Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 19 recordsLinked to original sources

PubMed1999

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Arginase↗

PubMed1999

Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.

The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head-on. However, head-on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery. Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase.

Bacillus Phages↗

PubMed1999

Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase.

Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.

Catalysis↗

PubMed1999

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi29 early-late transcriptional switch.

Protein p6 is a nonspecific DNA-binding protein occurring in high abundance in phage phi29-infected cells. Here, we demonstrate a novel role for this versatile histone-like protein: its involvement in regulating the viral switch between early and late transcription. p6 performs this role by exhibiting a reciprocal functional interaction with the regulatory protein p4, also phage encoded, which is required for repression of the early A2b and A2c promoters and activation of the late A3 promoter. On the one hand, p6 promotes p4-mediated repression of the A2b promoter and activation of the A3 promoter by enhancing binding of p4 to its recognition site at PA3; on the other, p4 promotes p6-mediated repression of the A2c promoter by favoring the formation of a stable p6-nucleoprotein complex that interferes with RNA polymerase binding to PA2c. We propose that the observed interplay between proteins p6 and p4 is based on their DNA architectural properties.

Bacillus Phages↗

PubMed1999

Oligomeric structures of the phage phi29 histone-like protein p6.

Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro. Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core. Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding. In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing. The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure. The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process. The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described. We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone.

Bacillus Phages↗

PubMed1999

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Bacillus Phages↗

PubMed1999

The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G.

The transcription program of the Bacillus phage GA-1, a distant relative of phage Phi29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the Phi29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the Phi29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased significantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to Phi29 regulatory protein p4, and was named p4G. In Phi29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4Gwas localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4Gprevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in Phi29, had no effect on the expression of the late A3 promoter: RNA polymerase could efficiently bind and initiate transcription from the A3 promoter in the absence of protein p4G. Therefore, activation of late transcription occurs differently in GA-1 and Phi29. We propose that protein p4Gis an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain.

Amino Acid Sequence↗

PubMed1999

A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.

Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx2SLYP". Mutants in phi29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two million-fold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase.

Bacteriophages↗

PubMed1999

Confounding by indication: an example of variation in the use of epidemiologic terminology.

Confounding by indication is a term used when a variable is a risk factor for a disease among nonexposed persons and is associated with the exposure of interest in the population from which the cases derive, without being an intermediate step in the causal pathway between the exposure and the disease. However, in the literature, the term confounding by indication is not always used consistently. The authors found three different situations in which the term has been applied or might have been used but was not: confounding by indication as protopathic bias, as confounding by severity, or as a form of selection bias. It might be helpful to limit use of the term confounding by indication to the situation in which the disease that forms the indication acts as a confounder irrespective of its severity and to apply the term confounding by severity if the severity of this disease acts as a confounder. Protopathic bias and selection bias should not be confused with these terms. The use of appropriate terms ultimately will improve communication among researchers and contribute to the clarity of their papers.

Confounding Factors, Epidemiologic↗

PubMed1999

Phage phi29 terminal protein residues Asn80 and Tyr82 are recognition elements of the replication origins.

Initiation of phage phi29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the phi29 terminal protein (TP) and the phi29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various phi29 TP mutants were generated at conserved amino acid residues from positions 61 to 87. In vitro phi29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with phi29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.

Amino Acid Sequence↗

PubMed1999

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.

Catalysis↗

PubMed1999

Effect of mutations in the "extended -10" motif of three Bacillus subtilis sigmaA-RNA polymerase-dependent promoters.

The "extended -10" motif described originally in Escherichia coli promoters occurs frequently in other bacterial promoters. Most Bacillus subtilis bacteriophage o29 promoters contain this motif. To analyse the influence of the motif on sigmaA-RNA polymerase transcription, the 5'-TG-3' dinucleotide was changed to 5'-AC-3' in three o29 promoters. This change impaired RNA polymerase binding to the promoters; the yields of closed and open complexes were reduced independently of other differences inherent to each promoter. The mutation abolished transcription in vitro from a promoter lacking the consensus sequence at the -35 hexamer. In contrast, at other promoters with a -35 consensus sequence, the yield of run off transcription was not reduced by the mutation. Indeed an apparent interference phenomenon at high polymerase/DNA ratios was relieved. These results indicate that the extended -10 motif provides contact points for sigmaA-RNA polymerase with a role restricted to the first steps of transcription.

Bacillus subtilis↗

PubMed1999

Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication.

We have analyzed the functional significance of the phi29 DNA polymerase "YxGG/A" motif in initiation and replication reactions involving the terminal protein (TP) as a primer. This motif, located between the proposed limits of the polymerase and exonuclease domains, has been shown to be very important for the coordination between synthesis and degradation in phi29 DNA polymerase. Mutations in this region affected the polymerization/exonucleolysis (pol/exo) balance, due to its importance for DNA template binding stability at both active sites. Here, we show that the YxGG/A motif of phi29 DNA polymerase is necessary for the formation of a stable complex between TP and phi29 DNA polymerase, affecting initiation and transition during replication of phi29 TP-DNA. The phenotypes in TP-primed reactions in nine of 11 mutant polymerases, showed reduced initiation and/or replication activities using TP-DNA as template. High dATP concentrations allowed the reduced initiation activities of some of these mutant polymerases to reach the wild-type level. The reduction in their affinity for the initiating nucleotide is likely due to their reduced interaction with the TP. Besides, the YxGG/A motif of phi29 DNA polymerase controls the pol/exo balance in the transition step immediately after TP-primed initiation, before DNA polymerase and TP dissociate. Thus, from the first elongation step, the phenotypes of the mutant polymerases parallel those obtained in DNA-primed replication: wild-type, high and low pol/exo balance. A detailed analysis of different transition intermediates suggests that mutants at the YxGG/A motif switch from interaction with TP to DNA once the TP has been extended with six nucleotides.

