Search PubMed⌕ Search

Biomedical subjects

N Morin

Publications and source records attributed to N Morin.

At least 73 records · Page 4Linked to original sources
PubMed1994

Transcriptional regulation of plasminogen activator inhibitor-1 expression in human synovial fibroblasts by prostaglandin E2: mediation by protein kinase A and role of interleukin-1.

Differential expression of PAI-1 in connective tissues has been associated etiologically with some forms of arthritis. Our objective was to delineate the mechanisms by which PGE2 and IL-1 beta, inflammatory mediators commonly found at sites of inflammation, regulate the expression and synthesis of PAI-1 in human synoviocytes. PGE2 (and PGE1) inhibited PAI-1 mRNA expression and secretion in a dose-dependent manner with an IC50 (for antigen secretion) of 4.6 x 10(-10) M and 8.7 x 10(-10) M, respectively. Cyclic AMP agonists forskolin, Sp-cAMP, and IBMX mimic the effects of the PGEs. rhIL-1 beta stimulated the secretion of PAI-1 in a dose-dependent fashion under basal culture conditions; the effect was reversed by actinomycin D and the protein kinase inhibitors H7 and staurosporine but not KT-5720. PMA, an activator of protein kinase C, transiently increased (maximum 3 h) the expression of PAI-1 mRNA by approximately 10-fold, especially the 3.2 kb species. However, there was no significant increase in PAI-1 antigen secreted into the culture medium after PMA (100-300 nM) treatment. The half-life (t1/2) of PAI-1 mRNA, both the 3.2 and 2.2 transcripts was about 9.6 h (mean n = 3) and PGE2 has no affect on the stability of both messages. PGE2 reduced the rate of PAI-1 gene transcription as judged by run-off assays. The NSAID naproxen (30 micrograms/ml) induced the expression of PAI-1 mRNA over basal levels and super-induced the inhibitor's expression above rhIL-1 beta stimulated levels. Our results suggest that PGE2 suppresses PAI-1 expression and synthesis by activation of the cAMP/PKA system and inhibition of the rate of gene transcription. Data concerning the activation of PKC suggest that the expression, synthesis and release of the PAI-1 may be differentially regulated in normal human synoviocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Expression of fibronectin and a fibronectin-binding molecule during preimplantation development in the mouse.

Using an indirect immunofluorescent technique, expression of cell surface fibronectin and a cell surface fibronectin-binding molecule was studied during mouse embryo preimplantation development. We also studied the expression of fibronectin on immunosurgically isolated inner cell masses (ICMs) and regenerated mouse blastocysts. Fibronectin and the fibronectin-binding molecule were not detected at the morula stage. From the early to late blastocyst stage, fibronectin expression increased on the trophectoderm. Expression of the fibronectin-binding molecule was found only in the polar trophectoderm region of the early blastocyst, then in the polar and mural trophectoderm regions of the middle blastocyst. In the late blastocyst stage, this fibronectin-binding molecule was only present in the mural trophectoderm. Fibronectin expression by ICMs of early blastocysts was more intense than that of late blastocysts. After 24 h of culture, 10% of ICMs isolated from early blastocysts regenerated a trophectoderm which stained intensively for fibronectin in the mural and polar trophectoderm regions. After 48 h of culture, regenerated blastocyst-like structures closely resembled the normally obtained late blastocysts and stained for fibronectin in the mural and polar trophectoderm regions. The significance of the results is discussed in relation to mouse embryo development, trophectoderm formation and blastocyst implantation.

Amino Acid Sequence↗
PubMed1994

Temporal regulation of a complex and unconventional promoter by viral products.

The DNA polymerase (dnapol) gene of Autographa californica nuclear polyhedrosis virus presents a complex promoter organization. It lacks the usual TATA box and start site, and its RNA accumulation initially increases and then decreases dramatically during infection. We investigated dnapol temporal regulation. Transiently expressed dnapol gene was transcribed at a low level from minor start sites. Coexpression with ie0 and/or ie1 immediate-early genes dramatically enhanced dnapol transcription, specifically from a new start site. Moreover, the ie1 transactivation required little or no information in front of this nonconventional proximal promoter. We showed that IE0 and IE1 proteins were stably expressed during infection and that the dnapol mRNA level decrease was not a consequence of the disappearance of these proteins. The dnapol promoter region contains a putative overlapping open reading frame (ORF) in the opposite direction. We showed that ORF-2 was indeed highly expressed late, when the dnapol mRNA level decreased, and that during that time, dnapol mRNA stability was not significantly altered, excluding a destabilizing antisense effect. Additionally, we showed that the dnapol promoter was inhibited late but not early during the infection of cells transiently expressing constructs carrying either the intact or the altered ORF-2 promoter. Therefore, ORF-2 initiation of transcription and dnapol promoter inhibition are two coincidental nonrelated phenomena. Finally, we showed that both IE1 transactivation and late inhibition occurred in the same limited region around the dnapol promoter.

