Integration of the PiGMaP and USD A maps for porcine chromosome 14

In order to align two previously published genetic linkage maps, a set of four of the United States Department of Agriculture (USDA) microsatellite linkage markers was mapped in the International Pig Gene Mapping Project (PiGMaP) reference families. Two-point linkage analysis was used between these USDA markers and the set of genes and markers previously mapped on the PiGMaP chromosome 14 map-Markers with threshold lod scores of three or greater were used for multipoint map construction. The USDA and PigGMaP linkage maps of chromosome 14 were aligned using the four USDA microsatellite markers along with three markers that are common to both maps. The PiGMaP genetic linkage map order for chromosome 14 was confirmed and the map was expanded to 193 cM with addition of the new markers.     

CXCL14 is no direct modulator of CXCR4

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.     

A de novo 2.3 Mb deletion in 2q24.2q24.3 in a 20-month-old developmentally delayed girl

We report a 20-month-old girl ascertained at the age of 11 months for developmental delay. She presented with hypotonia and delayed motor development. The patient had severe language impairment and showed behaviour consistent with autism spectrum disorder. She was microcephalic with mild dysmorphic features and had joint hyperlaxity. We detected a 2.3 Mb de novo deletion in 2q24.2q24.3 on her paternal chromosome.     

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14‐3‐3 proteins

Alkaline/neutral invertases (A/N‐Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At‐A/N‐InvG or AT1G35580). We demonstrate that Ser547 at the extreme C‐terminus of the AtCINV1 protein is a substrate of calcium‐dependent kinases (CPK3 and 21) and that phosphorylation creates a high‐affinity binding site for 14‐3‐3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14‐3‐3 proteins enhances its activity. The analysis of three quadruple 14‐3‐3 mutants generated from six T‐DNA insertion mutants of the non‐epsilon family shows both specificity as well as redundancy for this function of 14‐3‐3 proteins. The strong reduction in hexose levels in the roots of one 14‐3‐3 quadruple mutant plant is in line with the activating function of 14‐3‐3 proteins. The physiological relevance of this mechanism that affects A/N‐invertase activity is underscored by the light‐induced activation and is another example of the central role of 14‐3‐3 proteins in mediating dark/light signaling. The nature of the light‐induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca⁺⁺ changes that activate calcium‐dependent kinases, await further study.     

靶向基质金属蛋白酶14类血红素结构域特异活性位点的确定及反义肽虚拟筛选

目的:确定基质金属蛋白酶14(MMP-14)类血红素结构域(HPX14)特异活性位点,虚拟筛选获得能与HPX14特异结合的短肽。方法:用metaPocket预测HPX14的活性口袋,多序列比对分析活性口袋的氨基酸残基特异性;基于M-I和R-B理论设计以特异活性位点为正义肽的反义肽库,并进行虚拟筛研究及结合特异性确定。结果:生物信息学分析确定了KGRGLTD为HPX14的特异活性位点,并构建了其1036条反义肽;通过2轮虚拟筛选,获得10条得分居前的与HPX14结合较好的反义肽,它们与HPX14具有较高的亲和力,并可能影响MMP-14同源二聚体的形成和MMP-14活性的抑制;VSETAPF、IGEPPPF是对接打分最好的2条短肽,与HPX14的结合具有特异性。结论:KGRGLTD是HPX14的特异活性位点,虚拟筛选得到的VSETAPF、IGEPPPF等反义肽与HPX14具有较高的亲和力,这为基于MMP-14的HPX结构域的靶向小分子多肽先导药物的发现提供了重要的前期工作基础与新思路。     

人GST-14-3-3σ融合蛋白的原核表达、纯化及活性检测

目的:构建人14-3-3σ基因的原核表达载体,获得其原核表达产物,并对融合蛋白进行纯化及活性检测。方法:采用PCR技术从人乳腺文库中扩增14-3-3σ基因编码序列,将其克隆到pGEX-KG载体中,重组质粒转化大肠杆菌Rossate后表达重组蛋白,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,并通过SDS-PAGE和Western印迹检测融合蛋白的表达,采用GST pull-down技术检测已纯化的蛋白与已知体外相互作用蛋白AKT之间的相互作用。结果:从人乳腺文库中扩增获得约750 bp的DNA片段,并成功克隆至pGEX-KG载体上,经双酶切鉴定得到与预期片段大小相符的外源基因插入片段,测序与目的基因序列完全一致;在Rossate菌株中诱导表达出相对分子质量约52 000的目的蛋白,SDS-PAGE和Western印迹结果表明融合蛋白表达成功,并纯化得到GST-14-3-3σ融合蛋白;通过GST pull-down技术检测证实GST-14-3-3σ融合蛋白可以和AKT在体外结合,并证实其具有生物学活性。结论:获得了原核表达的活性较好的GST-14-3-3σ蛋白,为后续研究细胞周期蛋白调控机制奠定了实验基础。