Biomedical subjects

K R Marotti

Publications and source records attributed to K R Marotti.

At least 37 records · Page 2Linked to original sources

PubMed1993

Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator.

Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.

Amino Acid Sequence↗

PubMed1993

Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates the exchange of neutral lipids among the lipoprotein. Because the principal core lipid of very-low-density lipoprotein (VLDL) is triglyceride and that of high-density lipoprotein (HDL) is cholesterol ester, CETP mediates a 'heteroexchange' of cholesterol ester for triglyceride between those lipoproteins. As a result, animals that express CETP tend to have higher VLDL and low-density lipoprotein (LDL) cholesterol levels, whereas those with no CETP activity tend to have high HDL cholesterol levels. Because VLDL and LDL are associated with the progression of atherosclerosis, and HDL are considered anti-atherogenic, CETP could be an 'atherogenic' protein, that is, given the other conditions required for atherosclerosis to develop, expression of CETP would accelerate the rate at which the arterial lesions progress. We report here that transgenic mice expressing CETP had much worse atherosclerosis than did non-expressing controls, and we suggest that the increase in lesion severity was due largely to CETP-induced alterations in the lipoprotein profile.

Animals↗

PubMed1992

Structure of the murine gene encoding apolipoprotein A-I.

A 1.6-kb DNA fragment containing the gene encoding apolipoprotein A-I from the mouse, Mus musculus, has been cloned and sequenced. It contains three exons separated by two introns and encodes a secreted polypeptide of 262 amino acids (aa), 238 of which constitute the mature protein. Comparisons with the rat and human proteins indicate moderate levels of shared identity (71 and 66%, respectively), although the overall aa compositions yield proteins with identical pIs (5.4). Kyte-Doolittle analyses of the three proteins indicate that there is no significant difference in the structure of these apolipoproteins.

Amino Acid Sequence↗

PubMed1992

Nucleotide sequence of the cynomolgus monkey apolipoprotein A-I gene and corresponding flanking regions.

The cynomolgus monkey (Macaca fascicularis) apolipoprotein A-I (apo A-I) gene and corresponding flanking regions have been isolated from a cynomolgus monkey genomic library and completely sequenced. Comparison with the human sequence shows a greater than 90% homology with the monkey sequence, overall. The monkey apo A-I structural gene (1856 bp) is representative of the general apolipoprotein gene structure and consists of four exons and three introns. Minor differences exist between the monkey and human structural gene sequence but do not result in significant changes. Sequences preceding the putative monkey apo A-I promoter are virtually identical to the human apo A-I enhancer region with an exact match to the three enhancer DNA binding sites.

Amino Acid Sequence↗

PubMed1992

The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Insights from studies with transgenic mice.

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r = -0.81 or higher, p less than 0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r = 0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL1 and HDL2) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p less than 0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Apo B metabolism in the cynomolgus monkey: evidence for post-transcriptional regulation.

Previous studies have shown that hepatic apo B mRNA levels do not increase in animals fed high cholesterol diets, even though plasma apo B concentrations increase markedly. As a result, it has been suggested that the diet-induced increase in plasma apo B levels was due solely to an inhibited clearance of those lipoproteins. The present study was undertaken to test that hypothesis. Hepatic apo B mRNA levels were measured in liver biopsies taken from five male cynomolgus monkeys before and twice after, they began to consume a high cholesterol diet. The diet had no effect on hepatic apo B mRNA levels, even though it caused a 7-fold increase in the plasma apo B levels. However, measurements of the apo B secretion rate in eight separate monkeys (four chow-fed and four cholesterol-fed) by isotope dilution showed that apo B secretion by the liver was increased 4-fold in the cholesterol-fed monkeys. These data, taken together, indicate that apo B secretion is not regulated by the rate at which the apo B gene is transcribed, but at some point further along in the secretion pathway.

Amino Acid Sequence↗

PubMed1991

Evidence that the nonparenchymal cells of the liver are the principal source of cholesteryl ester transfer protein in primates.

Previous studies showed that 90% or more of the cholesteryl ester transfer protein (CETP) mRNA is contained in the liver of cynomolgus monkeys. The purpose of this study was to determine if the parenchymal cells (hepatocytes) were the hepatic cell type that contained that mRNA. The parenchymal and nonparenchymal cells were separated by standard methods, and the CETP, apoA-I, apoB, and apoE mRNA content of the preparation determined at each step in the purification process. ApoA-I and apoB are produced only in the parenchymal cells; apoE is produced by both cell types. The mRNA measurements showed that the CETP mRNA: apoA-I mRNA and the CETP mRNA: apoB mRNA ratios were more than 2500-fold greater in the nonparenchymal cell preparation than in the starting material, and that the purified parenchymal cell fraction was virtually devoid of CETP mRNA. In situ hybridization studies showed that, whereas the apoA-I mRNA signal was evenly distributed over the tissue section, the CETP mRNA signal was associated with the hepatic sinusoids, suggesting that it was the hepatic sinusoidal cells that were principally responsible for the high CETP mRNA levels in the liver. We conclude that the nonparenchymal cells are the principal source of CETP in the cynomolgus monkey.

