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P García

Publications and source records attributed to P García.

At least 73 records · Page 4Linked to original sources
PubMed1998

Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells.

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.

Amino Acid Sequence↗
PubMed1998

Pneumococcal bacteriophage Cp-1 encodes its own protease essential for phage maturation.

The major capsid protein of the pneumococcal phage Cp-1 that accounts for 90% of the total protein found in the purified virions is synthesized by posttranslational processing of the product of the open reading frame (ORF) orf9. Cloning of different ORFs of the Cp-1 genome in Escherichia coli and Streptococcus pneumoniae combined with Western blot analysis of the expressed products led to the conclusion that the product of orf13 is an endoprotease that cleaves off the first 48 amino acid residues of the major head protein. This protease appears to be a key enzyme in the morphopoietic pathway of the Cp-1 phage head. To our knowledge, this is the first case of a bacteriophage infecting gram-positive bacteria that encodes a protease involved in phage maturation.

DNA-Binding Proteins↗
PubMed1998

Morphometric and proliferative effects of growth hormone on radiation enteritis in the rat.

UNLABELLED: Radiation enteritis is a common occurrence after radiotherapy in patients with abdominal tumors. Growth hormone may modify the response of the intestinal mucosa to radiation through its effects on the cell cycle or by increasing cell mass. The aim of this study is to determine the effects of growth hormone in the radiation-induced morphoproliferative changes in the intestinal mucosa. MATERIAL AND METHODS: An intestinal mucosal lesion was induced in adult male Wistar rats by means of abdominal irradiation with a lethal dose (LD50) of 1200 cGy. All animals received treatment with either saline or growth hormone for 7 days after irradiation. The animals were sacrificed on day 7. Body weight was determined the morphoproliferative status of the intestinal mucosa was assessed and the disaccharidase activity was measured. RESULTS: Growth hormone reduced body weight loss and increased mucosal length in irradiated rats. Mucosal proliferation was incremented in both irradiated and nonirradiated growth hormone-treated rats. Disaccharidase activity levels were similar to or higher than control values in all treated groups. CONCLUSION: Administration of growth hormone to irradiated rats reduces intestinal injury, probably as a consequence of an earlier recovery of intestinal morphology and functional status.

Animals↗
PubMed1998

Growth hormone reduces bacterial translocation in radiation enteritis in the rat.

BACKGROUND: Radiotherapy may be considered as one of the most effective treatments for digestive tumours. This procedure has major side effects, especially in fast growing tissues like intestinal mucosa. The administration of drugs that reduce or avoid radiation injury of the intestinal mucosa may be clinically advantageous. Growth hormone is a peptide suitable for this purpose by modifying cell proliferation within the intestinal crypt. MATERIAL AND METHOD: Adult male Wistar rats were used in a model of abdominal irradiation. Each irradiated animal received 1200 cGy under anaesthesia and was sacrificed four and seven days later. The animals were treated with either saline or growth hormone (1 mg/kg/day) beginning immediately after the irradiation treatment. On the day of sacrifice, intestinal samples were taken for morphometric measurements and mesenteric lymph nodes for bacterial translocation. RESULTS: Mortality was of 50% approximately and was not affected by growth hormone treatment in irradiated animals. Bacterial translocation increased (p < 0.05) in irradiated animals whereas no significant increase was observed in rats treated with growth hormone. Growth hormone promotes an earlier growth of intestinal villi in irradiated animals (p < 0.05). CONCLUSIONS: Growth hormone promotes the morphologic adaptation of intestinal mucosa after abdominal irradiation, reducing bacterial translocation in rat.

Animals↗
PubMed1997

A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells.

Natural killer (NK) cell-mediated lysis is negatively regulated by killer cell inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules. In this study, we characterize a novel inhibitory MHC class I receptor of the immunoglobulin-superfamily, expressed not only by subsets of NK and T cells, but also by B cells, monocytes, macrophages, and dendritic cells. This receptor, called Ig-like transcript (ILT)2, binds MHC class I molecules and delivers a negative signal that inhibits killing by NK and T cells, as well as Ca2+ mobilization in B cells and myelomonocytic cells triggered through the B cell antigen receptor and human histocompatibility leukocyte antigens (HLA)-DR, respectively. In addition, myelomonocytic cells express receptors homologous to ILT2, which are characterized by extensive polymorphism and might recognize distinct HLA class I molecules. These results suggest that diverse leukocyte lineages have adopted recognition of self-MHC class I molecules as a common strategy to control cellular activation during an immune response.