Amino Acid Sequence↗

PubMed1999

Persistence with treatment for hypertension in actual practice.

BACKGROUND: Despite the existence of efficacious medications, many patients in actual practice remain with uncontrolled hypertension. Randomized clinical trials, cannot address this issue well given their highly restricted environment. This paper examines persistence with antihypertensive therapy among patients in actual practice. METHODS: Cohort study of patients who received a diagnosis of hypertension and were treated between 1989 and 1994 identified through the Saskatchewan Health databases. Patients with concurrent diagnoses likely to affect initial treatment choice were excluded. The resulting population of 79,591 subjects was grouped into those with established hypertension (52,227 [66%]) and those with newly diagnosed hypertension (27,364 [34%]). The initial antihypertensive prescription, subsequent changes in treatment and persistence with antihypertensive therapy were analysed. RESULTS: Persistence with antihypertensive therapy decreased in the first 6 months after treatment was started and continued to decline over the next 4 years. Of the patients with newly diagnosed hypertension, only 78% persisted with therapy at the end of 1 year, as compared with 97% of the patients with established hypertension (p < 0.001). Among those with newly diagnosed hypertension, older patients were more likely than younger ones to persist, and women were more likely than men to persist (p < 0.001). INTERPRETATION: This analysis of actual practice data indicates that barriers to persistence occur early in the therapeutic course and that achieving successful therapy when treatment is started is important to maintaining long-term persistence.

Adult↗

PubMed1999

Effect of initial drug choice on persistence with antihypertensive therapy: the importance of actual practice data.

BACKGROUND: Rational medical decisions should be based on the best possible evidence. Clinical trial results, however, may not reflect conditions in actual practice. In hypertension, for example, trials indicate equivalent antihypertensive efficacy and safety for many medications, yet blood pressure frequently remains uncontrolled, perhaps owing to poor compliance. This paper examines the effect of initial choice of treatment on persistence with therapy in actual practice. METHODS: The authors examined all outpatient prescriptions for antihypertensive medications filled in Saskatchewan between 1989 and 1994 by over 22,000 patients with newly diagnosed hypertension whose initial treatment was with a diuretic, beta-blocker, calcium-channel blocker or angiotensin-converting-enzyme (ACE) inhibitor. Rates of persistence over the first year of treatment were compared. RESULTS: After 6 months, persistence with therapy was poor and differed according to the class of initial therapeutic agent: 80% for diuretics, 85% for beta-blockers, 86% for calcium-channel blockers and 89% for ACE inhibitors (p < 0.001). These differences remained significant when age, sex and health status in the previous year were controlled for. Changes in the therapeutic regimen were also associated with lack of persistence. INTERPRETATION: A relation not seen in clinical trials--between persistence with treatment and initial antihypertensive medication prescribed--was found in actual practice. This relation also indicates the importance of real-world studies for evidence-based medicine.

Adult↗

PubMed1999

Development of play behavior in neonatally undernourished rats.

The effects of neonatal food and sensory deprivation on play social behavior (boxing, wrestling, and pinning) were studied in male and female Wistar strain rats from 20 to 60 days of age. Data showed that the mean frequency of total play was markedly increased in neonatally underfed subjects. Play did also increase in the females and during the interaction in pairs and in the play that occurred during the prepuberal period. These findings suggest that early food restriction and the unavoidable sensory deprivation associated to the undernourishing procedure, interfere with the neuroendocrine maturational processes of central and peripheral modulatory mechanisms underlying play behavior.

Aging↗

PubMed1999

Effects of neonatal undernutrition and binaural ear occlusion on neuronal development of the superior olivary complex of rats.

Golgi-Cox-impregnated neurons of the superior olivary complex were investigated in control, binaural ear-occluded control, undernourished, and binaural ear-occluded undernourished Wistar rats of 12, 20, and 30 days of age. In neonatally undernourished and binaural ear-occluded animals, a significant reduction in the number of dendrites and dendritic intersections in the medial trapezoid body neurons was observed. Inconsistent effects upon the dendritic arbor lateral superior olivary cells were also detected. Control and undernourished rats with binaural ear occlusion exhibited a significant increase in the dendritic arbor measurements of lateral superior olivary neurons as compared with the cells of the medial trapezoid body. The cytoarchitectonic imbalance between the modulatory efferent control of these systems and the sensory auditory input to the cochlea may be important for the integration of complex auditory physiological phenomena.

Aging↗

PubMed1998

Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication.

The gene 17 of the Bacillus subtilis phage phi29 is known to be involved in the viral DNA replication in vivo. In this paper, we show that the presence of protein p17 is required when phage infection occurs at a low multiplicity of infection (moi), which is probably the natural condition for infection, but is dispensable at a high moi. Gene 17 has been cloned in an Escherichia coli expression vector and protein p17 purified. A stimulatory effect of protein p17 was demonstrated under in vitro conditions required to amplify phi29 DNA, starting with a low amount of input DNA. We propose that p17, which is synthesized early after infection, is required at the very beginning of the phage amplification, conditions in which a low number of viral DNA molecules enter the host cell, possibly to recruit the limiting amount of initiation factors at the replication origins. Once the infection process is established and the other replication proteins reach optimal concentration, p17 becomes dispensable.

Bacillus Phages↗