A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

The in vitro metabolism of androgens in rat epididymis during sexual development

The in vitro activity of epididymal 5α-reductase and 3-hydroxy-steroid dehydrogenase was studied in rats during sexual development (ages 15 to 50 days).It was found that total activity (per organ) of 3-hydroxysteroid dehydrogenase reached an absolute maximum in 25 day-old rats. At this age there was also a marked increase in the specific activity (per mg weight) of both enzymes studied. While at all other ages 5α-DHT was the main product from testosterone, in the 25 day-old rat, 5α-androstandiol accounted for 73% of the metabolites formed. The subcellular distribution of both enzymes in the epididymides of adult and prepuberal rats was found to present only minor differences. Kinetic parameters (K_M and V_max) were measured in the subcellular fractions of the immature epididymis.These results indicate the existence of a marked, but transient, shift in the activity of the enzymes which metabolize androgens in the epididymis, towards the formation of 5α-androstandiol during the discrete period between 20 and 30 days of life.     

Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.     

19-hydroxycoronaridin und 19-hydroxyibogamin,zwei antibiotisch wirksame alkaloide vom ibogamin-typ

During the separation of an extract from Tabernaemontana glandulosa, small amounts of a new alkaloid were obtained for which the structure 19-hydroxycoronaridine was deduced. This new alkaloid and 19-hydroxyibogamine, which can be obtained from it, show marked antibiotic activity in the agar plate diffusion test.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

Stereoselective synthesis of β-arabino glycosyl sulfones as potential inhibitors of mycobacterial cell wall biosynthesis

A series of β-arabino glycosyl sulfones with varying alkyl chain lengths were synthesised in a stereoselective fashion as putative mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which was maximal for a C-12 chain.     

Protozoa as Agents Responsible for the Decline of Xanthomonas campestris in Soil

A streptomycin-resistant mutant of Xanthomonas campestris was used to assess the persistence of the plant pathogen in soil and the changes in populations that might be important for its survival. In soil into which large numbers of the organism were introduced, a marked decline in its abundance occurred, but after about 1 week its population density reached a level of about 10⁵ and did not continue to fall during the test period. No such marked decline was evident in sterile soil inoculated with X. campestris. The bacterium did not lose viability if starved for carbon or inorganic nitrogen. Although abundant in soil, the numbers of propagules capable of producing antibiotics or lytic enzymes active against X. campestris did not increase coincident with the pathogen's decline, and no increase in tartrate-extractable toxins was observed. Neither bdellovibrios nor bacteriophages active against the xanthomonad were found in the soil, but a marked increase in the frequency of protozoa paralleled the phase of rapid diminution in the X. campestris population. In actidione-treated soil, in which protozoan activity was severely limited, the high cell density of the pathogen was maintained. On the basis of these data, it is concluded that predation by protozoa is responsible for the abrupt fall in frequency of the bacterium in natural soil.     

Enzymic oxidation of linolenate by Ginkgo leaves: Fractionation and characterization of active fractions

An aqueous extract of defatted, macerated leaves of Ginkgo biloba L. catalysed the oxidation of linolenate. Extracts prepared from quickly frozen Ginkgo leaves had almost the same activity as the extract from fresh leaves but no trans-2-hexenal was formed. The activity was surprisingly stable at 100° and to acids. However, at pH 12 a marked loss of activity was observed, particularly when the soln was heated, and Pronase, also destroyed most of the activity.     

Eimeria Spp.: Influence of coccidia on digestion (amylolytic activity) in broiler chickens

Loss in pancreatic weight and an overall decrease in amylolytic activity in the pancreas were seen in broiler chickens infected with Eimeria acervulina, E. maxima, and E. necatrix. Only E. acervulina and E. maxima reduced amylolytic activity in the surface mucosa of the regions they infected. Surface-bound amylolytic activity decreased as the severity of infection (as measured by lesion score) with E. acervulina increased. This decrease in activity was not related to the decreased feed consumption in infected birds. Little decrease in amylase activity was found in the lumenal contents with the exception of E. acervulina in the jejunum. When the effect of pH on enzyme activity was studied in vitro, a marked reduction in amylolytic activity was observed as the pH went below 5.0.     

