Expression patterns and association analysis of the porcine DHX58 gene

RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH (Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of 1ipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron, was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine.     

Serine-palmitoyl transferase activity in cultured human keratinocytes

Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We report here the initial characterization of serine-palmitoyl transferase (EC 2.3.1.50; SPT), the rate-limiting enzyme in the synthesis of sphingolipids, from cultured human neonatal keratinocytes. Subcellular fractionation studies demonstrated that 79% of the total cellular SPT activity was associated with the microsomes. The specific activity of keratinocyte SPT was 270 +/- 20 pmol/min per mg of microsomal protein, a level significantly higher than activities reported in other tissues. Keratinocyte SPT showed an apparent Km for L-serine of 0.40 (+/- 0.04 mM, with an alkaline pH optimum (8.2 +/- 0.4). Keratinocyte SPT utilizes palmitoyl-CoA preferentially over other saturated or unsaturated acyl-CoA substrates; increasing acyl-CoA chain lengths above C16 by one or two carbons was less detrimental to activity than similar decrements in chain length. Finally, the mechanism-based inhibitors L-cycloserine and beta-chloro-L-alanine, demonstrated potent inhibition of keratinocyte SPT activity, with 50% inhibitory concentrations of approximately 3.0 and 25 microM, respectively. In summary, we have found that cultured human neonatal keratinocytes contain unusually high levels of serine-palmitoyl transferase activity, and that the substrate specificity of keratinocyte SPT may determine the base composition of epidermal sphingolipids.     

二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用

本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行ＰＣＲ扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记ＰＣＲ产物,简便、快速,分辨率可达１ｂｐ,并可用多对引物同时进行多重ＰＣＲ分析。用此方法对ＤＭＤ家系成员ｄｙｓｔｒｏｐｈｉｎ基因的５＇－脑型外显子止游区和３＇－非翻译区的两个（ＣＡ）。位点进行了扩增片段长度多态性分     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

冬小麦根表面氧化还原活力的研究

证实了两个不同品种的冬小麦根系表面存在着氧化ＮＡＤＨ和还原Ｋ３Ｆｅ（ＣＮ）６的氧化的活力。还原铁氰化物活力在ＰＨ５．５到８．５范围内随着ＰＨ值升高而增大,温度在１５℃到４５℃范围内随温度升高还原活力增强,４５℃达最高值,５５℃时活力急剧下降。     

Melatonin and the pea aphid,Acyrthosiphon pisum

Pea aphids, Acyrthosiphon pisum, were fed on artificial diet containing various concentrations of melatonin. Under long-day conditions (16h light:8h dark) their progeny included males and virginoparous/oviparous (asexual/sexual) intermediate females, which normally occur only in short days or around critical night-length. Endogenous melatonin in pea aphids was measured by radioimmunoassay and verified by parallelism with a melatonin standard curve and by thin layer chromatography. However, melatonin titres showed large variations and although they tended to be higher during the scotophase than during the photophase they were not significantly different. The possibility of melatonin being involved in photoperiodism is discussed.     

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges.

A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation.