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Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 181 records · Page 10Linked to original sources
PubMed1991

[Pancreas transplantation in 132 insulin-dependent diabetic patients].

A total of 140 pancreatic transplants have been performed in 132 insulin-dependent diabetic patients at Lyon (France). Most cases received a double pancreatic and renal transplant. The duct obstruction technique was utilized in 113 segmental transplants. A total pancreatic transplant was performed in 27 patients, with duct obstruction in 27, GI tract derivation in 14, urinary tract derivation in 11 and intraductal obstruction in 2 patients. Utilizing quadruple immunosuppression, the one year survival rate was 90% and the transplant survival was 60%.

Actuarial Analysis↗
PubMed1991

[Treatment of cystine calculi using intra-urologic methods and extracorporeal lithotripsy].

The hardness and frequent recurrence of cystine stones represent a special challenge for the urologist. Fifteen cystinuric patients were treated in our department and followed over a mean period of 30 months. Most patients had a previous history of open surgery (1.5 pyelolithotomy/patient). Diagnosis of cystinuria was confirmed by metabolic studies and stone analysis. Over the follow-up period recurrence was observed in 23 instances in 11 patients thus leading to 38 stone treatments on 74 cystine stones. Stone size was less than 10 mm: 35 (47%); 10-20 mm: 21 (28%); 20-30 mm: 14 (19%); 30 mm: 4 (staghorn stones). A percutaneous approach was used in 9 cases as monotherapy (55% success) and in association with ESWL in 10 cases (50% success). ESWL was employed 18 times as monotherapy (39% success). Medical treatment included high fluid intake, alkalinisation and thiola in 6 patients. In conclusion, results obtained are poor in terms of stone clearance when compared to non-cystine stones. Recurrence rate is very high. Instrumental treatment should not be used excessively and is only indicated in symptomatic stones or refractory to intensive medical therapy.

Adolescent↗
PubMed1990

Short N-terminal deletions in the phage phi 29 transcriptional activator protein impair its DNA-binding ability.

The expression of Bacillus subtilis phage phi 29 late genes from the A3 promoter requires the viral protein p4. This protein is a transcriptional activator which binds to a region of the A3 promoter located between nucleotides -56 to -102, relative to the transcription start point. Mutants at the N terminus of protein p4 have been constructed and their function investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation and DNase I footprinting assays. The results indicate that the N terminus of protein p4 could be involved in its binding to the A3 promoter, suggesting that it may not be a typical Cro-like helix-turn-helix DNA-binding protein.

Amino Acid Sequence↗
PubMed1990

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.

Coliphages↗
PubMed1990

Deletions at the N terminus of bacteriophage phi 29 protein p6: DNA binding and activity in phi 29 DNA replication.

Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage phi 29 protein p6. The activity of such proteins in the in vitro phi 29 DNA replication system, their capacity to interact with the phi 29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied. The initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa were removed. The mutant proteins were unable to specifically interact with the phi 29 DNA ends. These results indicate the need of an intact N terminus for the activity of protein p6. However, such N-truncated proteins inhibited both the specific binding of the wt protein p6 to the phi 29 DNA ends and its activity in phi 29 DNA replication.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains amino acid consensus sequences common to other alpha-like DNA polymerases. Using site-directed mutagenesis we have studied the functional significance of the most conserved C-terminal segment mainly represented by the YCDTDS motif. A series of single point mutants has been constructed and the corresponding proteins have been overproduced and characterized. Measurements, on crude fractions, of the activity of the mutant proteins in the formation of the protein p3-dAMP initiation complex and in an in situ DNA polymerase assay, indicate that the YCDTDS domain is involved both in initiation and in elongation reactions.

Amino Acid Sequence↗
PubMed1990

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.

Amino Acid Sequence↗
PubMed1990

A novel nucleoprotein complex at a replication origin.

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.

Bacillus subtilis↗
PubMed1990

Functional domain for priming activity in the phage phi 29 terminal protein.

By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication.

Amino Acid Sequence↗
PubMed1990

Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation.

Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.

Amino Acid Sequence↗
PubMed1990

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.

Amino Acid Sequence↗
PubMed1990

Continent cutaneous Ileostomy (Kock pouch) prior to renal transplantation.