Preparation and properties of an o-diphenol: O2 oxidoreductase from cocoa husk

A particulate preparation from cocoa husk which shows o-diphenol: O₂, oxidoreductase activity contains a copper protein moiety linked to a partially formed insoluble polyphenol polymer. The particles are easily stained with osmium tetroxide for electron microscopy and show marked o-diphenol-polymerisation properties when incubated with substrate. The activity and kinetic parameters of the particles against a number of substrates and inhibitors have been determined.     

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these animals.     

SKLB-163, a new benzothiazole-2-thiol derivative,exhibits potent anticancer activity by affecting RhoGDI/JNK-1 signaling pathway

Small-molecule inhibitors are an attractive therapeutic approach for most types of human cancers. SKLB-163, a novel benzothiazole-2-thiol derivative, was developed via computer-aided drug design and de novo synthesis. MTT assay showed it had potent anti-proliferative activity on various human cancer cells. Treatment of cancer cells with SKLB-163 induced obvious apoptosis and inhibited proliferation in vitro. SKLB-163 administered p.o. showed a marked antitumor activity in vivo. Proteomic techniques were employed to identify possible drug target proteins. The data showed molecular mechanism of action might be involved in downregulation of RhoGDI, which finally contributed to increased apoptosis and inhibited proliferation. These findings provided the potential value of SKLB-163 as a novel candidate antitumor drug.     

Purification and properties of the melanization and reddish colouration hormone (MRCH) in the armyworm,Leucania separata (Lepidoptera)

Larvae of Leucania separata show a marked black colouration under crowded conditions due to the action of the neurohormone termed melanization and reddish colouration hormone (MRCH). MRCH was extracted with 80% ethanol from larval brains (Br) or suboesophageal ganglia (SG) of L. separata and Br-SG of the silkworm moth, Bombyx mori. Approximately a 16,300-fold purification of the extract derived from 192,000 Bombyx heads afforded two active fractions, each of which caused a remarkable melanization of the cuticle of an L. separata larva. The MRCH activity contained in each fraction is due to a peptide (mol. wt. 6400–8000). Partially purified MRCH also caused melanization in Leucania loreyi, Spodoptera litura, and Mamestra brassicae, but had no diapause hormone activity in B. mori.     

Purification and Properties of β-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced β-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher β-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had β-N-acetylgalactosaminidase activity equivalent to about 40% of its β-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for β-N-acetylglucosaminidase activity and 5.5 to 6.5 for β-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

Differential Induction of β-1,3 Glucanase in Resistant and Susceptible Varieties of Cicer arietinum L. Following Infection with Ascochyta rabiei

Infection of chickpea (Cicer arietinum L.) by Ascochyts rabiei resulted in marked induction of β-1, 3 glucanase activity. The induction of enzyme activity was two fold higher in resistant variety as compared to the susceptible variety. Slot blot analysis of total RNA using glucanase cDNA clone from tobacco as probe revealed that the induction of enzyme activity is at mRNA level.     

Acid phosphatase activity in maize leaves as related to their evolution and phosphorus deficiency

The effect of phosphorus deficiency on the activity of acid phosphatase of the first, second and third leaves of maize plants was followed. The supernatant obtained by centrifuging the homogenate of plant tissue at 1500 ×g was further centrifuged at 18 000 ×g, the sediment marked as fraction II and the supernatant as fraction III. Acid phosphatase activity of fraction II of the first to third leaves was for the whole period of culture higher in plants grown in the nutrient solution without phosphate. In fraction III this relation was established in the first leaf, after 3 days of culture in the second leaf and after 5 days in the third leaf. In all leaves higher enzyme activity was unambiguously determined in fraction III when compared with fraction II. Higher acid phosphatase activity was established in those leaves which were younger in their development, particularly in the first days of culture. With the ageing of leaves the enzyme activity decreased.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

Upregulation of <Emphasis Type="Italic">Slc38a1</Emphasis> Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1–100 μM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.