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Biomedical subjects

R Muñoz

Publications and source records attributed to R Muñoz.

At least 73 records · Page 4Linked to original sources
PubMed1997

Mechanism and location of atrial flutter in transplanted hearts: observations during transient entrainment from distant sites.

OBJECTIVES: This study was designed to elucidate the location and mechanism of typical atrial flutter in the transplanted heart. BACKGROUND: Although the F wave morphology in atrial flutter is similar in nontransplanted and transplanted hearts, the surgical incision needed for the atrial anastomosis may create a distinct electrophysiologic substrate of atrial flutter. METHODS: Entrainment from the lateral wall of the right atrium and interatrial septum was used to determine the location of atrial flutter in five patients with a transplanted heart and six patients with a nontransplanted heart. The difference between the first postpacing interval (FPPI) and the flutter cycle length (FCL) was used as an index of proximity to the circuit. RESULTS: In the transplant group, the FPPI was equal to the FCL at sites located close to the tricuspid annulus (TA); the mean differences (+/-SD) were 1 +/- 5 and -1 +/- 2 ms at the lateral wall and interatrial septum, respectively. However, from sites close to the surgical incision at the lateral wall and at the interatrial septum, these differences were significantly longer (29 +/- 12 and 27 +/- 9 ms, respectively, p < 0.05). In the nontransplant group, the FPPI was similar to the FCL at points in the lateral wall and interatrial septum close to the TA (mean difference 7 +/- 6 and 6 +/- 11 ms, respectively) and at sites close to the crista terminalis (CT) in the lateral wall (mean difference 4 +/- 4 ms). However, in sites separated from the TA at the interatrial septum the difference was markedly longer (35 +/- 11 ms, p < 0.05). CONCLUSIONS: Atrial flutter in transplanted hearts may best be explained by macroreentry around the tricuspid ring. In non-transplanted hearts a different structure (perhaps the CT?) may be the basis for atrial flutter at the lateral wall.

Atrial Flutter↗
PubMed1997

Molecular organization of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae: formation of binary encapsulated pneumococci and identification of cryptic dTDP-rhamnose biosynthesis genes.

We report here the molecular organization of the capsular locus (cap1) of the type 1 pneumococcus. This locus is located between dexB and aliA and flanked by IS1167 insertion elements. Sequence analysis showed that the cluster contains 11 genes (cap1A to cap1K), which are apparently arranged as a single transcriptional unit. The presence of a functional promoter (cap1p) located upstream of cap1A has been demonstrated and the transcription start point was mapped by primer-extension analysis. A 14.3 kb fragment containing the genes cap1ABCDEFGHIJK and including cap1p was sufficient to allow the synthesis of a type 1 capsule in Streptococcus pneumoniae. An internal deletion of cap1E leads to an unencapsulated phenotype demonstrating that this gene is essential for capsular production. The cap1K gene has been expressed in Escherichia coli resulting in UDP-glucose dehydrogenase (UDP-GlcDH) activity. Moreover, this gene was able to restore the synthesis of type 3 capsule when cloned into a plasmid and introduced by transformation into S. pneumoniae cap3A mutants deficient in UDP-GlcDH. In marked contrast with what was previously thought, recombination between cap1K and cap3A does occur. We provide data on the molecular mechanism that leads to the formation of binary encapsulated pneumococcal cells, i.e. strains that simultaneously produce type 1 and type 3 capsules. Downstream of cap1K, one truncated and three complete open reading frames homologous to those involved in the biosynthesis of dTDP-rhamnose, a monosaccharide that does not participate in the formation of type 1 polysaccharide, have been identified in all the clinical strains of type 1 pneumococcus tested. Our results provide new insights into the generation of capsule diversity in pneumococci.

Bacterial Capsules↗
PubMed1997

Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance.

Streptococcus pneumoniae is uniquely sensitive to amino alcohol antimalarials in the erythro configuration, such as optochin, quinine, and quinidine. The protein responsible for the optochin (quinine)-sensitive (Opts, Qins) phenotype of pneumococcus is the proteolipid c subunit of the FzeroF1 H(+)-ATPase. OptR/QinR isolates arose by point mutations in the atpC gene and produce different amino acid changes in one of the two transmembrane alpha-helices of the c subunit. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opts) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis (OptR) revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin, quinine, and related compounds specifically inhibited the membrane-bound ATPase activity. Equivalent differences between Opts/Qins and OptR/QinR strains, both in growth inhibition and in membrane ATPase resistance, were found. Pneumococci also show a characteristic sensitivity to coumarin drugs, and a relatively high level of resistance to most quinolones. We have cloned and sequenced the gyrB gene, and characterized novobiocin resistant mutants. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates. This residue position is equivalent to Val-120 of Escherichia coli ryGB, a residue that lies inside the ATP-binding domain but is not involved in novobiocin binding in E. coli, as revealed by crystallographic data. In addition, the genes encoding the ParC and ParE subunits of topoisomerase IV, together with the region encoding amino acids 46 to 172 (residue numbers as in E. coli) of the pneumococcal ryGA subunit, were characterized in respect to fluoroquinolone resistance. The gyrA gene maps to a physical location distant from the gyrB and parEC loci on the chromosome. Ciprofloxacin-resistant (CpR) clinical isolates had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level CpR), or in both resistance-determining regions of ParC and GyrA (high-level CpR). Mutations were found in residue positions equivalent to Ser-83 and Asp-87 of the E. coli GyrA subunit. Transformation experiments demonstrated that topoisomerase IV is the primary target of ciprofloxacin, DNA gyrase being a secondary one.

