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Biomedical subjects

A Garcia

Publications and source records attributed to A Garcia.

At least 181 records · Page 10Linked to original sources
PubMed1996

Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

Acinetobacter↗
PubMed1995

Effect of deoxycholate on guanine-nucleotide-dependent carbachol stimulation of phosphoinositidase C in mouse brain cortical membranes.

Demonstration of guanine-nucleotide-dependent neurotransmitter stimulation of phosphoinositide breakdown in brain membranes has generally required the presence of the detergent, deoxycholate (DOC), in the assay medium. In the present study, by using mouse brain cortical membranes labelled with [3H]inositol in the presence of CMP through the reverse PtdIns synthase reaction, we have been able to show guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent carbachol (CCh) stimulation of the formation of [3H]inositol phosphates in the absence of DOC and have studied how the detergent affects the response. The results of our study indicate that DOC affects the muscarinic receptor-G-protein-phosphoinositidase C (PIC) transduction system in several ways. First, it enhances agonist-induced PIC activity towards [3H]PtdInsP and [3H]PtdInsP2 and, secondly, it decreases the potency for GTP[S] stimulation of PIC, thus enhancing the agonist-induced leftward shift of the dose-response curve for GTP[S]. Additionally, DOC appears to increase the activity of the enzymes of the phosphoinositide cycle, PtdIns 4-kinase, Ins(1,4,5)P3 5-phosphatase and Ins(1,4)P2 1-phosphatase, thus altering the proportion of phosphoinositide substrates and inositol phosphate products. These observations advise caution in drawing conclusions about PIC substrate specificity and the potency of both guanine nucleotides and agonists from experiments performed in membranes in the presence of DOC or related bile salts.

Animals↗
PubMed1995

Evaluation of T-lymphocyte subsets present in semen and peripheral blood of healthy donors: a report from the heterosexual transmission study.

The purpose of this study was to accurately determine the T-lymphocyte subsets found in semen from healthy volunteers, to evaluate the impact of repeated ejaculation on the frequency or type of immune cells present in semen, and to compare subset analysis in semen to that in the peripheral blood. To accomplish this, a flow cytometric method was developed to identify and count immunophenotypically distinct cells present in semen. Fresh semen samples and peripheral blood were collected over three consecutive days from nine healthy donors. Donors had normal ejaculate volume, sperm count, sperm motility, morphology, and leukocyte count. No significant intra-donor differences were seen in these parameters over time. No significant differences were observed in the percentage of CD3+ cells, CD4+ cells, CD8+ cells, and the CD4:CD8 ratio in semen on consecutive days. However, within the CD4+ subset, when naive and memory CD4+ cells were measured, some day to day variability was suggested. No significant differences in CD3+, CD4+, CD8+, CD4/CD8 ratio, or naive and memory subsets were seen in the peripheral blood between sampling days. When semen was compared to peripheral blood some differences in immune subset values were observed, with an increase in the percentage of memory CD4+ cells in semen being the most striking. This finding may be relevant to HIV transmission, since others have shown that this cell may be preferentially infected with HIV and is the primary reservoir for virus in infected individuals.

Adult↗
PubMed1995

Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A.

The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5' flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-kappa B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5' of a luciferase reporter gene revealed that the 5' flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis.

Amino Acid Sequence↗
PubMed1995

Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

Base Sequence↗
PubMed1995

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

Amino Acid Sequence↗
PubMed1995

The light-harvesting complex II (B800-850) of Rhodobacter sulfidophilus: characterization and formation under different growth conditions.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

Aerobiosis↗
PubMed1995

Complex segregation analysis of familial diseases with variable age of onset: comparison of different methods by a simulation study.

Standard methods of segregation analysis, such as originally developed in the unified model (UM) or the regressive logistic model (RLM) do not account for age of onset, and use instead age at examination. To take into account age of onset, models should be formulated using survival analysis concepts, as it was recently proposed with a model based on a logistic hazard function (LHM). A simulation study was conducted to compare the performances of the three methods (UM, RLM, and LHM) in analyzing generated familial data with variable age of onset. When the data were simulated under a polygenic hypothesis, all analysis models were robust with respect to the false conclusion of a major gene, if the tests of transmission probabilities were performed properly. When the data were generated under a major gene hypothesis, two main results were observed: 1) the use of the LHM markedly increased the power to detect a major gene, in particular when a genotype by age interaction was introduced in the model; and 2) in the situation of disease-specific mortality, the use of either UM (whether specific mortality was accounted for or not) or RLM led to both spurious conclusions and bias in parameter estimates. These latter results obtained with the UM and the RLM can be explained by the violation of one constraint of both models observed in a situation of disease-specific mortality, i.e., given all covariates, the probability of being affected and that of not being affected should sum to 1. The use of methods based on survival analysis concepts is recommended in the study of familial diseases with variable age of onset, especially in the case of a correlation between age of onset and age at examination which is induced by disease-specific mortality.

Adult↗
PubMed1995

Role of leukocyte activation in patients with venous stasis ulcers.

Alteration in leukocyte activation has been implicated as an etiological factor in the development of chronic venous stasis ulcers (CVSU). The purpose of this study was to determine differences in expression of cell surface activation markers on circulating leukocytes and systemic, soluble, serum cytokine levels between healthy controls and patients with CVSU. Twenty-three patients were separated into two groups. Group I consisted of 12 healthy, adult, age-matched male patients with no venous disease. Group II consisted of 11 adult male patients with CVSU who underwent air plethysmography (APG) and duplex scanning to determine the severity of venous insufficiency. All patients had measurements of systemic, serum-based, soluble IL-1 beta, IL-2, IL-6, TNF-alpha, and beta 2 microglobulin levels. Using fluorescence flow cytometry, we measured the percentage of lymphocytes (CD3), monocytes (CD14), and granulocytes (CD15) expressing various cell surface activation markers. By APG and duplex scan, all group II patients exhibited venous insufficiency, with a mean venous filling index of 6.9 +/- 3.9 sec. Relative to group I, group II patients demonstrated a decreased expression of the CD3+/DR+ (13.3 +/- 1.5, P < or = 0.01) and CD3+/CD38+ (31.1 +/- 2.1, P < or = 0.04) markers on T-lymphocytes and an increased expression of CD14+/CD38+ (99.6 +/- 0.2, P < or = 0.008) markers on monocytes. Circulating neutrophils showed no evidence of activation. In addition, a significant elevation in the T-helper to T-suppressor ratio (2.9 +/- 0.6, P < or = 0.0001) between groups I and II was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Hydration in protein crystallography.

Water in close proximity to the protein surface is fundamental to protein folding, stability, recognition and activity. Protein structures studied by diffraction methods show ordered water molecules around some charged, polar, and non-polar (hydrophobic) amino acids, although the later are only observed when they are at the interface between symmetry related molecules in the crystal. Water networks surrounding the protein have been observed for small proteins. Crystallographically observed water molecules are referred to as bound structural water molecules. During crystallographic data analysis, bound water molecules are often treated as though they belong to the protein. Recent developments in the treatment of the bulk solvent contribution to the low order diffraction data allow a better evaluation of the surface structure of the protein and a better localization of bound waters. The mobility of bound waters is studied by means of temperature and occupancy factors. The bulk solvent has relatively large disorder (liquid like) which is represented by liquidity factors. Within this context water layers surrounding the protein have little mobility.

Biophysical Phenomena↗
PubMed1995

Identification and biotyping of Acinetobacter spp. isolated in Chilean hospitals.

Two hundred and eighty-one isolates of Acinetobacter spp. obtained from clinical specimens in hospitals from five Chilean cities were identified to species level and biotyped. Respiratory tract and wound secretions were the main sources of the isolates. Acinetobacter baumannii was the most frequent species (96.8%), followed by Acinetobacter genospecies 3 (2.8%). Twelve different biotypes of A. baumannii strains were found of which biotypes 9, 8 and 6 were the most frequent. Isolates of other biotypes were rare. These results differ from most other Latin American and European countries.

Acinetobacter↗
PubMed1995

Differential MAGE-1 gene expression in two variants of an erythroleukemic cell line (K562).

The MAGE-1 gene encodes an antigen recognized on melanoma cells by autologous cytotoxic T cells. This gene shows a wide range of expression in many human tumors but not in normal tissues except for testes. We used reverse transcription polymerase chain reaction assays to analyze the expression of the MAGE-1 gene in two variants of an erythroleukemic cell line, K562. Comparison of two variants of the K562 cell line in different stages of differentiation showed different patterns of expression of the MAGE-1 gene. The more undifferentiated cell line (K562A) expressed high levels of specific MAGE-1 mRNA, in contrast to K562B, which features of erythroid differentiation, without MAGE-1 expression. Interestingly, we could not modulate MAGE-1 gene expression when in vitro differentiation of K562A was induced with Ara-C. Finally, our data indicate that MAGE-1 expression is not necessary for the maintenance of the transformed phenotype.

Antigens, Neoplasm↗
PubMed1995

Diminished mononuclear cell function is associated with chronic venous insufficiency.

PURPOSE: With clinical progression of chronic venous insufficiency (CVI), dermal infiltration of mononuclear cells increases. Because these cells regulate chronic inflammatory responses and modulate wound healing, cellular dysfunction could explain alterations in wound healing with CVI. The purpose of this study was to determine whether monocytes in patients with CVI are dysfunctional. METHODS: Mononuclear cell function was measured as the degree of proliferation in response to a mitogenic challenge. Fifty patients were separated into four groups: group 1, 14 patients with normal limbs; group 2, 10 patients with class 2 CVI; group 3, 15 patients with active venous ulcers; group 4, 11 patients with healed venous ulcers and current evidence of lipodermatosclerosis. Duplex scanning and air plethysmography correlated with the clinical classification of CVI. Systemically circulating monocytes and lymphocytes were obtained by antecubital venipuncture from groups 1 to 4. Cells were cultured in the presence of staphylococcal enterotoxins A, B, C1, D, and E (mitogens) at 1, 8, 31, and 125 micrograms/well on the basis of previous dose-response experiments. Phytohemagglutinin (PHA), 5 micrograms/well, served as a control mitogen. The dose-response curves indicated that 8 micrograms/well elicited the greatest degree of cell proliferation. Proliferative responses at 8 micrograms/well were analyzed for statistical significance among groups 1 to 4. Comparisons among groups were performed by use of the nonparametric Mann Whitney U post tests and a one-tailed unpaired t test. Results were considered significant at p < or = 0.05. RESULTS: Proliferative responses to PHA indicate that lymphocytes and monocytes from patients with CVI are not globally depressed. However, patients in group 2 did not exhibit the same degree of proliferation to PHA as did groups 1, 3 and 4. Proliferative responses between groups 2 and 1 (44.38 +/- 43.9 vs 118.87 +/- 27.1, p < or = 0.05) and groups 2 and 3 (44.38 +/- 43.9 vs 105.95 +/- 60.99, p < or = 0.05) were significant. Challenges with staphylococcal enterotoxin A and B reveal significant diminution of proliferative responses in groups 2 (42.73 +/- 11.55, p < or = 0.05) and 3 (45.57 +/- 9.1, p < or = 0.05) and groups 3 (36.81 +/- 6.9, p < or = 0.05) and 4 (35.04 +/- 7.5, p < or = 0.05), compared with staphylococcal enterotoxin A controls (68.68 +/- 9.9) and staphylococcal enterotoxin B controls (66.25 +/- 13.56), respectively. A trend of diminished mononuclear cell function with progression of CVI was observed with staphylococcal enterotoxins B, C1, D, and E, strongly suggesting biologic significance. Furthermore, patients with lipodermatosclerosis uniformly exhibited the poorest proliferative responses. CONCLUSIONS: Deterioration of mononuclear cell function is associated with CVI. A trend of diminishing proliferative responses with clinical disease progression is observed and suggests biologic significance. The decreased capacity for mononuclear cell proliferation in response to various challenges may manifest itself clinically as poor and prolonged wound healing.

