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M Salas

Publications and source records attributed to M Salas.

At least 109 records · Page 6Linked to original sources
PubMed1996

Effect of (8-32) salmon calcitonin, an amylin antagonist, on insulin, glucagon and somatostatin release: study in the perfused pancreas of the rat.

1. The 8-32 fragment of salmon calcitonin ((8-32) sCT) has been proposed as a highly selective amylin receptor antagonist. 2. In the present study, we have studied the influence of (8-32) sCT on the inhibitory effect of both amylin and its structural congener, calcitonin gene-related peptide (CGRP), on insulin secretion in the rat perfused pancreas. 3. Both amylin and CGRP, at 75 pM, clearly inhibited glucose-induced insulin release (by 80% and by 70%, respectively). Simultaneous infusion of 10 microM (8-32) sCT reversed the inhibitory effect of amylin (by 80%; P < 0.05 vs. amylin experiments) but did not significantly affect the inhibition of glucose-induced insulin output elicited by CGRP. Furthermore, at the same concentration (10 microM), (8-32) sCT alone potentiated the insulin response to 7 mM glucose (2.5 fold; P < 0.05) whilst it did not affect glucagon or somatostatin secretion. 4. The observation that infusion of an amylin antagonist into the rat pancreas potentiates the insulin response to glucose, favours the concept of endogenous amylin as an inhibitor of insulin release. 5. Finally, as an amylin antagonist at the level of the beta-cell, (8-32) sCT might be considered of potential interest in experimental and clinical pharmacology.

Amyloid↗
PubMed1996

Effect of cyclosporin weaning on glomerular filtration rate in renal transplantation.

The objective of this study was to determine the effect of weaning cyclosporin on glomerular filtration rate in 55 renal transplant patients from the Renal Transplant Unit at the Royal Victoria Hospital. Men or women, older than 20 years of age, who received cadaveric or living-related renal transplant and who were treated with the complete cyclosporin protocol, were included. Weaning of cyclosporin was started 15 days after the prednisone dose was reduced to 15 mg/day and this weaning process was completed at day 105 posttransplant. The daily cyclosporin dose was decreased by 25 mg once every 2 weeks, and completely withdrawn after treatment when 50 mg/day was tolerated for 2 weeks. The duration of the weaning process varied because of fixed dose reduction. Renal graft function was assessed by plasma creatinine concentrations. We used the time point of cyclosporin discontinuation as time zero. Serum creatinine decreased over the time from 150 +/- 61 mmol/l at time zero to 100 +/- 18 mmol/l in the last determination, while GFR had a significant increment from 66 +/- 18 to 77 +/- 20. A total of 4 rejection episodes were observed 6 months after cyclosporin discontinuation. There were no graft failures and deaths. To determine predictors of improvement, we carried out multiple regression analysis and we found that prophylactic antilymphocyte globulin and the onset of graft function after transplantation were predictors.

Adult↗
PubMed1996

[Causes of postpartum intrauterine device rejection].

The objective of this study was to determine the causes of rejection to intrauterine device postpartum (IDP) in puerperal patients before they left the hospital. Two hundred puerperal patients who accepted IDP and two hundred patients who rejected IDP were studied. They received a questionaire to know the motives to accept or not IDP and degree of knowledge about it. There was not statistical differences between groups in relation to age, scholarity, religion, marital status, occupation and degree of knowledge, 51% had prenatal control, 62% of puerperal patients who rejected IDP did not have information about this contraceptive method, in 17% husband did not accept it and 9.5% have been influenced by negative rumors about IDP. In conclusion puerperal patients reject IDP mainly for lack of knowledge in relation to IDP and because their husbands did not accept it.

Adult↗
PubMed1995

A novel kinetic analysis to calculate nucleotide affinity of proofreading DNA polymerases. Application to phi 29 DNA polymerase fidelity mutants.

Amino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3'-->5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by phi 29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleotide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that phi 29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide.

Amino Acid Sequence↗
PubMed1995

Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication.