Animals↗
PubMed1993

Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae.

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from alpha-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100-1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.

Amino Acid Isomerases↗
PubMed1993

Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.

Amino Acid Sequence↗
PubMed1992

Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast.

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest. Here we show that FK506 bound to yeast FKBP-12 appears to form a complex with yeast calcineurin. Moreover, recovery from mating factor arrest is highly sensitive to FK506 or CsA, and this sensitivity requires the presence of FKBP-12 or cyclophilin, respectively. These results define a key physiological target of an FK506- and CsA-sensitive signal pathway in yeast, suggest a high degree of mechanistic conservation with mammalian cells, and indicate that further examination of the yeast system should provide insight into the same process in T cells.

Alleles↗
PubMed1992

Yeast FKBP-13 is a membrane-associated FK506-binding protein encoded by the nonessential gene FKB2.

The immunosuppressants FK506 and rapamycin prevent T-cell activation and also inhibit the growth of certain strains of the yeast Saccharomyces cerevisiae. It has previously been shown that yeast contains a 12-kDa cytosolic FK506-binding protein (yFKBP-12), which also possesses peptidylprolyl cis-trans isomerase activity, and that fkb1 strains lacking yFKBP-12 are resistant to rapamycin and sensitive to FK506. The absence of yFKBP-12 permitted the detection and isolation of a second FK506- and rapamycin-binding protein, which is about 13 kDa in size (yFKBP-13) and membrane-associated. Purified yFKBP-13 binds FK506 with 15-fold lower affinity than yFKBP-12 and has peptidylprolyl cis-trans isomerase activity with a similar substrate profile. The sequence of the first 37 N-terminal amino acids was determined, and the yFKBP-13 gene (FKB2) was cloned and sequenced. A hydrophobic putative signal sequence precedes the N terminus of the mature protein. yFKBP-13 most closely resembles the membrane-associated human FKBP-13, which also possesses a signal peptide, whereas yFKBP-12 most closely resembles human FKBP-12. fkb2 and fkb1 fkb2 mutants are viable and unaltered in their sensitivity to FK506, suggesting that yeast possesses an additional target for this drug. Furthermore, fkb2 null mutations confer no change in rapamycin sensitivity. These findings show that yFKBP-13 and yFKBP-12 have distinct functions within the cell.

Amino Acid Sequence↗
PubMed1992

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads.

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23 degrees C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO(3) was added to the growth media. There were strong interactions between CaCO(3) and media, CaCO(3) and pH level, and CaCO(3), media, and pH level. In media with CaCO(3), all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO(3) increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 x 10/g). In CaCO(3)-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.

Journal Article↗
PubMed1990

Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1.

The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E. coli and is readily transferred between the two species by transfection and transformation. The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage. Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable. Some of the deletion mutants fail to lysogenize S. cattleya (Lyg- phenotype). The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis. The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S. cattleya.

Bacteriophages↗
PubMed1990

Protein synthesis and acrosome reaction-inducing activity of human cumulus cells.

The acrosome reaction of human spermatozoa is a prerequisite to fertilization. It has been hypothesized that secretions of cumulus cells could be involved in the induction of this sperm reaction. A source of variation in the in-vitro fertilization of human oocytes could be the maturational differences in the cumulus cells which affect the acrosome reaction. In order to investigate this possibility, we have studied the capability of secretions obtained from cumulus cells of different maturational stages to induce the acrosome reaction. Capacitated spermatozoa were exposed to the culture medium containing cumulus cell secretions and their acrosomal status was evaluated using the triple stain technique. The maturational status of the cumulus or fertilization of the enclosed oocyte did not influence the percentage of acrosome-reacted live spermatozoa. In order to further document these observations, cumulus cells were submitted to [35S]methionine incorporation and total and secreted proteins were analysed by SDS-PAGE electrophoresis. Neither quantitative nor qualitative changes were detected in the electrophoretic patterns of the proteins obtained from cumulus cells of different maturational status. Thus, variation of the acrosome reaction-inducing activity of cumulus cells does not appear to be involved in the variable fertilization of oocytes obtained from follicles of differing maturity.

