Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 127 records · Page 7Linked to original sources

PubMed1994

In vivo functional relationships among terminal proteins of Bacillus subtilis phi 29-related phages.

Gene 3 of the Bacillus subtilis phage phi 29 encodes the terminal protein (TP), which acts as a primer in the initiation of viral DNA replication. We have developed an in vivo functional assay for the phi 29 TP based on the ability of TP-producing B. subtilis non-suppressor (su-) cells to support DNA replication of a phi 29 sus3 mutant phage. This trans-complementation assay has been used to study in vivo functional relationships between the TP of phi 29 and related phages. Our results demonstrate that phi 29 TP functionally substitutes the TP of phage PZA, whereas replication of phage Nf DNA cannot take place in vivo using the phi 29 TP as a primer.

Bacillus Phages↗

PubMed1994

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these 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.

Amino Acid Sequence↗

PubMed1994

Complex formation between phage phi 29 single-stranded DNA binding protein and DNA.

Bacteriophage phi 29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB) which stimulates viral DNA replication. In the present study, a structural characterization of the complex between ssDNA and the phi 29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of phi 29 SSB upon binding. Phage phi 29 SSB behaves as a monomer in solution and forms complexes with ssDNA which have a homogeneous structure, as if they consist of a continuous array of protein bound to DNA. Interaction of phi 29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of phi 29 SSB bound to the ssDNA and the maximal quenching upon binding was very high (Qmax = 94.6 +/- 3.5%). Direct titration experiments have allowed us to estimate that the stoichiometry (n) of binding to ssDNA was 3.4(+/- 0.3) nucleotides per phi 29 SSB monomer. Both Qmax and n are independent of the salt concentration, suggesting the existence of only one major binding mode. At low salt concentrations, the effective binding constant (Keff = K omega) to poly(dT) was 2.2 x 10(5) M-1, the intrinsic binding constant (K) and the cooperativity parameter (omega) being 4.3 x 10(3) M-1 and 51, respectively. At increasing salt concentrations, the Keff exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of phi 29 SSB during viral DNA replication is discussed.

Bacillus Phages↗

PubMed1994

Requirement for an A-tract structure at the binding site of phage phi 29 transcriptional activator.

The Bacillus subtilis phage phi 29 transcriptional activator, protein p4, binds to the 5'-AACT-TTTT-15 base-pair spacer-AAAATGTT-3' inverted repeat. In this communication, we study the influence in protein p4 binding of the DNA helical structure within the protein p4 recognition sequences, 5'-AAAATAG-3'. Protein p4 could efficiently bind to a modified target in which the A-tracts had been changed into T-tracts (a different sequence with a similar structure). Binding was lost when the structure of the binding site was modified by an interrupting C residue. The results suggest that the DNA helical structure of the A-tracts is critical for p4 binding. Two models are described that would explain how protein p4 recognized its target sequences on the DNA.

Bacillus Phages↗

PubMed1994

Amylin (islet amyloid polypeptide) inhibition of insulin release in the perfused rat pancreas: implication of the adenylate cyclase/cAMP system.

Amylin inhibits glucose-induced insulin secretion in the rat pancreas. To study the mechanism by which amylin acts on the B-cell, we have investigated, in the perfused rat pancreas, the effect of synthetic rat amylin (75 pM) on insulin release elicited by secretagogues acting on the B-cell via the adenylate cyclase/cAMP system, i.e., glucagon (10 nM), gastric inhibitory polypeptide (GIP, 1 nM), forskolin (1 microM) and isobutylmethylxanthine (IBMX, 75 microM). In addition, we examined the effect of amylin on GIP-induced insulin release in pancreata from rats pretreated with pertussis toxin, an agent which inactivates certain Gi proteins coupled to adenylate cyclase. Amylin inhibited the insulin response to glucagon (approx. 70%), GIP (approx. 90%), IBMX (approx. 75%) as well as the early phase of forskolin-induced insulin output (approx. 74%). However, amylin failed to modify GIP-induced insulin release in pancreata obtained from pertussis toxin pretreated rats. These results would indicate that the inhibitory effect of amylin on insulin secretion could be, at least in part, attributed to its interfering with the adenylate cyclase/cAMP system. Furthermore, prevention of the inhibitory effect of amylin on GIP-induced insulin output by pertussis toxin pretreatment, supports the concept that amylin can inhibit insulin release via a pertussis toxin-sensitive Gi protein coupled to the adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.

Bacillus Phages↗

PubMed1994

Assessing pain threshold in the rat: changes with estrus and time of day.

