Immobilization of Rhodococcus equi DSM 89-133 onto porous celite beads for cholesterol side-chain cleavage

Summary Rhodococcus equi cells immobilized onto porous celite beads were active in side-chain cleavage of cholesterol. The effect of bead size, adsorption time and washing cycles were examined. The degradation of cholesterol side-chain using adsorbed cells was studied in batch and semi-continuous systems. Compared to free cells (80 mol%), the end-product (1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione) profile was lower (70 mol%) with the celite-adsorbed system. Correspondence to: B. N. Johri     

Nucleotide sequences of three different isoforms of beta-tubulin cDNA from Chinese hamster ovary cells.

The complete cDNA sequences of two clones encoding beta-tubulin isotypes and the partial sequence of a third isoform from Chinese hamster ovary cells have been determined. The deduced amino acid sequences of the three isoforms show extensive homology to each other as well as with other alpha and beta-tubulin sequences from various species. These results provide evidence for the expression of three different isoforms of beta-tubulin in Chinese hamster ovary cells.     

Mammalian pyruvate carboxylase: effect of biotin on the synthesis and translocation of apo-enzyme into 3T3-L adipocyte mitochondria

The effect of biotin on the induction (and possible requirement for uptake into mitochondria) of apopyruvate carboxylase has been examined in 3T3-L adipocytes. Cells fed biotin-sufficient medium contained only holoenzyme in mitochondria and no apoenzyme was detected. The amount of apoenzyme elaborated in biotin-deficient 3T3-L adipocytes was comparable to the holopyruvate carboxylase protein found in cells maintained on biotin-sufficient medium. Like the holoenzyme, the apoenzyme was detected exclusively in the mitochondrial fraction of 3T3-L adipocytes. This indicates that the synthesis of apopyruvate carboxylase and its translocation into mitochondria occur independently of the cofactor, biotin.     

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, -sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2-dipyridyl, an inhibitor of ring cleavage.S. Ahmad and B.N. Johri are with the Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantriagar 263 145, Nainital, UP, India. P.K. Roy, A.W. Khan and S.K. Basu are at Fermentation Technology Division, Central Drug Research Institute, Lucknow, India.     

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.