Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Production of fuel alcohol from oats by fermentation

Very high gravity (>30 g dissolved solids per 100 ml) mashes were prepared from hulled and hulless oats and fermented at 20° C with active dry yeast to produce ethanol. Excessive viscosity development during mashing was prevented by hydrolyzing -glucan with crude preparations of -glucanase or Biocellulase. Both these preparations possessed endo--glucanase activity. By using these enzymes and by decreasing the water to grain ratio, very high gravity mashes with low viscosity were prepared. Unlike wheat and barley mashes, oat mashes contained sufficient amounts of assimilable nitrogen to promote a fast rate of fermentation. The free amino nitrogen (FAN) content of oat mash could be predicted by the equation, mg FAN L^–1=8.9n wheren is the number of grams of dissolved solids in 100 ml of mash supernatant fluid. Ethanol yields of 353.2±3.7 L and 317.6±1.3 L were obtained per tonne (dry weight basis) of hulless (59.8% starch) and hulled (50.8% starch) oats respectively. The efficiency of conversion of starch to ethanol was the same in normal and very high gravity mashes.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Biochemical and bioassay analysis of resistance of potato (Solanum tuberosum L.) cultivars to attack by the slug Deroceras reticulatum (Muller)

A method is described for using young field slugs Deroceras reticulatum (Muller) in a bioassay study of biochemical resistance of potato (Solanum tuberosum L.) cultivars to slugs. Tuber parts or an artificial diet were provided as food sources. Comparisons were made of feeding, survival and weight gain between the susceptible cultivar Maris Piper and the resistant cultivar Pentland Dell. Biochemical analyses were made of these two cultivars and the resistant cultivars Stormont Enterprise and Majestic. Comparisons of tuber sections and peelings as food sources indicated factors affecting growth were located in the surface layers of the tubers. Phenolics and glycoalkaloids were concentrated in the surface layers but the amounts were similar in the susceptible and resistant cultivars and the bioassays indicated that neither acting alone could explain resistance. The amounts and distribution of free amino acids also did not correlate with resistance although when supplied in the artificial diet they partly inhibited feeding. Proteinaceous inhibitors of slug gut proteolytic enzymes were present throughout the tubers but were not concentrated in the surface layers and the amounts were similar in the different cultivars thus they too did not explain the difference in susceptibility between the cultivars. Bioassays using acetone extracts (low molecular weight substances) and acetone powders (high molecular weight substances) either alone or in combination indicated that the resistant cultivar Pentland Dell contained a high molecular weight substance which together with a low molecular weight substance from either the same cultivar or the susceptible Maris Piper could confer resistance. Bioassays using protein extracts supplied in the presence or absence of chlorogenic acid indicated that this mechanism could comprise enzymic oxidation of phenolics. Assays of phenolase confirmed this since activity was highest in the outer layers of the tubers and was highest in the three resistant cultivars. Thus the chief resistance factor identified was high phenolase activity acting rapidly on phenolics when the slug first bites the tuber surface. The quantity of phenolics per se did not control the resistance. Thus while phenolics must be available, resistance is compatible with low blackening on cutting the tuber.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter^−1), and BAP (1 mg · liter^−1) plus NAA (0.1 mg · liter^−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.