Amino Acid Sequence↗
PubMed1997

A functional analysis of the Streptococcus pneumoniae genes involved in the synthesis of type 1 and type 3 capsular polysaccharides.

Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a beta (1-->3) linkage. Cellobiuronic acid is a disaccharide consisting of D-glucuronic acid (GlcA) beta (1-->4) linked to D-glucose (Glc). The genes implicated in the biosynthesis of the type 3 capsule have been cloned, expressed, and biochemically characterized. The three type 3-specific genes--designated as cap3ABC--are transcribed together. However, the two complete open reading frames located upstream of cap3A are not transcribed and, consequently, are not required for capsule formation. The promoter of the cap3 operon was localized by primer extension analysis. The products of cap3A, cap3B, and cap3C were biochemically characterized as a UDP-Glc dehydrogenase, the type 3 polysaccharide synthase, and a Glc-1-P uridyltransferase, respectively. The Cap3B synthase was expressed in Escherichia coli, and pneumococcal type 3 polysaccharide was synthesized in this heterologous system. When a recombinant plasmid (pLSE3B) containing cap3B was introduced by transformation into encapsulated pneumococci of types 1, 2, 5, or 8, the lincomycin-resistant transformants displayed a binary type of capsule, this is, they showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2) laboratory strains of S. pneumoniae also synthesized a type 3 capsule when transformed with pLSE3B. On the other hand, we have cloned and sequenced seven type 1-specific genes (designated as cap1A-G), and their functions have been preliminarily assigned based on sequence similarities.

Amino Acid Sequence↗
PubMed1997

Pneumocystis carinii infection in heart transplant recipients. Efficacy of a weekend prophylaxis schedule.

Most series of heart transplant patients report incidences of Pneumocystis carinii pneumonia (PCP) below 5% but do not individually describe the cases. From August 1988 to March 1994, 138 patients received 1 or more heart transplants at our institution. No anti-PCP chemoprophylaxis was provided, and 5 (3.6%) patients developed PCP. Incidence for listeriosis was 0.7% and for nocardiosis, 3.6%. We found descriptions of 14 more heart transplant patients with PCP in the medical literature. Data from the 19 patients follow. Mean age was 52 years, and PCP was diagnosed a median of 75 days after heart transplant (range, 37-781 d). Clinical presentation was acute (less than 48 h) with fever (89%), shortness of breath (84%), dry cough (74%), and hypoxia (63%). Cytomegalovirus was isolated from lung or blood in 74% of patients. Chest X-ray usually showed interstitial pneumonia (84%). Three patients required ventilatory support. All patients were treated with trimethoprim-sulfamethoxazole (TMP/SMX) (4 also with corticosteroids and 5 with ganciclovir). Mortality was 26%. Older age was the only significant poor prognostic factor (61 versus 49 years; p < 0.03). From March 1994, 50 heart transplant patients were given TMP/SMX prophylaxis at our institution (1 double-strength tablet, 160/800 mg, every 12 hours on Saturdays and Sundays), and no new cases of PCP, Listeria or Nocardia have been detected since then. Tolerance has been excellent. Heart transplant recipients are at a substantial risk of PCP pneumonia, which presents with an abrupt onset and a high mortality. Weekend TMP/SMX chemoprophylaxis was very effective at our institution.

Adult↗
PubMed1996

Cytogenetic study of neuroendocrine carcinoma of Merkel cells.

We describe the cytogenetic study of a neuroendocrine tumor of Merkel cells which appeared in a patient following a heart transplant. An abnormal karyotype was observed in a metastatic lymph node. The abnormality includes two markers derived from the long arm of chromosome 1, while maintaining two normal chromosomes 1.

Aneuploidy↗
PubMed1996

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.

Amino Acid Sequence↗
PubMed1996

Ciprofloxacin-resistant Haemophilus influenzae strains possess mutations in analogous positions of GyrA and ParC.

The nucleotide sequences of the quinolone resistance-determining regions of the gyrA and parC genes from five ciprofloxacin-resistant strains of Haemophilus influenzae (MICs, 2 to 32 micrograms/ml) isolated from patients with cystic fibrosis and three ciprofloxacin-susceptible strains of H. influenzae (MICs, < or = 0.1 micrograms/ml) were determined. Four of the five resistant strains possessed at least one amino acid substitution in each of the GyrA and ParC fragments studied. The mutations identified in GyrA were a serine at residue 84 (Ser-84) to Leu or Tyr and Asp-88 to Asn or Tyr. ParC mutations were in positions exactly analogous to those identified in GyrA, namely, Ser-84 to Ile and Glu-88 to Lys. The Glu-88 to Lys ParC substitution was identified only in high-level ciprofloxacin-resistant strains. These mutations have been shown to be the origin of the observed resistance after transformation into ciprofloxacin-susceptible H. influenzae isolates. These results suggest that H. influenzae isolates require at least one amino acid substitution in both GyrA and ParC in order to attain significant levels of resistance to quinolones.

