Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

The effect of postpartum uterine lavage on foal heat pregnancy rate

Mares (n = 37) were treated on Days 2 and 4 post partum with a uterine lavage of 10 l of warm, sterile NaCl (0.9%) solution. Endometrial cytology and culture were performed on Day 7. Mares were bred on the first postpartum estrus by artificial insemination. Pregnancy rates were determined by ultrasound examination at Day 16 post ovulation. No differences were noted in degree of uterine inflammation or presence of uterine bacteria at Day 7 post partum between treated (n = 18) and control (n = 19) mares. Pregnancy rates at the first postpartum estrus for treated mares (55.5%) was not statistically different from that of control mares (68.4%). No advantage was noted in the use of intrauterine lavage with 10 l of warm sterile NaCl (0.9%) at Days 2 and 4 post partum as a means of improving foal heat pregnancy rate.     

Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund's incomplete adjuvant. Immunized mares ovulated a significantly larger (P < 0.01) number of follicles per estrous cycle (2.8 +/- 1.1; range 1 to 8 ovulations) than 14 nonimmunized control mares (1.1 +/- 0.1; range 1 to 2 ovulations). Day-7 embryo recovery rates tended to be higher (P < 0.1) in immunized mares (1.6 +/- 0.5 embryos per flush) than in control mares (0.7 +/- 0.2 embryos per flush). No differences in interovulatory intervals were found between the 2 groups. These results indicate that immunization against inhibin may be useful in inducing development and ovulation of multiple follicles for embryo transfer in the mare.     

Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Gene expression and the evolution of phenotypic diversity in social wasps

Background  

Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.     

Clusterin regulates vascular smooth muscle cell nodule formation and migration

Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall where they exist in a differentiated state to maintain vascular tone. However, VSMC are not terminally differentiated and can be induced to dediffentiate, proliferate, and migrate. In fact, smooth muscle cell migration from the vascular wall into the lumen of the vessel is a central feature of occlusive vascular pathologies including atherosclerosis and intimal hyperplasia. In vitro, in the presence of an extracellular matrix, cultured vascular smooth muscle cells can migrate and invade the underlying gelatinous matrix, form multicellular nodular aggregations, and secrete the glycoprotein clusterin. Nodular cultures appear to mimic some of the properties of differentiated VSMC, in vivo. Here, to test the hypothesis that clusterin functions to modulate the formation of VSMC nodules and to facilitate cell migration a clusterin negative VSMC clone, SM-CLU13AS (Moulson and Millis, 1999, J Cell Physiol 180:355), was transiently transfected with plasmid pRcCMVCLU that contains the full-length porcine clusterin cDNA sequence under control of the CMV promoter. The transiently transfected VSMC culture expressed and secreted clusterin and formed nodules. To determine if clusterin regulates VSMC migration we used modified Boyden chamber assays. Clusterin, at 10 microg/ml, clearly promotes VSMC migration. In addition, a 15 amino acid synthetic peptide, representing amino acids 118-132 [KQTCMKFYARVCRSG] of the mature clusterin polypeptide, inhibits VSMC attachment to gelatinous substrate. Finally, clusterin appears to have a role in regulating endogenous clusterin expression in the clusterin negative clone. These results clearly establish that clusterin has functional role in VSMC nodule formation and support the conclusion that clusterin is a critical component of smooth muscle cell phenotypic modulation.     

The current status of equine embryo transfer

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.     

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles > or = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.     

Identification of a novel class in the alpha/beta hydrolase fold superfamily: the N-myc differentiation-related proteins

The alpha/beta hydrolases constitute a large protein superfamily that mainly consists of enzymes that catalyze a diverse range of reactions. These proteins exhibit the alpha/beta hydrolase fold, the essential features of which have recently been delineated: the presence of at least five parallel beta-strands, a catalytic triad in a specific order (nucleophile-acid-histidine), and a nucleophilic elbow. Because of the difficulties experimentally in identifying protein structures, we have used a Bayesian computational algorithm (PROBE) to identify the members of this superfamily based on distant sequence relationships. We found that the presence of five sequence motifs, which contain residues important for substrate binding and stabilization of the fold, are required for membership in this superfamily. The superfamily consists of at least 909 members, including the N-myc downstream regulated proteins, which are believed to be involved in cell differentiation. Unlike most of the other superfamily members, the N-myc downstream regulated proteins have never been proposed to possess the alpha/beta hydrolase fold and do not appear to be hydrolases.