The single-stranded DNA (ssDNA)-binding protein (SSB) of bacteriophage phi 29 is one of the virus-encoded proteins required for viral DNA replication. We have found that phi 29 SSB has helix-destabilizing activity since it removes secondary structure of the ssDNA in phi 29 replicative intermediates, as revealed by electron microscopy, and displaces oligonucleotides annealed to M13 ssDNA. To investigate the mechanism of the SSB-dependent stimulation of phi 29 DNA replication we have characterized the helix-destabilizing activity of phi 29 SSB and measured its effect on the DNA elongation rate by phi 29 DNA polymerase, which does not require an accessory helicase. The use of replication reactions where strand displacement is either required (phi 29 DNA replication) or not (conversion of primed M13 ssDNA into double-stranded DNA (dsDNA)) has allowed us to find that (1) strand displacement DNA replication was affected by lowering the temperature or by increasing the salt concentration, since the DNA elongation rate on the phi 29 template was three to fourfold slower than on primed M13 ssDNA, (2) under those conditions, addition of phi 29 SSB stimulated to different extents the DNA elongation rate during phi 29 DNA replication, whereas it had a marginal effect on primed M13 ssDNA replication, and (3) phi 29 SSB increased four to sixfold the phi 29 DNA elongation rate by phi 29 DNA polymerase strand displacement mutants, reaching approximately 50% the rate of the wild-type enzyme. The implications of the helix-destabilizing properties of the phi 29 SSB under conditions in which DNA opening is impaired are discussed.

Bacillus Phages↗
PubMed1995

Transcription regulation in Bacillus subtilis phage phi 29: expression of the viral promoters throughout the infection cycle.

Transcription of the genome of Bacillus subtilis phage phi 29 is tightly controlled, taking place in two stages, early and late. We have analyzed the abundance of the transcripts produced from each viral promoter throughout the infection cycle. We compare the relative strength of each promoter, as well as get a better understanding of the regulatory events, finding a new promoter regulated by the viral protein p4. The two strong early promoters, A2b and A2c, responsible for the expression of genes 6 to 1, are coordinately repressed by the viral protein p4, although repression is not complete: both promoters are still active at late times of infection. Since repression by protein p4 was very efficient in vitro, and affects its own synthesis, it is likely that this protein is produced in limiting amounts, not being bound to all viral DNA molecules present in the cell at a given time. Protein p4, also known to activate the late promoter responsible for the expression of all the structural and morphogenetic genes, is the key regulator of phage phi 29 development.

Bacillus Phages↗
PubMed1995

Primer terminus stabilization at the phi 29 DNA polymerase active site. Mutational analysis of conserved motif KXY.

phi 29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. A conserved amino acid motif, located in the C-terminal portion of the polypeptide and characterized by the amino acid sequence KK(K/R)Y, is conserved in the group of eukaryotic-type DNA polymerases. In the subgroup of DNA polymerases that have a protein-priming mechanism, this motif is restricted to the sequence KXY, X never being a positively charged amino acid. Residues Lys498 and Tyr500 form this conserved motif in phi 29 DNA polymerase. Mutant K498T, in which the positive charge of the motif has been eliminated, was strongly affected both in initiation (terminal protein-dAMP formation, using terminal protein as primer) and DNA polymerization reactions. Mutants K498R and Y500S were able to carry out the initiation reaction to a higher or similar extent, respectively, than wild-type phi 29 DNA polymerase but were affected in DNA polymerization reactions. All of the mutations severely affected the stable binding of the polymerase to a primer-template DNA. In addition, all of the mutant polymerases analyzed in this work showed an unusually strong 3'-5' exonuclease activity both under polymerization or non-polymerization conditions. The results obtained suggest a role of the conserved residues of the KXY motif in stabilizing the primer terminus at the polymerization active site, the positive charge of residue Lys498 being critical for the synthetic activities of phi 29 DNA polymerase.

Amino Acid Sequence↗
PubMed1995

Interaction of arginase with metal ions: studies of the enzyme from human liver and comparison with other arginases.

As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.

Arginase↗
PubMed1995

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication.

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.

Bacillus Phages↗
PubMed1995

Dendritic branching of claustral neurons in neonatally undernourished rats.

Golgi-Cox-impregnated neurons of the claustrum were studied in control and undernourished Wistar strain rats at 12, 20, and 40 days of age. A reduced cross-sectional somatic area was observed in the 20-day-old undernourished rats and a significant reduction in the dendritic area was observed in the three ages studied. Dendritic arbor alteration was mainly observed in the number of high order and in the total number of dendritic branches of undernourished rats throughout the study. The data suggest vulnerability of the dendritic claustral growth process during the postnatal period to neonatal undernutrition. These alterations may be associated with telencephalic integrative impairments described in perinatal undernourished rats.

Age Factors↗
PubMed1995

Measurement of clinical activity of systemic lupus erythematosus and laboratory abnormalities: a 12-month prospective study.

