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L Valenzuela

Publications and source records attributed to L Valenzuela.

At least 19 recordsLinked to original sources
PubMed2001

TOR modulates GCN4-dependent expression of genes turned on by nitrogen limitation.

In Saccharomyces cerevisiae, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability. One of the mechanisms by which TOR regulates cell proliferation is by excluding the GLN3 transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein. We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability. The results presented in this paper indicate that GCN4 plays a role in the rapamycin-sensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls GCN4 activity by regulating the translation of GCN4 mRNA. This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.

DNA-Binding Proteins↗
PubMed1999

Composition and antimicrobial activity of the essential oil of Peumus boldus leaves.

The composition and the antimicrobial activity of the essential oil from the leaves of Peumus boldus is investigated. Analyses of the oil obtained by hydrodistillation were carried out by GC and GC-MS using columns of two different stationary phases. Fractionation of the essential oil by column chromatography on silica gel was performed to improve identification of some constituents. More than 90% of the total oil (46 components) was identified, major constituents being monoterpenes (90.5%), among which limonene (17.0%), p-cymene (13.6%), 1.8-cineole (11.8%), and beta-phellandrene (8.4%) reached the highest percentages. Determination of the minimal bactericidal or fungicidal concentration against several microorganisms showed interesting activities towards Streptococcus pyogenes, Micrococcus sp., and Candida sp.

Anti-Infective Agents↗
PubMed1998

Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.

Base Sequence↗
PubMed1997

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.

Ammonium Sulfate↗
PubMed1996

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase.

Glutamate synthase (GOGAT) and glutamine synthetase play a crucial role in ammonium assimilation and glutamate biosynthesis in the yeast Saccharomyces cerevisiae. The GOGAT enzyme has been purified and the GOGAT structural gene (GLT1) has been cloned, showing that this enzyme is a homotrimeric protein with a monomeric size of 199 kDa. We report the GLT1 nucleotide sequence and the amino acid sequence of its deduced protein product. Our results show that there is a high conservation with the corresponding genes of Escherichia coli, Medicago sativa (alfalfa) and Zea mais (maize). Binding domains for glutamine, cofactors (FMN and NADH) and the cysteine clusters (which comprise the iron-sulfur centres) were tentatively identified on the basis of sequence comparison with GOGAT sequences from E. coli, alfalfa and maize.

Amino Acid Sequence↗
PubMed1995

A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

Amino Acid Sequence↗
PubMed1995

Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide.

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

Amino Acid Sequence↗
PubMed1987

Synthetic antigens as immunogens: Part II: Antibodies to synthetic T antigen.

Antibody to the carbohydrate moiety of T antigen was developed. The synthetic antigen (Gal beta 1----3 GalNAc alpha 1----OC6H4N = N-BSA) was prepared by coupling the diazonium salt of the disaccharide derivative Gal beta 1----3 GalNAc alpha 1----OC6H4NH2 (o) with bovine serum albumin. Specificity of the antibody produced was examined with structurally related synthetic saccharides using the enzyme immunoassay technique. The presence of a glycosyl group at 0-6 of either the Gal or the GalNAc residue of the disaccharide Gal beta 1----3 GalNAc did not prevent binding of the antisera to the saccharide moiety. However, the antisera did not bind either the trisaccharide moiety NeuAc2----3 Gal beta 1----3 GalNAc alpha 1----OC6H4NO2 (o) or GlcNAc beta 1----3 Gal beta 1----3 GalNAc alpha OBn. These observations indicate that antibody approach to the antigen is to the 0-3 side of the terminal galactose in the disaccharide Gal beta 1----3 GalNAc. We have also observed that the antibody prefers Gal beta 1----3 GalNAc alpha 1----to Gal beta 1----3 GalNAc beta 1----disaccharide derivatives in its binding capacity. The antibody was found to bind natural T antigen present on neuraminidase-treated red blood cells and, by immunohistochemical analysis, it was found to bind to naturally occurring T antigen on breast tumor cells.

Antibodies↗
PubMed1987

Synthetic antigens as immunogens: Part I. The combining site specificities of antibodies formed in rabbits to synthetic disaccharide alpha-L-Fuc-(1----3)-D-Gal.

Antibody to chemically synthesized Fuc alpha 1----3 Gal-1----OC6H4N = N bovine serum albumin was developed. Specificity analysis using related synthetic saccharides was performed. The antisera were shown to be specific for both sugars in the disaccharide, to the 1----3 positional linkage and to the alpha anomeric configuration. Immunohistochemical staining was performed on benign disease and adenocarcinoma tissue of the colon. 5/8 of the benign colon polyp tissue and 4/6 of the colonic adenocarcinoma tissue showed strong granular cytoplasmic staining of the epithelia cells lining the ducts. None of the six normal colon tissues tested showed any reactivity. Immunohistochemical staining was also performed on tissue from benign disease and from carcinoma of the mammary gland. 2/5 of the tissue from benign disease of the breast and 3/6 of the tissue from breast carcinoma showed strong granular cytoplasmic staining of the ductal epithelial cells.

Adenocarcinoma↗
PubMed1985

Immunohistochemical demonstration of prostate-specific antigen in metastases with the use of monoclonal antibody F5.

