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N Ruiz-Opazo

Publications and source records attributed to N Ruiz-Opazo.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differential interaction of the dual alpha tropomyosin/N5 enhancer with multiple DNA binding proteins: N5 is a putative novel z-ZIP DNA binding protein.

The alpha tropomyosin (TM)/N5 enhancer is an SV40-like mammalian enhancer comprised of a 99 bp repeat with modular cis-acting regulatory elements exhibiting apparent hierarchical organization. The enhancer differentially regulates the alpha TM and N5 transcription units which exhibit distinct tissue-specific expression patterns and interacts with multiple myotube-associated nuclear DNA binding proteins that varied in size and amount. To further characterize the interaction with multiple myotube nuclear factors, comparative southwestern blot analyses were done with a panel of strategic DNA probes representative of modular enhancer sequences in the alpha TM/N5 enhancer and respective alpha TM and N5 promoter regions. Results demonstrate that multiple DNA binding proteins, which vary in size and amount, can interact with a particular enhancer modular sequence (delimited to 18 bp- to 38 bp-long); and that likewise, a DNA binding protein can bind specifically to different DNA enhancer modular sequences with apparent different affinities. Results also demonstrate DNA binding proteins that differentially bind to both enhancer modular sequences and respective promoter regions supporting a putative parsimonious mechanism for the approximation of enhancer and promoter elements as an alternative to the multi-protein stereospecific enhancer complex. Cogent to this interesting "head to head"/shared enhancer gene arrangement, we investigated the primary structure of the "other" transcription unit, N5. Nucleotide sequence analysis of the N5 cDNA reveals that it is a putative DNA binding protein representing a new structural class of transcription factors exhibiting a novel combinatorial motif: single zinc finger (DNA-binding)-leucine zipper (dimerization)--making it a z-ZIP instead of a b-ZIP (basic region/leucine zipper) protein.

Amino Acid Sequence↗
PubMed1993

The alpha 1 Na(+)-K+ pump of the Dahl salt-sensitive rat exhibits altered Na+ modulation of K+ transport in red blood cells.

The properties of the alpha 1 Na(+)-K+ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive fluxes (mmol/liter cell x hr = FU, Mean +/- SE) in red blood cells (RBCs) and varying internal (i) and external (o) Na+ and K+ concentrations. Kinetic parameters of several modes of operation, i.e., Na+/K+, K+/K+, Na+/Na+ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n = 12 rats of each strain) into fresh cells incubated in 140 mM Na(+) + 5 mM K+, ouabain-sensitive K+ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na+ efflux and Nai were similar in both strains. Thus, the coupling ratio between unidirectional Na+:K+ fluxes was significantly higher in DS than in DR cells at similar RBC Na+ content. In the presence of 140 mM Nao, activation of ouabain-sensitive K+ influx by Ko had a lower Km and Vmax in DS as estimated by the Garay equation (N = 2.70 +/- 0.33, Km 0.74 +/- 0.09 mM; Vmax 2.87 +/- 0.09 FU) than in DR rats (N = 1.23 +/- 0.36, Km 2.31 +/- 0.16 mM; Vmax 5.70 +/- 0.52 FU). However, the two kinetic parameters were similar following Nao removal. The activation of ouabain-sensitive K+ influx by Nai had significantly lower Vmax in DS (9.3 +/- 0.4 FU) than in DR (14.5 +/- 0.6 FU) RBCs but similar Km. These data suggest that the low K+ influx in DS cells is caused by a defect in modulation by Nao and Nai. Na+ efflux showed no differences in Nai activation or trans effects by Nao and Ko, thus accounting for the different Na+:K+ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na+ and K+ fluxes, where the net ouabain-sensitive Na+ losses showed similar magnitudes in the two Dahl strains while the net ouabain-sensitive K+ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na+ influx and K+ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na+ influx by Ko was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, Ko could activate higher K+ influx in DR pumps without a complete inhibition of ouabain-sensitive Na+ influx.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1991

Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting.

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Alteration of alpha 1 Na+,K(+)-ATPase 86Rb+ influx by a single amino acid substitution.

The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension.

Amino Acid Sequence↗
PubMed1990

Regulation of alpha-tropomyosin and N5 genes by a shared enhancer. Modular structure and hierarchical organization.

We have identified a 99-base pair (bp) tandem repeat consisting of two modules/repeat: a 40-bp-long d(CA.GT)20 potential Z-DNA sequence and a 59-bp-long unique sequence. This repeat is located between and differentially regulates two transcription units, rat alpha-tropomyosin and N5, organized in "head-to-head" orientation. Transient expression experiments showed orientation- and position-independent enhancer activity of the 99-bp repeat in a tissue-specific and developmental manner. Deletion analysis revealed a hierarchical organization establishing the two modules as protoenhancers. Southwestern blot analysis of crude nuclear extracts identified at least six C2C12 myotube nuclear proteins binding to the 99-bp repeat. These observations demonstrate a mammalian gene regulatory sequence structurally and functionally organized like the simian virus 40 viral enhancer and confirm in a specific gene context the contribution of a d(CA.GT)20 tract in transcriptional regulatory mechanisms as a protoenhancer.

Animals↗
PubMed1988

Isoform-specific modulation of Na+, K+-ATPase alpha-subunit gene expression in hypertension.

Sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and myocardial contractility. By means of RNA blot analyses of cardiac, aortic, and skeletal muscle RNAs in two rat hypertensive models, Na+,K+-ATPase alpha-subunit messenger RNA isoforms (alpha 2 and alpha 3) were shown to be deinduced in response to increased intravascular pressure. The changes were observed after 48 hours or more of experimental hypertension. Under these conditions, there is coordinate induction of another alpha isoform (alpha 1) and of beta-subunit messenger RNAs, probably in response to alterations in sodium flux rather than to elevated blood pressure.

Angiotensin II↗
PubMed1988

Sequence and expression of Tangier apoA-I gene.

We have isolated and characterized the apoA-I gene from a lambda L47.1 genomic library constructed with DNA obtained from the lymphocytes of a Tangier disease patient. The DNA-derived protein sequence of Tangier apoA-I was found to be identical to normal apoA-I. Transfection of mouse C127 cells with a recombinant vector containing the Tangier apoA-I gene (pSV2-gpt apoA-I) allowed selection of stable clones resistant to aminopterin and mycophenolic acid. Analysis of these clones for apoA-I synthesis showed that the protein secreted by cells expressing the Tangier apoA-I gene was indistinguishable from the apoA-I secreted by HepG2 cells. These experiments establish that the Tangier apoA-I gene is structurally normal. It appears that the molecular basis of Tangier disease is not related to apoA-I structure or regulation of expression, but rather to other factors pertinent to apoA-I and high-density lipoprotein metabolism.

Amino Acid Sequence↗
PubMed1988

Expression of the human apolipoprotein A-I gene in rat myogenic L6E9 cells. DNA methylation and regulation of gene activity.

An 11-kilobase EcoRI fragment containing the human apoA-I gene was cloned in the pSV2gpt vector. The recombinant plasmid was then used to transfect a rat myogenic cell line (L6E9) and HeLa cells. Selection and analysis of cell clones resistant to hypoxanthine/aminopterin/thymidine and mycophenolic acid showed that the apoA-I gene is expressed in L6E9 but not in HeLa cells. The apoA-I mRNA transcribed by either the transfected L6E9 or control human liver cell line, HepG2, has a major transcription initiation site located 26 nucleotides downstream from the CATAAA box. The apoA-I synthesized by the L6E9 cells is secreted into the culture medium and is indistinguishable from the apoA-I secreted by HepG2 cells. These results demonstrate that the L6E9 cells contain the necessary factors required for the expression of the apoA-I gene. Further analysis showed that the endogenous rat apoA-I gene is not expressed in L6E9 cells and exists in a hypermethylated configuration. In contrast, the exogenous human apoA-I gene was found to be hypomethylated in the transfected L6E9 cells and in other apoA-I-producing cells. These findings are consistent with the hypothesis that the expression of the endogenous rat apoA-I gene in L6E9 cells is prevented by hypermethylation, suggesting that compared to other mechanisms which control initiation of transcription, DNA methylation may play a higher hierarchical role in the regulation of expression of eukaryotic genes.

Animals↗
PubMed1987

Cloning and tissue distribution of rat heart fatty acid binding protein mRNA: identical forms in heart and skeletal muscle.

A fatty acid binding protein (FABP) has been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.

Amino Acid Sequence↗
PubMed1987

Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms.

We have previously isolated and characterized cloned complementary DNAs (cDNAs) for striated and smooth muscle alpha-tropomyosin. The sequences of these cDNA clones suggested that these two isoforms were encoded by the same gene. Here, we have determined the complete structure of the alpha-tropomyosin (alpha-TM) gene, establishing that a single gene, with a sequence complexity of 28 kilobase pairs, is split into 12 exons and produces the smooth and striated muscle alpha-TM mRNA isoforms by alternative splicing of a minimum of five exchangeable isotype-specific exons. The elucidation of the intron/exon organization of alpha-TM suggests that this gene evolved from an ancestral gene encoding a 21-aa protein that might represent the primordial actin binding domain. Sequence comparison between the pairs of exons coding for the "isotype switch regions" and among the corresponding regions of tropomyosin genes in a variety of species ranging from insects to mammals, suggests that the alternatively spliced exons are very old and might have arisen before the radiation of the arthropods, more than 600 million years ago. Additionally, the examination of the intronic sequences has uncovered potential alternative intramolecular secondary structures (hairpin-loop structures) which might be involved in the tissue-specific expression of the duplicated and mutually exclusive alpha-TM isotype-specific exons.

Animals↗
PubMed1987

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications.

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

Amino Acid Sequence↗
PubMed1984

Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing.

Fast myosin light chains (MLCf) 1 and 3 are proteins associated with the thick filament in vertebrate fast muscle fibers. MLC1f and MLC3f have complete sequence homology for the first 141 amino acids from their COO- end, but they differ in length and amino acid sequence at their NH+3 ends (MLC1f = 49 amino acids, MLC3f = 8 amino acids), and they are translated from different mRNAs. To elucidate the structural relationship between mammalian fast myosin light chain 1 and 3 isoforms, cDNA clones have been isolated from rat skeletal muscle and their primary nucleotide sequences were determined. MLC1f and MLC3f mRNAs display complete sequence identity in the 3' untranslated sequences (285 base pairs) and in the codons specifying the 142 carboxyl-terminal amino acids. In contrast, the sequences encoding the amino-terminal 49 and 8 amino acids of MLC1f and MLC3f, respectively, as well as their 5' untranslated regions, are highly divergent. Using the cloned cDNAs as probes we have isolated the single gene locus encoding both MLC1f and MLC3f in four overlapping genomic clones spanning over approximately 25 kilobase pairs (kb) of DNA. The sequences encoding the common body of MLC1f and MLC3f are distributed in 4 separate exons at the 3' end of the gene. The start of transcription site and 5' untranslated region of MLC3f is found 5 kb upstream from the common body while the MLC1f mRNA transcription start site and the exon specifying the 5' untranslated region and amino acid 1-40 of MLC1f lies about 10 kb further upstream. Strikingly, a mini-exon that specifies amino acids 41-49 of MLC1f is located downstream from the two MLC3f-specific exons (5' untranslated and amino acids 1-8) and is separated from the upstream MLC1f exons by 12 kb of DNA. This bizarre gene organization implies a novel form of alternative promoter utilization and RNA splicing that is tissue-specific and developmentally regulated in order to generate the mature MLC1f and MLC3f mRNAs from a single gene.

Amino Acid Sequence↗
PubMed1984

Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix.

The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (iii) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.

Animals↗
PubMed1983

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1982

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection.

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.

Animals↗
PubMed1982

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection.

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.

Animals↗