Reversed-phase chromatographic method for specific determination of glutathione in cultured malignant cells

A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20 000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5′-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered.     

Synergistic interaction between immune serum and thoracic duct cells in the adoptive transfer of rapid expulsion of Trichinella spiralis in adult rats

Rapid expulsion of Trichinella spiralis could be transferred to naive adult rats with thoracic duct lymphocytes and immune serum. Thoracic duct cells collected from Days 3-5 and immune serum collected on Day 28, respectively, after infection were effective. Both cells and serum were unable to transfer rapid expulsion when given alone, even in large volumes. Recipients of immune serum and cells eliminated a significantly higher number of larvae than control rats by 1 hr after challenge with muscle larvae. Rapid expulsion produced 30-80% larval worm rejection but could not be increased by the transfer of more cells or immune serum. Mucus trappings did not appear to play a role in the rejection process. After transfer of 2 x 10(8) cells and 4.0 ml immune serum, rapid expulsion persisted for less than 1 week. However, after adoptive transfer of cells alone, the gut remained functionally receptive to the passive transfer of immune serum for 7 weeks. Therefore, the changes effected by transfer of cells were long lived in contrast to the 1 week, or less, of functional persistence by transferred immune serum. The data indicate that two separate processes, one cell mediated and the other immune serum mediated, interact synergistically in the intestine and lead to the expression of rapid expulsion.     

Biopharmaceutical studies of 3-substituted isatin derivatives

The metabolic fate of isatin hydrazone (Ia), isatin-3-thiosemicarbazone (Ib), isatin-3-semicarbazone (Ic), isatin-3-phenylhydrazone (Id), isatin oxime (Ie) and 3-hydroxy-3-acetonyl oxindole (II) was studied in rabbits. The compounds were administered orally in the dose of 300 mg/kg body wt. Isatin anthranilic acid, tryptophan and nicotinic acid were identified as the major metabolites excreted in urine. The 3-hydroxy-3-acetonyl oxindole (II) gave on additional metabolite, oxindole. The major metabolites were separated and identified unambiguously on thin layer silica gel plate. Metabolic pathways have been proposed to explain the biotransformation of the compounds investigated.     

Immunofluorescent localization of acetyl CoA carboxylase, fatty acid synthetase and pyruvate carboxylase during the adipocyte conversion of 3T3 fibroblasts

Three enzymes involved in the conversion of 3T3-L2 fibroblasts into fat cells, acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS) and pyruvate carboxylase (PC) have been localized by immunofluorescence techniques. The method enables the identification of cells undergoing the conversion while they are still fibroblastic in appearance, often before the obvious appearance of fat droplets. Specific fluorescence for each enzyme can be seen in "clones" of cells derived from single cells, which may undergo an event during logarithmic growth, which programs the cells to differentiate subsequent to confluence of and addition of induction medium.     

Acyl coenzyme A carboxylase of Propionibacterium shermanii: detection and properties.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.     

Characterization of a stable intermediate in the unfolding of diazoacetylglycine ethyl ester--pepsin by urea.

The irreversible unfolding of covalently inhibited swine pepsin by urea was studied by spectrophotometric and viscosity measurements. At pH 4.5 and 25 degrees C in 8 M urea, a stable intermediate form of the protein was detected. It differed from the native protein by a slight loss of secondary structure and an increased intrinsic viscosity ([pi] = 7.5 mL g-1), indicating the intermediate to have an increased molecular volume or to be more asymmetric in shape. The protein was transformed into a random coil form by increases of temperature and pH. Comparison with other results suggested that at pH 6 pepsin is less stable than its inactive precursor, pepsinogen, by about 3 Kcal mol-1 (1 cal = 4.187 J).     

Analysis of tadpole growth following gamma-ray irradiation

This investigation reports observations on the growth of Rana cyanophlyctis tadpoles following a whole body gamma-ray exposure. The effect on the body and hind limb size is perhaps the most significant of the period of latency. A dose of 2,000 R serves as a stressor and activates ACTH secretion in the tadpoles. The resultant somatotrophic hormone (STH) inhibition and subsequent recovery during the following days seem an integral part of the radiation syndrome.