Biomedical subjects

M Garcia

Publications and source records attributed to M Garcia.

At least 145 records · Page 8Linked to original sources

PubMed1996

Formation and dissolution of spinules and changes in nematosome size require optic nerve integrity in black bass (Micropterus salmoides) retina.

Teleost retinas adapted to light show numerous spinules invaginated in the cone pedicles and small nematosomes in the distal horizontal cells. Darkness induces the dissolution of spinules and the presence of large and numerous nematosomes. The aim of this work is to study the influence of optic nerve integrity on spinule formation/dissolution and changes in nematosome size during light or dark adaptation of black bass (Micropterus salmoides) retinas. Eyes from fish, dark- or light-adapted, were removed and the eyecups placed in oxygenated Ringer's solution and immediately exposed to light or dark, respectively, for 1 h. The number of spinules per pedicle and the nematosome diameter were measured on electron micrographs. Isolation of eyecups in the dark, impaired both spinule formation and nematosome size reduction when they were superfused in light. In the same way, isolation of eyecups in the light, impaired both spinule dissolution and nematosome size increase when they were superfused in dark. No significant differences in spinule number and nematosome size, following dopamine superfusion, were found in comparison to retinas superfused with Ringer's solution only. Our results suggest: (1) optic nerve integrity is necessary to yield spinule formation/disruption and changes in nematosome size during light or dark adaptation. (2) dopamine does not appear to be the primary agent responsible for spinule formation.

Adaptation, Ocular↗

PubMed1996

Biological and clinical significance of cathepsin D in breast cancer metastasis.

Cathepsin D (cath-D) is an aspartyl lysosomal protease expressed in all tissues. Most metastatic breast cancer cell lines, unlike normal cells, secrete high levels of pro-cath-D. This abnormal secretion is due to both overexpression of the cath-D gene and to an altered processing of the precursor protein. Cath-D gene transcription is increased by estrogen and growth factors in estrogen-receptor-positive breast cancer cells and by an unknown mechanism in estrogen-receptor-negative cells. A large number of independent clinical studies associated high cath-D concentrations in the cytosol of primary breast cancers with increased risk of subsequent metastasis. The amino acid sequence of cath-D analyzed in two breast cancer cell lines is normal, but glycosylation appears to be different with more acidic isoforms. To assess the potential role of this protease in cancer metastasis, we transfected a human cDNA cath-D expression vector in 3Y1-Ad12 embryonic rat tumorigenic cells which did not secrete the proenzyme. A moderate overexpression of human cath-D was sufficient to increase the metastatic potential of these cells in nude mice. The mechanism of cath-D-induced metastasis seems to require maturation of the proenzyme, in endosomes and in large acidic compartments identified as phagosomes. Rather than increase cancer cell escape from the primary tumor through basement membrane degradation as proposed for neutral proteinases, cath-D appears to facilitate cell growth at distant sites. The mechanism of this indirect mitogenic effect is discussed from results obtained in different models. Different cath-D substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.

Animals↗

PubMed1996

An Arg-Lys insertion at the hemagglutinin cleavage site of an H5N2 avian influenza isolate.

Recent isolations of H5N2 subtype avian influenza (AI) viruses in North America have raised questions concerning their origin, transmission to commercial poultry, and potential for virulence. One ratite-origin isolate of low pathogenicity, A/emu/TX/39924/93 (H5N2), was subjected to a procedure that rapidly selects and/or amplifies highly pathogenic (HP) strains. The resulting highly virulent derivative had an altered hemagglutinin (HA) gene containing an additional six nucleotides at position 970-975 in the HA1 coding region. This resulted in an arg-lys insertion near the proteolytic cleavage site of the HA protein. The remainder of the HA sequence differed by an additional seven amino acids from the parent. The HA precursor of the derivative, but not the parent, was readily cleaved during replication in cell culture without addition of trypsin. In experimentally infected chickens, the derivative produced lesions typical of highly pathogenic avian influenza. A reverse transcriptase-polymerase chain reaction (RT-PCR) primer set was designed to amplify exclusively from molecules with the inserted six nucleotides. The set yielded product only from the selected derivative samples and not the parent. Thus, the levels of the HP variants in the parent stock were undetectable, or the insertion occurred rapidly during the selection process.

Animals↗

PubMed1996

Retinoid regulation of human cathepsin D gene expression.

Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration-dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect.

Base Sequence↗

PubMed1996

Assessment of mitral annular dynamics during diastole by Doppler tissue imaging: comparison with mitral Doppler inflow in subjects without heart disease and in patients with left ventricular hypertrophy.

