Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris

ABSTRACT:   Until now, six crustacean hyperglycemic hormones (CHH) designated Pej-SGP-I, -II, -III, -V, -VI and -VII have been characterized in the kuruma prawn Penaeus japonicus . All CHH consist of 72 amino acid residues and have an amidated carboxyl (C)-terminus. In the present study, we expressed Pej-SGP-III in methylotrophic yeast Pichia pastoris in order to obtain a large quantity of recombinant CHH possessing biological activity. A cDNA encoding Pej-SGP-III that had been previously cloned was processed by polymerase chain reaction (PCR) and the resulting product was ligated into an expression vector. Pichia pastoris was transformed with this vector after which a recombinant Pej-SGP-III was expressed having an additional amino acid residue (glycine) at the C-terminus (rPej-SGP-III-Gly), a form considered to be a putative precursor of this hormone. rPej-SGP-III-Gly secreted into the culture medium was purified by reversed-phase HPLC, and amidated using a peptidylglycine alpha-amidating enzyme. The amidated rPej-SGP-III (rPej-SGP-III) showed hyperglycemic activity in in vivo bioassay almost comparable to that of the natural Pej-SGP-III. rPej-SGP-III thus obtained will be a useful tool not only for its physiological study but also for the determination of its 3-D structure.     

葡萄霜霉菌候选效应子RXLR5信号肽的鉴定

利用Getorf、SignalP 3.0、BLASTP等生物信息学软件和PERL语言从葡萄霜霉菌基因组中预测到一条编码RXLR-EER结构域的效应蛋白候选基因,将该基因编码的蛋白命名为RXLR5。将RXLR5信号肽SP5编码序列连接到pSUC2T7M13ORI载体并转化到酵母蔗糖酶缺陷菌株YTK12后,重组菌株能成功分泌蔗糖酶,促使棉籽糖分解成单糖,因此,能在YPRAA培养基上正常生长;同时生成的单糖能与TTC反应产生红色不溶于水的氯化三苯基四氮唑。由此推测,SP5具有信号肽活性,可能在RXLR5从葡萄霜霉菌细胞内泌到胞外的过程中起重要作用。     

壳聚糖/羧甲基纤维素钠负载苦瓜小分子多肽复合物的制备及其复合效果的表征

以壳聚糖盐酸盐（CSC）和羧甲基纤维素钠（CMC）为壁材,采用离子凝胶法制备苦瓜小分子多肽复合物,并对其制备工艺和特性进行研究。结果显示,复合物的最佳制备工艺为CSC浓度5.35 mg·mL^-1,CMC浓度1.60 mg·mL^-1,包埋时间33.10 min,此时包埋率可达到69.98%。红外光谱和X-射线衍射分析结果表明,芯材和壁材产生了离子交联作用,苦瓜小分子多肽被成功包埋在CSC/CMC复合壁材中。扫描电镜显示复合物表面疏松多孔,呈不规则形状。所制得的复合物在胃肠液中具有较好缓释特性,在室温下贮藏30 d后仍具有较强的DPPH自由基清除能力。     

白绒红蛋鸟巢菌发酵液提取物的抑菌活性

对白绒红蛋鸟巢菌[Nidula niveotomentosa(P.Henn)Lloyd]发酵液的正丁醇提取物进行抑菌试验,结果显示:正丁醇提取物对土曲霉、绳状青霉、产黄青霉、宛氏拟青霉和青霉均有较强的抑制作用。不同浓度处理时,提取物对霉菌的抑制作用呈极显著差异。且抑菌作用随浓度的增大而增强。结果还显示:提取物对细菌和植物病原菌无抑菌活性。且提取物对土曲霉、绳状青霉、产黄青霉的MIC为6.25 mg/mL,对宛氏青霉和青霉的MIC为12.5 mg/mL。     

Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

Antibiotic susceptibility of bacteria isolated from infections in cats and dogs throughout Europe (2002–2009)

A monitoring program of the pre-treatment susceptibility of clinical isolates of bacteria from diseased dogs and cats was active between the years 2002 and 2009. Susceptibility of each isolated strain to a panel of nine antibiotics (amoxicillin/clavulanic acid, ampicillin, penicillin, clindamycin, doxycycline, enrofloxacin, marbofloxacin, trimethoprim and trimethoprim/sulfamethoxazole) was assessed. The Minimum Inhibitory Concentration (MIC) of marbofloxacin was also determined by a standardized microdilution technique following CLSI recommendations. In total, 1857 bacterial strains were collected throughout Europe from cases of otitis, respiratory, urinary and dermatological infections. Although bacterial susceptibility varied for each of the antibiotics within the panel, patterns of susceptibility were similar to those described in the literature for comparable time periods and geographical areas. With a clinical resistance varying from 0 to 14.48% against the isolated strains, marbofloxacin susceptibility was very high and remains an effective antibiotic for the treatment of otitis, urinary, respiratory and dermatological infections in companion animals.     

Risk-based surveillance of antimicrobial residues in pigs – Identification of potential risk indicators

Around 20,000 samples are analysed each year for the presence of antibacterial residues in Danish finisher pigs, and between zero and five samples are detected positive above the maximum residue level (MRL). The intention was to develop a risk-based surveillance programme involving fewer samples while ensuring equal sensitivity. Therefore, risk indicators were searched for. Data were obtained from the Danish slaughterhouse database covering the period from July 2010 to December 2012. Residues were found or suspected in 17 incidents. In nine of these, the farmer had called in to prevent the pigs from being slaughtered. Hence, eight suspect cases were found through the surveillance programme, and two of these were above MRL. For these eight case herds, the number of pigs slaughtered and the number in which each of the following lesions were found were included in a statistical analysis: chronic pleuritis, tail bite, chronic pericarditis, chronic pneumonia, chronic peritonitis, osteomyelitis, abscess in hindquarters, abscess in leg/toe and abscess in forequarters. Only chronic pleuritis was associated with the presence of residues. Next, data from all herds delivering pigs for slaughter to the same abattoir were included covering a 3-month period prior to the residue finding. The prevalence of chronic pleuritis was on average 1.7 times higher in the eight case herds compared to all other herds. In two herds, the prevalence was significantly higher (P ≤ 0.05), and in one herd substantially higher, but only borderline significant (P = 0.1). In the remaining herds, the prevalence did not differ from the other herds delivering pigs to the abattoir. This indicates that chronic pleuritis might be considered as a risk indicator for use in surveillance. Other risk indicators/factors – reported in the cases where the farmers called in – were inadequate marking of treated animals and incorrect use of medication dispensers. These factors are not suited for use in surveillance and should be dealt with otherwise.     

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals.