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Biomedical subjects

L Blanco

Publications and source records attributed to L Blanco.

At least 19 recordsLinked to original sources
PubMed2004

[Analysis of respiratory muscle structure and tumor necrosis and insulin-like growth factor expression in chronic obstructive pulmonary disease: are samples valid if obtained during thoracotomy performed because of localized pulmonary neoplasia?].

OBJECTIVE: Various methods have been used to obtain samples to study the structure of human respiratory muscles and the expression of diverse substances in them. Samples are most often obtained from autopsies, from muscle biopsies during thoracotomy performed because of a localized pulmonary lesion (TLL), and from ambulatory thoracoscopic biopsy in patients free of comorbidity (AT). The disadvantage of the first 2 of these methods lies in the possibility of interference from factors related to the patient's death in the first case or from the disease that necessitated surgery in the second. Although AT is free from the disadvantages of the other 2 methods, it is impossible to obtain samples of the diaphragm the principal respiratory muscle with this procedure. The objective of this study was to analyze the fibrous structure of the external intercostal muscle of patients with chronic obstructive pulmonary disease and to quantify the expression of the principal inflammatory cytokine tumor necrosis factor alpha (TNF-alpha)- and of insulin-like growth factor (IGF-1) in the same muscle, comparing the results obtained with TLL and AT samples. METHODS: Prospective and consecutive samples were taken of the external intercostal muscle (fifth space, anterior axillary line) in 15 patients with chronic obstructive pulmonary disease (mean [SD] age 66 [6] years; forced expiratory volume in 1 second 49% [9%] of predicted; PaO2 75 [9] mm Hg). Samples were taken during TLL (8 patients, all with pulmonary neoplasms but carefully selected in order to rule out systemic effects) or TA (7 patients). Patients with serious comorbidity were excluded from the second group. Samples were processed for structural analysis of fibers (immunohistochemical and enzymatic histochemical) and genetic expression of TNF-alpha and IGF-1 (real-time polymerase chain reaction). RESULTS: No differences in the structure of fibers were found between the 2 groups. No differences were observed in the expression of TNF-alpha or IGF-1. CONCLUSIONS: Using rigorous criteria, the TLL method appears to be suitable for studying the structural characteristics and expression of inflammatory cytokines and growth factors in the external intercostal muscle. Moreover, it can also be inferred that TLL is probably also useful for obtaining samples of the diaphragm, a muscle which cannot currently be sampled by any alternative method.

Aged↗
PubMed2003

In vitro susceptibility of 4903 bacterial isolates to gemifloxacin--an advanced fluoroquinolone.

The in vitro activity of gemifloxacin against over 4900 bacterial isolates was determined by microbroth dilution with interpretation in accordance with NCCLS guidelines. Susceptibility results were compared with those for ciprofloxacin, gatifloxacin, levofloxacin and moxifloxacin. Gemifloxacin and the other fluoroquinolones were not affected by either beta-lactamase production or penicillin-resistance in Streptococcus pneumoniae. The MIC90 values for gemifloxacin were: S. pneumoniae 0.063 mg/l; Haemophilus influenzae 0.016 mg/l; Moraxella catarrhalis 0.008 mg/l, methicillin-susceptible Staphylococcus aureus 0.063 mg/l; methicillin-susceptible Streptococcus pyogenes 0.031 mg/l; Enterobacteriaceae 0.031-0.16 mg/l; Pseudomonas aeruginosa 4 mg/l; Neisseria meningitidis 0.008 mg/l. The MIC90 for gemifloxacin was lower than those for the other quinolones tested against S. pneumoniae (ciprofloxacin 2-4 mg/l, gatifloxacin 0.5 mg/l, levofloxacin 1-2 mg/l, moxifloxacin 0.25 mg/l). This study confirms the enhanced potent activity of gemifloxacin against Gram-positive pathogens, its broad-spectrum, Gram-negative activity and indicates that gemifloxacin is likely to have an important role in treating patients with Gram-positive and/or Gram-negative infections.

