Biomedical subjects

F Ramirez

Publications and source records attributed to F Ramirez.

At least 109 records · Page 6Linked to original sources

PubMed1987

Organization of the human pro-alpha 2(I) collagen gene.

Sixty kilobases of cloned DNA containing the entire human pro-alpha 2(I) collagen gene and 22 kilobases of flanking sequences have been isolated. Like the homologous avian gene, the 1366 amino acid residues of the human pre-pro-alpha 2(I) chain are encoded by 52 exons, whose relative locations and sizes have been determined. Analysis of the 5'- and 3'-untranslated regions have confirmed their exact lengths, as well as conclusively established the nature of five polymorphic mRNA transcripts.

Amino Acid Sequence↗

PubMed1987

DNA sequences in the first intron of the human pro-alpha 1(I) collagen gene enhance transcription.

A chimeric gene was constructed in which sequences between 253 base pairs (bp) upstream of the start of transcription of the human pro-alpha 1(I) collagen gene and 117 bp downstream of that site were fused to the human alpha 1-globin gene, at a site immediately upstream of the globin initiation codon. Expression of this and subsequent chimeric gene constructions was investigated in microinjected Xenopus laevis oocytes. Presence of 253 bp of pro-alpha 1(I) collagen gene promoter sequences, containing a CAAT box and a TATA box, resulted in a relatively modest level of expression of the linked globin sequences. Lengthening of the pro-alpha 1(I) collagen gene promoter region to include 2400 bp of upstream sequences, increased transcription of the marker gene to some extent. Strong activation of transcription was obtained when a 782-bp fragment of the first intron of the collagen gene was introduced in chimeric constructions containing only 240 bp of the collagen promoter. An enhancing effect of the intron segment on expression of the marker gene was observed from downstream or upstream positions relative to the initiation site of transcription. Sequencing of the intron segment revealed the presence of four decanucleotide consensus sites for binding of the constitutive transcription factor Sp1, as well as an enhancer "core" motif. Our experiments also showed that the cis-acting region strongly enhance the activity of the pro-alpha 1(I) promoter in transfected fibroblasts. On the basis of these observations we conclude that the segment broadly located between +700 and +1300 in the first intron of the human pro-alpha 1(I) collagen gene contains cis-acting sequences with an enhancer effect on transcription of the gene.

Base Sequence↗

PubMed1987

Presence of different types of procollagen messenger RNAs in human hepatoma cell lines.

Human hepatoma cell lines were shown for the first time to contain various types of procollagen mRNAs. The amounts and types of procollagen mRNAs differed depending on the cell lines. Pro alpha 1 (III) and pro alpha 1 (IV) collagen mRNAs were present in PLC/PRF/5, a hepatocellular carcinoma cell line, whereas pro alpha 1 (I), pro alpha 2 (I), pro alpha 1 (IV) and pro alpha 2 (V) collagen genes contrast, HepG2 cells derived from hepatoblastoma contained little, if any, mRNAs for these types of procollagens we had examined.

Actins↗

PubMed1987

Role of platelet-activating factor in adriamycin-induced nephropathy in rats.

The effect of steroids, heparin and specific PAF-acether antagonists (BN 52021 and triazolobenzodiazepines) on proteinuria and renal histological changes induced in rats by adriamycin was studied. Adriamycin evoked a marked proteinuria that was unaffected by methylprednisolone and slightly reduced by heparin. In contrast, adriamycin-injected rats treated with PAF-acether antagonists had a low proteinuria, if any, and no ultrastructural glomerular alterations. These data suggest that PAF-acether could play a major role in the occurrence of proteinuria and that PAF-acether antagonists might provide a new therapeutic approach in certain human nephropathies.

Animals↗

PubMed1987

The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens.

A number of overlapping cDNA clones, covering 5.2 kb of sequences which code for the human pro alpha 2(V) collagen chain, have been isolated. Analysis of the structural data have indicated a close evolutionary kinship between the pro alpha 2(V) chain and the major fibrillar collagen types. Isolation and analysis of an 8 kb genomic fragment has further supported this notion by revealing a homologous arrangement of nine triple-helical domain exons. These studies have therefore provided conclusive evidence which categorizes the Type V collagen as a member of the Group 1 molecules, or fibrillar-forming collagens.

Amino Acid Sequence↗

PubMed1987

DNA-polymorphism of type I collagen gene detected with Bgl II in the genetically isolated Finnish population.

The pro alpha 2(I) collagen gene has been screened for Bgl II restriction fragment length polymorphisms (RFLPs) in the Finnish population which has a long background of genetic isolation. As genetically isolated populations tend to demonstrate deviations in the degree of heterozygosity, the RFLP markers described in more heterogeneous populations can not be utilized as such in the former. This proved to be the case with Bgl II polymorphism within the pro alpha 2(I) collagen gene, the gene involved in different disorders of connective tissue. This report describes the presence of three new RFLP-loci and the absence of one RFLP-locus, which had been earlier reported from a population with a genetic background remote from the population studied here.