Two patients in end-stage renal failure and with neurogenic bladders due to spina bifida complicated by myelomeningocele were considered for renal transplantation. One patient had had a right nephrectomy and urinary diversion via an ileal conduit; the other, after various external drainage procedures (cystostomy, bilateral nephrostomy), had had a tubular ileocystoplasty. Both underwent 2 surgical procedures prior to renal transplantation: in case 1 we performed a left nephrectomy and then ileal conduit removal + Kock pouch; in case 2 a bilateral nephrectomy was performed via 2 posterior incisions and then we removed the ileocystoplasty and formed the pouch. The continent ileostomy was formed according to the original technique with slight modifications. The patients have been followed up for 12 and 15 months after transplantation.

Acute Kidney Injury↗
PubMed1990

[Autotransplantation in the treatment of isolated dissection of the renal artery].

Isolated dissection of renal artery is uncommon; 150 cases were reported in the literature, whose 2/3 diagnosed during patient's life and 1/3 post mortem, during autopsy. From 1976 to 1988 4 cases of isolated dissection of the renal artery were diagnosed and treated in our Institution. Three men and one woman with age ranging from 39 to 46 yrs (mean age 41.75) were seen at our Institution for intense abdominal pain and blood hypertension (mean blood pressure 196.25/113.75 mmHg) of recent occurrence. In 2 cases angiography showed dissection of the left renal artery, with involvement of peripheral branches; in 1 case the dissection involved the right renal artery with complete occlusion of an upper pole branch and upper pole infarction; our youngest patients presented a bilateral dissection, limited to the main trunk on the right side and involving prepelvic and retropelvic branches on the left side. Surgical treatment consisted of renal autotransplantation in the iliac fossa after extracorporeal reconstruction of the arterial pedicule; a branched hypogastric graft was used in 3 patients; in the patient with bilateral dissection an hypogastric graft was used on the right side and a sapehenous graft on the left side (3 months later). Partial nephrectomy was also performed in the patient having right upper pole infarction. Histologic examination showed that dissection always started from lesions of the lamina media. All patient were alive from 6 to 48 months after renal revascularization, with normalization of blood pressure (mean BP 125/80 mmHg); postoperative angiographic control showed thrombosis of 2 small branches in 1 case and partial stenosis of 3 anastomoses in another patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

[Antibiotic prophylaxis with cefotiam in transurethral resection of the prostate].

UNLABELLED: Over a period of 7 months, 50 patients underwent a trans-urethral resection of the prostate (TURP), this intervention is classified as "clear-contamined". The antibiotic prophylactic protocol, the subject of this study, was the following: cefotiam 2 g IV with the induction of anesthesia, 1 g 3 hours later and 1 g IV with the removal of the urethral catheter. RESULTS: there were 3 early post-operative urinary tract infections, one of these patients was symptomatic, at the time of removal of the urethral catheter. Most of these infections were due to Enterococcus faecalis bacteria. There was 4 late post-operative symptomatic urinary tract infections at days 15, 20, 50, and 120 respectively. All these late infections were due to Streptococcus faecalis. In total, there was a 10% rate of urinary tract infections in the critical 30 days period following the TURP. All the blood cultures were negative. These results lead us to conclude that the short term preventive treatment using the cefotiam, cephalosporin which has a strong prostatic tissue penetration, reduces the rate of post TURP urinary tract infections.

Aged↗
PubMed1989

Functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 DNA polymerase and with DNA.

Deletion mutants at the amino- and carboxyl-ends of the phi 29 terminal protein, as well as internal deletion and substitution mutants, whose ability to prime the initiation of phi 29 DNA replication was affected to different extent, have been assayed for their capacity to interact with DNA or with the phi 29 DNA polymerase. One DNA binding domain at the amino end of the terminal protein has been mapped. Two regions involved in the binding to the DNA polymerase, an internal region near the amino-terminus and a carboxyl-terminal one, have been also identified. Interaction with both DNA and phi 29 DNA polymerase are required to led to the formation of terminal protein-dAMP initiation complex to start phi 29 DNA replication.

Bacteriophages↗
PubMed1989

Effects of internal deletions on the priming activity of the phage phi 29 terminal protein.

A series of internal deletions of gene 3, coding for the phage phi 29 DNA terminal protein, have been constructed and characterized. In addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained. The priming activity of the substitution mutant protein, in the formation of the protein p3-dAMP initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function. Deletions of 8 to 33 aa, from aa residue 48 towards the N terminus of the substitution mutant, further decreased the priming activity of the protein. The activity of deletion mutants lacking 15 or 21 aa from residue 57 towards the C terminus, and also containing a point mutation at position 56, was greatly reduced, and no activity was seen when 24 aa were lacking.

Amino Acid Sequence↗
PubMed1989

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.

Amino Acid Sequence↗