OBJECTIVE: To assess flares in outpatients with systemic lupus erythematosus (SLE) using SLAM (systemic lupus activity measure) and to determine laboratory abnormalities as predictors of disease activity. METHODS: Fifty-three Mexican patients were assessed using SLAM scale. They were evaluated monthly for a total of at least 572 months. The SLAM scale was applied at each visit. Samples were drawn for complete blood cell count, erythrocyte sedimentation rate, urinalysis, 24-h protein and creatinine clearance, anti-DNA, C3 and C4. An SLE flare was defined as the occurrence of new clinical manifestations or worsening compared to the previous month that usually required restarting or increasing prednisone or immunosuppressive drugs. RESULTS: Thirty-three patients had flares, mainly in minor organs. The incidence of flares was 0.69/patient/year of followup. Active nephritis and extrarenal manifestations correlated with high levels of dsDNA and low complement levels. We found an odds ratio (OR) = 3 (CI = 1.7-5.7) for flare in asymptomatic patients with high dsDNA and OR = 2 (CI = 1.3-4.5) for low C3 levels. CONCLUSION: Flares are frequent in patients with SLE and they occur independent of disease duration and the time the disease has been under control. Flares are apparently predictable and are related to serologic abnormalities.

Adult↗
PubMed1995

Inhibitory effect of amylin (islet amyloid polypeptide) on insulin response to non-glucose stimuli. Study in perfused rat pancreas.

Amylin, also called islet amyloid polypeptide (IAPP), can inhibit the glucose-induced insulin secretion in perfused rat pancreas at 75 pmol/l, a concentration comparable to that found in the effluent of this experimental model. To further explore the influence of amylin on insulin release, we investigated the effect of synthetic rat amylin (75 pmol/l) on insulin response to non-glucose secretagogues. These agents stimulate B-cell secretion via different mechanisms, such as a dihydropyridine derivative (BAY K 8644, 10 mmol/l) which activates Ca(2+)-channels, a sulfonylurea (tolbutamide, 0.2 mmol/l) which blocks ATP-dependent K(+)-channels, KCL (11 mmol/l) which depolarizes B cells and the 26-33 fragment of cholecystokinin (8-CCK, 1 nmol/l) which increases phospholipid turnover. The study was performed in perfused rat pancreas. Amylin significantly inhibited insulin response to BAY K 8644 (65%), KCI (60%) and 8-CCK (80%) as well as the early phase of tolbutamide-induced insulin output (70%). Thus, amylin can inhibit insulin release induced by secretagogues that interact at different levels of B-cell stimulus-secretion coupling. This inhibition may be due to a multifarious influence of amylin on the B-cell secretory mechanism and/or a disturbing effect on a distal, crucial step in the insulin-releasing mechanism, e.g. by affecting exocytosis of the secretory granule or by inhibiting an essential metabolic pathway within the B cell.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

3'-->5' exonuclease active site of phi 29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism.

The residues forming the 3'-->5' exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3'-->5' exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3'-->5' exonuclease.

Bacillus Phages↗
PubMed1994

Terminal protein-primed DNA amplification.

By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.

Bacillus Phages↗
PubMed1994

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage phi 29.

Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication. This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

Bacillus Phages↗
PubMed1994

Primer-terminus stabilization at the psi 29 DNA polymerase active site. Mutational analysis of conserved motif TX2GR.

The functional significance of the conserved motif TX2GR, included in one of the six main regions of amino acid sequence similarity identified in the C-terminal portion of both Escherichia coli DNA polymerase I-like and eukaryotic-type DNA polymerases (Blanco, L., Bernad, A., Blasco, M.A., and Salas, M. (1991) Gene (Amst.) 100, 27-38) has been studied by site-directed mutagenesis in the psi 29 DNA polymerase. A revised multiple alignment of this region, including 61 DNA polymerases belonging to these two superfamilies, is presented. In addition, based on amino acid sequence comparisons and by extrapolation to the crystal structure of T7 RNA polymerase, a similar motif (DX2GR) is predicted to be structurally and functionally equivalent in RNA polymerases, the other class of DNA-dependent polymerases. The severe defect in polymerization displayed by two of the psi 29 DNA polymerase mutants used in this study (T434N and R438I) is interpreted as the consequence of a decreased capacity to stabilize the binding of primer-template DNA structures in a polymerization-competent conformation. These mutants were also severely affected in the formation of terminal protein (TP)-dAMP initiation complex, a reaction in which psi 29 DNA polymerase is able to use the TP as primer.

Amino Acid Sequence↗