The purpose of this study was to determine the normal pattern and magnitude of mitral annular velocities in diastole by Doppler tissue imaging (DTI) and to assess whether this is altered in patients with left ventricular hypertrophy. Mitral annulus velocities were measured by DTI. Peak and time-velocity integral were measured from the DTI tracings and the timing of the velocities in relation to electrocardiogram. DTI was compared with M-mode echo of the annulus and mitral inflow Doppler velocities. Integrated annular velocities by DTI correlated with the annular displacement. Early diastolic velocities decreased with age and in patients with left ventricular hypertrophy. In the hypertrophy group, early diastolic velocities were significantly lower than normal even after correcting for age. Patients with left ventricular hypertrophy also showed a delay in peak early diastolic mitral annular velocity (5.5 +/- 21 msec after the E wave). In conclusion, mitral annular velocity in diastole is readily recorded by DTI. The magnitude and the pattern of these velocities are significantly altered by age and by left ventricular hypertrophy. This method provides a new insight into diastolic filling events and may prove useful in detecting abnormal diastolic function.

Adult↗

PubMed1996

Synthesis, lipophilic derivatization and interaction with liposomes of HAV-VP3 (102-121) sequence by using spectroscopic techniques.

Hepatitis A virus (HAV) is composed mainly of three structural capsid proteins: VP1, VP2 and VP3. Our group has reported the synthesis and the immunogenic evaluation of VP3 (110-121) peptide sequence. In the present work, in order to stimulate a T-cell immune response, we have selected the HAV-VP3 (102-121) peptide which has maximum amphipathicity. Its synthesis was carried out manually in the solid phase and semipreparative HPLC was used for purification of the crude peptide. Finally the purified peptide was characterized by analytical HPLC, amino acid analysis and MS. A palmitoyl derivative of VP3 (102-121) was synthesized to modify the hydrophobicity of the peptide. Both free and lipophilically derivatized peptides were incorporated into multilamelar liposomes. Physicochemical studies of the HAV-related peptides described above were carried out using monolayers as membrane models. Compression isotherms, surface activity and penetration kinetics into dipalmitoylphosphatidylcholine monolayers were determined. Moreover, changes in the fluidity of bilayers induced by these peptides were determined by means of polarizable probes such as 8-anilino-1-naphthalenesulfonic acid and 1,6-diphenyl-1,3,5-hexatriene. The integrity of the membranes has also been ascertained with the carboxyfluorescein.

Amino Acid Sequence↗

PubMed1996

Investigation of a multiyear multiple critical care unit outbreak due to relatively drug-sensitive Acinetobacter baumannii: risk factors and attributable mortality.

From 1990 to 1993, an outbreak of respiratory Acinetobacter baumannii infection occurred in five intensive care units (ICUs) of a tertiary care center. A. baumannii was subsequently isolated from disinfected temperature probes and ventilator circuits. Pulsed-field gel electrophoresis suggested that a single strain accounted for 93% of patient isolates and 88% of environmental isolates. Univariate risk factors for A. baumannii acquisition were tracheostomy (P < .01), ventilation >3 days (P < .01), dialysis (P = .03), Stenotrophomonas maltophilia respiratory colonization (P = .02), parenteral nutrition (P = .05), and enteric feeding (P < .01). Logistic regression analysis showed duration of ventilation and enteric feeding to be independent risk factors. The outbreak strain was relatively antibiotic-susceptible, but the mortality attributable to respiratory A. baumannii acquisition was 23%. Only the APACHE II score was independently associated with increased mortality. Multifaceted control measures, including gas sterilization of temperature probes, terminated the outbreak.

Acinetobacter Infections↗

PubMed1996

Phenotypic characterization and regulation of the nolA gene of Bradyrhizobium japonicum.

Two Bradyrhizobium japonicum nolA mutants were constructed and used to test the functional role of NolA in nodulation. Contrary to the previous hypothesis that NolA acts as a repressor of nod gene transcription, the expression of a nodD1-lacZ or nodY-lacZ fusion in the nolA mutant strains was similar to that found in the wild type. However, NolA does appear to act as a transcriptional regulatory protein since it is required for its own expression, as well as that of nodD2. Expression of NodD2 from a constitutive promoter led to a significant reduction in nodC-lacZ activity. Therefore, the repression of nod gene expression by NolA is likely an indirect effect, perhaps mediated by other genes (e.g., nodD2) that are regulated by NolA. When inoculated onto soybean roots, the nolA mutant strains showed only a slight delay in nodulation as compared to the wild type. However, the mutant strains were grossly defective in nodulation and nitrogen fixation on cowpea plants. Microscopic examination of soybean nodules induced by the nolA mutant strains showed developmental and morphological characteristics similar to nodules formed by the wild type with only a slight delay in bacteroid maturation. In contrast, cowpea nodules induced by the nolA mutant strains contained fewer infected cells and bacteroids were not found in a typical symbiosome structure. These results indicate that NolA is a transcriptional activator required for the expression of genes that play a role not only in the early stages of infection, but also during the later stages of bacteroid development and maintenance.

Bacterial Proteins↗

PubMed1996

PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats.

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.

Animals↗

PubMed1996

Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin.

In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.

Caco-2 Cells↗

PubMed1996

Alterations and role of human cathepsin D in cancer metastasis.

Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.

Amino Acid Sequence↗

PubMed1996

Interaction of cytotoxic T lymphocytes and guinea pig ventricular myocytes. Pharmacological modulation by blocking K+ currents in cytotoxic T lymphocytes.

Infiltrating cytotoxic T lymphocytes (CTLs) are important immune effectors that damage the myocardium during heart transplant rejection as well as in cardiomyopathy and Chagas' heart disease. We have previously shown that in an in vitro model of murine-derived peritoneal exudate CTL (PEL)-guinea pig ventricular myocyte interaction, PEL induced in conjugated myocytes reduction of resting membrane potential and action potential (AP) amplitude, shortening of AP duration, delayed afterdepolarizations (DADs), and myocyte contracture and destruction. Since these findings indicated that cytotoxicity was largely caused by [Ca2+]i overload, in the present study we tested the hypothesis that blocking the L-type Ca2+ current (ICa,L) in the myocyte will eliminate the trigger for Ca2+ release from intracellular stores and will reduce [Ca2+]i overload and subsequent myocyte deterioration. CoCl2 (3 mmol/L) prevented PEL-induced AP changes, induction of DADs, and myocyte destruction. Since verapamil (2 mumol/L) was ineffective, indicating that the CoCl2 protection was not due to block of ICa,L, we tested whether the different action of these Ca2+ channel blockers was due to their differential effect on the PEL's K+ current (IK), previously shown to participate in lymphocyte activation and cytotoxicity. In agreement with their protective efficacy, CoCl2 but not verapamil blocked IK in PELs, suggesting that this is the mechanism for the protection provided by CoCl2. To support this notion, we tested the effect of the scorpion-derived peptide margatoxin (10 nmol/L), a specific K+ channel blocker in lymphocytes, on PEL-myocyte interaction and on PEL's IK; margatoxin prevented PEL-induced cytotoxicity and also blocked IK in PEL. Based on these findings, an alternative modality for attenuating CTL-induced lymphocytotoxicity is proposed.

Animals↗

PubMed1996

Detection of Mycoplasma pneumoniae by polymerase chain reaction in lung aspirates from patients with community-acquired pneumonia.

STUDY OBJECTIVE: This study was designed to evaluate the usefulness of polymerase chain reaction (PCR) to detect Mycoplasma pneumoniae DNA in samples obtained by transthoracic needle aspiration (TNA). DESIGN: Prospective study of cases. SETTING: A university hospital in Lleida, Spain. PATIENTS: A total of 101 unselected patients, admitted between January 1993 and March 1994 in the emergency department, with a clinical and radiologic picture of community-acquired pneumonia, and without contraindications for TNA application. INTERVENTIONS: Patients were studied with conventional diagnostic techniques for community-acquired pneumonia. In addition, a sample obtained by TNA was processed by the following methods: culture in standard media, culture in selective media for Legionella, detection of capsular antigens for Streptococcus pneumoniae and Haemophilus influenzae, and detection of M pneumoniae specific genome by PCR. RESULTS: Serologic data were not available in eight patients and were excluded from this analysis. M pneumoniae PCR amplification was possible in eight cases, well correlated with serologic responses indicating current infection. Samples from ten additional patients, negative by PCR, were found to be demonstrative of recent M pneumoniae infection by serologic study. Finally, in all the remaining 75 cases, including the 59 patients for whom a different microbial diagnosis was established, M pneumoniae PCR test gave negative results. CONCLUSION: This study indicates that PCR, applied to samples obtained by TNA, appears to be a moderately sensitive and highly specific method for rapid detection of M pneumoniae lung infection.

Adult↗

PubMed1996

Immunological detection of lipopolysaccharide antigens of thermophilic campylobacters captured on polymyxin-coated polyester cloth.

Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth. The captured antigens were detected by sequential reactions with rabbit anti-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate. A polyclonal rabbit antibody elicited against a single Campylobacter-jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme. Moreover, LPS antigens of six C. Jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes. The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.

Animals↗

PubMed1996

[Value of echocardiography in the study of aortic valve homografts and autografts].

Although increasingly used, the echocardiographic parameters of aortic valve homografts and autografts have not been extensively investigated. The aim of this study was to assess the value of transthoracic (TTE) and transoesophageal (TOE) echocardiography in the preoperative assessment of these patients, especially with regards to prediction of homograft size and to describe the normal and pathological echocardiographic appearances of this type of prosthesis. Thirty-seven consecutive patients were studied. Before surgery, the diameter of the aortic ring (24.1 +/- 3 mm), correlated well with the value measured by the surgeon (24.4 +/- 2 mm; r = 0.9), but in 2 cases, this measurement was impossible and in 3 cases inaccurate, the difference between the 2 measurements being greater than 2 mm. It was possible to measure the aortic ring diameter in all cases by TOE. After surgery, TTE showed normal function of 33 of the 37 prosthetic valves with minimal or no obstruction (mean gradient 5.9 +/- 4 mmHg, aortic surface area = 2.8 +/- 0.5 cm2) with no significant difference between the homografts and autografts. Better visualisation of the homograft leaflets was possible by TOE and detected minimal central regurgition in 16 (84%) of the homografts examined, the persistence of an annular abscess in 2 patients and a minimal aorto-left atrial fistula in 1 patient. In conclusion. TTE coupled with Doppler examination is usually adequate for preoperative selection of the homografts and haemodynamic evaluation and follow-up of these patients. TOE should be performed preoperatively in patients with unsatisfactory TTE studies but is mainly useful for preoperative evaluation and postoperative follow-up of patients operated for aortic endocarditis with paravalvula abscess.

Adolescent↗

PubMed1996

Cell cycle-independent regulation of p21Waf1/Cip1 and retinoblastoma protein during okadaic acid-induced apoptosis is coupled with induction of Bax protein in human breast carcinoma cells.

Okadaic acid (OA) is a serine/threonine protein phosphatase inhibitor and has been shown to induce apoptosis in a number of different tumor cell lines, including human breast carcinoma (HBC) cells. The molecular basis of OA-induced apoptosis remains to be investigated. Here, we demonstrate that the OA concentration that inhibits only protein phosphatase 1 and 2A was sufficient to induce apoptosis in HBC cells. In MCF-7 cells, the OA-induced apoptosis was coupled with the overexpression of endogenous p53, p21Waf1/Cip1, and Bax proteins, whereas the Rb protein levels were decreased. OA also induced apoptosis and concomitantly enhanced the p21Waf1/Cip1 and Bex levels in human papilloma virus protein E6-transfected variants of MCF-7 cells, in which p53 function had been disrupted. OA, by contrast, had no effect on the levels or the subcellular localization of Gadd45 and Bcl2 proteins in either wild-type of E6-transfected MCF-7 cells. Bcl-xL, Bcl-xS, and Bak levels were also unchanged after OA treatment in both cell types. OA-induced apoptosis and its effect on the expression of the above molecular markers occurred in the absence of any detectable changes in the cell cycle phase distribution. On the basis of our findings, we conclude the following: (a) OA-induced apoptosis in HBC cells occurs independently of cell cycle arrest; (b) the wild-type p53 function is not an absolute prerequisite for OA-induced cell death; and (c) OA-induced apoptosis is associated with up-regulation of endogenous p21Waf1/Cip1 and Bax protein levels.

Apoptosis↗

PubMed1996

[Abnormal origin of the left coronary artery from the pulmonary artery. Diagnostic contribution of ultrasonographic technique].

We report a case of abnormal origin of the left coronary artery from the pulmonary artery trunk. The original features of this case were its discovery in a young pregnant woman, the normality of basal left ventricular function and the electrocardiogram, the diagnosis by transoesophaegeal echocardiography, and the demonstration of severe myocardial ischaemia by stress echocardiography.

Adult↗

PubMed1995

Overexpression of p21WAF1/CIP1 induces growth arrest, giant cell formation and apoptosis in human breast carcinoma cell lines.

The p21WAF1/CIP1 protein was shown to be a critical downstream effector of p53 and a potent inhibitor of cyclin-dependent kinases. We investigated the effects of exogenous p21WAF1/CIP1 in two different human breast carcinoma (HBC) cell lines MCF-7 and T47D. p21WAF1/CIP1 transfected cells formed significantly reduced number of drug-resistant colonies than their mock-transfected counterparts. The majority of the p21WAF1/CIP1 transfected MCF-7 cells acquired 50-100-fold increase in their sizes and persisted as single giant cells for several days without cell division, while the remainder underwent nuclear division (multiple nuclei) but were unable to complete cytokinesis, and finally all succumbed to apoptosis. Only few p21WAF1/CIP1 transfected T47D cells displayed increase in their sizes while a number of them formed small sized drug-resistant colonies which diminished in size due to cell death. The cells in these shrinking colonies exhibited characteristic signs of apoptosis such as chromatin condensation and fragmented nuclei while their membrane integrity was preserved. Thus in HBC cell lines the growth suppression due to enforced overexpression of p21WAF1/CIP1 is associated with giant cell formation and apoptosis.

Apoptosis↗