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.     

Interaction of poly(ADP-ribose)polymerase with DNA polymerase α

Homogeneously purified poly(ADP-ribose) polymerase (PARP) specifically stimulated the activity of immunoaffinity-purified calf or human DNA polymerase by about 6 to 60-fold. Apparently, poly(ADP-ribosyl)ation of DNA polymerase was not necessary for the stimulation. The effects of PARP on DNA polymerase were biphasic: at very low concentrations of DNA, it rather inhibited its activity, whereas, at higher DNA concentrations, PARP greatly stimulated it. The autopoly(ADP-ribosyl)ation of PARP suppressed both its stimulatory and inhibitory effects. By immunoprecipitation with an anti-DNA polymerase antibody, it was clearly shown that PARP may be physically associated with DNA polymerase . Stimulation of DNA polymerase may be attributed to the physical association between the two, rather than to the DNA-binding capacity of PARP, since the PARP fragment containing only the DNA binding domain showed little stimulatory activity. The existence of PARP-DNA polymerase complexes were also detected in crude extracts of calf thymus.     

The effects of warming by active and passive means on the subsequent responses to cold water immersion

Two experiments were undertaken to investigate the effects of warming the body upon the responses during a subsequent cold water immersion (CWI). In both experiments the subjects, wearing swimming costumes, undertook two 45-min CWIs in water at 15° C. In experiment 1, 12 subjects exercised on a cycle ergometer until their rectal temperatures (T _re) rose by an average of 0.73°C. They were then immediately immersed in the cold water. Before their other CWI they rested seated on a cycle ergometer (control condition). In experiment 2, 16 different subjects were immersed in a hot bath (40° C) until their T _re rose by an average of 0.9° C; they were then immediately immersed in the cold water. Before their other CWI they were immersed in thermoneutral water (35° C; control condition). Heart rate in both experiments and respiratory frequency in experiment 1 were significantly (P < 0.05) higher during the first 30 s of CWI following active warming. In experiment 1, the rate of fall of T _re during the final 15 min of CWI was significantly (P < 0.01) faster when CWI followed active warming (2.46° C · h^–1) compared with the control condition (1.68°C · h^–1). However, this rate was observed when absolute T _re was still above that seen in the control CWIs. It is possible, therefore, that if longer CWIs had been undertaken, the two temperature curves may have converged and thereafter fallen at similar rates; this was the case with the aural temperature (T _au) seen in experiment 1 and the T _au and T _re in experiment 2. It is concluded that pre-warming is neither beneficial nor detrimental to survival prospects during a subsequent CWI.     

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.     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

ISL2, a new mobile genetic element in Lactobacillus helveticus

Spontaneous, phenotypically stable mutations at the -galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 by long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1 % identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4–21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.     

Physiological effects of five months exposure to low concentrations of O3 and NH3 on Douglas fir (Pseudotsuga menziesii)

Three years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed lo filtered air, O₃ (day and night concentrations of 78 and 30 μgm^?3: respectively). NH₃ (54 μg m^?3) and to a mixture of NH₃+O₃ (day and night concentrations of 49 + 83 and 49 + 44 μg m^?3 respectively), for 5 months in fumigation chambers. Both gas exchange and chlorophyll fluorescence were measured on shoots which had sprouted at the beginning of the exposure period. After 4. 8, 10 and 20 weeks of exposure, light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. Net CO₂ assimiialion was measured at maximum light intensity of 560) μmol m^?2 S^?1 (P_n.560). After 8 and 10 weeks of exposure also light response curves of CO₂ assimilation were assessed. Shoots exposed to O₃ showed a reduction in net CO₂ assimilation as compared to the control shoots during the entire exposure period. The reduction was related lo a lower chlorophyll content and a lower electron transport rate, whereas no effect on quantum yield efficiency (qy) was observed. In contrast, shoots exposed to NH₃ showed a positive effect on photosynthesis. Shoots exposed to NH₃. + O₃ showed a rapid increase in P_n.560, in the period between 4 and 8 weeks to a level equal of that of the NH₃-treatment. After this period a decline in P_n.560 was observed. After 10 weeks of exposure shoots exposed to O₃ showed an increased transpiration rate in the dark as compared to the control shoots. In addition, water use efficiency (WUE) declined as a result of an increase in leaf conductance. Both observations indicate that the stomatal apparatus was affected by O₃. A high transpiration rate in the dark was also found for shoots esposed to NHX. However, shoots exposed to NH₃+ O₃ showed neither an effect on WUE, nor an effect on transpiration rate in the dark. The possibility that NH₃ delayed the O₃ induced effects on photosynthesis and stomatal conductance is discussed.     

The effects of tail autotomy on survivorship and body growth of Uta stansburiana under conditions of high mortality

We examined the effects of tail autotomy on survivorship and body growth of both adult and juvenile Uta stansburiana by directly manipulating tail condition. Tail loss decreased neither survivorship nor rate of body growth for individuals in two natural populations. Lack of an influence of tail loss on survivorship in these two populations may be the result of high mortality. Under high mortality any differential effects of tail loss will be lower than in populations facing lower mortality. Growth experiments in the laboratory demonstrated that, under conditions of minimal environmental variation and social interactions, there is no tradeoff between body growth and tail regeneration as has been suggested for other species of lizards. One possible reason for this difference is that U. stansburiana does not use the tail as a storage organ for lipids. The original and regenerated tails are composed mainly of protein. In general, any differential body growth between tailed and tailless individuals may be due to social interactions and not a diversion of limited energy into tail regeneration.