PubMed Health⌕ Search

Biomedical subjects

J V Castell

Publications and source records attributed to J V Castell.

At least 109 records · Page 6Linked to original sources
PubMed1987

Evidence for arrested G2 cell subpopulation in rat liver inducible to mitosis.

The intraperitoneal administration of several substances (biliverdin, heat-killed bacteria and diatomaceous earth) to rats caused the prompt appearance of a mitotic wave in the liver. Autoradiographic analysis of livers of treated animals showed no evidence of [3H]-thymidine uptake by mitotic hepatocytes. In addition, livers from xenobiotic-treated rats showed a very low thymidine kinase activity, close to that found in normal livers. This excludes the possibility that non-cycling cells move to mitosis through the S phase. The results suggest that mitosis could be derived from a hepatocyte subpopulation arrested in the G2 phase of the cell cycle, which is stimulated to divide by the xenobiotics.

Animals↗
PubMed1987

A cytochemical stain for glutathione in rat hepatocytes cultured on plastic.

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.

Animals↗
PubMed1986

Measurement of fibronectin in human body fluids.

Two facts must be taken into consideration when quantifying fibronectin in biological samples: the sensitivity of the assay method and the appropriate handling of samples. A three-antibody, non-competitive ELISA was used in the present study. This procedure offers a simple, inexpensive and very sensitive technique for evaluating fibronectin: reproducible standard curves in the range 5-100 micrograms/l, 3% variability in the assay, and a detection limit of 0.5 ng/well that allows accurate measurement of fibronectin in segmental bronchoalveolar lavage and in ascites liquid samples. Typical dilution ranged from 1/10000 for plasma to 1/100 for bronchoalveolar lavages. For long-term storage of samples two procedures are recommended. First, a rapid freezing with liquid nitrogen, storage at -20 degrees C and thawing once at 37 degrees C avoiding refreezing. Losses of immunoreactive fibronectin after 35 days storage were 15, 15 and 22% in plasma, bronchoalveolar lavages and ascites fluid, respectively. Alternatively, samples can be stored as ammonium sulphate precipitates. This allows the easy availability of the samples when assays have to be repeated, and avoids fractionation in vials and freeze-thawing cycles. Using this storage procedure, losses are, however, slightly higher (22, 25, 25%, respectively, after 35 days).

Enzyme-Linked Immunosorbent Assay↗
PubMed1985

Enzyme-linked immunosorbent assay to quantify fibronectin.

Fibronectin, a multifunctional soluble glycoprotein, is found in soluble form in blood and other body fluids. Fibronectin binds to biological substrata--collagen, gelatin, etc.--and to nonbiological substrata as well. An interesting finding was that fibronectin bound to polystyrene plastic quickly and tightly and further treatment of plastic with denaturing agents did not remove or denature it once it was adsorbed. Through the use of this property, a three-step noncompetitive enzyme-linked immunosorbent assay has been developed to quantify soluble fibronectin in biological samples. The following parameters were studied: selective attachment of fibronectin to polystyrene; interference caused by increasing amounts of external protein; and linear response and limit of sensitivity. The procedure has been applied to measure fibronectin from different animal sources.

Adsorption↗
PubMed1984

Biochemical functionality and recovery of hepatocytes after deep freezing storage.

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.

Animals↗
PubMed1984

Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone.

Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin. Specific hormone requirements are needed by hepatocytes to retain the biochemical pattern of mature cells. Insulin supplementation of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence of dexamethasone amplified the insulin-induced glycogen synthesis. Cytophotometric analysis showed differences in the way that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in culture are functionally heterogeneous.

Animals↗
PubMed1984

Isozyme pattern of thymidine kinase during liver regeneration.

Liver regeneration after partial hepatectomy is characterized by an active phase of cell proliferation that is associated with a marked increase in thymidine kinase activity. Using non-denaturing gel electrophoresis and different substrate specificity, two isozymes could be detected. One was identified as the adult isozyme while the other was the fetal one. Both forms were present during liver regeneration. When the regenerative process was completed, the total enzymatic activity dropped to normal values and the fetal isozyme was displaced by the adult type.

Animals↗
PubMed1983

Effect of glucocorticoids on the expression of gamma-glutamyltransferase and tyrosine aminotransferase in serum-free-cultured hepatocytes.

Glucocorticoids exert a known beneficial effect on cultured hepatocytes when present in culture medium, maintaining their polygonal morphology and ultrastructural organization throughout the days of culture. Parallel to this excellent morphology, hepatocytes cultured in serum-free conditions, but with continuous presence of Dexamethasone, retained after a week the ability to express tyrosine aminotransferase when stimulated by glucagon and glucocorticoids. The rise of gamma-glutamyltransferase was blocked in cultures supplemented by Dexamethasone.

