Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Lipase production byCandida rugosa: Fermentation behaviour

Summary Extracellular lipase production byCandida rugosa growth has been studied. The main growth parameters, and the lipase activity in the culture broth were determined in order to identify the maximum of enzyme activity.The effect of lipidic material and size and growth phase of the inoculum on enzymatic production have been studied. Maximum extracellular lipase activity was associated with an increase in enzyme production when the number of viable cells started to decrease.     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

Effect of artemisinin on proliferation and apoptosis-related protein expression in vivo and in vitro

Artemisinin is the first-line drugs for the treatment of malaria. In recent years, a large number of reports showed that artemisinin exhibit anti-tumor activity. In this study, we used C6 glioma cells and rat C6 brain-glioma model to study anti-tumor activity of artemisinin in vivo and in vitro. We found that artemisinin inhibited the proliferation in C6 cells and induced cell cycle arrest and a caspase-3-dependent cell apoptosis. It also inhibited the growth of C6 brain-glioma in vivo and enhanced living state of rat brain-glioma model. These results suggested that artemisinin had significant anti-tumor activities on C6 cells both in vitro and in vivo. Artemisinin might be exploited as a promising clinical anti-cancer drug in future.     

Overproduction of human insulin-like growth factor-II inEscherichia coli

Summary Human insulin-like growth factor II (hIGF-II) was produced inEscherichia coli as a protein fused to human growth hormone. High level expression of the fusion protein was attained with pIBL-1 plasmid. The hIGF-II obtained byin vitro cleavage of the fusion protein with cyanogen bromide was highly purified and its biological activity was assessed.     

Brassica grown gall tumourigenesis and in vitro of transformed tissue

A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8–10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

Optimization of growth conditions for the induction of tyrosine decarboxylase inStreptococcus faecalis

Summary Growth conditions were investigated for optimal tyrosine decarboxylase (EC 4.1.1.25) activity in acetone dried cells ofStreptococcus faecalis. A growth pH of 6.0 was found optimal for enzyme induction. The enzyme was also shown to be growth-associated which indicates that continuous fermentation is preferable for optimal process productivity.     

The Arabidopsis Mei2 homologue AML1 binds AtRaptor1B,the plant homologue of a major regulator of eukaryotic cell growth

Background  

TOR, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.     

Plasmodium chabaudi: efficacy of artemisinin + curcumin combination treatment on a clone selected for artemisinin resistance in mice

Recent studies have proposed curcumin as a potential partner for artemisinin in artemisinin combination therapies to treat malaria infections. The efficacy of curcumin alone and in combination with artemisinin was evaluated on a clone of Plasmodium chabaudi selected for artemisinin resistance in vivo. The addition of piperine as an enhancer of curcumin activity was also tested.Results indicated that curcumin, both alone and in combination with piperine had only a modest antimalarial effect and was not able to reverse the artemisinin-resistant phenotype or significantly affect growth of the tested clone when used in combination with artemisinin. This is in contrast with previous in vivo work and calls for further experimental evaluation of the antimalarial potential of curcumin.     

Production of active human interferon-β inE. coli II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid

Summary The maximum level of human interferon- activity was expressed under the control of theE. coli tryptophan promoter whenE. coli cells were induced at late logarithmic growth phase by 3,-indoleacrylic acid (IAA). The level is one order of magnitude higher than that obtained when the cells were induced at early logarithmic or stationary phase. When IAA was subsequently further added, the decrease in the activity observed at a latter period of fermentation was suppressed.     

Growth conditions of a pectinolyticAspergillus fumigatus for degumming of natural fibres

Summary Optimum growth conditions forA. fumigatus strain 4 when citric pectin was the sole carbon source were at a temperature of 45°C, pH 4.0 and an incubation time from 36 to 42h. Under these conditions no cellulase activity was found. When orange pulp was the sole carbon source, optimum polygalacturonase activities were found when the fungus was cultured for 36 h at 45°C and a pH 3.0 to 4.5.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

A new medium for growth and delta-endotoxin production by Bacillus thuringiensis var. kurstaki

Summary The influence of composition of media used for growth and delta-endotoxin production by B. thuringiensis var. kurstaki was studied with the idea of finding a cheap medium for attaining high yields of spore-crystal preparations. A new medium, based on malt sprouts is proposed. Data on growth and bioinsecticidal activity are given. A concentration of 2.1 × 10⁹ spores.ml^-1 was attained in a 48 h process. The spore-crystal preparations obtained present a LC₅₀ of 2.18 × 10⁸ spores.g^-1 against larvae of Galleria mellonella.     

Effect of media composition on the production of extracellular amylase from the thermophilic fungusThermomyces lanuginosus

Summary The production of extracellular amylase by the thermophilic fungusThermomyces lanuginosus was studied in shake flask cultures with different carbon sources in the growth medium. Maximum amylase production was obtained with dextran as carbon source. The greatest yield of amylolytic activity was found when low molecular weight dextrans were used as carbon source.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

Cumulative role of bioinoculants on growth,antioxidant potential and artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> L. under organic field conditions

Artemisia annua L. is mostly known for a bioactive metabolite, artemisinin, an effective sesquiterpene lactone used against malaria without any reputed cases of resistance. In this experiment, bioinoculants viz., Streptomyces sp. MTN14, Bacillus megaterium MTN2RP and Trichoderma harzianum Thu were applied as growth promoting substances to exploit full genetic potential of crops in terms of growth, yield, nutrient uptake and particularly artemisinin content. Further, multi-use of the bioinoculants singly and in combinations for the enhancement of antioxidant potential and therapeutic value was also undertaken which to our knowledge has never been investigated in context with microbial application. The results demonstrated that a significant (P < 0.05) increase in growth, nutrient uptake, total phenolic, flavonoid, free radical scavenging activity, ferric reducing antioxidant power, reducing power and total antioxidant capacity were observed in the A. annua treated with a combination of bioinoculants in comparison to control. Most importantly, an increase in artemisinin content and yield by 34 and 72 % respectively in the treatment having all the three microbes was observed. These results were further authenticated by the PCA analysis which showed positive correlation between plant macronutrients and antioxidant content with plant growth and artemisinin yield of A. annua. The present study thus highlights a possible new application of compatible bioinoculants for enhancing the growth along with antioxidant and therapeutic value of A. annua.     

Formation of anti-plant viral protein by Mirabilis jalapa L. cells in suspension culture

The extract of Mirabilis jalapa cultured cells and its precipitate fraction with 90% saturated ammonium sulfate showed an anti-plant viral activity comparable to that of the roots and leaves of the original plant. In the immunodiffusion experiment, the extract of cultured cells positively reacted with MAP (Mirabilis Anti-plant viral Protein) anti-serum. The changes in MAP formation during cell growth and the MAP content of roots and leaves were examined using enzyme-linked immunosorbent assay (ELISA). MAP formation proceeded almost in parallel with cell growth. The MAP content of cultured cells reached the highest level (0.6 mg/g dry weight) on the 9th day after inoculation, which was less than one-third of the content of the roots but three times larger than that of the leaves.Abbreviations MAP Mirabilis anti-plant viral protein - TMV tobacco mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - ELISA enzyme-linked immunosorbent assay Studies on the production of anti-plant viral substances of higher plant cells in suspension culture. Part 1     

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.