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Biomedical subjects

A Cid

Publications and source records attributed to A Cid.

32 records · Page 2Linked to original sources
PubMed1996

Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry.

Flow cytometric measurements were used to investigate the toxic action of copper on some Phaeodactylum tricornutum membrane systems. Throughout the time of metal exposure, the percentage of viable cells decreased as copper concentration increased. The forward scatter signal increased as a result of copper exposure. After 72 h of metal exposure, cultures with 0.5 and 1 mg l-1 of copper showed an important increase in the peroxidase activity in comparison with control cells. Cells cultured with copper presented alterations in the membrane potential, increasing as copper concentration increased, after 96 h of metal exposure. Results obtained in this work showed that copper induced a degenerative process in P. tricornutum cells, closely related with alternations or disorders in membrane systems.

Cell Membrane↗
PubMed1995

Flow cytometry determination of acute physiological changes in a marine diatom stressed by copper.

Flow cytometry (FCM) was used to determine changes in cellular volume, transmembrane potential, mitochondrial membrane potential and intracellular pH in the marine microalga Phaeodactylum tricornutum immediately (5 to 10 seconds) after the addition of selected concentrations of copper. An acute increase in the forward scatter signal of this diatom was detected after the addition of 10 mg 1-1 of copper. Stress produced by the copper addition resulted in various physiological alterations that can be easily and quickly detected by FCM: (i) the hyperpolarization of the cell membrane, as a result of an immediate increase in the cytoplasmic membrane potential, (ii) the increase of the mitochondrial membrane potential, being maximum at the higher copper concentration assayed, and (iii) the increase in the intracellular pH with the highest copper concentration assayed (10 mg 1-1).

Adaptation, Physiological↗
PubMed1994

Different responses of the marine diatom Phaeodactylum tricornutum to copper toxicity.

Different responses of the marine diatom Phaeodactylum tricornutum (Bohlin) to toxic copper concentrations were investigated. Besides the classical variables applied to toxicity studies in microalgae, such as growth or chlorophyll a content, other variables analyzed by flow cytometry were used. Toxic effects due to copper concentration were observed. Cell density reached in the stationary phase was reduced to 50% in cultures with 20 mg Cu/l, with respect to control cultures without copper. Cell light scatter properties (related to cell volume and intracellular granularity) and chlorophyll a fluorescence of microalgal cells were determined by flow cytometry analysis at the beginning of growth, 1 h after copper exposure, and when cultures reached the stationary phase (72 h). After 1 h of exposure to metal, no differences were observed, but when cultures reached the stationary phase, a gradual increase in the variables analyzed by flow cytometry was observed as the copper concentration increased. The increase in chlorophyll a fluorescence detected by flow cytometry was not correlated with an increase in the cell content of this photopigment, thus indicating an inhibitory effect of copper on photosystem II.

Chlorophyll↗
PubMed1993

Cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region in transgenic mice and its modulation by tetradecanoyl phorbol acetate and glucocorticoids.

The upstream regulatory region (URR) of human papillomavirus type 18 (HPV-18) harbors transcriptional promoter and enhancer elements which are thought to determine the cell-type specificity of the virus. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice carrying the bacterial lacZ gene under the control of the HPV-18 URR. Analysis of beta-galactosidase activity by histochemical staining of tissue sections of four independent transgenic mice showed that the viral promoter was specifically active in epithelial cells within a variety of organs (e.g., tongue, ovary, uterus, testis, and small intestine). Very strong staining was observed in newborn transgenic mice in contrast to a weak activity found during fetal life. Determination of beta-galactosidase activity in crude extracts from tissues of three lines of transgenic mice proved to be a useful tool for a quantitative analysis of transgene expression. In mice from two different transgenic lines treated with dexamethasone such measurements revealed a biphasic effect of the hormone on the activity of the enzyme in the stratified epithelium of the tongue (transient increase followed by a decrease). Northern (RNA) blot analysis showed similar changes in beta-galactosidase mRNA in that tissue. Treatment with tetradecanoyl phorbol acetate (TPA) led to a twofold increase in both enzymatic activity and mRNA levels. Finally, combined treatments with dexamethasone and TPA showed that both factors interfered with each other in their respective effects on transgene expression, suggesting that a cross-talk mechanism between transcription factors could be involved in the regulation of the HPV-18 URR.

Animals↗
PubMed1988

Mutations of the yeast plasma membrane H+-ATPase which cause thermosensitivity and altered regulation of the enzyme.

Four random mutations of the plasma membrane H+-ATPase of Saccharomyces cerevisiae which result in thermosensitive growth have been sequenced. All of the mutations map in regions conserved by the family of ATPases which form a phosphorylated intermediate. The Gly254----Ser mutation affects a glycine residue conserved in all of the sequenced ATPases. The Thr212----Ile and Ala547----Val mutations do not affect conserved amino acids, but their replacements are not found in any of the sequenced ATPases. Thr212 and Gly254 occur in the proposed phosphatase domain, whereas Ala547 is located within the putative ATP-binding site. The other mutation is a double change (Asp91----Tyr and Glu92----Lys) in the N-terminal domain, in which the altered glutamate is conserved in fungal and protozoan H+-ATPases. Proton efflux from whole cells and ATP hydrolysis by purified plasma membranes are more thermolabile in cells carrying the ATPase mutations than in wild-type yeast. Therefore, the defects in growth and proton transport at the nonpermissive temperature can be attributed to impairment of ATPase activity. Incubation of wild-type yeast cells with glucose before homogenization induces changes in the specific activity, Km, pH optimum, and vanadate sensitivity of the plasma membrane ATPase. The Ala547----Val mutation results in an enzyme from starved cells with the kinetic parameters of the glucose-activated wild-type ATPase. Therefore, a single amino acid change mimics the poorly understood regulatory mechanism triggered by glucose.

