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Biomedical subjects

B Oliva

Publications and source records attributed to B Oliva.

At least 37 records · Page 2Linked to original sources
PubMed1997

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

Animals↗
PubMed1997

A mathematical model to determine the optimal number of fragments for comparison of bacterial chromosomic macrorestriction patterns.

To our knowledge, although comparison of chromosomic macrorestriction patterns has become one of the most feasible molecular tools of the current microbial taxonomy, a mathematical approach to optimize the choice of a restriction enzyme among the endonucleases tested for such comparison has not been previously described. The coincidence of restriction patterns for two tested bacterial strains with this chosen endonuclease will ensure a high genetic relatedness between them. We report a mathematical model to determine the probability of hazardously obtaining a particular chromosomic macrorestriction pattern by PFGE and to calculate the optimal number of fragments for its comparison. The model presented allows us to determine the optimal number of fragments in order to compare chromosomic restriction patterns. The model calculates this values as a function of the chromosome size and the restriction site length. The model is not useful for choosing a restriction enzyme previous to experimental steps, but as a tool for the choice of the restriction enzyme that yields the lowest probability of hazardously obtaining coincidences of chromosomic patterns. The applicability of this model has been exemplified by determining the optimal number of fragments for some well-characterized bacteria and by comparing these values with those that have been experimentally used.

Bacteria↗
PubMed1997

An automated classification of the structure of protein loops.

Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a non-redundant set of 233 proteins with less than 25% sequence homology (X-ray resolution better than 2.5 A). Loops have been classified into five types (alpha-alpha, beta-beta links, beta-beta hairpins, alpha-beta and beta-alpha) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (phi, psi) are used to define the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 alpha-alpha, 11 beta-beta links, 14 beta-beta hairpins, 13 alpha-beta and 9 beta-alpha) were identified with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and finds other clusters that have hitherto not been described. Consensus sequence patterns were identified for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.

Crystallography, X-Ray↗
PubMed1997

'TransMem': a neural network implemented in Excel spreadsheets for predicting transmembrane domains of proteins.

MOTIVATION: Genomic sequences from different organisms, even prokaryotic, have plenty of orphan ORFs, making necessary methods for the prediction of protein structure and function. The prediction of the presence of hydrophobic transmembrane (HTM) stretches is a valuable clue for this. RESULTS: The program. TransMem, based on a neural network and running on personal computers (either Apple Macintosh or PC, using Excel worksheets), for the prediction and distribution of amino acid residues in transmembrane segments of integral membrane proteins is reported. The percentage of residue predictive accuracy obtained for the set of proteins tested is 93%, ranging from 99.9% for the best to 71.7% for the worst prediction. The segment-based accuracy is 93.6%; 63.6% of the protein set match any of the predicted and observed segment locations. AVAILABILITY: TransMem is available upon request or by anonymous up: IP address: luz.uab.es, directory/pub/ TransMem. It is also placed on the EMBL file server (ftp:/(/)ftp.ebi.ac.uk/pub/software/mac/TransMem ).

Animals↗
PubMed1996

On the sensitivity of MD trajectories to changes in water-protein interaction parameters: the potato carboxypeptidase inhibitor in water as a test case for the GROMOS force field.

A critical evaluation is presented of the sensitivity of the results of molecular dynamics simulations of proteins to changes in the parameters describing water-protein and protein-protein van der Waals interactions in the GROMOS force field. The origin of the van der Waals and electrostatic parameters of the GROMOS standard force field is reviewed, and possible weaknesses are discussed. Four alternate sets of van der Waals parameters for the oxygen types of the GROMOS force field that have been suggested by different authors are then tested against the original force field. Six 500 ps molecular dynamics simulations of the potato carboxypeptidase inhibitor (PCI) in solution using the different parameter sets are analyzed and the results compared with the available X-ray and NMR data. It is shown that the behavior of the molecular system is very sensitive to changes in the van der Waals parameters of the oxygens, especially when affecting the interactions between water and aliphatic or aromatic groups. It is also shown that correction of just the repulsive van der Waals parameter of the water oxygen for its interactions with nonpolar groups is sufficient to correct the main deficiency of the original GROMOS parameter set. Nevertheless, the present study suggest that further refinement of the current parameters is still needed for a proper representation of nonbonded interactions.

