Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine;and we haven't found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰlocus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.     

粒细胞系集落刺激因子对慢性肾功能衰竭患者影响的研究

为研究粒细胞系集落刺激因子对慢性肾功能衰竭的影响，方法；以酶标免疫测定法检测了３１例ＣＲＦ患者血清及尿－ＧＣＳＦ水平及４０例健康人血清及尿Ｇ－ＣＳＦ水平，并进行了相关分析。结果：ＣＲＦ患者血清及尿－ＧＣＳＦ水平分别是２４．１４±２．１ｐｇ／ｍｌ和３０．７２±１．８３ｐｇ／ｍｌ，高于健康对照组，Ｐ〈０．００１；ＣＲＦ血清Ｇ－ＣＳＦ与ＢＵＮ，Ｓｃｒ均无相关性，但血Ｇ－ＣＳＦ与尿Ｇ－ＣＳＦ水平呈正相关。     

Analysis of a single nucleotide polymorphism that controls the cooking quality of rice using a non-gel based assay

The waxy gene encoding granule-bound starch synthase (GBSS) is responsible for the synthesis of amylose in developing grain. Recent work has shown that a G-T polymorphism in the leader intron 5' splice site of GBSS plays a key role in determining the cooking and processing quality of rice. Cultivars with sequence AGGTATA at this location splice GBSS pre-mRNA efficiently and produce relatively large amounts of amylose. These varieties generally a have firm texture when cooked and the grains remain separate. In contrast, GBSS pre-mRNA splicing is temperature sensitive and generally less efficient in cultivars with the sequence AGTTATA. As a result, these cultivars generally have lower amylose content and produce soft and sticky cooked rice. We have used the READIT^TM assay, anon-gel based assay that uses the ability of DNA polymerase to perform pyrophosphoralysis, the reverse of DNA polymerization, to screen the critical G-T polymorphism in more than 750 samples from U.S. and Asian germplasm. We observed complete concordance between the results obtained using DNA sequencing or restriction enzyme digestion and the READIT^TM assay. It also gave accurate results with both heterozygous plants and with complex mixtures as might result when grain from advanced generation plants is pooled to obtain larger samples. This revised version was published online in August 2006 with corrections to the Cover Date.     

荧光定糖法测定纤维素酶活力的条件研究

研究结果表明 ,荧光定糖法测定纤维素酶活力的反应最佳条件 :反应液浓度为46.7mg·g-1,反应时间为10min,反应温度为150℃。将荧光定糖法与滤纸酶活测定法相结合应用于纤维素酶蛋白组分及土壤纤维素酶活力的研究与测定。     

“肿瘤消”诱导肿瘤细胞凋亡的作用研究

研究“肿瘤消”的抗肿瘤活性和作用,为临床用药提供依据。通过 MTT 法检测“肿瘤消”对多种肿瘤细胞株和正常细胞存活率的影响;采用 Hoechst 染色法和 DNA 片段化分析对“肿瘤消”诱导 MDCC-MSBl 细胞凋亡的作用进行检测;Western blot 检测“肿瘤消”诱导 MDCC-MSBl 细胞凋亡相关蛋白因子表达情况。MTT 法检测结果显示,“肿瘤消”对 MDCC-MSBl、C6、SP2／0和 A549细胞的增殖具有明显抑制作用,对293A 细胞增殖无显著影响;Hoechst 染色和 DNA 片段化分析均证实“肿瘤消”能诱导 MDCC-MSBl细胞凋亡;Western blot 检测发现“肿瘤消”诱导 MDCC-MSBl 细胞凋亡与凋亡因子 caspase 3、caspase 8和caspase 9的活化有关。证实“肿瘤消”具有诱导肿瘤细胞凋亡的作用。     

Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteins

Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. flesh. The methods included gel filtration chromatography, displacement of a hydrophobic probe and ultrafiltration. With gel filtration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein–astaxanthin complexes. Displacement of the fluorescent probe 8‐anilino‐1‐naphthalenesulphonate (ANS) was not effective as astaxanthin quenched the fluorophore so that displacement could not be observed. An ultrafiltration method was developed using 200‐mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30‐kDa filter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of different fractions was assessed using the ultrafiltration method. The significant difference (P<0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect differences in affinity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon flesh. This method can be used for the identification of astaxanthin‐binding proteins in salmon flesh and other tissues.     

Fish hepatocyte glycogen phosphorylase – a sensitive indicator for hormonal modulation

The absence of a reproducible method for the assay of glycogen phosphorylase (GPase) in isolated fish hepatocytes has made the interpretation of hormone-induced glycogenolysis data difficult. This study presents such an assay and demonstrates its sensitivity to hormonal activation. The enzyme is assayed in the reverse direction using glucose 1-phosphate (G1-P) and glycogen as substrates and uses standard methods for the quantification of the liberated inorganic phosphate. The assay is highly reproducible, sensitive, and provides an excellent means to follow small and rapid changes in enzyme phosphorylation status following the addition of hormones. We show for hepatocytes isolated from rockfish (Sebastes caurinus) and brown bullhead (Ameiurus (Ictalurus) nebulosus) that small concentrations of three model hormones, namely epinephrine (catfish), norepinephrine, and prostaglandin E₂ (rockfish), lead to the rapid, concentration and time-dependent conversion of existing GPase into the active GPase a form. Some of the enzyme seems to be impervious to hormonal activation, as the highest %GPase a never reaches 100%. We provide evidence that changes in enzyme phosphorylation status provide a better short-term insight into hormone-dependent activation than estimates of glucose or some other end products, that usually must accumulate for long periods before detection is possible. Our data also show that GPase in freshly isolated hepatocytes is already in an activated state and cells should be given a period of rest for several hours before hormonal studies involving glycogen breakdown or the cAMP cascade are initiated.     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.