Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

灰飞虱体内类酵母共生菌异常毕赤酵母的分离培养及其对杀菌剂的敏感性

【目的】随着杀虫剂等化学农药的大规模使用,灰飞虱Laodelphax striatellus的抗药性不断增强,防治效果下降,因此寻找新的防治方法尤为重要。本研究探索使用杀菌剂来减少灰飞虱体内共生菌的数量,以降低灰飞虱的活力,为"抑菌防虫"提供理论依据。【方法】对灰飞虱体内的类酵母共生菌(yeast-like symbiotes,YLS)进行离体培养并鉴定,测定离体培养YLS对不同浓度不同种类杀菌剂的敏感性,将对其抑制作用明显的杀菌剂喷施于麦苗,待灰飞虱取食后统计灰飞虱死亡率、体重,并通过荧光定量PCR检测体内共生菌的数量变化。【结果】离体培养得到1株YLS,经碳源同化实验和18S r DNA分子鉴定,将该菌株归为异常毕赤酵母Pichia anomala,以变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术验证了该菌株在灰飞虱体内的存在。对杀菌剂敏感性的测定结果显示,70%丙森锌和50%戊唑醇+25%肟菌酯对P.anomala抑制效果明显,而氟吡菌胺+霜霉威盐酸盐(687.5 g a.i./L)与40%嘧霉胺对P.anomala的抑制效果较差。用对P.anomala抑制效果明显的两种杀菌剂(70%丙森锌和50%戊唑醇+25%肟菌酯)以800 mg/L浓度喷施麦苗,灰飞虱取食喷施麦苗后的死亡率分别达到46.7%与63.3%,显著高于对照,而体重比对照组显著低。荧光定量检测结果表明,灰飞虱体内3种共生菌(Noda菌、季也蒙毕赤酵母和异常毕赤酵母)的数量在使用了各种杀菌剂后均显著降低。【结论】结果说明,体外喷施杀菌剂对灰飞虱体内共生菌有抑制作用,进而对灰飞虱的生长存活产生影响,这为杀菌剂与杀虫剂的协同使用防治灰飞虱奠定基础。     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994     

Phenotypic and genotypic intra-diversity among Anatolian durum wheat “Kunduru” landraces

Kunduru is an important Anatolian landrace having peculiar traits that are appreciated by farmers and breeders. 33 accessions known as Kunduru collected by ICARDA from six geographical provinces of Turkey, were used to study the phenotypic and genotypic intra-diversity. Kunduru landraces exhibited high intra-diversity for most of the studied morphological traits. GPC (12.10–14.90%), vitreousness (75–100%), TKW (31.80–56.70 g), YP (4.70–8.00 ppm), b*-value (14.30–19.50), ash content (1.60–2.0%) and gluten strength (14–60 ml) showed marked variations. Gliadin and glutenin banding patterns showed high polymorphism. 65 alleles were detected with 14 SSR markers, giving a mean of 6.77 alleles per locus. The average PIC value was 0.44 and ranged from 0.11 to 0.70. The average genetic distance between pairs of landraces was 0.47 and ranged between 0.11 and 0.72. This study showed that Kunduru landraces maintains high allelic variation. PCoA indicated that eco-geographical variables have a significant effect on SSR diversity as well as morphological traits. Many of the landraces studied are in danger of disappearing from the local farmers' fields; this study demonstrates the importance of maintaining and conserving this precious genetic resources.     

Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.     

The stiffness‐controlled release of interleukin‐6 by cardiac fibroblasts is dependent on integrin α2β1

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2^{tm1.1Tkun/tm1.1Tkun} than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.     

Insulin stimulates phosphorylation of a heat-stable protein in rat adipose tissue

Insulin in rat adipose tissue acts to increase the phosphorylation about 2.5-fold of a low molecular weight protein in the cytosol designated phosphoprotein m. Isoproterenol had no effect on the phosphorylation of phosphoprotein m. Some of the properties of phosphoprotein m are: soluble in 1% trichloro acetic acid, heat-stable and has a molecular weight of 23,000 on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphoserine and phosphothreonine are the phosphorylated amino acid residues of phosphoprotein m. The physical and chemical properties of phosphoprotein m are similar to those of previously described inhibitor and modulator proteins.     

Eimeria tenella: utilization of a nasal vaccine with sporozoite antigens incorporated into Iscom as protection for broiler breeders against a homologous challenge

The aim of this study was to evaluate a nasal vaccine using antigens derived from sporozoites of Eimeria tenella incorporated into Iscom to protect broiler chicks. Forty-five one-day-old chickens (Cobb), unvaccinated against coccidiosis, were used in this experiment. The birds were maintained in separated battery cages and divided into three groups: G1 (n = 15), G2 (n = 15), and G3 (n = 15). G1 received 50 μg of sporozoites + Iscom vaccine, G2 received Iscom without antigens, and G3 received only PBS. The treatments were administered by nasal route on days 0, 7, and 21 of the experiment. On the 28th day, all birds were challenged with 105 sporulated oocysts of E. tenella. On the challenge day, three birds from each group were euthanized to evaluate lymphocyte proliferation. Lesion scores were obtained from five birds from each group, 7 days after challenge. The remaining animals were euthanized on the 50th day. The mean lymphocyte proliferation responses were significantly different (P = 0.03); G1 was 2.3-2.6 times more elevated than G2, and G3 (P < 0.001). 83% of the birds from G1 showed an IgY antibody reaction by ELISA at challenge. The means for oocysts shedding were 16,890 ± 20,511, 48,080 ± 50,047, and 65,020 ± 74,461, for G1, G2 and G3 birds, respectively. There was no significant difference (P > 0.17) in oocysts shedding between groups. However, the G1 and G2 chicks demonstrated reduction in percentage of oocyst shedding when compared to control birds (G3) by 74.02% and 26.05%, respectively. The average lesion scores were G1 = 0.4, G2 = 1, and G3 = 2. This study demonstrated that the lowest lesion score and oocyst shedding were observed in the birds from the group that received antigens derived from sporozoite with an Iscom adjuvant (G1). These results suggest that this vaccine can induce protection against avian coccidiosis.