Algorithms↗
PubMed1995

SB 205952, a novel semisynthetic monic acid analog with at least two modes of action.

The biological properties of SB 205952, a nitrofuryl oxazole derivative of monic acid, differ from those of the closely related antibacterial agent mupirocin. Compared with mupirocin, SB 205952 has increased antimicrobial potency, an extended spectrum including mupirocin-resistant staphylococci, and rapid bactericidal activity. SB 205952, like mupirocin, is a potent inhibitor of bacterial isoleucyl-tRNA synthetase (IRS) in mupirocin-susceptible organisms but does not inhibit IRS from mupirocin-resistant staphylococci, indicating that SB 205952 has more than one mechanism of action. SB 205952 rapidly inhibits protein, RNA, and DNA syntheses in mupirocin-susceptible and mupirocin-resistant staphylococci. In each case, the effect on RNA synthesis is relaxed by treatment with chloramphenicol, indicating that inhibition of RNA synthesis is probably a secondary consequence of stringent control. It is proposed that SB 205952 possesses one or more mechanisms of action in addition to IRS inhibition, probably mediated by its nitrofuryl component.

Anti-Bacterial Agents↗
PubMed1995

Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci.

The stringent response in Escherichia coli and many other organisms is regulated by the nucleotides ppGpp and pppGpp. We show here for the first time that at least six staphylococcal species also synthesize ppGpp and pppGpp upon induction of the stringent response by mupirocin. Spots corresponding to ppGpp and pppGpp on thin-layer chromatograms suggest that pppGpp is the principal regulatory nucleotide synthesized by staphylococci in response to mupirocin, rather than ppGpp as in E. coli.

Adaptation, Biological↗
PubMed1994

C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase A.

Although the carboxypeptidase A-potato carboxypeptidase inhibitor (CPA-PCI) complex is a well known example of protein-protein interaction, little was known about the basis of its thermodynamic stability. Site-directed mutagenesis has been used to identify key residues in the PCI tail and estimate the contribution of their chemical groups to the binding to CPA. Two deletion mutants were created, one lacking the C-terminal residue of the tail (Gly39) and another one lacking the two C-terminal residues (Val38, Gly39). The last mutant had an inhibition constant for CPA 10(4)-fold higher than that of wild-type PCI, indicating that Val38 is a key residue. The interactions of Val38 with CPA residues contribute 5.4-5.7 kcal mol-1 to the overall stability of the CPA-PCI complex (11.9-12.1 kcal mol-1). A series of PCI point mutants at valine 38 were created, and their inhibition constant for CPA was measured. Two of these mutants with smaller side chains, V38G and V38A, allowed us to estimate that the contribution of the three side chain aliphatic groups of valine 38 to the overall stability of the complex is 3.4-4 kcal mol-1. Another two mutants with larger side chains, V38L and V38I, were constructed, the first being a significantly worse inhibitor than the wild type. These results suggest that only aliphatic groups in positions beta and gamma of residue 38 in PCI (but not those in delta) can establish van der Waals interactions with atoms of the active center of CPA and participate in binding. The energetic contribution of each methyl/methylene group in those positions can be estimated as 1-1.5 kcal mol-1. Our hypothesis is supported by computer simulation analysis.

Carboxypeptidases↗
PubMed1993

Mode of action of the cyclic depsipeptide antibiotic LL-AO341 beta 1 and partial characterization of a Staphylococcus aureus mutant resistant to the antibiotic.

