采食后奶牛瘤胃短链脂肪酸及其吸收相关蛋白表达的研究

本试验旨在研究奶牛采食前后瘤胃中短链脂肪酸(SCFA)浓度的变化及其吸收相关蛋白表达量的差异。试验选用3头体重(720±30)kg且装有瘘管的健康荷斯坦牛(动物伦理审查编号为SXAU-EAW-2019-C002013),采食精粗比为40:60的日粮(10kg),试验预试期10d,于第11天饲喂前开始取样,采用气相色谱法检测奶牛采食前(0 h)和采食后(1、2、3、4、5、6、7、8 h)瘤胃液中SCFA浓度;并采用荧光定量PCR方法检测瘤胃上皮组织中与SCFA吸收相关的蛋白表达量。结果表明:在采食后1 h奶牛瘤胃中SCFA浓度最高(P<0.05);在采食后一段时间内(2~5h)与SCFA吸收相关蛋白表达量上调(P<0.05),AE2、MCT1基因表达量均在5 h最高,PAT1、NHE3基因表达量均在4 h最高,MCT4基因表达量在4、5、6h均较高,NHE1基因表达量在2h达到最高;AE2、MCT1、MCT4、NHE1基因表达量与SCFA浓度负相关或正相关(P<0.05),AE2、MCT1、MCT4基因表达量与瘤胃内pH正相关(P<0.05)。以上结果初步揭示,在采食后一定时间内,瘤胃中与SCFA吸收相关蛋白表达受SCFA浓度和pH的调节。     

Identification of differential protein expression and putative drug target in metacyclic stage of Leishmania major and Leishmania tropica: A quantitative proteomics and computational view

Cutaneous leishmaniasis (CL) is an infectious disease that commonly caused by Leishmania (L.) major and L.tropica. Recently there has been a growing interest in proteomics analysis on Leishmania for drug target discovery. Therefore, we aimed to distinguish proteins which might be characteristic for each of the species from those shared by both to the detection of drug targets, which may become helpful for designing new drugs for CL. To identify differences in protein profiles of L. major and L. tropica, we conducted a Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) analysis. Totally 67 differentially expressed proteins (DEPs) (fold change> 2 and p < 0.05) were identified between species. Of these, 42 and 25 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process of up-regulated proteins. Furthermore, the small molecule metabolic process and translation were detected as significant biological processes for up-regulated proteins in L. major, while translation was identified for L. tropica. Also, KEGG analysis has revealed glycolysis/gluconeogenesis and translation as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Finally glycosomal malate dehydrogenase was identified as putative drug target using network and homology analyses. The DEPs between the species are essential in host-pathogen interactions and parasite survival in the macrophage. Furthermore, L. major and L. tropica possibly uses different pathogenicity mechanisms that leads to anthroponotic or zoonotic CL. Our results may help in the drug discovery and chemotherapeutic interventions.     

灌水对强筋小麦籽粒产量及营养品质的影响

为明确水地强筋冬小麦高产、优质、高效的灌溉技术，试验设3个灌水时期8个灌溉处理［越冬期灌1水（W₁），拔节期灌1水（W₂），孕穗期灌1水（W₃），越冬期和拔节期灌2水（W₁₂），越冬期和孕穗期灌2水（W₁₃），拔节期和孕穗期灌2水（W₂₃），越冬期、拔节期和孕穗期灌3水（W₁₂3），全生育期不灌水处理（CK）］,于小麦成熟期测定籽粒产量、总蛋白及其组分含量和淀粉含量。结果表明，与不灌水的CK比较，所有灌水处理的籽粒产量、有效穗数、穗粒数、千粒重、蛋白质产量以及籽粒淀粉含量均显著增加，但籽粒的总蛋白及其组分含量均呈不同程度降低（W₁处理除外）。越冬期灌水对有效穗数、籽粒产量、总蛋白及其组分含量、淀粉含量的提升作用较大；拔节期灌水对穗粒数的提升作用较大，但对淀粉含量的提升作用较小，对总蛋白及其组分含量的降低作用较大；孕穗期灌水对千粒重的提升作用较大，对蛋白质产量的提升作用较小。随着灌水次数增加，小麦籽粒产量显著提高，淀粉含量先显著提高后基本不变，而籽粒总蛋白及其组分含量降低。W₁₂3处理籽粒产量最高，其次是W₁₃处理；W₁处理籽粒蛋白质及其组分含量最高，其次是W₁₂及W₁₃处理；W₂₃处理淀粉含量最高，其次是W₁₂或W₁₃处理。综合各项指标，最好的灌水组合是越冬期和孕穗期灌2水（W₁₃）。     

