Studies on the growth of a thermotolerant yeast strain,Kluyveromyces marxianus IMB3, on sucrose containing media

A thermotolerant alcohol-producing yeast strain, Kluyveromyces marxianus IMB3 was shown to grow on sucrose (10% [w/v]) containing media at 45 °C. Under such conditions the organism reached stationary phase within 20 hours and yielded ethanol concentrations in the region of 33g/L. During growth on sucrose containing media the organism was found to produce a cell- associated activity capable of hydrolysing sucrose. This activity was shown to have a Km of 5.0mM when sucrose was used as the substrate. In addition the enzyme was shown to have a pH optimum of 5.0 and a temperature optimum of 50–55 °C and under those conditions the enzyme was shown to be relatively thermostable.     

Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.     

The rate of protein degradation in developing brain. Methodological considerations.

Recently we reported that the rate of protein breakdown decreases during development. Breakdown rates were calculated from the rates of protein synthesis and the changes in brain protein content with age. A different study, measuring breakdown by monitoring the loss of label from brain protein after an H14CO3- pulse, came to the opposite conclusion: that the rate of breakdown is low in immature brain and increases during development. We have now investigated some of the factors (the distribution of label in protein and the potential for recycling) that might introduce errors into these measurements. The specific radioactivities of both protein-bound and free amino acids were determined in the brains of young rats several days after an intraperitoneal pulse of H14CO3-. For a number of amino acids the specific radioactivity of the free amino acid is high compared with that of the protein-bound amino acid, and therefore recycling could result in an underestimate of the degradation rate. Because glutamic acid had a relatively low specific-radioactivity ratio, [1-14C]glutamic acid was used in a pulse-labelling experiment to measure degradation. The rate so obtained, 0.6% . h-1, is twice the rate found with H14CO3- labelling (based on total protein-bound radioactivity). Insofar as recycling is a possible complication, 0.6% . h-1 may be a minimum value. Although somewhat higher degradation rates are found after labelling with an intracranial pulse, which was considered as a possible route to limit recycling, there are difficulties in interpreting these data.     

Strategic use of gonadotrophin-releasing hormone (GnRH) to increase pregnancy rate and reduce pregnancy loss in lactating dairy cows subjected to synchronization of ovulation and timed insemination

The objective of this study was to determine the effect of GnRH (100 microg i.m.) treatment 5 and 15 days after timed insemination (TAI) on pregnancy rate and pregnancy loss in lactating dairy cows subjected to synchronization of ovulation. The study included 831 lactating dairy cows subjected to a Presynch-Ovsynch protocol for first service. On the day of TAI (Day 0), cows were randomly assigned to one of four experimental groups. Cows in Group 1 (n = 214) were treated with GnRH on Day 5; cows in Group 2 (n = 209) were treated with GnRH on Day 15; cows in Group 3 (n = 212) were treated with GnRH on both Day 5 and Day 15; cows in Group 4 (n = 196) were not treated. Pregnancy rate was evaluated at Day 27 and Day 45 after TAI. The interestrus interval and the proportion of cows diagnosed not pregnant based on expression of estrus and insemination before pregnancy diagnosis on Day 27 were determined. The results of this study are: (1) GnRH treatment on Day 5 or Day 15 did not increase pregnancy rate, or reduce pregnancy loss between Day 27 and Day 55 after TAI; (2) cows treated with GnRH on both Day 5 and Day 15 had a lower (P < 0.01) proportion of cows diagnosed not pregnant based on expression of estrus before ultrasonography on Day 27 (26.5%) compared to control cows (52.9%), and these cows had an extended (P = 0.05) interestrus interval (23.4 days vs. 21.5 days); and (3) GnRH treatment on both Day 5 and Day 15 after TAI reduced pregnancy rate on Day 27 (36.8% vs. 44.4% for control cows; P < 0.03) and Day 55 (28.3% vs. 36.2% for control cows; P < 0.01). Therefore, strategies to stimulate CL function using multiple doses of GnRH during the luteal phase need to consider potential negative effects.     

