Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.     

Heparan sulfate-independent infection attenuates high-neurovirulence GDVII virus-induced encephalitis

The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HS-deficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.     

Functional analysis of immune response genes in Drosophila identifies JNK pathway as a regulator of antimicrobial peptide gene expression in S2 cells

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.     

Black currant seed oil and fish oil supplements differ in their effects on fatty acid profiles of plasma lipids, and concentrations of serum total and lipoprotein lipids, plasma glucose and insulin

European diets provide a suboptimal intake of eicosapentaenoic (20:5n3) and docosahexaenoic (22:6n3) acids, which are derived mainly from fish oils. The present study indicates that black currant seed oil, which contains 14.5% alpha-linolenic (18:3n3), 12.6% gamma-linolenic (18:3n6), 47.5% linoleic (18:2n6) and 2.7% stearidonic (18:4n3) acids, could potentially serve as alternative to fish oil as a n3 fatty acid source. Fifteen healthy females participated in a randomized, double-blind, crossover study including two 4-week periods with either 3 g/day of black currant seed oil or 2.8 g/day of fish oil separated by a 4-week washout period. The results show that black currant seed oil supplementation increased the proportion of 18:3n6 in triacylglycerols (TAG) and cholesteryl esters (CE), and that of dihomo-gamma-linolenic (20:3n6) in TAGs, CEs and glycerophospholipids (GPL) (P<.05). Proportion of 18:3n6 was higher (P<.05) after black currant seed oil than after fish oil in TAGs and CEs, and that of 20:3n6 in TAGs, CEs and GPLs. Black currant seed oil supplementation caused only minor changes in the proportions of 20:5n3 or 22:6n3. Serum levels of LDL cholesterol were lower (P<.05) after black currant seed oil compared to fish oil. Plasma glucose concentration decreased during the fish oil supplementation (P<.05).     

Thermophilic aeration of cattle slurry with whey and/or jam wastes

Thermophilic aeration of cattle slurry and food industrial by-products was studied with the aim to improve hygienic qualities of the slurry so that it could be used as a safe fertiliser for berries to be eaten raw. We also wanted to study if the process would be energetically favourable in an arctic climate. Cattle slurry alone or with whey and/or jam waste was treated. The tests were done in a well heat-insulated reactor with a 10 m(3) volume. Temperature increases up to over 70 degrees C could be recorded in 19 days even though some processes were carried out in winter time when the ambient air temperature was less than 0 degrees C. The heat energy formed was higher than the electrical energy needed to carry out the aeration. The hygienic qualities of the aerated product were good with only minor nitrogen losses. The end product could be useful as a fertiliser and soil improving compound to increase the organic matter content of agricultural soil. Cattle slurry alone was well suited as the raw material if attaining a high temperature was the main goal. A part of slurry could be replaced with food-industrial side products. Whey waste suited better for co-composting than jam waste but the mixture of whey, jam waste, and slurry was optimal for composting.     

Ant predation and the cost of egg carrying in the golden egg bug: experiments in the field

Golden egg bug ( Phyllomorpha laciniata ) females lay eggs on the backs of both female and male conspecifics. Bugs receive eggs voluntarily and involuntarily, and even when males carry eggs, many eggs are not fertilised by the carrier. Carrying the eggs of another individual is unexpected, particularly if egg carrying bears a cost in survival. We examined the predation risk associated with egg carrying experimentally in the field. P . laciniata individuals were enclosed with workers of one of two ant species, Pheidole pallidula or Cataglyphis piliscapus, which co-occur with the bug in the wild. Pheidole pallidula workers preyed on golden egg bugs and their eggs, but Cataglyphis piliscapus workers did not . In P. pallidula enclosures, golden egg bugs carrying larger egg loads were eaten first. These results suggest that golden egg bugs experience high predation pressure and that egg carrying increases the risk of predation. Due to the direct survival costs associated with egg carrying and the lack of relatedness between the eggs and the carrier, we suggest the golden egg bug as the first known intraspecific parasite in which parasitism is not related to active parental care.