoprotein genes as well as exogenous apolipoprotein genes introduced by transfection.

Animals↗
PubMed1987

Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator.

It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells.

Animals↗
PubMed1987

Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA.

The serine protease tissue-type plasminogen activator (t-PA) is synthesized by murine oocytes undergoing meiotic maturation, but not by arrested primary oocytes. Dormant, stable t-PA mRNA accumulates during oocyte growth, so that fully grown, arrested primary oocytes contain in their cytoplasm approximately 10,000 copies of this molecule. Translation of t-PA mRNA is triggered upon resumption of meiosis and is accompanied by a progressive and concerted increase in its size. This structural change can be accounted for by increased polyadenylation at the 3' end of the molecule. Following its translation, t-PA mRNA is degraded; it is no longer detectable in fertilized eggs. The identification of a dormant mRNA in murine oocytes and the demonstration that its translational activation is accompanied by elongation of its poly(A) tail may provide insights into the control of gene expression during meiotic maturation and early mammalian development.

Animals↗
PubMed1987

Hormone binding and coupled response relationships in systems dependent on the generation of secondary mediators.

The relationship between hormone binding and biological response curves is discussed for instances in which the response is dependent upon the generation of a secondary mediator of hormone action. In the simplest model, where the rate of mediator generation is directly proportional to the level of hormone receptor occupancy, and degradation of the secondary mediator follows first-order kinetics, the form of the biological response curve is identical to that for hormone receptor occupancy, but the curve is displaced to the left such that a half-maximal biologic response occurs at less than 50% hormone receptor occupancy (spare-receptor phenomenon). The same is shown to be the case when the model is iterated to include a sequence of coupled intermediate binding reactions intervening between initial hormone binding and the final biological response. When, in contrast, the degradation of one or more of these coupled intermediates is not strictly first order, but instead shows standard Michaelis-Menten kinetics, the response curve, while again remaining parallel to the curve for hormone binding, can now move to the right of the binding curve such that a high threshold is observed for the biological effect, and a half-maximal response may not occur until the level of hormone receptor occupancy is well over 50%. Some consequences of this model are discussed with special reference to the sensitivity and speed of reversibility of the biological response, implications for responses at pharmacological as opposed to physiological concentrations of hormone, and parallels which can be extended to coupled enzymatic reactions of the Michaelis-Menten type.

Calcium↗
PubMed1986

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen.

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.

Animals↗
PubMed1986

Expression of a secreted transplantation antigen gene during murine embryogenesis.

We examined the midgestation mouse embryo for transcripts related to the secreted transplantation antigen Q10 and show here that this gene is transcribed in the endoderm of the visceral yolk sac. Its level of expression is highest at day 14 and then declines as development proceeds. Concurrently with the decrease in yolk sac expression, the amount of transcripts accumulating in the fetal liver increases during late embryogenesis.

Animals↗