吡啶衍生物研究(IX):N-(1-甲氧羰基)乙基-N-[5-(2-氯吡啶-4-基)-1,3,4-噻二唑-2-基]酰胺的合成及除草活性

为了进一步研究前期发现的除草先导化合物2-仲丁氨基-5-(2-氯吡啶-4-基)-1，3，4-噻二唑(BCPT)的结构-活性关系并提高其除草活性，设计并合成了一系列N-(1-甲氧羰基)乙基-N-[5-(2-氯吡啶-4-基)-1，3，4-噻二唑-2-基]酰胺类化合物。其苗后除草活性测定结果表明，所有化合物的活性都远低于BCPT本身。说明BCPT可能具有与传统酰胺类除草剂不同的作用机制。     

鼠层粘蛋白α5链E3、LG4、LG5片段的克隆及表达

从鼠的肝脏组织中提取总 RNA,采用 RT- PCR获得了鼠层粘蛋白 ( laminin)α5链的 E3 ( L G4 - 5)、L G4 和 L G5等 3个基因片段 ,与 NCBI数据库参考序列相比 ,同源性在 99%以上。将 E3 ( L G4 ,5)、L G4 和 L G5定向克隆到原核高效表达载体 Pet- 2 8a中 ,构建 Pet- 2 8a- E3 、Pet- 2 8a- L G4 、Pet- 2 8a- L G5等 3个原核表达质粒。将表达质粒转化表达受体菌 BL2 1中 ,IPTG诱导表达。收集菌液进行 SDS- PAGE电泳、Western- blotting分析 ,结果显示 ,这 3段基因在原核细胞中成功表达 ,薄层扫描显示表达蛋白占细胞总蛋白的 2 0 %以上。大量提取包涵体 ,纯化后免疫家兔 ,8周后得多克隆抗体 ,间接 EL ISA在抗体稀释到 1∶ 1 2 80 0时仍为阳性     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

西安荷斯坦奶牛群5个基因座位遗传多态性的PCR-RFLP分析

应用PCR-RFLP方法对西安荷斯坦牛的κ-en、β-lg、β-lg5′侧翼区、CSN1S2、IGFBP-3共5个基因座位进行了多态性分析。结果表明，在西安荷斯坦牛群中，没有发现携带CSN1S2^P等位基因的个体，其多态信息含量为0。κ-en基因座位呈现低度多态(PIC=0．2366)，β-lg、β-lg5′侧翼区、IGFBP-3基因座位的多态信息含量分别为0．3168、0．3689、0．4439，均呈现中度多态。κ-en、β-lg、β-lg5′侧翼区、IGFBP-3、CSNIS2基因座位的杂合度和DNA多态度分别为0．2742、0．3947、0．4879、0．4891、0和0．0255、0．0116、0．0333、0．0112、0。而且，在西安荷斯坦牛群中，κ-en、β-lg、β-lg5′侧翼区、IGFBP-3共4个基因座位均处于Hardy-Weinberg平衡状态，CSN1S2基因座位处于纯合状态。     

亚洲璃眼蜱唾液腺一新功能基因的克隆与测序

HaB1是亚洲璃眼蜱雌成蜱唾液腺差异表达基因文库中的一个片段,根据其序列设计引物HaB1-GSP1和HaB1-GSP2,以唾液腺总RNA为模板,RACE法扩增获得HaB1的未知3′-末端。测定该末端序列,进行序列拼接,设计全长引物5-′CCAGTCCGAAGGAAGGGCG-3′和5-′TCTC-CGGGCACGTGAAGTGTC-3′。以cDNA第一链为模板,扩增获得基因全长。经核查表明,该基因为一新基因(登录号AY803896)。     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.     

Interaction between the sequence of feeding of hay and concentrate,and boiling of barley on feed intake,the activity of hydrolytic enzymes and fermentation in the hindgut of Arabian mares

The interaction between the sequence of feeding of hay and concentrate and the hydrothermal processing of barley in alleviating concentrate effects on intake, and hindgut fermentation in horses was tested. Six Arabian mares (4–10 years of age, 410 ± 35 kg body weight) were used to evaluate the effects of feeding sequence (FS) and type of barley (TB) on intake, and faecal volatile fatty acids (VFA), activities of α‐amylase (AA: EC 3.2.1.1), carboxymethyl cellulase (CMCase: EC 3.2.1.4), microcrystalline cellulase (MCCase: EC 3.2.1.91) and general filter paper degrading activity (FPD). Mares were offered a ration of air‐dried alfalfa and concentrate (70:30 as‐fed) in four subsequent periods of 14 days including 8 days of adaptation and 6 days of sampling. In each period and each meal, mares received concentrate either 30 min after (HC) or 30 min before (CH) alfalfa hay. Barley was either milled or boiled in water. Rectal samples were grabbed directly from rectum once per period. Mares subjected to CH had higher dry matter intakes than mares under HC regime. The acetate:propionate ratio (A:P ratio) in rectal content was higher with CH than HC. The AA activity was higher under CH than under HC. Mares fed boiled barley had lower rectal concentrations of VFA and propionate and a higher A:P ratio than mares fed milled barley. Furthermore, the rectal content showed a higher MCCase activity but a lower AA activity when mares were fed boiled compared with milled barley. Interactions between FS and TB were observed with respect to CMCase activity, and concentrations of propionate and valerate. In conclusion, the present results suggest that both, feeding concentrate before hay and boiling the barley, might improve the hindgut environment in Arabian mares, and that the two measures were mostly additive and sometimes even synergistic.