Animals↗

PubMed1991

Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression.

Sodium butyrate was used to enhance expression levels and thereby facilitate the generation of analytical quantities of nine different tissue plasminogen activator (tPA) analogues expressed under the control of the cytomegalovirus immediate early (CMV IE) promoter by the Chinese hamster ovary (CHO) mammalian expression system. Production involved growth in roller bottles, using serum free or low serum media formulations, together with repetitive, sodium butyrate inductions. Average inductions in the presence of sodium butyrate ranged from 2 to 9-fold relative to uninduced controls, using cell lines with no previous butyrate exposure. Retardation of growth rate by butyrate minimized the need to split cells during the production runs, extending longevity of roller bottles containing cells secreting at induced levels. SDS-PAGE analyses indicate a consistently high percentage of single-chain material. Measurements of specific activity and fibrinogen fragment enhancement for one of the analogues demonstrate that neither of these two critical parameters are affected by production in the presence of butyrate. Induction kinetic data and growth curves for the expression of sCD4 under control of the SV40 early promoter demonstrate that the benefits of butyrate can be realized with different promoters and heterologous genes, and are additive when used in conjunction with an amplified cell line constitutively expressing at elevated levels. This work demonstrates the practical application of sodium butyrate in the production of analytical quantities of protein from the CHO expression system, and suggests a role for sodium butyrate in commercial scale processes as well.

Animals↗

PubMed1991

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.

Animals↗

PubMed1991

Secretion of pre-beta-migrating apoA-I by cynomolgus monkey hepatocytes in culture.

Cynomolgus monkey hepatocytes that had been stored frozen were thawed, established in culture, and used to study apoA-I secretion. Protein synthetic activity was low at first, but increased with time, approaching what appeared to be the constitutive levels of the intact liver by day 7. During the first week, cellular RNA levels increased from 5.3 +/- 0.3 to 18.6 +/- 1.0 micrograms/10(6) cells; albumin secretion rates increased from undetectable to 55.4 micrograms/10(6) cells per day; apoA-I mRNA levels increased from 174 +/- 12 to 564 +/- 145 ng/10(6) cells; and apoA-I secretion rates increased from undetectable to 2.11 +/- 0.27 micrograms/10(6) cells per day. Analysis of day 7-conditioned media by agarose electrophoresis, gradient gel electrophoresis-immunoblotting, and column chromatography, showed that the apoA-I produced by the cells was present in three distinct forms. One had an apparent molecular mass greater than 1 million Da, migrated pre-beta, and accounted for 11 +/- 3 (mean +/- SD)% of the total; one had an apparent molecular mass of 104 kDa, had alpha migration, and accounted for 27 +/- 2% of the total; and one had an apparent molecular mass of 50 kDa, migrated pre-beta, and accounted for 46 +/- 9% of the total. These data support the proposition that the pre-beta-migrating, 50 kDa, apoA-I-containing particles identified in the plasma of cynomolgus monkeys are nascent hepatic HDL.

Albumins↗

PubMed1990

Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene.

The fibrinolytic potential of the vasculature is modulated primarily by the availability and activity of plasminogen activators, which convert the zymogen plasminogen into the active fibrin-degrading enzyme plasmin. The activities of these key regulatory enzymes are directly neutralized by their primary endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1). Although some individuals with a tendency to develop thrombotic disorders exhibit elevated levels of PAI-1 in their plasma, the cause-and-effect relationship between increased PAI-1 and thrombosis is still unclear. Specifically, it is not known whether chronic depression of fibrinolytic activity results in the development of thrombosis. To address this question we developed transgenic mice in which the contribution of PAI-1 to thrombus formation could be evaluated. The results presented in this report indicate that elevated levels of PAI-1 contribute to the development of venous but not arterial occlusions.

Animals↗

PubMed1990

Apo A-I metabolism in cynomolgus monkeys: male-female differences.

Female cynomolgus monkeys have significantly higher plasma apo A-I concentrations than males (P = 0.04) and are able to maintain higher levels than the males even after consuming a high-cholesterol diet that severely depresses the apo A-I concentration in primates (P less than 0.05). The mechanism responsible for this difference was investigated by comparing apo A-I turnover (synthesis and catabolism) in males and females consuming monkey chow and in a separate group of males and females that had consumed the high-cholesterol diet for several weeks. The average length of time an apo A-I molecule remained in the plasma compartment of chow-fed monkeys was 2.62 days but decreased to 1.52 days (P less than 0.01) in animals fed the HC diet. There were no male-female differences in the residence times. The absolute turnover rate (mg/day) of apo A-I was not statistically affected by diet or sex; however, the females were substantially smaller than the males (3.8 vs. 4.8 kg; P less than 0.01) and their plasma volumes were significantly smaller than those of the males, even after correction for differences in body wt. (32.6 vs. 37.0 ml/kg, respectively; P less than 0.01). Taken together, the data indicate that females cynomolgus monkeys have higher apo A-I synthesis rates than males of comparable plasma volume (P = 0.03), which we would propose accounts for the higher plasma apo A-I concentrations evident in females.

Animals↗

PubMed1990

A novel screening system for yeast strains capable of secreting tissue plasminogen activator.

We have developed a simple screening procedure that allowed us to identify Saccharomyces cerevisiae strains able to secrete human tissue plasminogen activator (tPA) into the culture medium. The screen can be used to isolate more efficient secretor strains and to look for novel tPA analogs. Employing one of these strains to study the effect of glycosylation on secretion, we show that glycosylation in the catalytic domain of tPA plays an important role in folding and/or secretion of the molecule. Removing this glycosylation site resulted in a 3-5-fold reduction in the level of tPA secretion. We anticipate that this system will prove useful in studying yeast secretory pathway as well as structure-function relationships in the tPA molecule.

Biotechnology↗

PubMed1990

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis.

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.

Amino Acid Sequence↗

PubMed1990

An improved method for precise quantitation of cellular and tissue apolipoprotein A-I mRNA levels by use of an internal standard.

We have developed a method for the quantitation of apolipoprotein A-I (apoA-I) mRNA by using a variation of traditional S1 nuclease analysis. This method uses an internal standard RNA that allows a level of precision not obtainable with traditional S1 nuclease analysis. The internal standard RNA is synthesized in vitro using the T7 promoter from the transcription vector, pApoAI, which contains the full length apoA-I cDNA. This RNA molecule is identical to authentic apoA-I mRNA except for the addition of 48 bases at the 5'-end which are derived from the vector. A labeled ssDNA probe is produced from pApoAI in such a way that solution hybridization of the probe to a mixture of total RNA and internal standard RNA followed by S1 nuclease digestion results in the protection of DNA fragments from authentic and internal standard RNA, which differ in size by 48 bases. The DNA fragments can be resolved by gel electrophoresis and quantitated. The addition of internal standard RNA to each hybridization reaction allows for correction of variations in hybridization and other sources of experimental error. Using this method we demonstrate a sevenfold increase in precision (the 90% confidence interval was reduced from +/- 186% to +/- 26% of the mean value) when compared to traditional S1 nuclease analysis of apoA-I mRNA in liver biopsies and hepatocytes in culture. The internal standard/S1 nuclease method can be adapted to the analysis of any mRNA.

Animals↗

PubMed1990

Design of novel thrombolytic agents via domain modifications.

Generation of plasmin in the vicinity of a blood clot has proven to be an effective approach for treating thrombotic disorders, particularly myocardial infarction. Conceptually, the ideal thrombolytic agent would initiate the formation of plasmin, primarily in association with fibrin incorporated into the occlusive thrombus. Thus, thrombolytic agents that exhibit relative fibrin specificity and, thus, presumably clot selectivity (e.g., tissue plasminogen activator) were expected to have a marked clinical benefit compared to agents that do not display affinity for fibrin (e.g., streptokinase). However, results obtained recently from clinical trials indicate that these 2 agents essentially were equally effective in treating myocardial infarction. With these findings in mind, efforts are being made to develop novel thrombolytic agents that might achieve more rapid and specific thrombolysis than that achieved by presently available agents and, thus, could be administered earlier because of an improved margin of safety. The available data suggest that tissue-type PA (tPA) mutants possessing resistance to endogenous inhibitors, altered fibrin affinity, and/or slower rates of clearance may prove beneficial in this regard. In addition, adjunctive therapies (i.e., anti-platelet and anti-thrombin compounds) have been found to decrease the time necessary to achieve reperfusion and have reduced rates of reocclusion. These efforts are expected to yield therapeutic agents in the 1990s and beyond that, when administered in combination, would exhibit increased efficacy in the treatment of myocardial infarction and other thrombotic disorders.

Binding Sites↗

PubMed1989

Production and secretion of porcine urokinase in Saccharomyces cerevisiae: characterization of the secreted gene product.

The properties of porcine urokinase plasminogen activator (u-PA), produced and secreted by Saccharomyces cerevisiae, were studied to evaluate processing of the enzyme by yeast. Porcine u-PA cDNA was positioned behind the triosephosphate isomerase promoter and the yeast alpha-mating factor secretion signal sequences in a yeast expression vector, pZV125. Greater than 99% of the secreted PA activity was found to be single chain (pro-urokinase). The secreted gene product could be converted to two-chain (tc) with plasmin and then purified to homogeneity on benzamidine sepharose. Plasmin cleavage resulted in the formation of high Mr (HMW) and low Mr moieties representing HMW tc and free catalytic domain, respectively, as detected by N-terminal amino acid sequence analysis. Approximately 60-70% of the secreted activity was found to be associated with hyperglycosylated fractions from G-75 sizing columns. Approximately 30% of the total activity was secreted into the culture medium, where levels of activity approached 200 I.U./ml.

Animals↗

PubMed1989

Purification, cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-II: comparison to the human sequence.

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-II. Sequence analysis indicated that the monkey apo C-II cDNA was 200 bp longer than the human and the difference in size was all in the 5 degrees untranslated region of mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-II cDNA sequence encoding a preprotein of 101 amino acids - identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-II amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-II and this is confirmed by isoelectric focusing and immunoblotting.

Amino Acid Sequence↗