Aged↗
PubMed1995

Involvement of different types of voltage-sensitive calcium channels in the presynaptic regulation of noradrenaline release in rat brain cortex and hippocampus.

Transmitter release at the nerve terminal is mediated by the influx of Ca2+ through voltage-sensitive calcium channels (VSCCs). Many types of VSCCs have been found in neurons (T, N, L, and P), but uncertainty remains about which ones are involved in neuronal excitation-secretion coupling. Specific ligands for the L- and N-type VSCCs were used to determine which of these subtypes might be involved in the K(+)-evoked [3H]noradrenaline release from superfused rat brain cortical and hippocampal synaptosomes. In cortical presynaptic terminals the 1,4-dihydropyridine agonist Bay K8644 enhanced the K+ (15 mM)-evoked [3H]noradrenaline release. This effect was reversed by the 1,4-dihydropyridine antagonists nimodipine and nitrendipine. The L-type VSCC ligands had no effect on hippocampal synaptosomes. In contrast, the N-type VSCC blocker omega-conotoxin markedly reduced the K(+)-evoked [3H]noradrenaline release in nerve terminals from both regions. Inhibition was greater in hippocampal synaptosomes. When applied together the inhibitory actions of nimodipine and omega-conotoxin were approximately additive. These findings indicate that both L- and N-type VSCCs participate in noradrenaline release in rat brain cortex and suggest that noradrenergic terminals in the two regions examined may have distinct populations of VSCCs: L type in cortex and N type in hippocampus.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

An in vitro procedure for studying enzymic dephosphorylation of phytate in maize-soyabean feeds for turkey poults.

An in vitro method was developed to predict inorganic P release from maize-soyabean poultry feeds containing supplemental phytase (EC 3.1.3.8), and to quantify the effect of acid phosphatase (EC 3.1.3.2), fungal protease (EC 3.4.23.6) and Aspergillus niger cellulase (EC 3.2.1.4) on phytate dephosphorylation. Pepsin (EC 3.4.23.1) and pancreatin digestion periods were preceded by a 30 min pre-incubation at pH 5.25 to simulate digestion in the crop of poultry. Pancreatin digestion was carried out in dialysis tubing, with a ratio of about 1:25 (v/v) between the digesta and dialysing medium, to simulate gradient absorption from the duodenum. The feed:water ratio was kept within physiological limits and a constant proportion of feed weight to digestive enzymes was maintained. There was a linear response to increasing dosages of phytase up to 1000 phytase units (FTU)/kg feed, and to increasing phosphate concentration in feeds. In vivo validation was performed with growing turkeys (1-3 weeks) fed on diets containing 12 g Ca/kg and 0, 500 or 1000 FTU phytase/kg in a factorial arrangement with 0, 1, 2 or 3 g supplemental phosphate/kg (from KH2PO4). After a simple transformation (variable/in vitro P = f (in vitro P)), amounts of P hydrolysed from feed samples by in vitro digestions correlated with 3-week body-weight gain (R 0.986, P < 0.0001), toe ash (R 0.952, P < 0.0001), feed intake (R 0.994, P < 0.0001) and feed efficiency (R 0.992, P < 0.0001). The dephosphorylating ability of phytase in vitro was significantly enhanced (P < 0.05) by the addition of acid phosphatase. Fungal acid protease and Aspergillus niger cellulase also enhanced the dephosphorylation process in vitro.

Acid Phosphatase↗