Amino Acid Sequence↗
PubMed1996

Quinine specifically inhibits the proteolipid subunit of the F0F1 H+ -ATPase of Streptococcus pneumoniae.

Streptococcus pneumoniae is uniquely sensitive to quinine and its derivatives, but only those alkaloids having antimalarial properties, i.e., those in the erythro configuration, also possess antipneumococcal activity. Quinine and related compounds inhibit the pneumococcal H+ -ATPase. Quinine- and optochin-resistant pneumococci showed mutations that change amino acid residues located in one of the two transmembrane alpha-helices of the c subunit of the F0F1, H+ -ATPase.

Amino Acid Sequence↗
PubMed1996

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.

Cell Division↗
PubMed1996

Recent advances in the uses and applications of ribosome-inactivating proteins from plants.

Plant ribosome-inactivating proteins (RIPs) are inhibitors present in all parts of plants that irreversibly inactivate eukaryotic ribosomes, thus impairing protein synthesis. RIPs are enzymes with N-glycosidase activity on the large rRNA. Their powerful inhibitory activity has been made use of advantageously to construct conjugates with suitable carriers targeted to altered specific cells. RIPs may be used to inhibit replication of both animal and plant viruses. The introduction of genes coding for RIPs into the genome of plants leads to an increase in resistance towards fungal pathogens and viruses. RIPs are important tools for the treatment of cancer and AIDS and for the protection of crop production.

Antiviral Agents↗
PubMed1996

Differential sensitivity of HELA cells to the type 2 ribosome-inactivating proteins ebulin l, nigrin b and nigrin f as compared with ricin.

The new type 2 RIPs ebulin l, nigrin b and nigrin f present in Sambucus display toxicity to HELA cells several orders of magnitude lower than that displayed by ricin. Despite N-terminal amino acid homology between the three RIPs in both the A and the B chains, these compounds display very different degrees of toxicity to HELA cells that does not seem to be paralleled by immunologic correlations. It is suggested that small changes in the protein structure are most probably responsible for the different degrees of toxicity.

Cross Reactions↗
PubMed1995

Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

3T3 Cells↗
PubMed1995

Tuberculosis in heart transplant recipients.

We present an analysis of the incidence, clinical presentation, and evolution of tuberculosis in heart transplant recipients at a 2,200-bed tertiary care center in Madrid and review the world literature. During a 5-year period (1989-1993), active extrapulmonary tuberculosis was diagnosed in three of the 144 patients who survived heart transplantation, resulting in an incidence of 1.35 cases per 100 heart transplant-years (> 20-fold the national average). The mean age of the patients was 52 years, and two were male. The mean time to development of tuberculosis after transplantation was 76 days (range, 55-102 days). All of the patients had had previous episodes of rejection and infection and had had initially negative tuberculin tests (one converted to positive during therapy). Clinical manifestations were mild or absent in two of the patients, and Mycobacterium tuberculosis was isolated in association with other microorganisms from two patients. All patients were successfully treated with antituberculous chemotherapy while they were receiving immunosuppressants. A severe drug interaction between cyclosporine and rifampin in the first case necessitated withdrawal of rifampin and precluded its use in subsequent patients. During a mean follow-up of 2 years, no recurrence of tuberculosis has been detected in any of the patients. Tuberculosis was diagnosed in a fourth patient before transplantation, which was performed while the patient was receiving antituberculous therapy. Our data support the conclusion that heart transplantation should be considered an unheralded risk factor for tuberculosis, particularly in countries where this disease is prevalent.

Antitubercular Agents↗
PubMed1995

Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance.

We report the cloning of the gyrB gene from Streptococcus pneumoniae 533 that carries the nov-1 allele. The gyrB gene codes for a protein homologous to the gyrase B subunit of archaebacteria and eubacteria. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates of S. pneumoniae. This amino acid position is equivalent to Val-120 of Escherichia coli GyrB, a residue that lies inside the ATP-binding domain as revealed by the crystal structure of the protein.

Amino Acid Sequence↗
PubMed1995

A new procedure for the preparation of highly active melonin from the dry seeds of Cucumis melo L.

Melon (Cucumis melo L.) dry seeds contain melonin, a protein that strongly inhibits ribosomes from different prokaryotic and eukaryotic sources including those from melon. The protein was purified by a new method to yield highly active and stable protein preparations that involves chromatography through S-Sepharose Fast Flow, CM-Sepharose, Superdex 75 and Mono-S. Melonin shows important functional properties: 1) its inhibitory effects on translation were irreversible; 2) it is a single unglycosylated polypeptide chain with an apparent M(r) of 22000; 3) it degrades RNA in a dose-dependent way without affecting DNA. In the light of present results melonin can be considered as a new plant RNase of unusual properties.

Animals↗