草鱼NK-lysin基因的克隆、原核表达与活性分析

为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。     

Quantity of IL‐2, IL‐4, IL‐10, INF‐γ, TNF‐α and KC‐Like Cytokines in Serum of Bitches With Pyometra in Different Stages of Oestrous Cycle and Pregnancy

The occurrence of the pyometra is most common in the first half of the dioestrus when there is decreased cellular immunity associated with increased serum concentration of progesterone in females. The aim of this study was to determine the immunological profile of bitches with pyometra, studying serum levels of IL‐2, IL‐4, IL‐10, IFN‐γ, KC‐like and TNF‐α and comparing them with those of healthy bitches in anoestrus, dioestrus and pregnant. Forty females were divided into four experimental groups: group 1 (G1): with pyometra (n = 10); group 2 (G2): bitches in the second week of gestation (n = 10); group 3 (G3): in anoestrus (n = 10); and group 4 (G4): in dioestrus (n = 10). The serum levels for IL‐2, KC‐like, INF‐γ and TNF‐α were similar for all experimental groups. The values obtained for IL‐10 were found increased (p < 0.001) in animals in dioestrus and pyometra compared with females in anoestrus and pregnant, and the levels of IL‐4 observed were significantly greater (p < 0.001) in bitches with pyometra when compared with others. The cytokine profile in animals with pyometra is similar to bitches in dioestrus for IL‐10 and had increase in IL‐4 for bitches with pyometra, which represents an anti‐inflammatory these cases. This suggests the presence of an immunosuppressive state in both cases, which may explain the propensity of bitches in dioestrus to be affected by pyometra and the severity of the disease on these animals.     

Comparative Aspects of the Endotoxin- and Cytokine-Induced Endocrine Cascade Influencing Neuroendocrine Control of Growth and Reproduction in Farm Animals

Disease in animals is a well-known inhibitor of growth and reproduction. Earlier studies were initiated to determine the effects of endotoxin on pituitary hormone secretion. These studies found that in sheep, growth hormone (GH) concentration was elevated, whereas insulin-like growth factor-I (IGF-I) was inhibited, as was luteinizing hormone (LH). Examination of the site of action of endotoxin in sheep determined that somatotropes expressed the endotoxin receptor (CD14) and that both endotoxin and interleukin-Iβ activated GH secretion directly from the pituitary. In the face of elevated GH, there is a reduction of IGF-I in all species examined. As GH cannot activate IGF-I release during disease, there appears to be a downregulation of GH signalling at the liver, perhaps related to altered nitration of Janus kinase (JAK). In contrast to GH downregulation, LH release is inhibited at the level of the hypothalamus. New insights have been gained in determining the mechanisms by which disease perturbs growth and reproduction, particularly with regard to nitration of critical control pathways, with this perhaps serving as a novel mechanism central to lipopolysaccharide suppression of all signalling pathways. This pathway-based analysis is critical to the developing novel strategies to reverse the detrimental effect of disease on animal production.     

Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil

The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.     

Doppler and Contrast‐Enhanced Ultrasonography of Testicles in Adult Domestic Felines

The objective was to characterize the vascular patterns of testicular blood flow of adult cats, measuring the systolic velocity (SV), diastolic velocity (DV), resistance index (RI), gate time (wash‐in) peak enhancement and output time (wash‐out) of the contrast and addition of tissue fill characteristics. Forty‐five adult cats were selected, and the echotexture, echogenicity, size, contours and margins of testicles were assessed via ultrasound. By Doppler were evaluated the blood flow and determined of vascular index in testicular artery (SV, DV and RI) and via contrast‐enhanced ultrasonography determine the time for phases: wash‐in, wash‐out and peak enhancement. Sonographic findings presented normal. Testicular artery was observed in the spermatic cord with tortuous patter and showed monophasic‐patterned waves and low vascular resistance and with systolic peak evident. Values of indices vascular were as follows: SV = 6.73 cm/s, DV = 2.8 cm/s and RI = 0.54 for left testicles; and SV = 6.23 cm/s, DV = 2.77 cm/s and RI = 0.53 for right testicles. Contrast filled the subcapsular vascular structures and after a few seconds, a homogeneous moderate enhancement of the parenchyma, with parenchymal vessels still distinguishable and after the peak phase, a rapid homogeneous decrease in echogenicity. Values of time for contrast‐enhanced ultrasonography were as follows: wash‐in = 8.78 s, peak enhancement = 21.62 s and wash‐out = 75.36 for left testicles; and wash‐in = 10.76 s, peak enhancement = 21.50 s and wash‐out = 81.81 for right testicles. Doppler and contrast‐enhanced ultrasonography of the testicles in healthy adult cats was easily implemented and may provide baseline data for this organ to allow the use of these techniques as a diagnostic tool for evaluating testicular abnormalities in sick cats.     

Electrochemical principles for active control of liquids on submillimeter scales

Electrochemical methods were combined with redox-active surfactants to actively control the motions and positions of aqueous and organic liquids on millimeter and smaller scales. Surfactant species generated at one electrode and consumed at another were used to manipulate the magnitude and direction of spatial gradients in surface tension and guide droplets of organic liquids through simple fluidic networks. Solid microparticles could be transported across unconfined surfaces. Electrochemical control of the position of surface-active species within aqueous films of liquid supported on homogeneous surfaces was used to direct these films into periodic arrays of droplets with deterministic shapes and sizes.     

Photorefractive materials for nonvolatile volume holographic data storage

Optically gated recording and nonvolatile readout in a digital volume holographic data storage system that uses a pair of mutually incoherent light sources during recording and only one for readout were demonstrated recently. This approach used stoichiometric lithium niobate, which after post-growth processing gave rise to an at least two orders of magnitude improvement in sensitivity over the best materials reported previously. It is also shown that by adding certain dopants (iron and manganese) to near-stoichiometric lithium niobate, the dark storage time and gating efficiency can be increased compared with previous work. The underlying physical mechanisms of gated recording and the effectiveness of the gating process responsible for this manifold improved performance are discussed, and bipolarons and small polarons are identified as the responsible photorefractive species.