Glucose oxidase ofAspergillus niger

The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ _{O 2} of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg²⁺ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium.     

Cultivation of lymphatic cells in protein-free medium and chemical study of the products

Rabbits immunized withBrucella suis within a period of one week formed antibodies in high titres. These antibodies were of a macroglobulin character only. Cells from the spleen and popliteal lymph nodes were cultivatedin vitro in protein-free medium, in which the only macromolecular substance was dextran or carbowax. Physico-chemical analysis of secretion products was not succesful, since under the given cultivation conditions the cells started to disintegrate from the outset and the medium contained nucleic acids as well as proteins. Some of these substances were present in particles of the size of different subcellular particles. After cultivation of spleen and lymph node cells, antibodies were detected by an agglutination test in media containing dextran and carbowax. When particles substantially larger than the antibody molecule were removed by ultracentrifugation, the agglutination antibody titre in the medium fell.     

Photoheterotrophic callus culture <Emphasis Type="Italic">Ficus elastica</Emphasis>. The formation of polyisoprene synthesis

The ability to synthesize polyisoprene was studied for lines of Ficus elastica photoheterotrophic callus culture which results from the selection and prolonged cultivation of the culture in light in a medium containing 1% sucrose, 0.5 and 0.05 mg/l BAP or in the absence of the hormone. The results of microscopy study show that F. elastica lines are able to synthesize polyisoprene typical of intact plants. The substantial accumulation of polyisoprene was observed for the culture grown in the BAP-free media. Moreover, for some lines considered, tracheid structures were shown to be present in tissues. The formation of tracheid structures correlated directly with polyisoprene synthesis. We conclude that varying BAP content in the cultivation medium and selection allow the preparation of a culture exhibiting a certain functional specificity. Under optimal cultivation conditions, the culture develops toward the intensification of polyisoprene synthesis.     

Strain variability of <Emphasis Type="Italic">Irpex Lacteus</Emphasis> basidiomycetes in the synthesis of specific milk-clotting proteinases

The ability to synthesize milk-clotting (rennet) proteinases was studied in eight strains of Irpex Lacteus basidiomycetes. It was found that the studied I. lacteus strains are characterized by different enzyme activities in their culture liquid. For I. lacteus strains 2425, 2426, and 2427, the maximum milk-clotting activity was detected during the exponential growth phase on the 15th day of cultivation on glucose–peptone medium. These I. lacteus strains are the most prospective milk-clotting enzyme producers for further research and practical application. I. lacteus strains 2421, 2422, 2423, 2424, and 2428 showed lower values of the enzyme activity and require additional research to determine the optimal culture conditions.     

Sorption of some ions by algae related to their trophic conditions

The unicellular algaeScenedesmus obliquus (125),Chlorella pyrenoidosa (82) andCoccomyxa solorinae saccatae (111) were studied with respect to the form of uptake of potassium, phosphate, calcium and zinc ions and to the energy sources involved: light under autotrophic conditions, glucose under mixotrophic or heterotrophic conditions (in light and in darkness or together with yeast extract as an auxotrophic substrate). We respected the trophic conditions of algae when preparing the experimental material (precultivation). The following conditions were reached:

1. (1)
   The three algae grow faster in a glucose medium under mixotrophic conditions and are capable of growing on it also heterotrophically:Ch. pyrenoidosa andSc. obliquus grow substantially better thanC. solorinae saccatae. The first two algae grow more intensively in a glucose medium containing yeast extract whileCoccomyxa does not. After cultivation under mixotrophic conditions the first two diminish endogenous respiration, the third raises it. Glucose stimulates respiration in the first two when grown autotrophically, while after mixotrophic cultivation the effect of glucose is suppressed inCh. pyrenoidosa and in the other two only after growth on glucose with yeast extract.     
   
   

Studies on regulation of chorionic gonadotropin secretion in primates

The regulation of secretion of chorionic gonadotropin in primates has been studied using bothin vivo andin vitro models.In vivo studies using the pregnant bonnet monkey revealed that at the doses tested, the administration of progesterone or estradiol 17Β in combination or alone did not result in any appreciable change in the duration or magnitude of serum chorionic gonadotropin levels. However, administration of lutropin-releasing hormone by intravenous route resulted in significant increase in chorionic gonadotropin levels within 30–60 min and the extent of stimulation seemed to depend on the state of pregnancy. Forin vitro studies, explants or cells prepared from first trimester human placenta has been used. The functional integrity of these cells has been established by demonstrating the binding of [¹²⁵I]-labelled human chorionic gonadotropin antibody to the cells as well as the synthesis of [³H]-labelled human chorionic gonadotropin.In vitro studies using the cells revealed that addition of lutropin-releasing hormone caused a significant increase in chorionic gonadotropin and estradiol 17Β secreted into the medium. Thus bothin vivo andin vitro results suggest that lutropin-releasing hormone could be one of the factors involved in regulation of chorionic gonadotropin secretion in primates.     

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m²/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.     

Synthesis of L-lactate oxidaze in yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> during submerged cultivation

The biosynthesis of L-lactate oxidase in the Yarrowia lipolytica yeast during submerged cultivation in laboratory bioreactors ANKUM-2M has been studied. It has been shown under optimal conditions of yeast cultivation with L-lactate that 24.5 U/L enzyme accumulated in the medium and the yield was 2.0 U/(L h). An increase in the biosynthesis of L-lactate oxidase to 75 U/L and the yield to 3.2 U/(L h) was achieved in the medium with L-lactate (1%) and glucose (2%). The enzyme was purified 251 times to homogeneity by hydrophobic and ion exchange chromatography state with a yield of 45% and a specific activity of 55.3 U/mg. Techniques of gel filtration and denaturing electrophoresis showed that L-lactate oxidase from Y. lipolytica is a tetramer with a molecular mass of 200–230 kDa. The enzyme showed a strict specificity to L-lactate and did not oxidize fumarate, pyruvate, succinate, ascorbate, dihydroxyacetone, glycolate, D-lactate, D, L-2-hydroxybutyrate and D, L-alanine or D-serine.     

Optimization of date syrup as a novel medium for lovastatin production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> ATCC 20542 and analyzing assimilation kinetic of carbohydrates

Lovastatin is a statin drug, which lowers cholesterol level in blood due to inhibition of (S)-3-hydroxy-3-methylglutaryl-CoA reductase. Date syrup is a rich medium for microbial growth and metabolite production. The main carbohydrates present in the date syrup are glucose and fructose. In this study, date syrup was used as a complex and bioresource medium for lovastatin production by Aspergillus terreus in the submerged cultivation. Optimization of the date syrup medium in order to achieve the highest titers of lovastatin and biomass was carried out. Four factors were studied by response surface methodology including concentration of date syrup carbohydrates, yeast extract concentration, pH, and rotation speed of the shaker. Optimal conditions for these factors found were as follows: concentration of date syrup carbohydrates, 64 g/l; yeast extract concentration, 15 g/l; pH, 6.5; and agitation speed, 150 rpm. It gave lovastatin concentration of 105.6 mg/l. Next, batch cultures in the optimal conditions were performed in a 2.5-l working volume bioreactor and led to the lovastatin titer of 241.1 mg/l during 12 days. Aspergillus terreus showed diauxic growth in the optimized medium with a shift from glucose to fructose assimilation during the run. Glucose and fructose assimilation kinetic parameters revealed that more lovastatin is produced during glucose assimilation, while more biomass was formed during fructose assimilation.     

Antibody formation by cells isolated from donors of different ages after transfer to young rabbits and to X-irradiated adult rabbits

Antibody formation by spleen cells isolated from adult, non-immunized donors and mixed withBrucella suis antigenin vitro was compared after transfer to young rabbits and to adult recipients irradiated with a dose of 450 r. It was found that this dose did not produce complete inhibition and that the recipients still responded actively to the given antigen. Only young animals, therefore, are a suitable experimental model in which to study antibody formation by transferred isolated cells. Development of the antibody reaction in young rabbits was studied by transferring cells isolated from the spleen of animals aged 8–30 days and mixed with antigenin vitro. Antibodies were formed after the transfer of these cells to X-irradiated recipients, but by the active response of the latter to the antigen. Antibody formation by cells of donors of different ages can be determined after transfer to young animals. The youngest donors whose cells form antibodies after transfer are three-week-old rabbits, but demonstration of antibodies requires the transfer of quite a large number of cells. The results are discussed in relation to the experiments of other authors on the transfer of cells to young animals.     

Study of the conditions of ribosidation of 6-azauracil

When studying the optimal conditions for the ribosidation of 6-azauracil it was found that the optimal cultivation time for the strainEscherichia coli 556 was 6–8 hours. Prolongation of the cultivation time slowed down the rate of ribosidation. On using 3-hour cells concentrated to the optimal extinction value 0.95 (corresponding to a 6-hour culture), ribosidation of 6-azauracil was only 50%. Either D-ribose itself or a saccharide capable of transformation in the presence of 6-azauracil, i.e. a saccharide which can be transformed by the enzyme system of the microorganism into D-ribose, should be used as the precursor of the riboside chain in the transformation of D-azauracil.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

<Emphasis Type="Italic">In vitro</Emphasis> growth profile and comparative leaf anatomy of the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> intermediate plant <Emphasis Type="Italic">Mollugo nudicaulis</Emphasis> Lam.

Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C₃–C₄ intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel?. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel? and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.     

Optimization of C16 and C18 fatty alcohol production by an engineered strain of <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.     