Acrosome↗
PubMed1989

Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed.

Adenoviruses, Human↗
PubMed1989

Isolation and characterization of a viable adenovirus mutant defective in nuclear transport of the DNA-binding protein.

The isolation and characterization of an adenovirus mutant, Ad5dl802r1, containing two independent deletions in the 72-kilodalton (kDa) DNA-binding protein (DBP) gene is described. The two deletions remove amino acids 23 through 105 of DBP, resulting in the production of a 50-kDa product. Expression of this truncated DBP was delayed 12 to 24 h compared with that of the 72-kDa protein produced by wild-type adenovirus type 5. The DBP was located primarily in the cytoplasm of infected cells, whereas the wild-type product was predominantly nuclear. Therefore, DBP appears to contain a nuclear localization signal within the deleted region. Ad5dl802r1 DNA synthesis, viral late gene expression, and virus production were all delayed 12 to 24 h and were approximately 10-fold lower than with wild-type adenovirus type 5. These phenotypic properties can be accounted for by the delay in synthesis and the inefficient accumulation of the 50-kDa DBP within the nucleus of infected cells. The truncated DBP also lacks the majority of amino acids which are phosphorylated in the normal protein. The loss of these phosphorylation sites does not appear to seriously impair the ability of the protein to carry out its functions.

Adenovirus Early Proteins↗
PubMed1989

cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Adenoviruses, Human↗
PubMed1989

Nuclear localization of the adenovirus DNA-binding protein: requirement for two signals and complementation during viral infection.

The adenovirus DNA-binding protein (DBP) is an abundant multifunctional protein located primarily in the nuclei of infected cells. To define sequences involved in nuclear transport of DBP, a series of point and small deletion mutants were constructed via oligonucleotide-directed mutagenesis. Two short stretches of basic amino acids located in the amino-terminal domain (amino acids 42 to 46 and 84 to 89) were identified. Their importance, however, depended on the context in which DBP was expressed. Disruption of either site prevented nuclear localization after transient expression in transfected 293 cells, implying that two nuclear localization signals are necessary for transport of this nuclear protein. In contrast, the mutant DBPs synthesized during viral infection were located either primarily in the nucleus or in the nucleus and cytoplasm, depending on the mutation and the stage of the viral infection. Thus, the nuclear localization defect could be complemented by viral infection, perhaps through the interaction of the mutant polypeptide with a virus-encoded or -induced factor(s).

Adenovirus E2 Proteins↗
PubMed1986

Hexon trimerization occurring in an assembly-defective, 100K temperature-sensitive mutant of adenovirus 2.

Analysis of 100K-defective temperature-sensitive adenovirus mutants confirmed the multifunctional character of the nonstructural, virus-coded 100K protein. In addition to its function in hexon trimerization (altered in H5ts1), and its possible direct or indirect role in hexon transport to nucleus (mutated in H2ts118), genetic and biochemical evidence was presented that 100K play some critical role in the scaffolding process of adenovirus capsid. This function appeared to be defective in H2ts107 and to map between coordinates 69.0 and 69.9, leftward from the H5ts1 lesion (70-73 map units; Arrand, 1978). This corresponded to the central domain of the 100K protein, between amino acid 300 and 400 from the N end. DNA sequencing of cloned fragments of H2ts107 DNA overlapping the mutation revealed two point mutations on the same codon at nucleotide 25,082 and 25,083 (GAC----GCA), corresponding to a nonconservative amino acid change (aspartic acid----alanine) at position 324 in the 100K sequence. 100K of adenovirus 2 wild type (WT) was found to bind in significant amounts to novobiocin-affinity column, and to be coeluted with hexon, penton, IIIa, and cellular topoisomerase II activity, by novobiocin- or ATP-Mg2+-containing buffers. H2ts107 100K also bound to novobiocin column, but the elution pattern differed from that of WT, suggesting some alteration in the affinity of the mutated 100K for novobiocin. The same behavior on affinity column as H2ts107 100K was observed for 90K, a cleavage product of the 100K, found in great abundance in H2ts107 at 39.5 degrees and corresponding to the C-terminal moiety of the 100K molecule. This implied that the "novobiocin-binding" domain of the 100K was not confined at its N terminus, and was altered in the H2ts107 mutant.

Adenoviruses, Human↗
PubMed1986

Possible role of ADP-ribosylation of adenovirus core proteins in virus infection.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Adenosine Diphosphate Ribose↗
PubMed1985

Isolation and characterization of the Streptomyces cattleya temperate phage TG1.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Attachment Sites, Microbiological↗