FAM3C activates Akt to promote viability of oral squamous-cell carcinoma cells

AIM: To investigate the expression and roles of family with sequence similarity 3, member C (FAM3C) in oral squamous-cell carcinoma cells. METHODS: The mRNA and protein expression levels of FAM3C in dysplastic oral keratinocyte (DOK) and oral squamous-cell carcinoma WSU-HN6 cells were detected by RT-qPCR and Western blot. The WSU-HN6 cells were treated with siFAM3C or FAM3C antibody. After 24, 48 and 72 h, the viability of WSU-HN6 cells was measured by CCK-8 assay, and the activation of protein kinase B (Akt) was detected by Western blot. Adenovirus was used to mediate over-expression of FAM3C in the DOK cells. The DOK cell viability was measured by CCK-8 assay after adenovirus infection for 24, 48 and 72 h, and the activation of Akt was detected by Western blot. RESULTS: Compared with the DOK cells, the mRNA and protein levels of FAM3C were significantly increased in the WSU-HN6 cells (P<0.05). The viability of WSU-HN6 cells transfected with siFAM3C was significantly inhibited at 48 h and 72 h (P<0.05). siFAM3C treatment inhibited the activation of Akt (P<0.05). FAM3C antibody treatment also suppressed the viability of the WSU-HN6 cells at 48 h and 72 h and the activation of Akt (P<0.05). Over-expression of FAM3C in the DOK cells promoted the cell viability at 48 h and 72 h and activated Akt (P<0.05). CONCLUSION: FAM3C might promote oral squamous-cell carcinoma cell growth by activating Akt.     

The effects of protein kinase A catalytic subunit on sperm motility regulation in Pacific abalone Haliotis discus hannai

Protein kinase A plays a central role in the regulation of sperm motility from echinoderms to mammals, but the information about its regulatory role in molluscs is very limited. In this study, a protein kinase A catalytic subunit (designated as HdPKA‐C) was identified from Pacific abalone Haliotis discus hannai. The open reading frame of HdPKA‐C was of 1,077 bp, encoding a peptide of 358 amino acids with a typical protein kinase domain. HdPKA‐C shared 82%–87% sequence similarities with other PKA‐Cs, and it was clustered first with gastropod PKA‐Cs in the phylogenetic tree. The mRNA of HdPKA‐C was constitutively expressed in examined tissues, with the highest level detected in hepatopancreas. The phosphorylated form of HdPKA‐C (p‐HdPKA‐C) was localized at the acrosome, connecting piece and flagellum of spermatozoa with variable intensity. Its phosphorylated substrates were also detected in these regions with much lower intensity at the connecting piece. The inhibition of HdPKA‐C activity with H‐89 led to a significant reduction in the percentage of motile sperm and sperm velocities. p‐HdPKA‐C was detected by Western blot in strip‐spawned sperm, naturally spawned sperm and H‐89‐treated sperm with almost the same intensity. The intensity of p‐HdPKA‐C substrates in naturally spawned sperm was higher than that in strip‐spawned sperm, and it was roughly the same as that in H‐89‐treated sperm except for two bands at 50 and 60 kDa. These results collectively indicated that HdPKA‐C played an important role in the regulation of abalone sperm motility by altering its substrates phosphorylation.     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

Influence of low soil nitrogen and phosphorus on gluten polymeric and monomeric protein distribution in two high quality spring wheat cultivars

The effect of low levels of nitrogen, phosphorus and a combination of the two on the distribution of polymeric and monomeric proteins in two high quality spring bread wheat cultivars was investigated for two consecutive seasons. Size exclusion-high performance liquid chromatography (SE-HPLC) was used to determine the quantity and relationships of monomeric and polymeric proteins, and their relationship with flour protein content (FPC) and SDS sedimentation volume (SDSS). The low nitrogen and combined low nitrogen and low phosphorus treatments had a much larger effect on the protein fractions than the low phosphorus treatment alone. The SDS-soluble large monomeric protein fraction and the percentage SDS-insoluble monomeric proteins, were significantly increased under low nitrogen and a combination of low nitrogen and low phosphorus treatments. The percentage SDS-insoluble large and total polymeric proteins was significantly reduced under low nitrogen and a combination of low nitrogen and phosphorus treatments. The SDS-soluble and -insoluble small polymeric proteins were significantly increased under both low nitrogen and a combination of low nitrogen and low phosphorus treatments. The low nitrogen treatment consistently caused the lowest FPC and SDSS values. Under low nitrogen conditions, there was a significant positive correlation between the SDS-soluble gliadins and SDSS, and FPC.     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

Phenotypic and allelic variation for wort protein Z in Australian and Canadian barleys

Protein Z is a major component in beer foam. Two-dimensional electrophoresis was used to analyze wort proteins of two Australian (Buloke and Commander) and two Canadian (CDC Meredith and Bentley) varieties. The Canadian barley contained more abundant proteins from MW 40–45 kDa (pI 5 to 7). These proteins were identified as either protein Z4 or protein Z7 using liquid chromatography–mass spectrometry. Full-length gene of protein Z4 and Z7 were sequenced from Canadian and Australian barleys. Sequence differences were identified in the coding region and upstream regions of the two genes, resulting in protein sequence and expression variations. Molecular markers were designed according to the indels in the upstream regions of protein Z4 and Z7 genes. These markers were highly correlated to wort protein Z content in Canadian and Australian varieties. The Canadian barleys contained ‘high level’ genotypes for protein Z4 and Z7 while most Australian barleys had ‘low level’ genotypes for protein Z4, Z7 or both. The markers identified in this study provide a valuable tool for the selection of protein Z alleles in marker-assisted breeding. Total protein Z content was assessed using different steeping conditions, and increasing air-rest time increased protein Z content in 15 varieties.