Anti-Infective Agents↗
PubMed2001

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation.

We have identified a novel polymerase beta (Pol beta)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol beta, shows a nuclear localization that contrasts with the mitochondrial localization of Pol beta from Crithidia fasciculata, a closely related parasite, the only polymerase beta described so far in Trypanosomatidae. Li Pol beta, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol beta-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol beta, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol beta mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol beta at the infective phase of the parasite. The data suggest a role of Li Pol beta in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells.

Amino Acid Sequence↗
PubMed2001

Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair.

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.

DNA Repair↗
PubMed2001

DNA polymerase mu, a candidate hypermutase?

A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.

Amino Acid Sequence↗
PubMed2001

Effect of Salmonella-infected human monocytes on natural killer cell cytotoxicity. In vitro studies.

Various chemicals, including some bacteria-derived components, modulate natural killer cell (NKC) activity. We have analyzed the effect of wild-type Ty2 and of mutant strain TYT1231 Salmonella typhi-infected monocytes (U937 cells and human autologous monocytes) on NKC cytotoxicity of peripheral blood mononuclear cell (PBMC) and highly purified NKC (HPNKC; CD16+/56+ > 95%; the rest corresponding to CD3+ T-cells). PBMC's co-culture with either S. typhi strain infected U937 cells (medium or non-infected U937 cells as controls) resulted in the induction of lymphocyte activated killer (LAK) cell activity showing cytotoxicity against target human NKC-resistant lymphoblastoid Daudi cell line. Comparable experiments using autologous monocytes gave similar results. Co-culture of HPNKC preparations with either S. typhi strain infected U937 cells resulted in increased LAK cell activity against target Daudi cells in each and everyone of the five samples tested; paired Student's t-test p < 0.01 for both times (20 and 40 h) tested. Similar to the results observed in the experiments using PBMC, we did not find significant differences in the ability between medium and non-infected cells, or between wild-type S. typhi Ty2 and mutant strain TYT1231 infected U937 cells, to induce LAK activity in HPNKC preparations. PBMC co-incubation with either S. typhi strain infected U937 cells or autologous monocytes resulted in significant increases in IL-12, TNF-alpha, and IFN-gamma secretion. In similar experiments using HPNKC samples instead, infected U937 cells significantly increased IL-12 and IFN-gamma, but not TNF-alpha secretion. PBMC co-incubation with non-infected U937 cells, but not with non-infected monocytes, significantly increased supernatant IL-12 and TNF-alpha levels (no significant changes in IFN-gamma were recorded). Secreted cytokines remained essentially unchanged after co-incubating HPNKC preparation with non-infected U-937 cells. Incubation of PBMC or HPNKC preparations with either S. typhi strain infected U937 cells failed to produce significant changes in the expression of NKC lineage (CD16+/56+) or activation (CD28+, CD69+ and CD95+) markers. The ability of infected monocytes to induce LAK activity, release NKC cytokines and upmodulate NKC's CD95+ marker expression was essentially the same for both infecting Salmonella strains used. These results suggest a role for NKC in the physiological defensive response against intracellularly infected monocytes representing, perhaps one of the earliest antimicrobial mechanisms of the innate immune system.

Acid Phosphatase↗
PubMed2001

A multicentre evaluation of a new filtration protocol for leucocyte depletion of high-haematocrit red blood cells collected by an automated blood collection system.

BACKGROUND AND OBJECTIVES: A multicentre trial was set up to evaluate the performance of a new leucodepletion protocol. MATERIALS AND METHODS: Filtration at high haematocrit was started during collection of red blood cell (RBC) products by apheresis with Trima. SAG-M was added after filtration through the filter. Haematocrits and haemoglobin of the filtered RBCs were measured. Residual leucocytes were determined by Nageotte counting. RESULTS: One-hundred and forty seven procedures were carried out. The haematocrit and haemoglobin contents were 57.3 +/- 3.0% and 55.1 +/- 4.3 g/unit, respectively. All products showed low residual leucocyte levels (< or = 0.75 x 106/unit; 99.31% < 1 x 106). CONCLUSION: Immediate, on-line, high-haematocrit filtration of red cells collected on Trima resulted in leucoreduced RBCs, which met the AABB and Council of Europe criteria.

Blood Cell Count↗
PubMed2000

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

Amino Acid Motifs↗
PubMed2000

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis.

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.

Amino Acid Sequence↗
PubMed2000

DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells.

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.

Amino Acid Sequence↗
PubMed2000

Analysis of O29 DNA polymerase by partial proteolysis: binding of terminal protein in the double-stranded DNA channel.

ø29 DNA polymerase, which belongs to the family of the eukaryotic type DNA polymerases, is able to use two kinds of primers to initiate DNA replication: DNA and terminal protein (TP). By partial proteolysis we have studied the regions of ø29 DNA polymerase involved in primer binding. With proteinase K, no change in the proteolytic pattern was observed upon DNA binding, suggesting that it does not induce a global conformational change in ø29 DNA polymerase. Conversely, two of the three main cleavage sites obtained by partial digestion of free ø29 DNA polymerase with endoproteinase LysC were protected upon DNA binding, indicating that the DNA could be occluding these cleavage sites to the protease either directly by itself and/or indirectly by induction of local conformational changes affecting their exposure. Partial proteolysis with endoproteinase LysC of ø29 DNA polymerase/TP heterodimer resulted in a protection and digestion pattern similar to that obtained with DNA, suggesting that both primers, DNA and TP, fit in the same double-stranded DNA-binding channel and protect the same regions of ø29 DNA polymerase.

Bacillus Phages↗
PubMed2000

Effect of Salmonella typhi wild type and O-antigen mutants on human natural killer cell activity.

We investigated the effect of glutaraldehyde-fixed Salmonella typhi Ty2 (Vi(-)) wild-type (World Health Organization's vaccine strain) and mutant strains MEI028 (rough, O-antigen(-)) and MEI012 [smooth (O-antigen(+)95%), immunomagnetically isolated NK cell preparations. Incubation of PBMC with each and every one of the S. typhi strains studied consistently and significantly, increased this cellular immune function, as well as the supernatant level of the various cytokines tested e.g. IFN-gamma, TNF-alpha, IL-10 and IL-12 (ELISA). In similar experiments, a significant increase in the cytolytic activity of HPNK cells was elicited by S. typhi Ty2 but not by mutant strain MEI028; neither of the cytokines assayed (IFN-gamma and TNF-alpha) was detected in the supernatant. Our results suggest that S. typhi O-antigen plays an essential role in a mechanism resulting in the direct activation of NK cell activity in HPNK cell preparations. However, the relative quantitative significance of this antigen in the direct stimulation of NK cell cytotoxicity expression in PBMC samples is less clear, as it appears that in this case bacterial-induced monocyte-released cytokines plays a most important role. Incubation with S. typhi Ty2 or MEI028 elicited significant expression of CD69, an early marker of NK cell activation, in PBMC but not in HPNK cell samples (flow cytometry); in similar experiments, the expression of CD16/56 and activation marker CD25 remained essentially unchanged.

Adult↗
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

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

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

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

Diastereomeric Shape Recognition Using NMR Spectroscopy in a Chiral Liquid Crystalline Solvent.

Unambiguous assignment of the resonances of different diastereomers in a nonracemic mixture is possible with (1)H-decoupled (2)H NMR spectroscopy in a chiral liquid crystalline solvent. This is demonstrated with, for example, the alpha,alpha'-dideuterated diol shown in the picture. Even diastereomers with centers of chirality up to nine bonds apart can be discriminated.

Journal Article↗