Pain threshold was determined in female rats using the tail flick test. Latency to respond depended on the locus of the tail heated, with the most distal sites resulting in the shortest response times (Experiment 1). Tail flick latency also varied according to the time of day, with shorter response times recorded around the middle of the dark phase than near its beginning or after its end. This was the case for intact, for ovariectomized, and for ovariectomized, estradiol-treated rats (Experiment 2). However, response times also varied across the estrous cycle, with significantly shorter latencies recorded during estrus and metestrus. Ovariectomy abolished these fluctuations, and whereas administration of estradiol increased response times, progesterone had little effect (Experiment 3). These results suggest that site of tail heating, time of day, and presence of ovarian hormones can influence tail flick latency independently, thus demonstrating the complexity of the mechanisms that may contribute to pain threshold even within the same paradigm.

Animals↗

PubMed1994

Neonatal capsaicin administration: effects on behavioral development of the rat.

The effect of a single dose of capsaicin administered neonatally on the development of six motor behavioral patterns (scratching, rearing, grooming, searching, remaining still, and sniffing) was examined in Wistar rats. Treated animals exhibited a significant increment in scratching, rearing, grooming, and searching. Capsaicin also provoked precocious eye opening of one to two days and a reduction in remaining still, sniffing, and body weight. These effects were accompanied by pelage dishevelment and presence of skin ulcers in the region of the head, neck, and shoulders. The distribution of skin ulcers suggests that the fifth cranial nerve and roots innervating C1-C3 spinal cord segments contain a high proportion of capsaicin-sensitive "C" and A delta fibers.

Animals↗

PubMed1994

Phage phi 29 protein p6: a viral histone-like protein.

Phage phi 29 protein p6 is one of the most abundant viral proteins in phi 29-infected B subtilis cells, constituting about 4% of the total cellular proteins (about 3 x 10(6) copies/cell) at late infection. Electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along phi 29 DNA, suggesting that protein p6 may have a role in genome packaging and organization. The low stability of the protein p6-phi 29 DNA complexes observed in vitro could reflect the dynamic nature of these complexes, to allow replication, transcription, and encapsidation of the genome. The protein p6-DNA complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. The DNA in this complex is strongly distorted and compacted. Protein p6 recognition signals have been mapped near the ends of the linear phi 29 DNA and act as nucleation sites for complex formation. Protein p6 does not recognize a specific sequence, but sequences with specific bendable properties that would favor the formation of the complex. Protein p6 represses transcription from the phi 29 C2 early promoter, and activates initiation of phi 29 DNA replication that occurs from both DNA ends. The formation of nucleoprotein complexes at the origins of replication, as well as the specific positioning of protein p6 with respect to the DNA ends are required for the activation of replication. This suggests that the proteins involved in the initiation step of phi 29 DNA replication, either directly interact with protein p6, or recognize a conformational change at a specific location in the DNA. The mechanism of activation could be the local and transient unpairing of DNA at specific sites, facilitated by the strong distortion of DNA conformation in the nucleoprotein complex.

Bacillus Phages↗

PubMed1994

DNA structure in the nucleoprotein complex that activates replication of phage phi 29.

Initiation of phage phi 29 DNA replication is activated by the viral protein p6 which forms a nucleoprotein complex at the replication origins, located at the linear genome ends. The complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. We have determined the superhelical path of the DNA in the complex, measuring the change in linking number induced by the protein, the surface-related helical repeat and the compaction of the DNA. One superhelical turn has approximately 63 bp (2.6 p6 dimers). Furthermore, we have determined that the DNA binding domain of protein p6 is located at the N-terminal region, predicted to form an amphipathic alpha-helix. We have obtained, by site-directed mutagenesis, protein p6 mutants in the polar side of the putative helix in which their DNA binding and replication activation properties were impaired or undetectable, in agreement with in vivo results.

Bacillus Phages↗

PubMed1994

Sex differences in the hypothalamo-pituitary-adrenal axis response to inflammatory and neuroendocrine stressors. Evidence for a pituitary defect in the autoimmune disease-susceptible female Lewis rat.

Susceptibility to inflammatory disease in infantile Lewis (LEW/N) female rats seems to be related to their impaired hypothalamo-pituitary-adrenal (HPA) axis response to different inflammatory stimuli, while the relative resistance to this type of disease in Fischer (F344/N) female rats is apparently due to their potent HPA axis response to the same stimuli. In the present study, we attempted to elucidate whether there is an impairment in the HPA axis response in the juvenile female LEW/N rat to inflammatory and noninflammatory stimuli, and also to determine whether the endogenous sex-steroid environment influences the HPA axis function in both strains of rats. For these purposes, juvenile F344/N and LEW/N rats of both sexes were submitted to different treatments: (a) inhalation of normal atmosphere or ether vapors for 1 min (Ether); (b) i.p. injection of vehicle alone or containing CRH (0.5 microgram/rat), arginine vasopressin (AVP; 5 micrograms/rat, angiotensin II (AII; 5 micrograms/rat), insulin (INS; 0.3 IU/rat), bacterial lipopolysaccharide (LPS; 100 micrograms/rat) or snake venom (SV; 100 micrograms/rat). Rats were then killed at different time intervals (in min) after treatments: 20 for Ether, AVP and CRH, 30 for AII, 45 for INS, 60 for SV and 120 for LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1994

Dendritic arbor alterations in the medial superior olivary neurons of neonatally underfed rats.