Survival and reproduction of some blue-green and green algae as affected by sewage water,fertilizer factory effluent,brassica oil,phenol, toluene and benzene

Fourteen blue-green and green algae survived for widely different time periods ranging between 22–102 d in control culture medium. Irrespective of their long or short survival period in control cultures, their pro- or eukaryotic nature, their different morphological types or natural habitats, they all survived for a short time period ranging between 3–8 d in sewage water, 5–10 d in fertilizer factory effluent, ¼-2 d in brassica oil, ½-2 d in phenol, 1–3 d in toluene, and 1–4 d in benzene (showing the relative toxicity of different chemicals to different algae, and the antialgal nature of brassica oil). Dilution decreased the toxicity of these agents very little, indicating that they all were very toxic to algae. None of the agent induced the formation of any reproductive or dormant cells. Sewage water, fertilizer factory effluent, brassica oil and/or benzene favored the formation of necridia cells in Phormidium bohneri, P. foveolarum, Microcoleus chthonoplastes, Lyngbya birgei, and L. major filaments. Scenedesmus quadricauda shed off all spines earlier, Hormidium flaccidum fragmented less or not at all, Scytonema millei formed no false branch and heterocyst, Aphanothece pallida and Gloeocapsa atrata cells did not divide, Cosmarium granatum cells did not form any zygospore and Oedogonium sp. not any oogonia-like cells under all or most of treatments with 25–100 % sewage water, 1–100 % fertilizer factory effluent, 1–100 % brassica oil, 25–100 % phenol, toluene and benzene.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Carbohydrate metabolism of the phase variants of purple photosynthetic bacteria

The R and M phase variants of Rhodobacter sphaeroides and Rhodobacter capsulatus were isolated. The growth rates in the dark and in the light in glucose-containing media were much higher for the Rba. sphaeroides R variant than for the M variant. For the Rba. capsulatus R and M variants, growth rates in the dark and in the light in fructose- or glucose-containing media differed insignificantly. The cells of Rba. sphaeroides and Rba. capsulatus phase variants growing in media with glucose and fructose exhibited differences in activity of the key enzymes of the Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways. The oxidative pentose phosphate pathway (PPP) does not participate in glucose and fructose metabolism in the studied bacteria. Specific activity of the ED pathway enzymes was higher in dark-grown R and M variants of both Rba. sphaeroides and Rba. capsulatus than in the cells grown under light. Specific activity of the EMP enzymes was higher for the R and M variants of both cultures grown in the light than for those grown in the dark. Activities of the 2-keto-3-deoxy-6-phosphogluconate and fructose bisphosphate aldolases, the key enzymes of the ED and EMP pathways in Rba. sphaeroides M variant grown in the medium with glucose in the light or in the dark, were approximately twice those of the R variant. In the medium with fructose activities of these enzymes in both R and M variants did not change significantly depending on growth conditions. Activities of the enzymes of the EMP and ED pathways in the extracts of the Rba. capsulatus R and M cells grown with glucose or fructose did not change significantly. Cultivation of Rba. sphaeroides and Rba. capsulatus phase variants in the medium with fructose resulted in a considerably increased synthesis of 1-phosphofructokinase. Induction of 1-phosphofructokinase synthesis in Rba. sphaeroides occurred only in the light, while in Rba. capsulatus induction of this enzyme in the medium with fructose was observed both in the dark and in the light. Thus, under aerobic conditions in the dark the phase variants of both bacteria probably assimilated glucose and fructose via the ED pathway, while in the light the EMP pathway was active.     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.     

Physiology-biochemical properties of the cyanobacterium <Emphasis Type="Italic">Oscillatoria deflexa</Emphasis>

The optimal cultivation conditions for the cyanobacterium Oscillatoria deflexa were studied: temperature (25–27°C), pH (9.0–11.0), and illumination (7 klx). A nutrient medium providing for optimum cyanobacterium growth was selected, as well as media containing an aqueous extract of human urine ash and an inedible material of wheat and vegetables with added nitrates and bicarbonate. The chemical composition (macro and microelements, content of proteins, lipids, carbohydrates, and vitamins, amino acid and fatty acid composition, ash residue) of O. deflexe was studied for the first time. An analysis of the results indicates that O. deflexa is not inferior to the cyanobacterium Spirulina platensis and the green laver Chlorella vulgaris in practical use, for its mineral composition, content of vitamins, essential amino acids and fatty acids, and exceed them in its content of vitamin E and microelements, such as Fe, Mn, Ni. The bacterium’s ability to transport NaCl up to 30 g/l within the medium was studied, and its unique ability for survival and long-term storage was shown. The enhibitory effect of the biomass of O. deflexa on the germination of wheat grains, and growth of daphnids and rotifers was shown.