Chloris gayana (Rhodes grass), a better host for the mas production ofGlomus fasciculatum inoculum

Seven different hosts,Panicum maximum, Chrysopogon fulvis, Themeda triandra, Chloris gayana, Brachiaria brizantha, Paspalum scrobiculatum andEleusine coracana were screened in order to select a better host for mass multiplication ofGlomus fasciculatum inoculum. Of these,Chloris gayana (Rhodes grass) was found to be the best host on the basis of root colonization and spore production and of the infective propagules of the potball.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Batch production and growth kinetics of hybridomas

Batch cultures of mouse-mouse hybridoma cell lines were carried out and their growth and production kinetics investigated. Three main cell specific production patterns (expressed as pg IgG/cell x hour) were found, which can be used as a classification system for hybridoma cell lines (groups I–III). Cells showing the highest IgG-production at the beginning of the batch culture (during the lag and the onset of the log-phase) were classified as either group I and II. The difference was that cell lines of group II showed a second high cell specific production at the onset of the stationary and death phases. Cell lines of group III had a quite constant production of antibodies during their growth; but IgG secretion completely stopped after the beginning of the stationary phase. The implications of these three production patterns on the design of a production process are discussed.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid