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Biomedical subjects

M Garcia

Publications and source records attributed to M Garcia.

At least 253 records · Page 14Linked to original sources
PubMed1989

Cell-wall-defective variants of Fusobacterium.

The activity of antimicrobial agents against Fusobacterium species has been reported as variable in the literature. For some strains, the inconsistency arises from difficulty in determining the endpoint of growth in agar dilution susceptibility tests. Certain strains persist as a subtle haze beyond the levels of antibiotic that permit conventional colonial growth. We have determined by light and electron microscopy that this haze represents the colonial growth of cell-wall-defective (CWD) variants of the parent Fusobacterium. The CWD forms could be propagated indefinitely in hypertonic medium containing the antibiotic inducing agent. However, when the antibiotic was eliminated, the organisms would revert to their native morphology. Formation of CWD variants was observed in the presence of cell-wall-active drugs (e.g., beta-lactam agents) but not with drugs that work by a different mechanism (e.g., clindamycin or chloramphenicol). Fourteen of 22 F. varium strains, 8 of 11 F. mortiferum strains, 2 of 10 F. gonidiaformans strains, and 1 of 4 of F. necrophorum strains could be induced to a CWD form in vitro in the usual agar dilution susceptibility test. Although the clinical significance of CWD variants of Fusobacterium is unknown, they may be a source of confusion in interpreting agar dilution susceptibility tests.

Anti-Bacterial Agents↗
PubMed1989

Survival of Azorhizobium caulinodans in the Soil and Rhizosphere of Wetland Rice under Sesbania rostrata-Rice Rotation.

The survival of indigenous and introduced strains of Azorhizobium caulinodans in flooded soil and in the rice rhizosphere, where in situ Sesbania rostrata was incorporated before the rice crop, is reported. The azorhizobia studied were both root and stem nodulating. In a pot experiment, two crop cycles each of inoculated and noninoculated Sesbania-rice were compared with two crop cycles of flooded fallow-rice. In a field experiment, the effect of repeated incorporation of in situ S. rostrata in the Sesbania-rice sequence was studied. Soils in which inoculated S. rostrata was incorporated contained about 3,000 times more azorhizobia than did soils in the flooded fallow treatment and about 50 times more azorhizobia than did soils in the noninoculated Sesbania treatment. Azorhizobial numbers in the inoculated Sesbania treatment declined toward rice harvest but remained much higher than in the flooded fallow-rice treatment. Repeated incorporation of S. rostrata increased the population density of indigenous soil azorhizobia, whereas the population of inoculated strain ORS571 (Str Spc) declined to an undetectable level; this finding suggested low competitiveness by the introduced strain. In the incorporated Sesbania treatment, the rice rhizosphere harbored significantly more A. caulinodans and supported higher nitrogenase activity per plant than did the rhizosphere of the flooded fallow-rice treatment. Sterile rice seedlings inoculated with A. caulinodans showed nitrogenase activity comparable to that of seedlings inoculated with Azospirillum lipoferum 34H, a rice root isolate. Rhizobia from Sesbania aculeata, Sesbania sesban, a Trifolium sp., and Vigna unguiculata did not support appreciable nitrogenase activity.

Journal Article↗
PubMed1989

Regulation of cathepsin-D and pS2 gene expression by growth factors in MCF7 human breast cancer cells.

In MCF7 human breast cancer cells, cathepsin-D and pS2 mRNAs are specifically and directly induced by estrogens at the transcriptional level. We studied the regulation of expression of these two genes by growth factors that are also mitogenic in this cell line. We show that pS2 mRNA, like cathepsin-D mRNA, is rapidly induced 2- to 4-fold by epidermal growth factor. The effect of epidermal growth factor on these two mRNAs was dependent upon de novo protein synthesis, indicating a different mechanism of regulation than with estradiol. Other peptide growth factors, such as insulin, insulin-like growth factor I, and basic fibroblast growth factor, also increased up to 3-fold the steady state levels of the two mRNAs in MCF7 cells. The pS2 mRNA, but not cathepsin-D mRNA, was also induced up to 8-fold by protein kinase-C activation with 12-O-tetradecanoylphorbol-13-acetate, suggesting the possible involvement of this transduction pathway in pS2 mRNA induction. The effect of 12-O-tetradecanoylphorbol-13-acetate was time and dose dependent and required protein synthesis. In addition, treatment by agents elevating cAMP increased pS2 mRNA accumulation 4-fold, whereas it had no effect on cathepsin-D mRNA levels. These results demonstrate that cathepsin-D and pS2 genes are under complex regulation in MCF7 cells, since growth factors stimulate their expression via indirect mechanisms contrasting with the primary transcriptional effects of estrogens.

Breast Neoplasms↗
PubMed1989

Two-site immunoenzymometric assay for the 52-kDa cathepsin D in cytosols of breast cancer tissues.

We developed a solid-phase two-site immunoenzymometric assay (IEMA) of the estrogen-induced 52-kDa cathepsin D (EC 3.4.23.5) and its processed forms (48-kDa and 34-kDa proteins) in cytosols of breast cancer tissues, using two monoclonal antibodies directed against two different epitopes of these antigens. The first antibody is bound to a polystyrene microtiter well; the second is labeled with alkaline phosphatase. The assay involves a simultaneous incubation of the antigen with both antibodies, because we observed signal loss during sequential incubations. Alkaline phosphatase was chosen because other enzymes (peroxidase, beta-galactosidase) were inhibited by cytosol extraction buffers. The measurable range of 52-kDa-related proteins is from 0.3 to 6 nmol/L with precision (CVs) within and between runs of 3.9% and 15.8%, respectively. The sensitivity, accuracy, and rapid turnaround time of the two-site IEMA should facilitate the clinical evaluation of this new marker in oncology.

Alkaline Phosphatase↗
PubMed1989

Effect of adenosine in some models of inflammation and anaphylaxis in experimental animals.

Adenosine has controversial effects. The inhalation of this substance causes bronchoconstriction in asthmatic patients, but possesses a protective effect in human inflammatory disease states. We have evaluated the effect of chronic treatment with adenosine (100 micrograms/kg/day), on anaphylactic shock in sensitized mice and guinea pigs and on ozone-induced changes of lung function in rats, taking into account that in these models inflammatory cells and mediators are involved. Adenosine is able to inhibit the increase of wet lung weight to body weight ratio induced by ozone, and to reduce the mortality induced by anaphylactic shock in guinea pigs and mice.

Adenosine↗
PubMed1989

HPLC analysis of supernatant soluble factors from tumor cell variants with defined metastatic status.

In the present study, we report a simple and sensitive procedure to study supernatant soluble factors of short time cultured cells by high performance gel permeation chromatography (HPGPC). This procedure enables a direct analysis with no preparatory procedures prior to application in the chromatographic column and eliminates tedious and often low reproducible techniques, required during conventional assay of culture medium. In addition, we show that HPGPC analysis is suitable for discriminating between two variants of a rhabdomyosarcoma with different metastatic potential, while two distinct cell lines (3LL carcinoma and B16F10 melanoma), which do not differ in their metastatic potential, do not present noticeable qualitative differences in their chromatographic secretion pattern.

Animals↗
PubMed1989

Body weight, tick burden (Boophilus microplus), physiological parameters and reproductive efficiency of crossbred zebu cattle.

Body weight at calving, degree of tick load (Boophilus microplus), rectal temperature, respiratory frequency and heart rate were evaluated and correlated to reproductive efficiency in 63 F1 Brown Swiss x Nellore milked animals reared in the tropical region of Peru. Cows were bred by free natural service, and first ovulation was estimated by progesterone measurements. The physiological parameters were registered at morning milking (04.00 to 06.00) at an average air temperature of 22.4 +/- 0.9 degrees C. The intervals from parturition to first ovulation and conception were significantly affected when the body weight at calving was lower than 400 kg (p less than 0.01). The degree of tick burden, rectal temperature (38.4 +/- 0.2 degrees C, mean +/- s.e.), respiratory rate (25.2 +/- 0.3 breaths/min) and heart rate (66.7 +/- 0.4 beats/min) were not related to post-partum reproductive efficiency. The average intervals from parturition to first ovulation and to conception were 56.3 +/- 55.5 and 77.7 +/- 46.0 days mean +/- s.d.), respectively. The fertility rate was 85.7%. The results indicate the F1 cross to be well adapted to the given environment. It remains to be determined whether higher levels of temperate blood can be used without an accompanying decrease in reproductive performance.

Animals↗
PubMed1988

Sequential study of the synaptonemal complex in rat (Rattus norvegicus) oocytes by light and electron microscopy.

The progression of first meiotic prophase and synaptonemal complex (SC) formation in female rats, Rattus norvegicus S.D., is described through the analysis of the different stages of the first meiotic prophase, and confirms the high synchrony of the process in this species. Leptotene is a stage of very short duration and since pairing of the homologues begins very early, only a leptotene-zygotene stage can be distinguished. The progression of pairing during zygotene is asynchronous. The morphology of the SCs is similar to that described in other species. During diplotene and before disintegration of the lateral elements, desynapsis takes place. In some oocytes a double or even multiple nature of lateral elements was seen. Associations between SCs and nucleoli or nucleolar filaments are frequent. The presence of fragmented SCs can be interpreted as a technical artifact.

Animals↗
PubMed1988

Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells.