With the use of a murine monoclonal antibody (F5), a panel of metastatic tumors was evaluated for the expression of prostate-specific antigen (PA) under immunoperoxidase staining procedures. Specimens studied included 25 of prostatic origin and 73 originating from nonprostatic primary sites. Regardless of the site of dissemination or the malignancy grade, all metastases from the prostate were antibody-reactive. In contrast, nonprostatic metastases were negative in each case, including those originating from other genitourinary neoplasms. Thus, PA expression as detected with monoclonal antibody F5 is a stable characteristic of disseminated prostatic tumors.

Antibodies, Monoclonal↗
PubMed1984

Reactivity of monoclonal antibody F36/22 with human ovarian adenocarcinomas.

Monoclonal antibody F36/22 recognizes high-molecular-weight glycoprotein components associated with neoplastic development of the ovary. Indirect immunoperoxidase staining techniques were performed on a panel of nonmalignant ovarian tissues, primary ovarian tumors, exfoliated ascitic tumor cells, and metastatic lesions. Normal ovarian tissue components (n = 20) failed to exhibit detectable levels of antigen, whereas benign ovarian tissues show a low incidence of immunostaining (three of 26) restricted to some ductal elements. One hundred % (19 of 19) of the immunopositive primary malignant tumors were histologically classified as adenocarcinomas. Each of the predominant adenocarcinoma histotypes consistently showed expression of the antigen with 30 to 100% of the tumor cells scored as immunopositive. Ascitic tumor cells obtained from all of the ovarian adenocarcinoma patients examined (47 of 47) displayed immunopositive reactions, whereas normal mesothelial cells in these specimens exhibited undetectable staining. In addition, ovarian adenocarcinoma metastases (12 of 12) exhibited very intense immunoreaction products. No detectable antigen was expressed by nonadenocarcinoma ovarian tumor cells.

Adenocarcinoma↗
PubMed1982

Hybridoma antibody to breast cancer immune complexes.

Hybridoma clones were established by fusing spleen cells from mice immunized with purified breast cancer-related immune complexes to drug-resistant nonproducing myelomas. Hybridoma cultures were evaluated for antibody production using immunofluorescence, radioimmunoassay and immunoperoxidase techniques. One antibody demonstrated strong reactivity against breast tumor cells (BT-20) in culture and also was found to bind ductal epithelial cells within sections of human mammary tissue. Antigen was detected within the soluble fraction of breast tumors with molecular weight greater than 500 K. The possible relationship between antigen detected in breast tumor cells and in immune-complexed form is discussed.

Antibodies, Monoclonal↗
PubMed1981

Expression of prostatic acid phosphatase in human prostate cancer.

By a specific immunochemical measurement, the activity of prostatic acid phosphatase (PAP) in prostate cancer was found to be about 25%, on average, based on micrograms DNA or per cell, of that in normal prostate or benign prostatic hypertrophy (BPH). The reduction of PAP in prostate cancer was further revealed by a decrease in PAP protein. The 125I-labeled anti-PAP IgG specifically bound to nascent peptides on PAP-synthesizing polysomes showed no qualitative differences among cancerous prostate, normal prostate and BPH. However, the quantitative binding of 125I-labeled anti-PAP IgG to polysomes of cancerous prostate was half that of normal prostate of BPH. These data suggest that a significant amount of PAP and its synthesizing polysomes was reduced in prostate cancer as a result of PAP gene suppression.

Acid Phosphatase↗
PubMed1981

Prostate antigen: a marker for human prostate epithelial cells.

Specificity of a previously reported prostate antigen (PA) was assessed by several immunologic procedures. This antigen, restricted in distribution to the prostate gland, was detected within ductal epithelial cells. Continuous established cell lines LNCaP and PC-3 of malignant prostate origin retained the expression of PA. Tumor cells released the antigen in vitro into the culture fluid and also in vivo into the circulation of nude mice preinoculated with LNCaP cells. Prostate cells in culture also specifically accreted immunoglobulin fragments of PA antiserum.

Animals↗
PubMed1980

Quantitation of prostate-specific antigen in serum by a sensitive enzyme immunoassay.

A sensitive sandwich-type enzyme immunoassay has been developed for quantitation of a human prostate-specific antigen (PA). With this method, PA at a concentration as low as 0.10 ng/ml can be detected. The assay was reproducible as within and between assays yielded a coefficient of variation of 5.7% and 4.6%, respectively. Only human prostate tissues (n = 31) were shown to contain PA. No PA was detected in other human normal or tumor tissues (n = 13). PA was not detectable in sera from normal females (n = 17) or female cancer patients (n = 25). A mean +/- S.D. of 0.47 +/- 0.661 ng/ml (ranging from less than 0.10 to 2.6) ws obtained from a group of 51 normal males. Sera from male patients with nonprostatic cancer contained a similar range of PA as that of normal males. Patients with prostate cancer (371 of 442) and benign prostatic hypertrophy (13 of 19) were shown to have elevated levels of circulating PA. Although no quantitative difference in PA levels was found between the benign prostatic hypertrophy group and Stage A of prostatic cancer, patients with Stages C and D prostatic cancer exhibited significantly elevated levels of PA qualitatively and quantitatively. These results therefore indicate that PA is a histiotypic product of the prostate and may be of use as an adjunctive tool in diagnostic procedures of prostate cancer.

Antigens↗