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[Kinetics and thermodynamics of heat inactivation of L-threonine-L-serine dehydratase from human liver].

It has been shown that heat inactivation of L-threonine- and L-serine dehydratase activities at 37; 45; 50 and 55 degrees C in human liver extracts (the liver was ectracted with buffer containing 1.10(-5) M of pyridoxal 5'-phosphate) in course of time is practically identical, and characterizes by the same of values of activation energy of heat inactivation process, activation enthalpy of this process, activation free energy of that and activation enthalpy of the heat inactivation process. A rise of pyridoxal 5'-phosphate concentration (to 2.10(-4) M) in the buffer used for the liver extraction and hence in the medium in which the heat inactivation process was carried out stabilises L-threonine- and L-serine dehydratase activities against the inactivation at 55 degrees C. It has been concluded thatL-threonine- and L-serine dehydratase activities in human liver belong to the single protein or to two proteins having very like physico-chemical properties, and that pyridoxal 5'-phosphate is essential for this enzyme not only as coenzyme but also it is necessary to support active and stable conformation of this oligomeric protein.

Adult↗

Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

Acetyltransferases↗

Allograft tolerance as in immunologically active process: analysis in a restricted H-2 incompatibility due to mutation (M504) in mouse strain B10.D2.

Cell-free spleen and liver extracts were used for attempted induction of allograft tolerance across a restricted H-2 barrier due to a chemically induced mutation in the B10.D2 mouse strain. When using mice of the latter strain as donors and the new congenic strain carrying the mutant H-2 hyaplotype as recipients, and with long-term administration of the cell-free antigens in adult life up to 100% yields (according to the dose of antigen) of permanently tolerant animals (particularly males) was obtained with spleen extract, but not with liver extract. This contrasts with a higher tolerance-inducing efficiency of liver extracts in various non-H-2 incompatibilities. In the reversed donor-recipient combination, neither spleen nor liver extracts gave positive results. The immune status of the permanently tolerant animals was analyzed and it was shown that with their bone marrow normal responsiveness can be conferred upon lethally irradiated syngeneic hosts whereas the specific allograft tolerance can be adoptively transferred to sublethally irradiated recipients by means of spleen or lymph node cells. It is concluded that in the present system the state of allograft tolerance is maintained by an immunologically active process of peripheral lymphocytes.

Animals↗

(3H)-estradiol binding by chick liver nuclear extracts: mechanism of increase in binding following estradiol injection.

Specific high affinity binding of [3H]-estradiol by 0.5 M KCl extracts of chick liver nuclei is substantially increased by estradiol injection of the immature chick. The effect is observed shortly after estradiol injection, while the estradiol-induced production of serum phosphoproteins (vitellogenic response) is not detectable until about 24 hr. Cycloheximide given 90 min before estradiol inhibits the increase in nuclear binding for 12-15 hr. At 24-48 hr the levels of nuclear binding are similar to those in the estradiol-treated animals not given cycloheximide, but serum phosphoprotein levels are depressed by about 80% at 48 hr. By 75 hr however the serum of the cycloheximide-treated estrogenized chicks contains about twice as much phosphoprotein as does serum of chicks given estradiol alone. It is suggested that the inhibition of protein synthesis for 12-15 hr delays the vitellogenic response until sufficient levels of nuclear [3H]-estradiol binding protein can be synthesized. A correlation between the levels of nuclear [3H]-estradiol binding at 24 hr and phosphoprotein at 48 hr is shown in a dose-response experiment. In vitro, nafoxidine-HCl (Upjohn 11,100 A) inhibits binding of [3H]-estradiol by the chick liver nuclear extracts. In vivo, a single injection of nafoxidine with estradiol inhibits phosphoprotein production. Injection of nafoxidine alone results in a small but significant increase in [3H]-estradiol binding by nuclear extracts, but it is not estrogenic. A possible interpretation is that nafoxidine transfers low levels of a putative cytosol receptor to the nucleus, but is unable to induce the amplification mechanism required to give the levels of nuclear estradiol-binding protein needed for the vitellogenic response.

Animals↗

Promotion of experimental breast carcinoma in rats treated with extract of liver from rats with portacaval shunt.

Liver homogenates or extracts of liver homogenates from rats in which portacaval shunt had been performed were found to have a significant growth-promoting effect on 7,12 dimethyl (a) benzanthracene (DMBA)-produced breast carcinoma in rats. Tumor potentiation was manifested by increased incidence of animals developing tumors, increased number of tumors, and increased tumor size, when compared with animals receiving injections of shunted or control liver. These observations suggest the existence of a tumor-stimulating factor in liver from which portal blood has been completely and chronically diverted by portacaval shunt. The demonstration of tumor growth stimulating factor(s) present in shunted liver, together with previously reported observations of the modification of the growth of various types of tumors in animals with a portacaval shunt, suggests that the liver is capable of playing an important role in tumor-host interactions. The portacaval shunt appears to be useful as a technique in elucidating ways that liver function may influence tumor growth.

Adrenal Glands↗

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography.

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.

Alcohol Dehydrogenase↗

In vitro proteolysis of endonucleases in rat liver nuclei extracts.

DNA endonucleases in rat liver nuclei extracts were examined by SDS-polyacrylamide gel electrophoresis followed by zymogram analysis. Four polypeptides of 120, 54, 31 and 28 kDa, which have DNA endonuclease activity, were shown to occur in the extract isolated in the presence of phenylmethanesulfonyl fluoride (PMSF), a proteinase inhibitor. Isolation without PMSF, as well as storage at -20 degrees C, or autodigestion, resulted in multiplication of active polypeptides in the extracts. Trypsin digestion led to the appearance of an active > 140 kDa polypeptide, indicating the existence of a potential endonuclease precursor in the nuclear extract.

Animals↗

Cell-free thymic extract from hypertensive rats induces hypertension in normotensive rats.

This study examined the role of the thymus in hypertension. Seven to eight week old, normotensive Wistar-Kyoto rats (WKYs) (systolic blood pressure 138 +/- 7 mm Hg) received a bolus injection of (1) WKY thymic extract (control), (2) spontaneously hypertensive rat (SHR) thymic extract, (3) SHR liver extract or (4) normotensive Sprague-Dawley rat (SD) thymic extract. Blood pressures of WKYs receiving SHR thymic rose significantly (p < 0.01) over an eight week period (168 +/- 6 mm Hg), while WKYs injected with WKY thymic extract (143 +/- 10), SHR liver extract (144 +/- 5) or SD thymic extract (138 +/- 4) showed no change in blood pressure. Thymuses from WKYs injected with SHR extract were significantly (p < 0.01) smaller than thymuses from WKYs injected with WKY extract. Aorta from WKYs administered SHR extract were significantly (p < 0.01) hyperresponsive to contractile agents, suggesting that immune dysfunction may lead to vascular damage as seen in several hypertensive models. The results suggest that hypertension can be transferred via an endogenous thymic factor, possibly a viral pathogen.

Animals↗

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.

Animals↗

Evidence for an intestinal mechanism of pancreatic polypeptide release.

The purpose of this study was to define the existence of an intestinal phase of pancreatic polypeptide (PP) release and to assess whether it was mediated by a cholinergic-sensitive mechanism. Four conscious dogs with 20-cm upper intestinal Thiry-Vella loops and chronic gastric fistulas were used. The Thiry-Vella (T-V) loops were perfused with 10% liver extract or 0.154 M NaCl at a rate of 1 ml/min for 120 min. In a separate experiment, 240 ml of 10% liver extract was infused over a 5-min period into the stomach via the gastric fistula. Basal PP levels were 29 +/- 4 fmol/ml. The gastric infusion of liver extract caused a significant increase of plasma PP levels to a peak of 215 +/- 29 fmol/ml (P less than 0.05). The perfusion of the T-V loop with liver extract significantly increased plasma PP levels over basal to a peak of 73 +/- 14 fmol/ml (P less than 0.05). This value was significantly less than that released by gastric infusion of liver extract (P less than 0.05). Perfusion of the loop with NaCl did not significantly alter basal plasma PP levels (P greater than 0.05). PP release by perfusion of the T-V loop with liver extract was abolished by atropine intravenous bolus (0.2 mg/kg). Although the combination of bethanechol (100 microgram/kg/hr intravenous) and liver extract consistently increased the plasma levels of PP, the values did not attain statistical significance when compared to liver extract alone (P greater than 0.05). The data presented are thus consistent with the hypothesis that there is an enteric phase of pancreatic polypeptide release and that this enteropancreatic reflex is mediated by a cholinergic-sensitive mechanism which might be hormonal or neural.

Animals↗

Protective effect of corticosteroid against the cytotoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells.

OBJECTIVES: Otic drops are commonly used not only for otitis externa but also for otorrhea in the presence of tympanic membrane perforation or tympanostomy tube. Many studies demonstrated the ototoxicity of aminoglycoside. In our previous study, we observed that gentamicin (GM), when activated with liver extract, demonstrated significant cytotoxicity. The purpose of this study was to assess the protective effect of corticosteroid against the cytotoxicity of GM and tobramycin drops using isolated cochlear outer hair cells (OHCs) in vitro with liver extract. METHODS: OHCs from adult chinchilla cochleae were exposed to standard bathing solution, liver extract alone, and aminoglycoside otic drops with and without corticosteroid and liver extract. All experiments were performed at an osmolality of 305 +/- 5 mOsm, at room temperature, and for up to 60 minutes. The images of OHCs were recorded using an inverted microscope and analyzed on the Image Pro-Plus 3.0 program. Time to cell death and change of cell length were measured and analyzed. RESULTS: The time to cell death and percent change in cell length observed was significantly longer in the GM + liver extract + dexamethasone group than the GM + liver extract group (P <.05). The Tobradex + liver extract group showed an insignificant increase in percent change of cell length (P >.05) and significantly increased time to cell death than the tobramycin + liver extract group (P <.05). CONCLUSION: This study demonstrated that dexamethasone significantly reduced aminoglycoside cytotoxicity.

Aminoglycosides↗

The misleading presence of glucose in lipid extracts from human liver.

Lipid extracts of two out of three liver samples examined contained appreciable quantities of an apparent glycolipid migrating with a chromatographic mobility intermediate between a tetrahexosylceramide standard and sphingomyelin. The substance was subsequently identified as free glucose. Its presence in the lipid extracts was probably related to the high levels of endogenous carbohydrate in the samples. To prevent possible confusion with glycolipids, care should be exercised in the interpretation of chromatographic data when organs containing high levels of endogenous carbohydrate are being examined.

Child↗