Role of IP(3) in modulation of spontaneous activity in pacemaker cells of rabbit urethra

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (<100 ms in duration) and "slow" (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store.     

Molecular cloning and functional expression of a Talaromyces emersonii derived alpha-amylase encoding genetic determinant in a human cell line

Summary A cDNA fragment encoding a Talaromyces emersonii acid stable alpha-amylase has been cloned into a mammalian cell expression vector system. When human HeLa cells were transformed with this DNA, functional enzyme could be detected in extracellular media and cell lysates derived from stable transformants. Expression of the T. emersonii derived cDNA was verified by northern dot blotting hybridisation analysis of mRNA extracted from transformants, using the labelled amylase encoding cDNA determinant as a probe. Results obtained suggest that expression of the fungus derived alpha-amylase in the transformed HeLa cells is glucocorticoid dependent.     

Foliose Ulva Species Show Considerable Inter‐Specific Genetic Diversity,Low Intra‐Specific Genetic Variation,and the Rare Occurrence of Inter‐Specific Hybrids in the Wild

Foliose Ulva spp. have become increasingly important worldwide for their environmental and financial impacts. A large number of such Ulva species have rapid reproduction and proliferation habits, which explains why they are responsible for Ulva blooms, known as “green tides”, having dramatic negative effects on coastal ecosystems, but also making them attractive for aquaculture applications. Despite the increasing interest in the genus Ulva, particularly on the larger foliose species for aquaculture, their inter‐ and intra‐specific genetic diversity is still poorly described. We compared the cytoplasmic genome (chloroplast and mitochondrion) of 110 strains of large distromatic foliose Ulva from Ireland, Brittany (France), the Netherlands and Portugal. We found six different species, with high levels of inter‐specific genetic diversity, despite highly similar or overlapping morphologies. Genetic variation was as high as 82 SNPs/kb between Ulva pseudorotundata and U. laetevirens, indicating considerable genetic diversity. On the other hand, intra‐specific genetic diversity was relatively low, with only 36 variant sites (0.03 SNPs/kb) in the mitochondrial genome of the 29 Ulva rigida individuals found in this study, despite different geographical origins. The use of next‐generation sequencing allowed for the detection of a single inter‐species hybrid between two genetically closely related species, U. laetevirens, and U. rigida, among the 110 strains analyzed in this study. Altogether, this study represents an important advance in our understanding of Ulva biology and provides genetic information for genomic selection of large foliose strains in aquaculture.     

Novel quantitative trait loci for partial resistance to Phytophthora sojae in soybean PI 398841

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is one of the most severe soybean [Glycine max (L.) Merr] diseases in the USA. Partial resistance is as effective in managing this disease as single-gene (Rps gene)-mediated resistance and is more durable. The objective of this study was to identify quantitative trait loci (QTL) associated with partial resistance to P. sojae in PI 398841, which originated from South Korea. A population of 305 F_7:8 recombinant inbred lines derived from a cross of OX20-8 × PI 398841 was used to evaluate partial resistance against P. sojae isolate C2S1 using a tray test. Composite interval mapping using a genome-wide logarithm of odd (LOD) threshold detected three QTL on chromosomes 1, 13, and 18, which individually explained 4–16 % of the phenotypic variance. Seven additional QTL, accounting for 2–3 % of phenotypic variance each, were identified using chromosome-wide LOD thresholds. Seven of the ten QTL for resistance to P. sojae were contributed by PI 398841. Seven QTL co-localized with known Rps genes and previously reported QTL for soil-borne root pathogens, isoflavone, and seed oil. Three QTL on chromosomes 3, 13, and 18 co-localized with known Rps genes, but PI 398841 did not exhibit an Rps gene-mediated resistance response following inoculation with 48 different isolates of P. sojae. PI 398841 is potentially a source of novel genes for improving soybean cultivars for partial resistance to P. sojae.