Amino Acid Sequence↗
PubMed1987

Tight control of the amount of yeast plasma membrane ATPase during changes in growth conditions and gene dosage.

The activity of the plasma membrane ATPase in growing yeast is increased by low pH and by glucose, conditions which result in a higher demand for proton pumping. The amount of enzyme is not significatively modified under these conditions. The amount of ATPase is only slightly increased by introducing extra copies of its gene in autonomous plasmids. In addition, the expression of the ATPase gene in a multi-copy plasmid causes a reduction of the copy number of the plasmid and slows growth. Therefore, overexpression of the ATPase is detrimental for the cell, justifying a regulatory mechanism based on increasing the catalytic activity and not the amount of enzyme.

Adenosine Triphosphatases↗
PubMed1987

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide.

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 M-1 min-1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.

Amino Acid Sequence↗
PubMed1987

Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences.

In order to probe the physiological role of the yeast plasma membrane ATPase we have replaced the constitutive promoter of its gene by a galactose-dependent promoter. The resulting cells stop growing on glucose medium when the preformed ATPase is diluted to 20% of normal. There is a correlation between ATPase activity and both proton efflux from the cells and amino acid transport. A large proportion of growth-arrested cells appear enlarged and with several buds containing nuclei.

Adenosine Triphosphatases↗
PubMed1979

Enzyme-linked immunosorbent choriomammotropin assay.

We have devloped an enzyme-linked immunosorbent assay for determining choriomammotropin (human placental lactogen) in serum. Unlabeled hormone competes with choriomammotropin-beta-galactosidase conjugate for antibody bound to polystyrene tubes. The entire assay can be performed in 2.5 h with good precision. The coefficient of variation for one sample with a mean concentration of 5.6 mg/L, assayed 10 times on the same day, was 5.7%. The coefficient of variation for nine samples (3.5 to 9.0 mg/L) assayed on five different days was 7.9%. Forty-eight clinical samples were assayed (y) and compared with results obtained by radial immunodiffusion (x). The resulting regression equation was: y = 1.05x + 0.78; r = 0.91.

Enzyme-Linked Immunosorbent Assay↗
PubMed1979

A new radioassay for serum vitamin B12 determination using chicken serum as a binder and an anion exchange resin column separation.

We present a new method for the determination of serum vitamin B12 concentration by competitive protein binding using 57Co labeled vitamin B12, chicken serum (CS) as the binding protein, and a sephadex column separation. We placed a mixture of extracted sample, labeled B12, and CS binder above an A-25 sephadex column, and after 1 hour incubation at room temperature drained the column and washed it twice with buffer. We counted the free fraction remaining in each column. Within run C.V.'s for low and high values in the normal range were 10.6% and 6.03%. The between run C.V.'s were 7.7% and 7.5% respectively. The values obtained for 95 clinical specimens correlated well with those for a reference method.

Animals↗
PubMed1978

A direct enzyme-linked immunosorbent assay (ELISA) for detection of antibodies for rubella virus in human sera.

A direct enzyme-linked immunoassay (ELISA), based on the "sandwich" principle on an antigen-coated plastic disc, was used for the rapid detection of rubella antibody. Results were obtained the same day, and the prior adsorption of sera to remove non-specific inhibitors was not necessary. The ELISA was compared to the hemagglutination-inhibition (HAI) test on 500 serum samples. There was general agreement between the two methods; most discrepancies occurred with low-titered HAI positive sera. There was excellent correlation between the tests with serum samples negative for rubella antibodies and those samples with HAI titers greater than or equal to 1:40.

Animals↗
PubMedDate not supplied

[Antibodies against hepatitis C virus in patients with liver diseases and in risk subjects. Preliminary report].

The recent discovery of an antigenic component of the causative agent of Non-A, Non-B hepatitis, has led to the characterization of this virus--Hepatitis C Virus (HCV)--and to the identification of an antibody present in infected subjects (anti-HCV) detected by means of the C-100 antigen derived from a nonstructural region of the viral genome. Using a commercial Kit (Ortho Diagnostic Inc.), the incidence of anti-HCV antibody was studied in the Military Hospital "Dr. Carlos Arvelo" of Caracas, Venezuela with the following results: Health personnel (doctors, nurses, laboratory staff): 102 persons studied, 2 positives (1.96%); 16 patients in chronic hemodialysis: 6 positives (33%); 20 subjects with antibodies against HIV virus, confirmed by Western Blot: 7 positives (35.4%). Of 10 patients with Surface Antigen negative Chronic Hepatitis, 7 (70%) positive for anti-HCV, of 25 patients with cirrhosis: 12 positive (48%), 2 patients with hepatocarcinoma 1 positive (50%). There was also a high incidence of total anti-core antibodies in the patients studied. The results suggest that the hepatitis C virus could be playing an important role as a causative factor of liver diseases in our Country.

Adult↗