Proteomic profiling of plasma-derived small extracellular vesicles: a novel tool for understanding the systemic effects of tick burden in cattle

Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

中华鲟神经内分泌多肽7B2基因克隆及组织特异性表达

以中华鲟(Acipenser sinensis)脑垂体总RNA为模板,采用RT-PCR和RACE方法,获得中华鲟神经内分泌多肽(7B2)基因的3个重叠片段,测序后拼接得到986 bp全长基因序列,其中包括5'端非翻译区(5′-UTR)14 bp、3′端非翻译区(3-′UTR)261 bp和开放阅读框711 bp。翻译编码236个氨基酸。其中前43个氨基酸为7B2的信号肽。经BLAST比对发现中华鲟7B2蛋白的同源性与斑马鱼(Danio rerio)的相似性最高为82%。系统发育分析表明,中华鲟与斑马鱼亲缘关系最近。半定量RT-PCR分析表明:在脑中7B2 mRNA表达量最高,心脏、性腺、胰等组织中表达次之,肠、肾、皮肤等组织中少量表达,肝和鳃几乎不表达。     

稻瘟菌基因组规模分泌蛋白的预测分析

利用信号肽分析软件SignalP v3．0，亚细胞器蛋白定位软件TargetP v1．01，GPI-锚定位点软件big—PI Predictor和跨膜螺旋结构软件THMM-2．0这4个软件分析预测稻瘟菌12595个蛋白序列中有1134个分泌蛋白。编码这些蛋白的可读框（ORF）最小值为78bp，最大值为7849bp，平均1231bp。引导它们的信号肽长度介于15—45个氨基酸之间，平均为21个氨基酸。435个分泌蛋白具有功能描述，主要是与代谢有关的酶类。分析了其中降解细胞壁组分和与致病相关的酶类，提示在稻瘟菌侵染水稻不同阶段产生的关键酶及基因的功能需进一步研究。     

长柱金丝桃(Hypericum ascyron L.)分泌结构的研究

对长柱金丝桃各类器官进行了整体透明和解剖学研究,确定合理的采收部位和采摘时间。结果表明,植株富含分泌囊和分泌小管道两种分泌结构;叶片中分泌囊(道)的发育属于裂生型;地上部分泌结构分布较多,尤以叶片中数量最多、分布密度最大;确定植物的最佳采收时期是每年8～9月的花果期、最佳采收部位是地上部。此研究结果为此药用植物合理的采收部位和采收时间的确定提供解剖学依据,同时为其他植物分泌囊(道)发育规律的研究提供理论依据。     

TAT-Apoptin基因原核表达载体的构建及其表达鉴定

为得到TAT-Apoptin 融合蛋白,以pMD-19T-Apoptin 为模板PCR 扩增出TATApoptin 基因,构建重组载体pET-32a-TAT-Apoptin,并对TAT-Apoptin 蛋白进行表达。PCR 和测序表明,成功构建重组载体pET-32a-TAT-Apoptin,进一步对其进行蛋白表达,发现该蛋 白能与Apoptin 多抗发生特异性结合。以上结果证明,TAT-Apoptin 基因原核表达载体构建成 功,并能在Rosetta 中进行分泌性表达。     

Localized hemibiotrophy in Colletotrichum: cytological and molecular taxonomic similarities among C. destructivum, C. linicola and C. truncatum

The infection process of hemibiotrophic isolates of Colletotrichum linicola (from flax, Linum usitatissimum ) and C . truncatum (from broad bean, Vicia faba and lentil, Lens culinaris ) was studied by light microscopy. Host surfaces were penetrated directly leading to a symptomless, biotrophic phase characterized by the elaboration of large multilobed, multiseptate, vesicular primary hyphae that were restricted to the initially infected epidermal cells. Biotrophy lasted for the first 48 h of the host-pathogen interaction and was rapidly succeeded by a necrotrophic phase during which narrow, secondary hyphae invaded the surrounding leaf tissues and water-soaked spreading lesions with sporulating, monosetate acervuli were produced on infected host surfaces. Molecular taxonomic analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed very close similarities (97–99%) between these isolates and those of C . destructivum obtained from cowpea ( Vigna unguiculata ) and lucerne ( Medicago sativa ), and also of C . truncatum obtained from pea ( Pisum sativum ). This association was consistent with results from a comparative assessment of some in-planta and in-vitro morphological and growth characteristics of these hemibiotrophic fungi. It was concluded that localized hemibiotrophy is an infection strategy utilized predominantly by a closely-related group of pathogens comprising C . destructivum , C . linicola and C. truncatum , and the formation of multilobed primary hyphae restricted to the first penetrated cell might therefore be a key taxonomic character which correlates consistently with ITS sequence data.     

Thyroxine-induced alterations in the testis and seminal vesicles of air-breathing catfish, Clarias gariepinus

Effect of experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish. T.N. Jacob and J.P. Pandey contributed equally     

芽孢杆菌植酸酶基因在毕赤酵母中的分泌表达

根据枯草芽孢杆菌WHNB02植酸酶phyC基因全序列设计一对引物,采用PCR法从含有该基因的pUC18-phyC质粒上获得不含有信号肽序列的phyC基因表达片段,亚克隆到毕赤巴斯德表达载体pPIC9K的多克隆位点,并电转化毕赤巴斯德酵母GS115.经MD和MM平板筛选、酶活性测定,获得了阳性转化子PP9KC.首次在毕赤巴斯德酵母中实现了有生物学活性芽孢杆菌植酸酶的分泌表达,去糖基化试验表明该植酸酶的分子量为53.5 kD和50.9 kD糖基化程度不同的两种糖蛋白.用麦芽汁半合成培养基对PP9KC进行诱导培养,培养48 h后酶活可达到2453 U/mL,表达量为出发菌株的10.5倍.酶学性质分析表明,其酶促反应最适pH值为7.0,最适反应温度为65℃,经90℃处理10 min,残留酶活性为37℃时的78.2%.     

Fat globule membrane of sow milk as a target for adhesion of K88-positive Escherichia coli

Membrane adhesion of K88-positive Escherichia coli was studied on intestinal brush-border membranes on 237 Finnish Landrace pigs. Forty-one per cent of the brush-border membrane preparations aggregated E. coli (positive adhesion). Similar dualism of adherence/nonadherence was observed on sow milk fat globule membranes. Washed milk fat globules (washed cream) can be used as a convenient source of material for adhesion studies. Bacterial adherence on to milk fat globules is evident as agglutination of the globules (dark-field microscopy). By this procedure the sows can be typed according to their receptor phenotype. This simple principle of fat globule agglutination due to receptors for K88-positive E. coli might be complicated by SigA-mediated bacterial adherence. Fat globule membranes were shown to contain SigA, which may act as a mediator of bacterial adherence onto fat globules. The significance of this adhesive property of milk fat globule might be to provide alternative receptors for E. coli thus preventing bacterial adhesion on to gastro-intestinal epithelium of the offspring. Sow milk fat globules can be used for typing E. coli for membrane adhesiveness. The adhesiveness of the strains showed a good correlation with the presence of the K88 antigen, as well as the hydrophobicity of the bacterial strain as determined by an association on Phenyl-Sepharose beads.