The antibacterial activity of the cyclic antibiotic LL-AO341 beta 1 was examined. The antibiotic was a narrow spectrum agent, effective principally against Gram-positive organisms. The intrinsic insusceptibility of Escherichia coli was due to exclusion of the drug by the outer membrane. The antibiotic was bactericidal against Staphylococcus aureus, and cell death was associated with lysis of the bacteria. The antibiotic did not specifically inhibit the synthesis of DNA, RNA, protein, lipid or peptidoglycan since these synthetic activities continued for several minutes after exposure to lethal concentrations of the antibiotic and then all abruptly ceased between about 8 and 15 minutes post antibiotic exposure. These results are consistent with the cytoplasmic membrane being the primary target for LL-AO341 beta 1. Mutants of S. aureus 8325-4 selected on 10- or 20-times the MIC of LL-AO341 beta 1 occurred spontaneously with a frequency of about 3 x 10(-6). A mutant expressing a 160-fold increase in the MIC of LL-AO341 beta 1 was obtained by exposing cultures to progressively increasing concentrations of the antibiotic. This mutant displayed no cross-resistance to other agents apart from telomycin (a structural analogue of LL-AO341 beta 1), apparently did not modify or degrade LL-AO341 beta 1 and had only a slightly longer doubling time than the parent strain.

Anti-Bacterial Agents↗
PubMed1993

Alpha 2-interferon in B-cell chronic lymphocytic leukemia: clinical response, serum cytokine levels, and immunophenotype modulation.

Fifteen patients with B-cell chronic lymphocytic leukemia (B-CLL) have been treated with alpha 2b-interferon (alpha IFN) for one year (3 mega units subcutaneously three times a week). The hematological response and the modulation of immunophenotype, serum levels of soluble interleukin-2 receptor (sIL-2R) and tumour necrosis factor (TNF) have been monitored. Hematologically 67% of cases were classified as responders, although no complete responses were observed; three cases progressed during treatment, and two patients showed stable disease. Both peripheral lymphocytes and CD24+ cell absolute number significantly decreased after twelve months of IFN treatment (40.7 +/- 17 x 10(9)/l versus 15.8 +/- 6 x 10(9)/l, mean values +/- sd, p < 0.01, and 30.4 +/- 5.5 x 10(9)/l versus 8.1 +/- 2.8 x 10(9)/l, p < 0.05, respectively), while CD24+ cell percentage did not change (72.1% +/- 4.6 versus 67.5% +/- 8.8, p not significant). In the majority of cases myelomonocytic markers (CD11c, CD14, CD11b) transitorily decreased during the treatment. Serum sIL-2R levels, elevated in all cases before IFN treatment, increased in responders. Serum TNF levels decreased in patients showing high values before the treatment. The explanation of these findings and their possible implication are discussed.

Aged↗
PubMed1992

Tet determinants provide poor protection against some tetracyclines: further evidence for division of tetracyclines into two classes.

Atypical tetracyclines were active against Escherichia coli and Staphylococcus aureus strains containing determinants that mediate resistance to typical tetracyclines by efflux (Tet B and Tet K) or ribosomal protection (Tet M) mechanisms. The results support recently published data that tetracyclines are divisible into at least two classes on the basis of their modes of action.

Escherichia coli↗
PubMed1992

Evidence that tetracycline analogs whose primary target is not the bacterial ribosome cause lysis of Escherichia coli.

The modes of action of atypical tetracyclines that do not directly inhibit bacterial protein synthesis were investigated. The analogs tested, chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, were bactericidal and caused the lysis of Escherichia coli accompanied by the release of the cytoplasmic enzyme beta-galactosidase into the supernatant. Examination by electron microscopy demonstrated that cells exposed to these analogs underwent marked morphological alterations that included the formation of numerous ghosts and the appearance of cellular debris in the culture medium. Although atypical tetracyclines promoted lysis in intact organisms, they did not cause lysis of E. coli spheroplasts, indicating that the analogs do not directly destroy the cytoplasmic membrane. These agents may promote cell lysis and death by interfering with the membrane's electrochemical gradient, which in turn leads to stimulation of autolytic enzyme activity and cellular lysis. The results support recently published data which indicate that tetracyclines are divisible into two classes on the basis of their modes of action.

Bacterial Proteins↗
PubMed1991

Stability and fluctuations of the potato carboxypeptidase A protein inhibitor fold: a molecular dynamics study.

