Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.     

Early events in the activation of Fc gamma RIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of Fc gamma RIIA

The signal transduction mechanisms associated with the ligation of FcgammaRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcgammaRIIA following its cross-linking. The results obtained indicate that cross-linking of FcgammaRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcgammaRIIA in the cells. The stimulated degradation of FcgammaRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcgammaRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by beta-octylglucoside. Sucrose gradient analysis of the distribution of FcgammaRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcgammaRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcgammaRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcgammaRIIA and provide new insights into the early events following FcgammaRIIA cross-linking.     

Effect of hyperthermia on blood constituents in the domestic rabbit (Lepus cuniculus)

1. 1. Effects of exposing rabbits to temperatures 37–50°C on body-core temperature and some blood constituents were investigated.

2. 2. Heat stroke death occurred at or above a critical core-temperature of 43.0°C.

3. 3. Plasma osmolality and levels of glucose, urea and lactate were significantly elevated in hyperthermia.

4. 4. Widespread tissue damage was indicated by increases in plasma activities of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine phosphokinase (CPK).

5. 5. The most sensitive indicators of impending heat stroke in heat stressed rabbits were plasma levels or urea, lactate and CPK.

Heterogeneity of mutations in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria

Summary Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. We have previously described two missense mutations in the UROIIIS gene, confirming that the primary defect responsible for CEP is a structural alteration of this gene. We have extended our work to 5 additional unrelated families. Two new point mutations, a deletion and an insertion have been found in the messenger RNA. Our study shows that a molecular heterogeneity of the mutations exists in Günther's disease. One mutation (C73R), however, appears to be more frequent than the others. Finally, the different normal and mutated proteins have been expressed in Escherichia coli to determine the consequence of the mutations on the enzyme activity.     

Joint analysis for heading date QTL in small interconnected barley populations

The purpose of the present work is to validate the effect of the main QTL determining heading date in a set of 281 doubled haploid lines of barley, derived from 17 small interconnected populations, whose parents are cultivars commonly used in the Spanish barley breeding program. We used 72 molecular markers distributed across the seven chromosomes, particularly in regions known to contain flowering time genes or QTL. A combined linkage map over the 17 populations was constructed. The lines were evaluated in four field trials: two autumn sowings and two winter sowings, and in two treatments at a greenhouse trial, under controlled conditions of photoperiod and temperature. We have found that it is possible to carry out QTL detection in a complex germplasm set, representative of the materials used in an active breeding programme. In most cases two alleles per QTL were detected, though polymorphism of flanking markers was notably higher. The results revealed that there is a set of QTL that accounts for an important percentage of the phenotypic variation, suitable for marker assisted selection. Also, the role of the regions carrying the photoperiod response genes Ppd-H1 and Ppd-H2, the vernalization response genes Vrn-H1 and Vrn-H2, and the earliness per se locus Eam6, of which allele-specific or closely linked markers were available, was confirmed. These results support the use of this kind of approach for the validation of QTL found in single cross population studies, or to survey allelic diversity in plant breeding sets of materials. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Uncoated endocytic vesicles require the unconventional myosin, Myo6, for rapid transport through actin barriers

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.     

Atypical recognition of particular DNA sequences by the archaeal chromosomal MC1 protein

The MC1 protein is a chromosomal protein likely involved in the DNA compaction of some methanogenic archaea. This small and monomeric protein, structurally unrelated to other DNA binding proteins, bends DNA sharply. By studying the protein binding to various kinds of kinked DNA, we have previously shown that MC1 is able to discriminate between different deformations of the DNA helix. Here we investigate its capacity to recognize particular DNA sequences by using a SELEX procedure. We find that MC1 is able to preferentially bind to a 15 base pair motif [AAAAACACAC(A/C)CCCC]. The structural parameters of this sequence are characterized by molecular dynamics simulation experiments, and the binding mode of the protein to the DNA is studied by footprinting experiments. Our results strongly suggest that the protein realizes an indirect readout of the DNA sequence by binding to the DNA minor groove.