Amino Acid Sequence↗
PubMed1997

p21WAF1/CIP1 and MDM2 expression in non-Hodgkin's lymphoma and their relationship to p53 status: a p53+, MDM2-, p21-immunophenotype associated with missense p53 mutations.

p53 is a tumour suppressor gene which is often found to be inactivated in most types of human cancer. p53 is a transcription factor, the inactivation of which may lead to significant variations in the levels of p53 downstream proteins, such as p21WAF1/CIP1 and MDM2. In view of the significance of p21WAF1/CIP1 and MDM2 as wild-type (wt) p53 targets, this study was undertaken to monitor the varying expression of these proteins in non-Hodgkin's lymphomas (NHLs) in relation to p53 gene status. A total of 57 cases of different histological types of NHL were included in this study. Proteins p53, p21WAF1/CIP1, and MDM2 were analysed by immunohistochemical techniques, taking the levels expressed in reactive lymphoid tissues as reference points. p53 gene point mutations (exons 5-8) were looked for using the PCR-SSCP technique and direct sequencing. Fifteen of the 57 cases studied showed 16 mutations at the p53 gene: 12 missense, one nonsense, two silent mutations, and one frameshift deletion. Most missense mutations were associated with high levels of p53 protein, while the nonsense mutations and frameshift deletion did not induce detectable levels of p53. All cases with mutation at the p53 gene (15) showed null or low levels of p21WAF1/CIP1 and MDM2 proteins, suggesting that null or missense mutations at this gene give rise to a protein that is unable to transactivate the p21WAF1/CIP1 and MDM2 genes. The association between missense p53 mutation and dissociate immunophenotype (p53+, MDM2-, p21-) was statistically significant (Fisher's exact test, P = 0.0024). This anomalous p53+, MDM2-, p21- phenotype was also found in a small group of five cases with wt p53; this could indicate that in these cases p53 transactivation capacity has been abrogated by a mechanism other than p53 mutation. Most cases with the wt p53 gene show simultaneous immunohistochemical expression of all three proteins and often display higher levels than those found in reactive lymphoid tissue. There is a tendency for EBV-positive cases to harbour high levels of p53+ and p21+, suggesting that EBV could be involved in the nuclear accumulation of p53 and p21WAF1/CIP1 in NHL.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1997

Copper deficiency with pancytopenia due to enteral nutrition through jejunostomy.

Copper deficiency is a rare complication of enteral nutrition. Haematologic abnormalities such as neutropenia and anaemia, but not pancytopenia, have been described associated to copper deficiency. We report the case of a patient requiring long term enteral nutrition through jejunostomy who developed copper deficiency and pancytopenia. In 1991, a 47-year-old woman was admitted with severe gastroesophageal mucositis after an attempted suicide with caustic intake. Enteral nutrition with a commercial, polymeric, fiber-containing formula was started. Twenty-eight months later, the patient developed anemia and neutropenia that did not respond to combined iron and parenteral vitamin B(12) supplementation. In 1996 the patient showed pancytopenia and low serum levels of copper and ceruloplasmin. Pancytopenia improved after copper supplementation. Possible mechanism causing copper deficiency and pancytopenia are discussed. We conclude that assessment of copper status is advisable in patients receiving long-term enteral nutrition by jejunostomy.

Journal Article↗
PubMed1997

[Nifedipine in the treatment of moderate and severe arterial hypertension. Long-term effect on arterial pressure and on the left ventricle].

INTRODUCTION AND OBJECTIVES: Moderate-to-severe hypertension is less prevalent than mild hypertension, but it is responsible for more incidences of complications. Its complex treatment requires several drugs, and is often inadequate. This study assessed the efficacy and safety of nifedipine GITS (oral release osmotic system) as monotherapy, in addition to the effects on left ventricular hypertrophy, after a long term follow-up (one year). PATIENTS AND METHODS: Thirty patients with diastolic blood pressure above 105 mmHg were studied after a short placebo phase. They received nifedipine GITS as monotherapy in a single daily dose of 30 mg; dose titration was made the first three months according to response and until they reached figures equal to or below 95 mmHg. By M-Mode echocardiogram, left ventricular mass index and systolic function were calculated at the end of the placebo phase and at 3, 6, 9 and 12 months. Hematological parameters, lipid profile, electrolytes and liver enzymes were assessed at the same periods. RESULTS: In 70% of the patients the blood pressure reached values of 140-90 mmHg. In 16 patients with adequate M-mode recordings, a 12% reduction in left ventricular mass was observed without modification in systolic function. Five patients were retired: two due to adverse events and three due to different reasons (drop out, evidence for secondary hypertension). There were no changes of clinical significance in the hematological or biochemical parameters and no hypertensive crisis occurred. CONCLUSION: The monotherapy with nifedipine GITS was effective in reducing high blood pressure, induced regression in ventricular hypertrophy and showed good tolerance in one year follow-up.