A 120ps non-inertial solvent (NIS) molecular dynamics (MD) trajectory of the potato carboxypeptidase A protein inhibitor (PCI) was calculated and analyzed. It is shown that, in spite of a very low content of regular secondary structure, the PCI fold has a large degree of stability, judged from the fairly good agreement between the average MD and X-ray structures. The N-terminal and C-terminal regions behave differently, both in their isoatomic positional shifts with respect to the X-ray structure, and in atomic fluctuation pattern. Positional shifts up to 9A are detected in the exposed N-terminal region as it folds back on the inhibitor's core. This large deviation is most likely caused by the absence of the receptor protein or by the lack of supporting solvent molecules. In contrast, the C-terminal region, which is the primary contact site with the enzyme, has an average structure similar to the X-ray conformation; this feature is probably due to a hydrogen bond network to the central core of PCI. The C-terminal tail shows larger fluctuations than the core. The secondary contact site retains its structure in this simulation. The results evidence an intrinsically stable PCI fold which favors a spatially well defined, fairly flexible, structuration of the primary and secondary contact sites that optimizes PCI's interaction with its target enzyme.

Binding Sites↗
PubMed1991

A molecular dynamics study of a model built Pro-36-Gly mutant derived from the potato carboxypeptidase A inhibitor protein.

A 120ps molecular dynamics (MD) trajectory was calculated and analyzed for a putative Pro-36-Gly mutant of the potato carboxypeptidase A (CPA) protein inhibitor (PCIm). The mutant protein's fold shows a large degree of stability, judged from its low alpha-carbon r.m.s. deviation from the X-ray structure of the wild type PCI (PCIw). The N-terminal tail of PCIm differs slightly less from the X-ray structure than it does in PCIw, while the mutant's C-terminal tail (the primary contact site with CPA) and residues 13-17 present deviations as they approach each other. Differences in fluctuation pattern exist between PCIm and PCIw in residues 2-4 (the N-terminal tail), 13-17, 22-23, 28-31 (the secondary contact site with CPA) and 37-38 (the C-terminal tail); the latter region is rigidified in PCIm. Results show that the MD method is able to sense local perturbative effects produced by amino acid substitutions in flexible regions of protein molecules. The simulation suggests that the conformation of the C-terminal tail is less favorable for interaction with the target protein in the mutant than it is in the wild type protein. The Pro-36-Gly mutant is predicted to be a less potent inhibitor.

Carboxypeptidases↗
PubMed1991

Diagnostic and prognostic relevance of the immunophenotype in acute myelocytic leukemia.

The immunophenotype of 72 cases with acute myelocytic leukemia was investigated with a panel of monoclonal antibodies. When the morphologic criteria of the FAB classification was compared with the normal myeloid and erythroid pathway of differentiation identified by MoAbs, a relationship was found with FAB M5 and M6. Moreover, a constant negativity to HLA-DR and CD15 antigens in M3 and the contemporaneous expression of HLA-DR and CD11b antigens on the M4 and M5 leukemic cells were observed. We identified phenotypically distinct groups of patients with different responses to therapy. In fact, patients whose leukemic cells did not express the HLA-DR antigen showed, in a univariate analysis, a significantly higher percentage of complete remissions than did HLA-DR-positive patients. Multivariate discriminant analysis, in line with this result, showed that the parameters of discriminant capacity were, in order of statistical significance, young age, low WBC count and the lack of DR expression, respectively. A trend for a better response to therapy, without any statistical relevance, was also observed in CD11b-negative and CD33-positive cases. Similar results were detected in patients who expressed either DR or CD11b, or none of these markers. These findings indicate that immunophenotype may identify some FAB subtypes. Moreover, in some cases the phenotypic profile can provide useful information about the clinical outcome.

Adult↗
PubMed1991

Beta-lactamase induction antagonizes beta-lactam susceptibilities in Citrobacter diversus and Enterobacter cloacae clinical isolates.

Inducible beta-lactamases were obtained after exposure to several beta-lactams in clinical isolates of Enterobacter cloacae and Citrobacter diversus. Enzyme production was related to the inducer and medium composition. beta-lactamase is able to inactivate only labile compounds, thus generating minimum inhibitory concentrations higher than in the absence of the inducer; imipenem susceptibilities usually were not changed.

Citrobacter↗
PubMed1991

Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome.

Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.

Bacteria↗