Embryo Transplantation in Cattle

Fourteen true repeat breeders with entirely normal oestrous cyclicity more than 1 year after calving and 14 control donor cows were superovulated with PMSG (2000 i.u.) and flushed non-surgically 6–8 days after the superovulatory heat. The superovulatory response was identical for the 2 groups such as assessed by the number of corpora lutea (9.4 ± 1.8 C.L. per repeat breeder and 9.1 ± 1.5 per control cow), occurrence of ovarian overstimulation (polycysts), presence of a non-countable amount of corpora lutea, negative outcome of the flushings and the number of recovered embryos (5.8 ± 1.0 embryos per repeat breeder and 6.0 ± 1.8 embryos per control cow). The most pronounced difference between the 2 categories of animals was related to the fertilization rate of embryos. In the repeat breeder group only 2.4 embryos per cow or 41 % were fertilized, whereas the control animals attained a fertilization rate of 4.9 embryos or 82 %. Since most factors liable to interfere with the fertilization process were identical for both groups (age, breed, nutritional and management conditions, semen quality, dose, AI-technician e.g.), it is believed that intraovarian, follicular, or follicular-dynamic conditions were responsible for producing a high proportion of non-fertilizable oocytes.     

Plant α-glucosidases of the glycoside hydrolase family 31. Molecular properties,substrate specificity,reaction mechanism,and comparison with family members of different origin

Plant Molecular Biology -     

Tachykinins May Mediate Capsaicin-Induced, But Not Vagally Induced Motility in Porcine Antrum

Schmidt, P. T., T. N. Rasmussen and J. J. Holst. Tachykinins may mediate capsaicin-induced, but not vagally induced motility in porcine antrum. Peptides 18(9), 1511–1516, 1997.—Tachykinins are thought to be involved in extrinsic control of motility in the gastrointestinal tract. Using the isolated perfused porcine antrum with intact vagal innervation, we studied the effects of substance P, neurokinin A and capsaicin infusion, and electrical stimulation of the vagus nerves on antral motility without or with infusion of non-peptide antagonists for NK-1 receptors (CP96345) and NK-2 receptors (SR48968). Substance P and neurokinin A stimulated antral motility in a dose-dependent manner. The effect could be inhibited by atropine or a combination of the NK-1 and NK-2 receptor antagonists. Electrical stimulation of the vagus nerves and infusion of capsaicin (10⁻⁵ M) stimulated antral motility. Vagally induced motility was not influenced by infusion of CP96345 and SR48968, whereas the effect of capsaicin was blocked. We conclude that tachykinins may be involved in regulation of antral motility through sensory nerves in the porcine antrum, but they do not seem to be involved in vagal regulation of antral motility.     

High Resilience in Heathland Plants to Changes in Temperature, Drought, and CO2 in Combination: Results from the CLIMAITE Experiment

Climate change scenarios predict simultaneously increase in temperature, altered precipitation patterns and elevated atmospheric CO₂ concentration, which will affect key ecosystem processes and plant growth and species interactions. In a large-scale experiment, we investigated the effects of in situ exposure to elevated atmospheric CO₂ concentration, increased temperature and prolonged drought periods on the plant biomass in a dry heathland (Brandbjerg, Denmark). Results after 3 years showed that drought reduced the growth of the two dominant species Deschampsia flexuosa and Calluna vulgaris. However, both species recovered quickly after rewetting and the drought had no significant effect on annual aboveground biomass production. We did not observe any effects of increased temperature. Elevated CO₂ stimulated the biomass production for D. flexuosa in one out of three years but did not influence the standing biomass for either D. flexuosa or the ecosystem as more litter was produced. Treatment combinations showed little interactions on the measured parameters and in particular elevated CO₂ did not counterbalance the drought effect on plant growth, as we had anticipated. The plant community did not show any significant responses to the imposed climate changes and we conclude that the two heathland species, on a short time scale, will be relatively resistant to the changes in climatic conditions.     

