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K R Marotti

Publications and source records attributed to K R Marotti.

53 records · Page 3Linked to original sources
PubMed1989

Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA.

Tumor-promoting phorbol esters stimulate tissue plasminogen activator (tPA) release from human endothelial cells, and simultaneous elevation of cyclic AMP potentiates this response 5-fold (Santell, L., and Levin, E. G. (1988) J. Biol. Chem. 263, 16802-16808). A similar effect on tPA mRNA was observed, with phorbol myristate acetate inducing a 3.5-fold increase in steady state tPA mRNA levels and forskolin enhancing that increase to 25-fold. Peak levels occurred at 8 h after agonist addition and returned to baseline levels by 16 h. As was found with tPA antigen secretion, delayed addition of forskolin reduced the level of potentiation, and, at 6 h after phorbol 12-myristate 13-acetate (PMA), forskolin was no longer effective. The protein synthesis inhibitor cycloheximide did not inhibit the rise in tPA mRNA levels in response to PMA/forskolin nor the decline in mRNA levels between 8 and 12 h. However, peak levels (8 h) were approximately 1.5-fold higher than in cultures not treated with cycloheximide. The effect of two inhibitors of protein kinases, H-7 and staurosporine, on PMA-induced tPA antigen secretion and tPA mRNA levels were examined. H-7 and staurosporine inhibited PMA, and PMA/forskolin induced tPA secretion in a dose-dependent manner. This effect was time-dependent; the inhibitory effect was reduced with delayed H-7 addition, and, by 6 h after PMA treatment, no inhibition was observed. H-7 and staurosporine also inhibited the PMA/forskolin-induced increase in tPA mRNA levels and were less effective the later they were added. The same time-dependent effect on the potentiation of PMA-induced tPA mRNA levels by forskolin was observed. Again, delayed addition reduced the effect, and, by 6 h, potentiation was absent. The results of this study indicate that changes in mRNA levels in response to PMA and PMA/forskolin precede and determine those that occur to tPA antigen secretion. In addition, the maximal response is dependent upon the prolonged activation of an H-7- and cAMP-sensitive pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins.

We have designed and constructed a DNA sequence encoding human tissue plasminogen activator (tPA) with convenient restriction sites that flank each of the domains of the heavy chain. To accomplish this, the first 1095 bases of the gene coding for the mature protein were synthesized with unique restriction sites engineered into the interdomainal regions. This synthetic construction was then ligated to a cDNA fragment of the tPA gene that encoded the active site, thus generating a full-length tPA gene. The gene products produced by Chinese hamster ovary (CHO) cells transfected with either the tPA cassette gene or the tPA cDNA gene were then compared with the tPA produced by Bowes melanoma cells to determine whether or not synthetic interdomainal amino acid changes had an effect on the biochemical characteristics of the molecule. Specifically, molecular weight, specific activity, enhancement by fibrinogen fragments and kinetic constants were analysed. None of the properties examined were significantly different from those of the native melanoma tPA. Therefore, the cassette gene described herein should provide considerable versatility and precision in the construction of tPA mutants by facilitating the manipulation of the finger, growth factor and kringle domains, and likewise should be useful in assessing the function of these domains within the tPA molecule. We present this cassette gene system as a model for the analysis of protein domain function applicable to other multi-domain proteins.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-1: a comparison with ovine trophoblast protein-1 and bovine interferon-alpha II.

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.

Amino Acid Sequence↗
PubMed1988

Interferon RNA of embryonic origin is expressed transiently during early pregnancy in the ewe.

Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.

Actins↗
PubMed1988

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector.

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.

Amino Acids↗
PubMed1987

Pseudorabies virus as a live virus vector for expression of foreign genes.

The cDNA coding for human tissue plasminogen activator (tPA) was cloned downstream from the promoter for pseudorabies virus (PRV) glycoprotein and flanked by downstream PRV DNA. After co-transfection with PRV DNA, this plasmid recombined to insert the tPA cDNA into the viral genome. In cells infected by this recombinant virus, tPA was detected by immunoprecipitation analysis and by enzymatic activity. Since it has a wide host range but does not infect humans, PRV is a possible vaccine vector for genes from animal pathogens.

Cloning, Molecular↗
PubMed1986

The primary structure of cynomolgus monkey apolipoprotein A-1 deduced from the cDNA sequence: comparison to the human sequence.

We have cloned and analyzed a cDNA containing the complete coding sequence for cynomolgus monkey apolipoprotein A-1 (apoA-1). This cDNA clone was found to share approx. 97% nucleotide sequence identity with the published human apoA-1 and encodes a protein of the same size as the human protein. Paired proline residues are present at positions 3 and 4 in the mature protein as has been reported for other primate species and the propeptide sequence is identical to the human propeptide. The amino acid content derived from the nucleotide sequence predicts a more basic protein than human apoA-1 and this was confirmed by isoelectric focusing analysis. In addition, we present evidence for two different transcriptional initiation sites for the cynomolgus monkey gene in contrast to only one for human.

Amino Acid Sequence↗
PubMed1985

Two-stage hormonal control of type IV collagen mRNA levels during differentiation of F9 teratocarcinoma cells.

A cDNA clone related to mouse Type IV collagen has been prepared from F9 teratocarcinoma cells induced to differentiate with retinoic acid and dibutyryl-cAMP. This cDNA clone has been used to investigate the regulation of Type IV collagen mRNA during differentiation. The level of this mRNA is very low in untreated F9 cells, increases substantially after treatment of the cells with retinoic acid, and is further increased by addition of dibutyryl-cAMP. In contrast, dibutyryl-cAMP has no effect on the mRNA level in cells that have not been previously exposed to retinoic acid. These results demonstrate that these two compounds regulate in a sequential manner the steady-state level of Type IV collagen mRNA. This cDNA clone should allow a detailed examination of the mechanism of the two-stage regulation of collagen expression by retinoids and cyclic AMP.

Animals↗
PubMed1981

LDHk, a uniquely regulated cryptic lactate dehydrogenase associated with transformation by the Kirsten sarcoma virus.

A novel isozyme of lactate dehydrogenase is detected in various cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme, designated LDHk, is strongly inhibited by physiological concentrations of oxygen, in an apparently cooperative fashion. LDHk is inhibited by guanosine triphosphate and related compounds, in a noncompetitive fashion. LDHk is found with both 35,000- and 22,000-dalton subunits, although these probably cleave from a 57,000-dalton precursor. In studies utilizing a temperature-sensitive transforming gene mutant of the Kirsten sarcoma virus, we find in vivo expression of LDHk is also temperature-sensitive. In studies using either crude cell-free extracts or purified LDHk, we find the enzyme from cells infected with a temperature-sensitive transforming gene mutant of KiMSV is thermolabile relative to that from wild type KiMSV-infected cells.

Animals↗
PubMed1980

Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP.

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.

Animals↗
PubMed1979

Apparent posttranscriptional block to anaerobic induction of endogenous leukemia virus.

Uninfected Fischer rat cells were induced by anaerobic stress to transcribe high levels of endogenous type C leukemia virus RNA. Complete 35S virus RNA with attached polyadenylic acid sequences was found associated with polysomes, indicating functional mRNA. Since no mature virus was released under these conditions, the presence of a posttranscriptional block to complete virus synthesis is strongly indicated.

Anaerobiosis↗
PubMedDate not supplied

Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

Animals↗
PubMedDate not supplied

Interferon-like sequence of ovine trophoblast protein secreted by embryonic trophectoderm.

In most species the length of a pregnancy exceeds that of the luteal phase of the ovarian cycle. The conceptus within the uterus, therefore, is believed to produce a substance or substances which directly or indirectly prolong the lifespan of the corpus luteum and prevent a return to ovarian cyclicity. This phenomenon is known as maternal recognition of pregnancy. The active substance implicated in signalling maternal recognition of pregnancy in the sheep is an embryonic secretory protein, known as ovine trophoblast protein-1 (oTP-1) (refs 2-4), which is targeted in a paracrine manner to the uterine epithelium of the mother. We report here the primary amino-acid sequence of oTP-1 as inferred from a cloned complementary DNA and demonstrate that the protein is most probably an interferon-alpha.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular cloning, sequence, and expression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlation between hepatic cholesteryl ester transfer protein mRNA levels and plasma high density lipoprotein levels.

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6 +/- 0.4 pg/micrograms total RNA (mean +/- SEM) to 4.1 +/- 0.8 pg/micrograms (p less than 0.005); mesenteric fat CETP mRNA increased from 0.4 +/- 0.1 pg/micrograms total RNA to 5.3 +/- 2.2 pg/micrograms (p less than 0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r = -0.85, p less than 0.001) and apoprotein A-I (r = -0.84, p less than 0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a function CETP gene.

Animals↗