Adaptation of feline immunodeficiency virus subtype B strain TM2 to a feline astrocyte cell line (G355-5 cells)

Based on receptor usage during infection, feline immunodeficiency virus (FIV) isolates can be divided into two groups; those that require feline CD134 (fCD134) as a primary receptor in addition to CXCR4 to enter the cells, and those that require CXCR4 only. Most primary isolates, including strain TM2, belong to the former group and cannot infect a feline astrocyte cell line (G355-5 cells) due to a lack of fCD134 expression. In a previous study, we found that G355-5 cells transduced with fCD134 (termed G355-5/fOX40 cells) were susceptible to strain TM2 and the inoculated cells became persistently infected. In this study, we examined the phenotype of the virus prepared from the persistently infected cells (termed strain TM2PI). Intriguingly, strain TM2PI replicated well in na?ve G355-5 cells and the inoculated G355-5 cells (termed G355-5/TM2PI cells) became persistently infected. The infection of TM2PI in G355-5 cells was inhibited by CXCR4 antagonist AMD3100 and TM2PI infected other fCD134-negative, CXCR4-positive cell lines, FeTJ and 3201 cells. Four amino acid substitutions were found in the Env protein of the strain TM2PI when compared with that of the parental strain TM2. Among the substitutions, the Env amino acid position at 407 of TM2PI was substituted to lysine which has been known to be responsible for the FIV tropism for Crandell feline kidney cells. The strain TM2PI will be useful for studying the receptor switching mechanism and FIV pathogenesis in cats.     

Variation in lysine,threonine, and tryptophan availability in meat and bone meal as estimated by the slope‐ratio growth assay technique in growing rats

Meat and bone meal (MBM) is an important protein source used in animal feeds. However, as the composition and availability of amino acids (AAs) in MBM fluctuate markedly, it is important to verify the magnitude of these parameters in different MBMs. In this study, the AA compositions of 19 MBMs were analyzed to confirm variations in lysine (Lys), threonine (Thr), and tryptophan (Trp), then which were compared with those of soybean meal (SBM) and fish meal (FM). Instability in Lys, Thr, and Trp availabilities in six MBMs were also considered after estimation using the slope‐ratio growth assay technique in rats. Variations in AA composition were evaluated using the coefficient of variance (CV: Standard deviation/Mean). CVs for Lys, Thr, and Trp content were 9.40, 11.83, and 18.12 in MBM, 2.71, 2.48, and 3.19 in SBM, and 10.09, 10.44, and 13.47 in FM. Furthermore, means and SDs for Lys, Thr, and Trp availabilities in MBM were 53.3 ± 10.4% (CV: 19.5), 65.9 ± 17.6% (CV: 26.6), and 83.2 ± 11.2% (CV: 13.5), respectively. These results provide the first evidence that variations in MBM AA compositions were 3.5 to 5.7 times higher than those in SBM, but similar to FM, and that the large variation in availability substantially existed.     

Population study and validation of paternity testing for Thoroughbred horses by 15 microsatellite loci.

Microsatellite 15 TKY System was characterized for parentage verification of horse registry. The Microsatellite 15 TKY System was constructed by using 15 microsatellites, TKY279, TKY287, TKY294, TKY297, TKY301, TKY312, TKY321, TKY325, TKY333, TKY337, TKY341, TKY343, TKY344, TKY374, and TKY394, to provide stringent PCR-based microsatellite typing specifically optimized for multicolor fluorescence detection. The Microsatellite 15 TKY System showed good resolutions for 250 unrelated Thoroughbred horses, and the probability of exclusion (PE) at each microsatellite ranged from 0.437 to 0.621, resulting in a total PE value of 99.998% for Thoroughbred horses. These results indicated that the Microsatellite 15 TKY System is useful for paternity testing of Thoroughbred horses. A paternity testing case for a Thoroughbred horse family, in which candidate sires had close relations, was analyzed using the Microsatellite 15 TKY System. In this case, the Microsatellite 15 TKY System excluded paternity of a false sire. We concluded that the Microsatellite 15 TKY System can give sufficient and reliable information for paternity testing.     

Application of the slope‐ratio growth assay technique to estimate tryptophan availability in soybean meal fed to young rats

The slope‐ratio assay for rat was used to determine whether tryptophan (Trp) availability in soybean meal (SBM) is affected by the presence of other amino acids (AAs). In a preliminary study, rats were fed graded levels of Trp‐supplemented diets to establish the Trp concentration range over which the weight gain response was linear. This range was found to be from 0.04% to 0.12% Trp. Subsequently, rats were fed basal (0.045% Trp) or Trp‐supplemented diets from three different sources: l ‐Trp alone, SBM, or l ‐Trp mixed with other AAs to reflect AA levels in the test SBM (AA‐mix). Weight gain in rats increased linearly with supplemental Trp intake (p < .05) for all Trp sources. Compared to the slope achieved with l ‐Trp alone, the estimated availability of Trp in SBM was 84.4%, while for the AA‐mix it was 93.4%. It is evident that the 6.6% reduction in l ‐Trp availability in AA‐mix is due to metabolic costs derived from excess levels of other AAs beside Trp, given that the absorption of crystalline l ‐Trp in the small intestine is 100%. In conclusion, the Trp availability of SBM was estimated to be around 90.4% (i.e., 84.4/93.4 × 100) after correcting for the effects of the other AAs in SBM.     

Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.     

Microbial conversion of bamboo hemicellulose hydrolysate to xylitol

A hemicellulose hydrolysate containing 19 g L^?1 xylose was prepared from the culm of bamboo (Phyllostachys pubescens) by hydrolysis with 3 % sulphuric acid with a liquor to solid ratio of 10 (g g^?1) at 121 °C for 1 h. After detoxification of the hydrolysate with a commercially available activated char followed by neutralisation with calcium carbonate, the resulting sugar solution was subjected to fermentation using the yeast, Candida magnoliae. The maximum xylitol production (10.5 g L^?1) and the maximum xylitol volumetric productivity (0.42 g L^?1 h^?1) were attained under agitation set at 400 min^?1 and aeration rate of 0.67 vvm (volume of air per volume of medium per minute). According to the results, a suitable control of the oxygen supply permits the xylitol formation from bamboo hemicellulose hydrolysate.     

Composition of inorganic elements and the leaching behavior of biomass combustion ashes discharged from wood pellet boilers in Japan

To evaluate the characteristics of wood ash as fertilizer, composition of inorganic elements and the leaching behavior were studied. The leaching behavior was evaluated by three leaching methods using different solvents (water, hydrochloric acid, and acetic acid: Japanese leaching test Nos. 18 and 19 and modified toxicity characteristic leaching procedure prescribed by the US Environmental Protection Agency). The ash composition varied according to raw materials for fuel and ash collection systems. Concentration of Na, Al, Si, and V became higher in bark ashes while that of K became higher in wood ashes. Pb, Cd, Se, and Zn were more volatile and enriched in fly ashes. Of the nutrient elements, K showed high water solubility and its phytoavailability also appeared high, although the amount of water-soluble K was low in bark ash. Ca and Mg had intermediate solubility while P was less soluble in both water and acetic acid. The amount of hazardous substances having leached was low for most, but not all the ashes, in terms of Pb, Cr and As. Nevertheless, the phytoavailability of these hazardous substances after their application to the forest appeared low, due to their amphoteric leaching behavior and the acidity of Japanese forest soils.     

Endogenous gibberellins and their effects on flowering and stem elongation in cabbage (Brassica oleracea var. capitata)

Summary

Endogenous gibberellins were extracted from cabbage shoots and were analysed using gas chromatography and mass spectrometry. Nine gibberellins (GA₁, GA₁₉, GA₂₀, GA₄₄, GA₁₂, GA₄, GA_15, GA₂₄ and GA₂₅) were identified. Two gibberellin biosynthesis pathways were suggested, an early-13-hydroxlyated pathway and a non-13-hydroxylated pathway, to operate in cabbage shoots. GA₁, GA₄ and prohexadione calcium, a gibberellin biosynthesis inhibitor, were applied to the shoot tip of cabbage ‘Sousyu’ and ‘Kinkei No.201’ with or without cold treatment. Without cold treatment, stem elongation was increased by gibberellins and was suppressed by prohexadione calcium in both cultivars. But prohexadione calcium treatment, followed by gibberellin, promoted stem elongation more than gibberellin alone. Flowering was not induced by gibberellin or prohexadione calcium without cold treatment. When gibberellin and prohexadione calcium were applied during a cold treatment, stem elongation after the cold treatment was increased by gibberellins and was suppressed by prohexadione calcium in both cultivars. Flower bud appearance was promoted by GA₁ and GA₄ in ‘Sousyu’, but in ‘Kinkei No. 201’ only GA₄ was markedly effective. Inhibition of stem elongation and delay of flower bud appearance by prohexadione calcium were overcome by applying GA₁ or GA₄. Neither gibberellin nor prohexadione calcium treatment changed the number of leaf nodes at anthesis. These results indicated that stem elongation and flower bud development are regulated by gibberellins, but gibberellins might have little effect on flower induction.     

Inducement of cytopathic changes and plaque formation by porcine haemagglutinating encephalomyelitis virus

ESK cells were shown to be a good medium for propagating the 67N strain of porcine haemagglutinating encephalomyelitis virus, although no cytopathic effect was observed. The virus induced a readily recognizable cytopathic effect in ESK cells, when a non-cytotoxic amount of diethylaminoethyl-dextran (DEAE-dextran) was incorporated in the culture medium. Based on this finding, a sensitive, practical assay method for the virus was developed. When DEAE-dextran was incorporated in the agar overlay medium, 67N virus formed plaques in ESK cell monolayers. The cytopathic effect as well as the plaque formation were specifically inhibited by antisera against the virus. Neutralization tests were developed on the basis of these findings. Neutralization and haemagglutination-inhibition tests on swine serum samples indicated a wide dissemation of haemagglutinating encephalomyelitis virus or antigenically-related viruses in Japanese pigs.