Golgi-Cox-stained bipolar cells of the medial superior olive (MSO) were analyzed in control and undernourished Wistar strain rats at 12, 20, 30 and 40 days of age. Undernutrition significantly reduced the number of dendrites and the extension of ipsilateral dendritic prolongations, with no effects upon the cross-sectional somal area and minimal alterations in the corresponding contralateral dendritic branches. The data suggest that in underfed rate, afferents from the receptors projecting to the MSO via the anteroventral cochlear nuclei may cause an imblance in the binaural interactions which occur between the axon terminals and the ipsilateral contralateral dendritic arbors of MSO neurons.

Animals↗

PubMed1993

Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid motif "Dx2SLYP" is critical for synthetic activities.

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif "Dx2SLYP" are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3' to 5' exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.

Amino Acid Sequence↗

PubMed1993

Residues of the Bacillus subtilis phage phi 29 transcriptional activator required both to interact with RNA polymerase and to activate transcription.

Regulatory protein p4 from Bacillus subtilis phage phi 29 activates transcription from the viral late promoter, PA3, by stabilizing the binding of RNA polymerase to the DNA as a closed complex. Protein p4-induced DNA bending and direct contacts between p4 and RNA polymerase have been proposed to play a role in P(A3) activation. By site-directed mutagenesis at the carboxyl end of protein p4 we have identified residues that are critical both to interact with RNA polymerase and to activate transcription. Substitution of arginine 120 gives rise to a p4 derivative unable to activate transcription, that can bind to DNA and induce a normal DNA bending, but does not stimulate the binding of RNA polymerase to the promoter and cannot form complexes with RNA polymerase. Modification of the closely located residue leucine 117 had a similar but milder effect. The results obtained suggest that arginine 120 and leucine 117 form part of the activating domain of the protein, and show that direct contacts between protein p4 and RNA polymerase play a critical role in transcription activation. The p4-induced DNA bending is therefore necessary but not sufficient for the activation of the PA3 promoter.

Amino Acid Sequence↗

PubMed1993

In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.

Bacteriophages↗

PubMed1993

Phi 29 DNA polymerase active site. The conserved amino acid motif "Kx3NSxYG" is involved in template-primer binding and dNTP selection.

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif "Kx3NSxYG" has been proposed to form part of the polymerization active site of alpha-like DNA polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.

Amino Acid Sequence↗

PubMed1993

Bacteriophage Nf DNA region controlling late transcription: structural and functional homology with bacteriophage phi 29.

The putative region for the control of late transcription of the Bacillus subtilis phage Nf has been identified by DNA sequence homology with the equivalent region of the evolutionary related phage phi 29. A similar arrangement of early and late promoters has been detected in the two phages, suggesting that viral transcription could be regulated in a similar way at late times of the infection. Transcription of late genes requires the presence of a viral early protein, gpF in phage Nf and p4 in phage phi 29, being the latter known to bind to a DNA region located upstream from the phage phi 29 late promoter. We have identified a DNA region located upstream from the putative late promoter of phage Nf that is probably involved in binding protein gpF. Furthermore, we show that the phage phi 29 protein p4 is able to bind to this region and activate transcription from the phage Nf putative late promoter. Sequence alignment has also revealed the existence of significant internal homology between the two early promoters contained in this region of each phage.

Bacillus Phages↗

PubMed1993

Reversal of the inhibitory effects of calcitonin gene-related peptide (CGRP) and amylin on insulin secretion by the 8-37 fragment of human CGRP.

The 8-37 fragment of human calcitonin gene-related peptide [(8-37)hCGRP] antagonizes the effects of calcitonin gene-related peptide (CGRP) and amylin in a number of tissues. We have studied the influence of (8-37)hCGRP on the effects of both CGRP and amylin on insulin secretion. In the perfused rat pancreas, homologous CGRP and amylin, at 75 pM, exerted comparable inhibitory effects on the insulin response to 9 mM glucose (ca. 70%; P < 0.025). These effects were antagonized by (8-37)hCGRP (1 microM). Our results suggest that CGRP and amylin act on the B-cell, at least in part, through a common receptor.

Amyloid↗