Bacterial Proteins↗

PubMed1987

Effects of immunoglobulin G from patients with systemic lupus erythematosus on human B cell function.

We examined the effect of systemic lupus erythematous (SLE) sera and Ig fractions on IgG and IgM release by cultured normal peripheral blood mononuclear cells (PBMC) when these cells were preincubated with serum dilutions or Ig fractions. Increases in both IgM and IgG (P less than 0.001 and less than 0.01) in cultured cell supernatants were recorded when PBMC were preincubated with SLE serum dilutions. IgG but not IgM from SLE was found to stimulate PBMC to release IgG (P less than 0.01). Similar results were obtained when SLE IgG was preincubated with adherent cell depleted cells (ADC) or isolated normal B cell fractions. When normal PBMC were preincubated with SLE serum or IgG and subsequently stimulated with pokeweed mitogen (PWM), a relatively blunted IgG release was observed (P less than 0.05); however, IgM release was significantly increased (P less than 0.001). This effect was not observed when PBMC were preincubated with SLE IgM, normal serum dilutions, or normal Ig fractions. Relative blunting of PWM response after PBMC were preincubated with SLE IgG was not reversed in PBMC depleted of adherent cells, OKT8+, or OKT9+ cells. Depletion of PBMC of LeuM1 cells increased IgG release in response to PWM when cells had been preincubated with SLE IgG. SLE serum or Ig fractions did not induce B cell growth factor release by T cells. SLE IgG appeared to act directly on B cell enriched populations to release IgG; this was not associated with significant increase in thymidine uptake, or apparent lysis of cells.

Antibodies, Monoclonal↗

PubMed1987

Genetic disorders of collagen.

Osteogenesis imperfecta, Ehlers-Danlos syndrome, and Marfan syndrome form a group of genetic disorders of connective tissue. These disorders exhibit remarkable clinical heterogeneity which reflects their underlying biochemical and molecular differences. Defects in collagen types I and III have been found in all three syndromes.

Collagen↗

PubMed1987

Prenatal prediction of osteogenesis imperfecta (OI type IV): exclusion of inheritance using a collagen gene probe.

Autosomal dominant osteogenesis imperfecta is caused by mutations in the COL1A2 and COL1A1 genes of type I collagen. In a family with OI type IV genetically linked to the COL1A2 gene, we attempted prenatal diagnosis in a pregnancy at risk by genotyping the DNA of the fetus for a COL1A2 gene associated RFLP. Our results showed that the fetus inherited the normal COL1A2 allele from her affected parent. Linkage analysis can thus be used in the prenatal diagnosis of dominantly inherited osteogenesis imperfecta.

Adult↗

PubMed1986

The two pathways for oxygen exchange by actomyosin and myofibrils and their dependence on temperature.

At an intermediate stage in the hydrolysis of MgATP by actomyosin there is an exchange of oxygen between water and the terminal phosphoryl group of MgATP, tightly bound to the myosin active site. This intermediate oxygen exchange results from the reversible hydrolysis of the bound MgATP. The rate of the exchange cycle (hydrolysis and the reverse) is assumed to be determined by the rate of reverse hydrolysis; and the average time available for exchange is determined by the post-exchange reaction that immediately follows the cycle. Past analytical studies of the exchange, using actomyosin mixtures and myofibrils at room temperature, have revealed two pathways for hydrolysis, operating at a comparable flux but differing greatly in the extent of exchange they support. It is shown here that these pathways also appear over a range of temperatures from 5 to 30 degrees C and that temperature had little effect on their relative fluxes. At each temperature, the flux ratio (%) for the low exchange pathway: high exchange pathway was near 50:50 for actomyosin mixtures and 60:40 for myofibrils. Apparently, the rate-limiting steps that determine the fluxes of the two pathways have a similar temperature dependence. However, the analysis indicates that one or both of the steps that determine the extent of exchange (reverse-hydrolysis and/or the post-exchange reaction) shows a different temperature dependence for the two pathways. We interpret this to reflect a difference in the temperature dependence of the post-exchange reaction, which we propose is exceedingly fast and independent of actin concentration along the low exchange route, but slow and dependent on the actin concentration along the high exchange route. Thus at all temperatures over a broad range of actin concentration there are two pathways of comparable flux that differ primarily in the time available for exchange.

Actins↗

PubMed1986

Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.

In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A2 on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A2 on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30 degrees C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.

Calcium↗

PubMed1986

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2.

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.

Anions↗

PubMed1986

The affinity of phospholipase A2 for the interface of the substrate and analogs.

