Bacterial cellulases and saccharification of lignocellulosic materials

Five locally isolated bacterial strains produced extracellular cellulase enzymes, primarily CMCase, when grown on different natural and commercial cellulosic substrates. Extracellular CMCase and avicelase activity was higher with the strain CLS-32, a Cytophaga sp., compared to four other strains. The whole-cell preparations of these isolates were found to saccharify cellulosic substrates to reducing sugars. Maximum release of reducing sugar (5.75 mg ml⁻¹) was obtained with CLS-32 using sugar cane bagasse as growth and hydrolysis substrates.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

In vitro propagation ofMitragyna parvifolia Korth.

Multiple shoot formation and their elongation from excised apical vegetative shoots of a 40-year old-tree ofMitragyna parvifolia Korth. was achieved in Murashige and Skoog's medium supplemented with 4.44 M benzyl adenine. The in vitro regenerated shoots rooted when cultured on modified Murashige and Skoog's medium containing low inorganic salts and the three auxins. Regeneration by this method was suitable for mass propagation of the plant.     

New prospects for deducing the evolutionary history of metabolic pathways in prokaryotes: Aromatic biosynthesis as a case-in-point

Metabolic pathways of prokaryotes are more biochemically diverse than is generally recognized. Distinctive biochemical features are shared by phylogenetic clusters. The hierarchical levels of characterstate clustering depends upon evolutionary events which fortuitously became fixed in the genome of a common ancestor. Prokaryotes can now be ordered on a phylogenetic tree. This allows the evolutionary steps that underlie the construction and regulation of appropriately complex biochemical pathways to be traced in an evolutionary progression of prokaryote types that house these pathways. Essentially the approach is to deduce ancestral character states at ever deeper phylogenetic levels, utilizing logical principles of maximum parsimony. The current perspective on the evolution of the biochemical pathway for biosynthesis of aromatic amino acids is developed as a case-in-point model for analyses that should be feasible with many major metabolic systems. Phenylalanine biosynthesis probably arose prior to the addition of branches leading to tyrosine and tryptophan. An evolutionary scenario is developed that begins with non-enzymatic reactions which may have operated in primitive systems, followed by the evolution of an enzymatic system that pre-dated the divergence of major lineages of modern eubacteria (Gram-positive bacteria, Gram-negative purple bacteria, and cyanobacteria).Florida Agricultural Experiment Station, Journal Series No. 8251.     

Luteolytic action of two antiprogestational agents (RU-38486 and ZK-98734) in the rat

RU-38486 or ZK-98734 treatment (3 mg/day, s.c.) to intact or hysterectomized adult female rats on Days 5-7 post coitum induced changes characteristic of luteolysis. Ultrastructurally, the luteal cells exhibited an extensive vacuolization of the cytoplasm and perinuclear areas, degeneration of mitochondrial cristae, massive accumulation of lipid droplets, increase in number of lysosome like granules and heterochromatinization of the nucleus. In general, RU-38486 induced more marked degeneration of the luteal cells than did ZK-98734. There was also a significant decrease in peripheral plasma progesterone concentrations in treated rats. We suggest that these antiprogestagens act via inhibition of luteal function in addition to their antagonism at the uterine progesterone receptor level.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 microM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.     

Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 μg/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigatedin vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2–3 min, but thereafter decreased with time. Under these incubation conditions, irreversible binding of catechol estrogen metabolite to cytochrome P450c increased for the first 2–3 min and then remained at this plateau level. It was concluded that enzyme destruction by a reactive estrogen metabolite or by lipid peroxides may be a major reason for the organ-specific decrease in cytochrome P450 enzymes in kidneys of estrogen-treated hamsters.     

Anaerobic growth on elemental sulfur using dissimilar iron reduction by autotrophic Thiobacillus ferrooxidans

Abstract Anaerobic growth on elemental sulfur using dissimilar iron reduction by Thiobacillus ferrooxidans has been demonstrated. The ferric ion reducing activity (FIR) of the anaerobic cells was double that of the aerobic cells. Significant differences in inhibition of FIR by respiratory inhibitors were observed between aerobic and anaerobic cells. A higher amount of cytochrome was detected in anaerobic cells compared to aerobic cells. Absorption minima developed with the addition of ferric sulfate in the dithionite reduced cell suspension demonstrated that the ferric ion could accept electrons from the cytochrome system of this bacterium. The possibility of two different electron transport chains in ferric ion reduction is discussed.