The estrogen-induced 52K protein secreted by human breast cancer cells is a lysosomal protease recently identified as a pro-cathepsin D by sequencing several cDNA clones isolated from MCF7 cells (Augereau et al., Mol. Endocr.). Using one of these clones, we detected, in MCF7 cells, a 2.2 kb mRNA whose level was rapidly increased 4- to 10-fold by estradiol, but not by other classes of steroids. Other mitogens, such as epidermal growth factor and insulin, also induced the 2.2 kb mRNA in a dose-dependent manner. Induction with epidermal growth factor was as rapid but was 2- to 3-fold lower than with estradiol. Antiestrogens had no effect on the 52K-cathepsin-D mRNA in MCF7 cells, but became estrogen agonists in two antiestrogen-resistant sublines R27 and LY2. The use of transcription and translation inhibitors and nuclear run-on experiments indicate that estradiol enhances transcription of the 52K-cathepsin-D gene in MCF7 cells.

Breast Neoplasms↗
PubMed1988

Immunoenzymatic assay of Mr 52,000 cathepsin D in 182 breast cancer cytosols: low correlation with other prognostic parameters.

The Mr 52,000 cathepsin-D-like protease induced by estrogens in MCF7 human breast cells was assayed in 182 primary breast cancer cytosols prepared for receptor assays from pre- and post-menopausal patients. Using two solid-phase sandwich immunoenzymatic assays, we quantified the total Mr 52,000 cathepsin D (52K-cath-D) (the Mr 52,000 precursor protein and its Mr 48,000 and 34,000 processed forms) and the Mr 52,000 precursor alone. The value of total 52K-cath-D varied between 3 and 165 pmol/mg protein and the proportion of the precursor varied from 0 to 28% of total 52K-cath-D. There was no correlation between the concentrations of 52K-cath-D and estrogen receptor, but the estrogen receptor status (greater than or less than 10 fmol/mg protein) was correlated to the 52K-cath-D status (greater than or less than 15 pmol/mg protein) according to the chi 2 test (P less than 0.001). The correlation with progesterone receptor concentrations and status was low (r = 0.43) and absent, respectively. There was no correlation with Scarff and Bloom stages, tumor size, or patient's age. The percentage of patients with invaded lymph nodes was significantly higher (80%) in the subgroup with the highest total 52K-cath-D levels (greater than or equal to 42 pmol/mg protein), representing only 12% of the population but not in the total population. On the basis of this prospective study, before clinical follow-up can be evaluated, we conclude that in the total population examined, the 52K-cath-D concentration was only correlated with estrogen receptor status, but not with any other prognostic parameter.

Aged↗
PubMed1988

Attempted suicide in youth: its relationship to school achievement, educational goals, and socioeconomic status.

Twenty-six suicide attempters, 9 to 18 years of age, were compared with 725 nonattempters on measures of school achievement, educational goals, socioeconomic status, and depression. As has been found in studies using clinical samples, suicide attempters in our community-based sample had significantly lower school achievement than nonattempters. The relationship between attempted suicide and low school achievement seemed to be explained by the effects of depression. Lower-SES youth tended to be more likely to attempt suicide than higher-SES youths. However, SES appeared to have little effect on the relationship between attempted suicide and school achievement. Results also suggest that children of mothers with low educational goals for them may be more at risk for attempted suicide than youths whose mothers have higher educational goals for their children.

Achievement↗
PubMed1988

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells.

In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1988

Cloning and sequencing of the 52K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11.

Two lambda gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screened by expression with monoclonal antibodies to the secreted 52K protein and with a 36-mer oligonucleotide derived from the N-terminal amino acid sequence of the secreted 52K protein. Four overlapping clones were sequenced, and found to be extensively homologous to the cathepsin D of normal human kidney, except for 5-point mutations resulting in one amino acid change (Ala to Val) in the profragment of cathepsin D. Northern blot analysis showed the 2.2 kilobase (kb) cathepsin D mRNA to be induced by estradiol in MCF7 cells and produced constitutively at high levels in the estrogen-receptor-negative BT20 cell line. A simple restriction pattern consistent with the restriction map of cathepsin D cDNA was obtained in Southern blot analysis of MCF7 cell DNA. In situ hybridization of the 52K-9 cDNA probe on normal lymphocytes assigned the 52K cathepsin D gene at the extremity of the short arm of chromosome 11, in the p15 band, close to the H-ras gene and in the region whose deletion increases the risk of invasive breast cancer. We conclude that the estrogen induced 52K protein has the same sequence as normal pro-cathepsin D and we propose that the 52K protein correspond to the only pro-cathepsin D expressed in MCF7 cells.

Breast Neoplasms↗
PubMed1988

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine.

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.

3,3'-Diaminobenzidine↗