Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Assembly and interactions of cotJ-encoded proteins, constituents of the inner layers of the Bacillus subtilis spore coat

During Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon ( cotJA and cotJC ) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA . Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.     

A study on the minimum number of loci required for genetic evaluation using a finite locus model

For a finite locus model, Markov chain Monte Carlo (MCMC) methods can be used to estimate the conditional mean of genotypic values given phenotypes, which is also known as the best predictor (BP). When computationally feasible, this type of genetic prediction provides an elegant solution to the problem of genetic evaluation under non-additive inheritance, especially for crossbred data. Successful application of MCMC methods for genetic evaluation using finite locus models depends, among other factors, on the number of loci assumed in the model. The effect of the assumed number of loci on evaluations obtained by BP was investigated using data simulated with about 100 loci. For several small pedigrees, genetic evaluations obtained by best linear prediction (BLP) were compared to genetic evaluations obtained by BP. For BLP evaluation, used here as the standard of comparison, only the first and second moments of the joint distribution of the genotypic and phenotypic values must be known. These moments were calculated from the gene frequencies and genotypic effects used in the simulation model. BP evaluation requires the complete distribution to be known. For each model used for BP evaluation, the gene frequencies and genotypic effects, which completely specify the required distribution, were derived such that the genotypic mean, the additive variance, and the dominance variance were the same as in the simulation model. For lowly heritable traits, evaluations obtained by BP under models with up to three loci closely matched the evaluations obtained by BLP for both purebred and crossbred data. For highly heritable traits, models with up to six loci were needed to match the evaluations obtained by BLP.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Increases in tobacco exposure biomarkers measured in non-smokers exposed to sidestream cigarette smoke under controlled conditions

National surveys of the exposure of non-smokers to secondhand smoke based on serum cotinine analyses have consistently identified certain groups within the population including children, males and non-Hispanic Blacks as having relatively greater exposure. Although these differences in mean serum cotinine concentrations probably represent differences in exposure of individuals in their daily lives, it is also possible that metabolic or other differences in response might influence the results. To better define the nature of those findings, we have examined the response of 40 non-smokers including both men and women and African-Americans and whites to sidestream (SS) cigarette smoke generated by a smoking machine under controlled conditions. In this study, participants were exposed to aged, diluted SS smoke (ADSS) generated in an environmental chamber with a mean air nicotine concentration of 140 μg m^?3 and 8.6?ppm CO for 4?h. Salivary cotinine was measured every 30?min, and serum cotinine samples were taken prior to, and 2?h after exposure. Urinary nicotine metabolites and NNAL, a tobacco-specific nitrosamine, and 4-aminobiphenyl (4-AB) haemoglobin adducts were also measured prior to and 2?h following the exposure. Under these uniform, controlled conditions, we found a similar response to ADSS smoke exposure among all the participants. In all cases a significant increase in biomarker concentration was noted following exposure, and the short-term increases in salivary cotinine concentration were quite similar at approximately 12?pg ml^?1 min^?1 among the groups. In this small study, no significant differences by gender or race were seen in the mean increases observed in cotinine, NNAL or 4-AB adducts following 4?h of exposure. Thus, our results are most consistent with a relatively uniform response in tobacco biomarker concentrations following short-term exposure to ADSS tobacco smoke, and suggest that biomarker measurements are capable of effectively indicating increases in exposure among groups of non-smokers.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.