Topological studies on the enzymes catalyzing the biosynthesis of Glc-P- dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2- P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P- Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.      

Mitochondrial gene divergence of Colombian Drosophila pseudoobscura

Isolated populations of drosophila pseudoobscura, separated from North American populations by about 2,400 km, were found in Colombia in 1960. We compared for sequences of the small ribosomal RNA (srRNA) gene on the mitochondria between North American and Colombian D. pseudoobscura in order to clarify the age of the Colombian isolates. The North American populations were not genetically different from each other but were genetically different from the Colombian populations. The Mexican strains represent the area from which the Colombian founders might have come. The estimated net nucleotide divergence between Mexican and Colombian D. pseudoobscura indicates that the Colombian population is not an ancient lineage. Phylogenies using both distance and parsimony methodologies reinforced this conclusion. The Colombian samples group together with both methods but, according to the bootstrap analysis, not significantly. It appears that the populations have not been separated long enough for their DNA sequences to show much divergence.      

Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

Background  

Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Quirks of dye nomenclature. 8. Methylene blue,azure and violet

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.     

A tritrophic signal that attracts parasitoids to host-damaged plants withstands disruption by non-host herbivores

Background  

Volatiles emitted by herbivore-infested plants are highly attractive to parasitoids and therefore have been proposed to be part of an indirect plant defense strategy. However, this proposed function of the plant-provided signals remains controversial, and it is unclear how specific and reliable the signals are under natural conditions with simultaneous feeding by multiple herbivores. Phloem feeders in particular are assumed to interfere with plant defense responses. Therefore, we investigated how attack by the piercing-sucking cicadellid Euscelidius variegatus influences signaling by maize plants in response to the chewing herbivore Spodoptera littoralis.     

Empirical and deterministic accuracies of across-population genomic prediction

Background

Differences in linkage disequilibrium and in allele substitution effects of QTL (quantitative trait loci) may hinder genomic prediction across populations. Our objective was to develop a deterministic formula to estimate the accuracy of across-population genomic prediction, for which reference individuals and selection candidates are from different populations, and to investigate the impact of differences in allele substitution effects across populations and of the number of QTL underlying a trait on the accuracy.

Methods

A deterministic formula to estimate the accuracy of across-population genomic prediction was derived based on selection index theory. Moreover, accuracies were deterministically predicted using a formula based on population parameters and empirically calculated using simulated phenotypes and a GBLUP (genomic best linear unbiased prediction) model. Phenotypes of 1033 Holstein-Friesian, 105 Groninger White Headed and 147 Meuse-Rhine-Yssel cows were simulated by sampling 3000, 300, 30 or 3 QTL from the available high-density SNP (single nucleotide polymorphism) information of three chromosomes, assuming a correlation of 1.0, 0.8, 0.6, 0.4, or 0.2 between allele substitution effects across breeds. The simulated heritability was set to 0.95 to resemble the heritability of deregressed proofs of bulls.

Results

Accuracies estimated with the deterministic formula based on selection index theory were similar to empirical accuracies for all scenarios, while accuracies predicted with the formula based on population parameters overestimated empirical accuracies by ~25 to 30%. When the between-breed genetic correlation differed from 1, i.e. allele substitution effects differed across breeds, empirical and deterministic accuracies decreased in proportion to the genetic correlation. Using a multi-trait model, it was possible to accurately estimate the genetic correlation between the breeds based on phenotypes and high-density genotypes. The number of QTL underlying the simulated trait did not affect the accuracy.

Conclusions

The deterministic formula based on selection index theory estimated the accuracy of across-population genomic predictions well. The deterministic formula using population parameters overestimated the across-population genomic accuracy, but may still be useful because of its simplicity. Both formulas could accommodate for genetic correlations between populations lower than 1. The number of QTL underlying a trait did not affect the accuracy of across-population genomic prediction using a GBLUP method.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.