In the intravesicle scooting mode of interfacial catalysis, the interfacial complex E*S is formed by the interaction of the membrane bound phospholipase A2 (E*) with the substrate monomer (S) in the interface. In the presence of nonhydrolyzable substrate analogs (I) the kinetics of interfacial catalysis is modified. If phospholipase A2 is added to a mixture of the vesicles of L-DMPMe ester and of DTPMe ether or D-DMPMe ester, the extent of hydrolysis, A, decreases and the interfacial scooting rate constant, ki, remains unchanged. On the other hand, when the enzyme is added to the vesicles prepared from premixed L-DMPMe ester with D-DMPMe ester or L-DTPMe ether, ki decreases but A remains constant. Qualitatively, these results are in excellent accord with the Scheme I for interfacial catalysis. However, a quantitative departure has been noted, which suggests that the interfacial dissociation constant for E*S is larger than that for E*I. These results are interpreted to suggest that the catalytic rate constant for decomposition of E*S to E* + P is larger than the rate constant for decomposition of E*S to E* + S. Broader implications of the scooting mode of interfacial catalysis are discussed.

Catalysis↗

PubMed1986

Use of R-loop mapping for the assessment of human collagen mutations.

R-loop mapping of DNA:RNA hybrids formed between mutant pro-alpha 2(I) mRNAs and appropriate human pro-alpha 2(I) genomic clones was employed to define the location of mutations which result in the synthesis of shortened pro-alpha 2(I) chains in skin fibroblasts from two variants of osteogenesis imperfecta. Hybridization of the genomic clone NJ-9 with pro-alpha 2(I) mRNA from a patient with a mild atypical form of the disease resulted in the identification of mutant pro-alpha 2(I) mRNA lacking the sequences which correspond to exon 11 of the pro-alpha 2(I) collagen gene. Exon 11, a 54-base pair exon, encodes amino acids 73 to 90 of the alpha 2(I) chain. Also, electron microscopy of R-loop structures formed between the genomic clone NJ-1 and mRNA from a variant with a perinatal lethal form of osteogenesis imperfecta visualized pro-alpha 2(I) mRNAs which did not hybridize to the sequences of exon 28, a 54-base pair exon coding for amino acids 448 to 465 of the alpha 2(I) chain. Moreover, nuclease S1 mapping of the variant's mutant pro-alpha 2(I) mRNA, employing the human pro-alpha 2(I) cDNA clone Hf-15, confirmed the location of the mismatch to the sequences corresponding to exon 28. Although the data do not determine the exact nature of the mutations, they illustrate the use of R-loop mapping as an alternative approach to S1 mapping analysis for the detection and localization of collagen mRNA deletions.

Base Sequence↗

PubMed1986

Immunoglobulin from systemic lupus erythematosus serum induces interferon release by normal mononuclear cells.

Ig fractions from patients with systemic lupus erythematosus (SLE) were tested with cultured normal peripheral blood mononuclear cells for induction of interferon release. Lymphocyte eluates, euglobulins containing IgG and IgM, and IgG or IgM from DEAE or sucrose gradients all induced interferon production. Lymphocytotoxic antibody in SLE sera showed a high correlation with capacity of isolated Ig fractions to induce interferon. Most interferon produced was of the gamma type. Monoclonal SLE IgM antilymphocyte antibody induced interferon synthesis.

Antibodies, Monoclonal↗

PubMed1986

Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen.

Ehlers-Danlos syndrome (EDS) type IV is a rare and catastrophic genetic disorder of the connective tissue. Individuals from two families with this disorder were studied for a restriction fragment length polymorphism (RFLP) associated with the COL3A1 gene. Our results suggested cosegregation of the EDS type IV phenotype with a COL3A1 RFLP allele. Biochemical studies in cultured skin fibroblasts indicated the presence of different mutations affecting the stability and secretion of the pro alpha 1(III) chains of type III procollagen in the two families, thus suggesting that EDS type IV is biochemically heterogeneous. Our data demonstrated the feasibility of molecular diagnosis in this condition using COL3A1 gene related RFLPs.

Alleles↗

PubMed1986

Assessment of small polymorphisms in defined human collagen gene segments.

Restriction fragment length polymorphisms (RFLPs) greater than 1.5 kb in size have been identified in all but one of the human fibrillar collagen genes. However, the number of informative RFLPs for these genes is still limited. Here we present the conjunct use of two techniques for the assessment of small length variations within defined segments of the genome. This strategy has the potential to be used for the allelic exclusion of cloned genomic fragments prior to sequencing. Moreover it can be a useful and sensitive tool to determine if genetic linkage exists between abnormal phenotypes and loci where conventional Southern blotting analysis has failed to detect informative RFLPs.

Chromosome Mapping↗

PubMed1986

The pro alpha 2(V) collagen gene (COL5A2) maps to 2q14----2q32, syntenic to the pro alpha 1 (III) collagen locus (COL3A1).

A recombinant probe specific for the pro alpha 2 chain of human Type V collagen has been used for the localization of the corresponding gene (COL5A2) to chromosome 2. Regional mapping by in situ hybridization and analysis of DNA from human X rodent cell lines indicated that COL5A2 is confined within the segment 2q14----2q32, thus syntenic to the pro alpha 1 (III) collagen gene (COL3A1).

Animals↗