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PubMed2006

Isolation of chebulic acid from Terminalia chebula Retz. and its antioxidant effect in isolated rat hepatocytes.

A hepatoprotective compound was isolated from the ethanolic extract of the fruits of Terminalia chebula Retz. by consecutive solvent partitioning, followed by silica gel and Sephadex LH-20 column chromatographies. The purified compound was identified as a mixture of chebulic acid and its minor isomer, neochebulic acid, with a ratio of 2:1 by spectroscopic analysis including 1D and 2D NMR and MS spectroscopy. To our knowledge, this is the first report on the protection of rat hepatocytes against oxidative toxicity by chebulic acid obtained from T. chebula Retz. This compound exhibited in vitro a free radical-scavenging activity and ferric-reducing antioxidant activity. Also, the specific ESR spectrum for the (*)OOH radical signals consisting of three-line ESR spectra was within the field of 0.27 mT, whereas 2.5 and 0.25 mg/ml of chebulic acid significantly reduced the signal intensity of the ESR spectra to 0.06 mT and 0.11 mT, respectively. Using isolated rat hepatocyte experiment, we demonstrated that the treatment of hepatocytes with chebulic acid significantly reduced the tert-butyl hydroperoxide (t-BHP)-induced cell cytotoxicity, intracellular reactive oxygen species level, and the ratio of GSSH, oxidized form of glutathione (GSH) to the over total GSH (GSH + GSSG) (4.42%) as compared to that with t-BHP alone (8.33%).

Animals↗
PubMed1986

A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources.

A rapid and simple procedure is described for obtaining CsCl-purified DNA from multiple small samples of cells or tissue. The DNA is recovered in a high-molecular-weight form (greater than or equal to 50 kb) that is readily cleaved with restriction enzymes. Sufficient quantities of DNA (10-50 micrograms) are recovered to allow multiple analyses by Southern blotting and most cloning procedures. The isolation procedure involves addition of intact cells or powders of frozen tissues directly to a simple lysis buffer containing detergent (sodium dodecyl sulfate or sodium sarcosinate) and high concentrations of EDTA. Ultra-high-speed centrifugation of CsCl gradients allows the isolation of DNA from 10 different samples in as little as 5 h. Applications are described for mammalian cells (HeLa cells), insect tissues (Drosophila melanogaster adults and pupa, Manduca sexta pupa, and Musca domestica pupa), higher plant tissues (Vicia faba leaves and meristems), algal cells (walled and wall-less Chlamydomonas reinhardi), yeast cells (Saccharomyces cerevisiae), and bacterial cells (Escherichia coli spheroplasts for preparation of both chromosomal and plasmid DNA). The procedure can be scaled up with larger sample sizes and longer centrifugation times to provide bulk quantities of DNA.

Animals↗
PubMed1989

Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half.

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

A comparison of UV induced DNA photoproducts from isolated and non-isolated developing bacterial forespores.

UV-induced photoproduct formation has been compared in non-isolated and isolated developing forespores. We have found that levels of spore type photoproducts are greatly affected by mother cell DNA. We have also observed the presence of the photoproduct 6-4'-(pyrimidin-2'-one)-thymine in developing forespores. We conclude from these and other data in the literature that the degree of hydration around the forespore DNA is reduced by the presence of dipicolinic acid which influences photoproduct formation without causing a change in conformational state.

Bacillus megaterium↗
PubMed1984

Isolation of a cigarette smoke fraction responsible for the inhibition of benzo[a]pyrene metabolism in the isolated perfused rabbit lung.

Among the several thousand components of cigarette smoke is a substance or substances capable of inhibiting pulmonary metabolism of nicotine and altering the metabolite profile of procarcinogens such as benzo[a]pyrene (BP). This substance(s) inhibits BP metabolism in the lung in amounts present in a few puffs of cigarette smoke. By a series of extractions and chromatographic methods an active subfraction containing only 1% of the total cigarette smoke condensate (CSC), was isolated. This fraction demonstrated the same inhibition of BP metabolism in the isolate perfused lung (IPL) as the whole smoke. The inhibitor(s) present in this fraction possess amphoteric characteristics. The acidic function is believed to be a phenolic one.

Animals↗
PubMed1995

Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate.

A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable. From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.

Amino Acid Sequence↗
PubMed1980

Isolation of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the isolated subpopulations.

Some methodological aspects of E-rosette gradient centrifugation for separation of human B and T lymphocytes are described. Treatment of sheep red blood cells (SRBC) with 2-aminoethylisothiouronium bromide (AET) accelerates and enhances E-rosette formation providing an effective and time saving method which takes less than 2 h to perform. Depletion of E-rosette forming cells (E-RFC) is almost complete, leaving less than 1% of the initial E-RFC at the interphase layer. By use of the lymphocyte donor's autologous serum containing naturally occurring anti-SRBC antibody and complement the SRBC in the rosetted T lymphocyte fraction are easily and completely lysed without damage to the lymphocytes. This procedure is shown to have little effect on the properties of the isolated T cells. The isolated lymphocyte subpopulations have been characterized by tests for lymphocyte and monocyte markers.

B-Lymphocytes↗
PubMed1992

Isolation and preliminary characterisation of a novel retrovirus isolated from a leukaemic dog.

A novel canine retrovirus was isolated from mononuclear cells of the peripheral blood of a leukaemic dog. The main clinical and pathological findings in this dog were lethargy, anorexia, weakness, dyspnoea, severe anaemia, thrombocytopenia and a high white blood cell count, practically all of which were lymphoblasts. The virus was isolated from mononuclear cells obtained from the blood, cocultivated with indicator cells. The virus particles encode a reverse transcriptase with Mg++ preference, have a density in sucrose gradients of 1.16 g ml-1, and induce syncytia in permissive cell cultures such as Himalayan tahr ovary and canine fetal thymus lines. This agent replicates to high titres. The virus exhibits a morphogenesis and morphology typical of lentiviruses. Immunoblotting and competitive radioimmunoassays failed to detect immunological crossreactivity with other representative lentiviruses and oncoviruses of the retrovirus family.

Animals↗
PubMed1989

Effects of toxin isolated from the sea anemone Bolocera tuediae on electrical properties of isolated rat skeletal muscle and cultured myotubes.

Electrophysiological effects of a polypeptide toxin isolated from the sea anemone Bolocera tuediae (BTTX II) on isolated fast and slow rat skeletal muscle and on cultured rat myotubes are described in this paper. Nanomolar concentrations of BTTX II cause a concentration-dependent depolarization. This effect is prevented by the presence of tetrodotoxin. Action potential duration is prolonged in all preparations investigated, with the fast muscle being the least sensitive. The positive overshoot and the maximum rate of rise of the action potential is decreased by BTTX II. Cultured myotubes which are less susceptible to the sodium channel blocking activity of tetrodotoxin have the highest sensitivity to all actions of BTTX II. It is further shown that the proportion of spontaneously contracting myotubes is diminished and the frequency of contractions is increased by the toxin. The effects of Bolocera tuediae toxin II resemble those observed with Anemonia sulcata toxin II on mammalian skeletal muscle. From the results it is concluded, that BTTX II may interfere with both the activation and inactivation of sodium channels of the muscle membrane.

Action Potentials↗
PubMed1991

Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes.

Venoms of nine different snake species of the genus Bothrops were fractionated using fast protein liquid chromatography (FPLC). Basic proteins with phospholipase A2 and/or myotoxic activities were isolated from venoms of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi. B. jararaca venom possessed very low concentrations of these proteins, which were undetectable in venoms of B. atrox, B. alternatus, B. cotiara and B. erythromelas. Basic proteins from B. moojeni and B. pradoi venoms were isolated in pure form. All active fractions possessed a common band of 15,000 mol. wt which caused a rise in serum creatine phosphokinase levels and histopathological changes in muscle cells following i.m. injection into mice. Levels of phospholipase A2 activity were variable. The implications of the possession of varying levels of myotoxins and phospholipase A2 in these venoms are discussed.

Animals↗
PubMed1986

Isolation and characterization of covalently closed circular proviral DNA molecules of several type D retroviruses isolated from human cell lines.

Infection of a human lymphoblastoid B cell line (Raji cells) with type D retroviruses, originally isolated either from subhuman primates (MPMV, LV) or from permanent human cell lines (PMFV, HeLaV, HEp-2V) led to the production of type D retrovirus particles. Subsequent cocultivation of uninfected and virus-producing Raji cells was employed for the generation of sufficient amounts of covalently closed circular DNA molecules (cccDNA). Highest amounts of cccDNA were obtained after cocultivation of virus-producing Raji cells and homologous uninfected cells at a ratio of 1 to 3 for 72 hr. The cccDNAs of type D retroviruses migrated at about 4.3 kbp compared to lambda DNA/HindIII markers. Digestion of cccDNAs with restriction endonucleases which have one recognition site generated molecules of approximately 8 kbp. The restriction endonuclease site analysis of the cccDNA of type D retroviruses revealed a genomic heterogeneity among the different isolates.

Cell Line↗
PubMed1987

Characterization of Tm-1 gene action on replication of common isolates and a resistance-breaking isolate of TMV.

Tm-1 is a gene which confers resistance to infection, in tomatoes, by tobacco mosaic virus (TMV). To investigate the biochemical mechanism of the resistance, we have established cell suspensions of three lines of tomatoes, i.e., +/+ (susceptible, wild-type, no Tm-1 gene), Tm-1/+ (heterozygous for the Tm-1 gene), and Tm-1/Tm-1 (homozygous for the Tm-1 gene). Protoplasts isolated from these cells were inoculated with RNA of the tomato strain L and Lta1 (a resistance-breaking strain which was recently isolated spontaneously from L) of TMV by means of electrophoration. The syntheses of all viral-coded proteins and TMV-specific RNAs could be detected in L-inoculated +/+ and Lta1-inoculated +/+, Tm-1/+, Tm-1/Tm-1 protoplasts, while their production was markedly reduced in L-inoculated Tm-1/+ protoplasts. L strain could multiply in Tm-1/+ protoplasts to a greater extent with less delay when a large amount of inoculum RNA was used. However, viral production was completely blocked in Tm-1/Tm-1 protoplasts even when a large amount of L-RNA was used for inoculation.

Cells, Cultured↗
PubMed1987

The first isolation of Newcastle disease virus (NDV) from free-flying birds in Israel: comparative studies on NDV strain isolated from migrating starlings (Sturnus vulgaris) wintering in Israel.

An NDV strain was isolated in Israel from a starling suffering from severe neurological affections. The strain appeared to be lentogenic to chickens, its hemagglutinin being thermostable and active toward horse erythrocytes. Comparative studies including LaSota and B1 NDV reference strains showed that the isolate is not a vaccinal strain used for vaccination in the country.

Animals↗
PubMed1986

Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes.

This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.

Animals↗
PubMed1984

Modification of glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15 degrees C--I. Changes in the activity of individual muscle isolates as evidence for variable adaptive responses.

The mean Km and Vmax values for G3PDH isolated from the lateral muscle of cold-adapted (5 degrees C) rainbow trout, Salmo gairdneri, were twice those of enzyme from warm-adapted (15 degrees C) trout when assayed at 7 degrees C but not at any other temperature. The entropy of activation of warm enzyme was about 3 times that of cold enzyme. However, enthalpy or free energy of activation among acclimation groups differed less or not at all. Individual G3PDH isolates within either adaptation group differed in kinetic characteristics.

Acclimatization↗
PubMed1984

Modification of glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12, isolated from rainbow trout during acclimation at 5 or 15 degrees C--II. Biochemical characterization of G3PDH muscle isolates.

G3PDH was isolated from the lateral muscle of rainbow trout (Salmo gairdneri) acclimated at 5 degrees C (cold) and 15 degrees C (warm). No differences were found in muscle concentration, molecular weights, isoelectric focusing patterns, amino acid compositions or peptide maps between cold and warm isolates. Cold and warm G3PDH contained mannose in variable concentration but no other prosthetic groups.

Acclimatization↗
PubMed1982

Pharmacological effects of ionophore A-23187 on isolated guinea-pig atria and isolated cat nictitating membrane.

1. The present paper is concerned with some pharmacological actions of ionophore A-23187 on both isolated guinea-pig atria and cat's nictitating membrane. 2. The drug showed an agonist effect on both tissues. 3. There is an indirect effect by releasing catecholamines which is antagonized by reserpine and beta receptor blockers in atria and alfa receptor blockers in nictitating membrane. 4. The ionophore exhibited a direct positive chronotropic effect on the atria. This effect is blocked by Ca2+ flux antagonist such as verapamil and pentobarbital and not by reserpine. 5. Ionophore produced no direct effect on the activity of the isolated tyrosine hydroxylase but showed an inhibitory action in intact tissues, mediated by released of endogenous catecholamines, since this effect is antagonized by reserpine. 6. Monoamine oxidase activity was unaffected by ionophore.

Animals↗
PubMed1993

Patch-clamp recording from Müller (glial) cell endfeet in the intact isolated retina and acutely isolated Müller cells of mouse and guinea-pig.

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K+ homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K+ conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K+ currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K+ currents by Ba2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K+ via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

Animals↗