Adult↗
PubMed1997

Haploidentical peripheral blood stem cell transplantation with a combination of CD34 selection and T cell depletion as graft-versus-host disease prophylaxis in a patient with severe combined immunodeficiency.

Severe combined immunodeficiencies are a group of rare genetic disorders characterized by a profound impairment in both cellular and humoral immune functions. This disorder is rapidly fatal without bone marrow transplantation. Unfortunately, most children lack a histocompatible donor. The development of T cell depletion allows for haploidentical transplantation with reduced risk of GVHD. In the present article, we discuss the case of a child diagnosed with X-linked severe combined immunodeficiency for whom haploidentical peripheral blood stem cell transplantation was performed by selecting CD34-positive cells followed by depletion of T cells. Both selection and purging were performed using immunoselection by the biotin-avidin system (CEPRATE System). The CD34-enriched T cell-depleted product contained 5.05 x 10(6)/kg CD34+ cells with only 0.01 [corrected] x 10(6)/kg CD3+ cells, achieving a T cell depletion of 4.2 log.

Antigens, CD34↗
PubMed1997

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region.

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.

Amino Acid Sequence↗
PubMed1997

Bacteriophages of Streptococcus pneumoniae: a molecular approach.

We have characterized four families of pneumococcal phages with remarkable morphologic and physiological differences. Dp-1 and Cp-1 are lytic phages, whereas HB-3 and EJ-1 are temperate phages. Interestingly, Cp-1 and HB-3 have a terminal protein covalently linked to the 5' ends of their lineal DNAs. In the case of Dp-1, we have found that the choline residues of the teichoic acid were essential components of the phage receptors. We have also developed a transfection system using mature DNAs from Dp-4 and Cp-1. In the later case, the transfecting activity of the DNA was destroyed by treatment with proteolytic enzymes, a feature also shared by the genomes of several small Bacillus phages. DNA replication was investigated in the case of Dp-4 and Cp-1 phages. The terminal protein linked to Cp-1 DNA plays a key role in the peculiar mechanism of DNA replication that has been coined as protein-priming. Recently, the linear 19,345-bp double-stranded DNA of Cp-1 has been completely sequenced, several of its gene products have been analyzed, and a complete transcriptional map has been ellaborated. Most of the pneumococcal lysins exhibit an absolute dependence of the presence of choline in the cell wall substrate for activity, and phage lysis requires, as reported for other systems, the action of a second phage-encoded protein, the holin, which presumably forms some kind of lesion in the membrane. The two lytic gene cassettes, from EJ-1 and Cp-1 phages, have been cloned and expressed in heterologous and homologous systems. The finding that some lysogenic strains of Streptococcus pneumoniae harbor phage remnants has provided important clues on the interchanges between phage and bacteria and supports the view of the chimeric origin of phages.

DNA Replication↗
PubMed1997

The pneumococcal cell wall degrading enzymes: a modular design to create new lysins?

Autolysins are enzymes that degrade different bonds in the peptidoglycan and, eventually, cause the lysis and death of the cell. Streptococcus pneumoniae contains a powerful autolytic enzyme that has been characterized as an N-acetylmuramoyl-L-alanine amidase. We have cloned the lytA gene coding for this amidase and studied in depth the genetics and expression of this gene, which represented the first molecular analysis of a bacterial autolysin. Two observations have been fundamental in revealing further knowledge on the lytic systems of pneumococcus: (a) The well-documented dependence of the pneumococcal autolysin on the presence of choline in the cell wall for activity, and (b) the early observation that most pneumococcal phages also required the presence of this amino-alcohol in the growth medium to achieve a successful liberation of the phage progeny. We concluded that choline would serve as an element of strong selective pressure to preserve certain structures of the host and phage lytic enzymes which should lead to sequence homologies. We constructed active chimeras between the lytic enzymes of S. pneumoniae and its bacteriophages using genes that share sequence homology as well as genes that completely lack homologous regions. In this way, we demonstrated that the pneumococcal lytic enzymes are the result of the fusion of two independent functional modules where the carboxy-terminal domain might be responsible for the specific recognition of choline-containing cell walls whereas the active center of these enzymes should be localized in the N-terminal part of the protein. The modular design postulated for the pneumococcal lysins seems to be a widespread model for many types of microbial proteins and the construction of functional chimeric proteins between the lytic enzymes of pneumococcus and those of several gram-positive microorganisms, like Clostridium acetobutylicum or Lactococcus lactis, provided interesting clues on the modular evolution of proteins. The study of several genes coding for the lytic enzymes of temperate phages of pneumococcus also highlighted on some evolutionary relationships between microorganisms. We suggest that lysogenic relationships may represent a common mechanism by which pathogenic organisms like pneumococcus should undergo a rapid adaptation to an evolving environment.