A stage‐wise approach for the analysis of multi‐environment trials

Plant breeders and variety testing agencies routinely test candidate genotypes (crop varieties, lines, test hybrids) in multiple environments. Such multi‐environment trials can be efficiently analysed by mixed models. A single‐stage analysis models the entire observed data at the level of individual plots. This kind of analysis is usually considered as the gold standard. In practice, however, it is more convenient to use a two‐stage approach, in which experiments are first analysed per environment, yielding adjusted means per genotype, which are then summarised across environments in the second stage. Stage‐wise approaches suggested so far are approximate in that they cannot fully reproduce a single‐stage analysis, except in very simple cases, because the variance–covariance matrix of adjusted means from individual environments needs to be approximated by a diagonal matrix. This paper proposes a fully efficient stage‐wise method, which carries forward the full variance–covariance matrix of adjusted means from the individual environments to the analysis across the series of trials. Provided the variance components are known, this method can fully reproduce the results of a single‐stage analysis. Computations are made efficient by a diagonalisation of the residual variance–covariance matrix, which necessitates a corresponding linear transformation of both the first‐stage estimates (e.g. adjusted means and regression slopes for plot covariates) and the corresponding design matrices for fixed and random effects. We also exemplify the extension of the general approach to a three‐stage analysis. The method is illustrated using two datasets, one real and the other simulated. The proposed approach has close connections with meta‐analysis, where environments correspond to centres and genotypes to medical treatments. We therefore compare our theoretical results with recently published results from a meta‐analysis.     

Targeted disruption of the mouse phosphomannomutase 2 gene causes early embryonic lethality

Mutations in the cytosolic enzyme phosphomannomutase 2 (PMM2), which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, cause the most common form of congenital disorders of glycosylation, termed CDG-Ia. It is an inherited multisystemic disease with severe neurological impairment. To study the pathophysiology of CDG-Ia and to investigate possible therapeutic approaches, we generated a mouse model for CDG-Ia by targeted disruption of the Pmm2 gene. Heterozygous mutant mice appeared normal in development, gross anatomy, and fertility. In contrast, embryos homozygous for the Pmm2-null allele were recovered in embryonic development at days 2.5 to 3.5. These results indicate that Pmm2 is essential for early development of mice. Mating experiments of heterozygous mice with wild-type mice could further show that transmission of the female Pmm2-null allele is impaired.     

“Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of “blind” mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.).     

Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment

Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80°C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with Firmicutes (Bacteria) and Thermococcales (Archaea). Potential heterotrophic nitrate reducers (Deferribacterales) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of dsrAB genes revealed that archaeal SRP (Archaeoglobus) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (Desulfacinum and Desulfotomaculum). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [³⁵S]sulfate incubations at 80°C. Sulfate reduction was found at 60°C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i) Archaeoglobus can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.During the process of secondary oil recovery in offshore oil fields, most often sulfate-rich seawater is injected into the reservoir to increase pressure and enhance recovery. The supply of large amounts of sulfate as an electron acceptor and the presence of oil organics and their degradation products as electron donors facilitate the enrichment and growth of sulfate-reducing prokaryotes (SRP) in the reservoir, as well as in piping and topside installations (51, 54). The activity of SRP causes severe economic problems due to the reactivity and toxicity of the produced hydrogen sulfide (H₂S). In addition to microbiologically influenced corrosion and reservoir souring, the efficiency of oil production is decreased due to plugging by SRP biomass and precipitated metal sulfides (12, 39). Besides the use of broad-spectrum biocides or inhibitors for sulfate reduction, the addition of nitrate effectively decreased the net production of H₂S in model column studies (15, 20, 38) and field trials (7, 53). The mechanisms by which nitrate addition might affect souring control are (i) the stimulation of heterotrophic nitrate-reducing bacteria (hNRB) that outcompete SRP for electron donors, (ii) the activity of nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), and (iii) the inhibition of SRP by the production of nitrite and nitrous oxides (21, 51).Identification and quantification of reservoir microorganisms, including NRB and SRP, has so far most frequently been assessed by cultivation-dependent methods (7, 12, 53), and cultivation-independent methods have only recently been introduced into the field of reservoir microbiology (11, 17, 28, 32). Considering the small number of these studies, information currently available on the microbial communities and especially on the abundance of nitrate and sulfate reducers present in oil reservoirs or production systems is sparse and, most notably, not quantitative.The goal of this study was to compare the diversity, abundance, and activity of SRP in production water (PW) from a nitrate-treated and an untreated oil reservoir using a combination of 16S rRNA and dsrAB gene-based analyses, newly developed quantitative PCR (qPCR) assays, and ³⁵SO₄²⁻ radiotracer incubations. The two analyzed oil reservoirs (Dan and Halfdan) share similar physicochemical characteristics with regard to injection water composition and reservoir conditions, but nitrate has only been added at Halfdan since the start of production. It is hypothesized that the addition of nitrate to the injection water favored the growth of hNRB and/or NR-SOB, thereby inhibiting the activity of SRP and reducing the concentration of H₂S, and is consequently reflected in a lower abundance of SRP and a more specialized prokaryotic community.     

Role of solvation barriers in protein kinetic stability

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well).