Animals↗
PubMed1981

Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli.

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.

DNA-Directed RNA Polymerases↗
PubMed1979

A fluorescamine-based sensitive method for the assay of proteinases, capable of detecting the initial cleavage steps of a protein.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of 'specific' proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a) the accumulation of N-amino termini in excess over that of the intact substrate; (b) a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.

Endopeptidases↗
PubMedDate not supplied

Toxic effects of the photoproducts of chlorpromazine on cultured hepatocytes.

The photodegradation of chlorpromazine, a drug frequently used in psychotherapy, was examined under different sets of experimental conditions. A primary culture of rat hepatocytes was used to evaluate the possible hepatotoxicity of the chlorpromazine photoproducts, keeping in mind the following criteria: leakage of cytosolic enzymes; attachment index to culture plates, and albumin synthesis. Cells exposed to concentrations greater than 10(-4) M of the photomixtures showed extensive leakage of GOT and GPT into the culture medium and, at the same time, the cell attachment was seriously impaired. A concentration of 10(-7) M of the photoproducts proved capable of inhibiting the synthesis of albumin (20%). Photoproducts obtained after aerobic irradiations were as toxic for hepatocytes as those found in anaerobic conditions. The implications of our results in connection with the relevance of oxygen-dependent photoreactions of chlorpromazine to its phototoxicity, and the possible appearance of hepatic alterations in patients treated with the drug after exposure to the sunlight, are discussed.

Animals↗
PubMedDate not supplied

S-adenosyl-L-methionine reverses the cholestatic effect of ethinylestradiol in rat hepatocytes by increasing its catabolism.

In this study, ethinylestradiol inhibited the uptake of taurocholate by cultured rat hepatocytes, increasing the Km while leaving the Vmax unchanged. S-Adenosyl-L-methionine (SAMe) had no effect on taurocholate uptake or release, but was able to reverse the competitive inhibition induced by ethinylestradiol. S-Adenosyl-L-homocysteine did not reverse this inhibition, which suggests that the methyl group of SAMe affects its activity. Several possible mechanisms for the action of SAMe were investigated. The methylation of cell membrane phospholipids was eliminated as a possible mechanism. The presence of SAMe greatly increased the catabolism of ethinylestradiol by hepatocytes and reduced its covalent binding to hepatocyte macromolecules. In culture supernatants, both highly polar (conjugated) and non-conjugated metabolites could be detected. Moreover, most of the metabolites were methylated. This suggests that SAMe may revert the effects of ethinylestradiol of taurocholate uptake by increasing its catabolic rate by hepatocytes.

Animals↗
PubMedDate not supplied

Mechanisms of drug photobinding to proteins: photobinding of suprofen to human serum albumin.

Photobinding of drugs to biomolecules constitutes an important early event in the onset of photoallergy. In the present work, UV irradiation of human serum albumin in the presence of either suprofen (SUP) or its major photoproduct, decarboxylated suprofen (DSUP), has been studied as a model system for drug-photosensitised protein binding. Both dark binding and binding in the presence of light were investigated since this will affect the mode, site and mechanism of drug interaction with the protein. In order to determine the binding features of SUP to albumin, competitive binding experiments were carried out using fluorescent probes specific for site I and II. Suprofen was found to selectively dark bind to site II on HSA. Photobinding of DSUP to HSA was more efficient than SUP. Parallel to this, the intrinsic tryptophan fluorescence of HSA decreased when the protein was previously irradiated in the presence of the photoactive compounds, again being DSUP more efficient compared with SUP. As fluorescence quenching involves electron transfer from the excited Trp to the ground state DSUP, it follows that the photoactive compound binding to HSA must be on (or in close proximity to) site I Trp(214) residue. It appears that photobinding of SUP is largely preceded by its photodecomposition to DSUP which, in turn, associates and photobinds to HSA.

Binding, Competitive↗
PubMedDate not supplied

In vitro studies on DNA-photosensitization by different drug stereoisomers.

The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated. The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis. Formation of single strand breaks was compared for each group of compounds. Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E. coli endonuclease III (revealing oxidized pyrimidines) and E. coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage. Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases. No pyrimidine dimers were observed. Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites. The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism. Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin. In the case of carprofen, a weak nucleoside degradation was obtained. The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin. In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer. However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

Anti-Infective Agents, Urinary↗