Bacteriolysis↗
PubMed1997

Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2.

Random Sau3A1 DNA fragments from the temperate Lactobacillus bacteriophage A2 were cloned into the promoter-probe plasmid pGKV210. Seven DNA fragments with promoter activity were selected, after transformation of Escherichia coli and Lactococcus lactis, subsp, lactis, through the chloramphenicol resistance they conferred to the corresponding clones. The seven promoters were functional in Lactobacillus casei. Their strength was analysed by measuring the levels of chloramphenicol resistance and chloramphenicol acetyltransferase activity induced in each host. The nucleotide sequences of these fragments were determined and primer extension analysis was used to locate the initiation site of transcription from each promoter in E coli. The promoters contained -10 and -35 regions similar to the consensus sequences of E. coli and Lactobacillus promoters.

Bacteriophages↗
PubMed1997

[Surveillance of hospital infections: validity of a selective system based on risk markers].

BACKGROUND: In order to achieve simultaneously a higher quality of hospital care and cost-containment, an objective assessment of alternative systems for surveillance of hospital infections is needed in our country. This assessment will allow to make a rational choice of the system to be used at each hospital. METHODS: 719 patients admitted to a Vascular Surgery ward were studied. Sensitivity, specificity, and predictive values for a selective system of surveillance of nosocomial infection (NI) were assessed. This selective system attempted to contain costs by means of limiting the revision of records to those patients who presented fever, prescription of antibiotics after admission or who required laboratory cultures. In this selective method charts revision was retrospectively performed by nurses. RESULTS: A low sensitivity (50.9%) and acceptable values for specificity and predictive values were found. CONCLUSIONS: Although this selective method requires only 20% of the reference method's time, its association with an important reduction in sensitivity precludes its systematic use and confines it eventually to low risk wards.

Anti-Bacterial Agents↗
PubMed1997

[Multiple hepatosplenic abscesses caused by Yersinia enterocolitica in a patient with hemochromatosis].

Yersinia enterocolitica is a gram-negative bacillus that thrives in conditions associated with iron overload. We describe an unusual case of a diabetic patient with a previously unrecognized hemochromatosis presenting with Y. enterocolitica septicemia. He was admitted because of a 10 day history of abdominal pain, fever and jaundice. Blood cultures grew Y. enterocolitica. The abdomen CT scan showed multiple liver and splenic abscesses. Antibiotic treatment with ciprofloxacin (2 months) resulted in a good clinical response. Serum iron studies showed iron overload. Liver biopsy revealed moderate fibrosis and early cirrhosis with large amounts of hemosiderin granules deposited in hepatocytes and bile duct epithelium. This report reviews the literature and highlights that iron overload must be ruled out in Yersinia septicemia patients.

Abscess↗
PubMed1996

Endoreplication in megakaryoblastic cell lines is accompanied by sustained expression of G1/S cyclins and downregulation of cdc25C.

In most eukaryotic cells, a link between S and M phases of the cell cycle must be assured in order to maintain the ploidy of newly divided cells. However, in some cell l/pes, e.g. the precursors of platelets megakaryocytes, extra S-phases can occur in the absence of concomitant mitoses, resulting in polyploidy. We have used two established cell lines with megakaryoblastic characteristics (HEL and MEG-01) to investigate the molecular events that lead these cells to bypass the regular control checkpoints that govern the interdependency of S and M phases. In the presence of the phorbol ester TPA, both cell lines stopped proliferating and displayed additional megakaryocytic features, including polyploidization. Analysis of key cell cycle regulatory factors implicated in the control of G1/S and G2/M transitions revealed a number of differences compared to normally cycling cells. Differentiating megakaryocytes were found to maintain high levels of cdk2, and cyclins E and A. This was accompanied by the appearance of the retinoblastoma protein in the hyperphosphorylated, functionally inactivated form. In addition, TPA-treated cells showed high levels of cyclin B and cdc2 proteins, however no activation of cdc2 was detected. This lack of cdc2 activation which should occur for entry into M phase appeared to be related to the down regulation of cdc25C phosphatase found in both differentiated HEL and MEG-01 cells. Together, our results suggest that in differentiating megakaryoblastic cells endoreplication is accompanied by sustained levels of cyclins A and E, and a lack of cdc2 activation, which is probably mediated through down regulation of cdc25C protein phosphatase.

CDC2 Protein Kinase↗