Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline → betaine aldehyde → betaine. Our previous experiments with intact chloroplasts, and in vivo¹⁸O₂ labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O₂ and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of ¹⁸O from ¹⁸O₂ by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg²⁺, had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

The development of epidermal feet in preparation for metamorphosis in an insect

Insect epidermal cell surfaces can be seen by scanning electron microscopy after removal of the basal lamina. This let us study surface changes in the 5th larval stage of Calpodes ethlius (Lepidoptera, Hesperiidae) in preparation for metamorphosis at the end of the stadium, in particular changes in the basal cell processes or feet, intercellular lymph spaces, filopodia and hemidesmosomes. The feet develop in three phases, initiation, elongation and contraction. Initial growth begins immediately after ecdysis and continues until commitment to pupation 66 hr later. During this phase the feet are randomly oriented. Elongation and orientation begin after commitment to pupation. Orientation is probably achieved by selective survival and growth of those feet that are axially oriented rather than by reorientation. As the larva shortens to the pupal form late in the stadium, contraction of the feet occurs and the cells become columnar. The feet finally disappear as the cells rearrange themselves into new positions in the pupal epidermis. The lateral margins of the feet are united by adhesions even when their interdigitations are most complex. The adhesions separate an intercellular lymph space from the haemolymph. The lymph space remains small through most of the stadium, but enlarges with the loss of lateral junctions as the feet contract and eventually extends along most of the length of the columnar cells. Filopodia then form and span the gaps between the cells as though they have been induced by the separation and loss of lateral cell to cell contact. Scanning electron microscopy also shows that hemidesmosomes reflect the axial alignment of the cells even before the orientation of the feet. The hemidesmosome plaques are linear structures having a constant width of 0.15 - 0.2 mum and variable length. They arise in short sections and lengthen by the linear addition of more sections with the same width. Late in the stadium they lose their axial alignment and may become branched.     

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.     

Influence of Water Potential on gamma-Decalactone Production by the Yeast Sporidiobolus salmonicolor

The influence of water potential on gamma-decalactone production by the yeast Sporidiobolus salmonicolor cultivated in a liquid medium was evaluated by gas-chromatographic analysis. Modifications in water potential led to a number of variations in the aroma production. Maximum extracellular production occurred at water activity (a(w)) with a value of 0.99. Further analyses revealed an important phenomenon of cellular accumulation of aroma for a(w) values between 0.97 and 0.99.     

Short-term instabilities and long-term community dynamics

Competition in a temporally variable environment leads to sequences of short-term instabilities that in some cases are the mechanism of long-term coexistence; in other cases they promote long-term instability. Recent work associates long-term stability with a positive relationship between environmental and competitive effects and with population growth rates that are buffered against jointly unfavorable environmental and competitive events. Buffered growth rates arise from population subdivision over life-history stages, microenvironments or phenotypes. A distinct but related mechanism of long-term stability relies on population growth rates that are nonlinear functions of competition. New ways of understanding and investigating species diversity follow from these results.     

A flow injection analysis system for fermentation monitoring and control

An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.     

alpha-Factor directed expression of the human epidermal growth factor in Saccharomyces cerevisiae

Expression kinetics of the human Epidermal Growth Factor (hEGF) from the alpha-factor prepro region in a 2-mum based plasmid was studied in Saccharomyces cerevisiae. Production of hEGF was highly medium de pendent as a chemically defined, nonenriched media had a significantly lower yield than did enriched media. Also cells grown on yeast nitrogen base without amino acids with casamino acids degraded the hEGF after cell growth as opposed to a yeast extract, peptone, and dextrose (YEPD) medium, which elicited no measurable extracellular proteolysis of the hEGF. alpha-factor directed production kinetics of hEGF on the YEPD medium were growth associated, secretion limitations and extracellular degradation were negligible, and the hEGF was nearly 100% selectively secreted. With sufficient agitation, shake flask experiments were representative of aerated controlled batch fermentations. No effect of high cell density was observed on cell growth or hEGF production kinetics. The hollow fiber bioreactor had no direct effect on the substrate or protein yields of S. cerevisiae, however the low oxygen transfer capacity of the membrane was not sufficient to support respiration.