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.     

猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究

旨在研究猪C1QTNF3基因可变剪接体的特性及miR-101通过C1QTNF3基因促进猪脂肪SV细胞成脂分化的机制。本研究采集3头30日龄健康马身猪仔公猪心、肝、脾、肺、肾、胃、股二头肌、腰大肌、皮下脂肪和背部脂肪组织样品,首先利用RT-PCR技术和生物信息学方法对C1QTNF3基因的不同转录本进行扩增和生物学特性分析,采用qRT-PCR技术检测C1QTNF3基因不同转录本在猪组织中的表达变化。随后,利用生物信息学预测发现,C1QTNF3上游的调控因子是miR-101,采用双荧光素酶报告试验验证miR-101对C1QTNF3的调控作用。最后,利用油红O染色和qRT-PCR技术检测过表达miR-101对猪脂肪SV细胞成脂分化的影响。基因克隆得到猪C1QTNF3存在两个转录本C1QTNF3和C1QTNF3-1,其中C1QTNF3-1为新鉴定的转录本;测序分析显示,与C1QTNF3相比,C1QTNF3-1的第1外显子区域缺失了219个碱基,少编码73个氨基酸。进化树分析显示,猪C1QTNF3和C1QTNF3-1蛋白序列与人和马来亚穿山甲等物种的亲缘关系较近。C1QTNF3和C1QTNF3-1在猪各组织中均有表达,且在各组织中C1QTNF3-1的表达量均极显著高于C1QTNF3（P<0.01）。过表达miR-101极显著下调C1QTNF3 3'UTR区域的荧光素酶活性（P<0.01）和C1QTNF3 mRNA的表达（P<0.01）,同时增加了脂肪细胞成脂分化关键基因PPARγ、C/EBPβ、SREBP-1c和FABP4的mRNA表达量（P<0.01）。本试验成功克隆了猪C1QTNF3基因的两个可变剪接体,其中C1QTNF3-1在猪不同组织中均高表达,推测该转录本为基因发挥功能的主要亚型。并深入研究了C1QTNF3基因的上游调控因子,揭示了miR-101靶向C1QTNF3促进成脂分化的机制,丰富了C1QTNF3的生物学作用和调控网络。     

Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA

In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.     

基于设计模板的快速变型设计方法

传统的变型设计方法存在变型过程复杂,容易产生约束冲突的问题.针对零部件之间依赖关系复杂,设计过程需要通过交互和人工干预的形式才能完成配置与变型的问题,提出了在模板框架下的快速变型设计方法.利用模板的灵活性和信息载体功能,提出了可变型单元的概念,通过可变型单元之间建立相互关联实现对象之间的参数联动,在模板定义域范围内实现产品零部件的快速变型.以变速箱为实例,通过产品的结构变型实现产品的系列化,在同系列产品中通过几何参数的变更实现产品的多样化.     

葡萄脯氨酸累积变异系CAT和SOD活性研究

研究了无胁迫和ＮａＣｌ胁迫条件下,同基因型二倍体葡萄４个脯氨酸累积变异系和对照系愈伤组织的过氧化氢酶（ＣＡＴ）和超氧物歧化酶（ＳＯＤ）活性。结果表明,在无胁迫条件下,葡萄脯氨酸累积变异系的ＣＡＴ和ＳＯＤ活性显著高于对照系,４个变异系的ＣＡＴ活性分别是对照的２．４１,２．３０,２．２９和２．１３倍,ＳＯＤ活性分别是对照的４．５１,３．６９,３．８４和３．７２倍。在一定浓度ＮａＣｌ胁迫下,对照系和变异系的ＣＡＴ和ＳＯＤ活性都有一定的增加,变异系的ＣＡＴ和ＳＯＤ活性仍高于对照系。脯氨酸累积变异系ＣＡＴ和ＳＯＤ活性高是其对ＮａＣｌ胁迫具有较强抗性的可能原因之一     

凡纳滨对虾肌钙蛋白Ⅰ型基因4种不同剪切体的序列分析

采用RT-PCR技术克隆了凡纳滨对虾肌钙蛋白Ⅰ基因全长编码序列,对其核苷酸序列和编码的氨基酸序列进行了生物信息学分析,并在对虾肌钙蛋白Ⅰ基因的编码区查找了单核苷酸多态性位点。结果显示：本研究克隆获得了凡纳滨对虾肌钙蛋白Ⅰ基因的4个可变剪切体,其编码的蛋白质均具有Troponin超家族结构域特征,同源性比对发现该蛋白的氨基酸序列在节肢动物中高度保守。系统进化分析发现凡纳滨对虾Tn-Ⅰ3和Tn-Ⅰ4与中国对虾和小龙虾的Tn-Ⅰ遗传距离较近,而Tn-Ⅰ1和Tn-Ⅰ2单独聚为一支,且与其他物种Tn-Ⅰ遗传距离较远;通过测序比较不同个体凡纳滨对虾Tn-Ⅰ基因序列发现在Tn-Ⅰ的4种不同剪切体编码区共同序列区第302位和472位碱基分别存在2个（A/G） SNP位点,其中位于第302位碱基的（A/G）错义突变使编码氨基酸由甘氨酸变为了谷氨酸（G/E）。凡纳滨对虾Tn-Ⅰ基因不同剪切体及编码区错义突变的发现为进一步探讨其基因功能奠定了基础。     

公猪精液PRRSV变异株检测及NSP2、ORF5基因序列分析

用荧光RT-PCR方法从某猪场精液中检测到猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)变异株(NSP2 1 594～1 680 bp缺失)核酸阳性.提取1份阳性样品病毒核酸测定和分析其NSP2和OBF5全基因序列,结果表明NSP2基因由950个氨基酸组成,与CH-1a、VR-2332等经典PRRSV相比481位缺失1个氨基酸、532～560位连续缺失29个氨基酸,与JXA1、HUN4等毒株具有相同的缺失特性;ORF5基因第13、151位为具有强毒特性的精氨酸(R),存在4个潜在的糖基化位点,分别位于30～32、35～37、44～46和51～53位氨基酸.遗传进化分析表明,测定序列与JX-A1、HUN4等毒株关系最近,与CH-1a、NVSL等同属一个大分支,而与BJ-4、P129、RespPRRS MLV、VR-2332等处于不同分支.研究证实公猪精液可携带PRRSV变异株,因此猪场(群)在人工授精和引进精液时必须强化检疫和生物安全措施.     

马尾松两个木材性状的变异规律和造纸原料林经营措施探讨

本文分析了马尾松管胞长度和木材密度这两个性状在树木个体内不同方向上及个体间的变异趋势,得出了管胞长度在个体高度方向上变异的几种数学模型。方差分析表明,两个性状在个体内的变异程度大于个体之间并且有极显著的统计学差异。同时还建立了胸径处两性状值与株加权平均值间的线性回归方程。讨论了不同年龄,不同林分密度对这两个性状的影响,结果是林分年龄的不同对管胞长度无显著影响,林分密度的不同,在23—24年左右时,对管胞长度有极显著影响。林分年龄和林分密度的不同,对木材密度无显著影响。     

‘冬枣’的五个自然变异材料评价

以‘冬枣’及其5个自然变异材料为试材,对其果实形态、果实营养、物候期、染色体倍性以及DNA指纹进行了比较分析,以期筛选出综合性状优异的‘冬枣’变异类型。结果表明:与普通‘冬枣’相比,变异1号材料为大果型,其单果质量显著增加,为25.22g,果实可溶性糖含量和糖酸比提高。在物候期上,变异1号和2号材料与‘冬枣’表现相近,但其它3个变异材料的果实成熟期比‘冬枣’晚约15d。流式细胞仪分析显示5个自然变异材料均为二倍体,15对SSR引物检测表明5个自然变异材料与‘冬枣’基因组DNA的SSR指纹相同,可能均为‘冬枣’的自然芽变。评价结果显示,变异1号属于优良大果变异,变异3、4、5号为晚熟变异。