牦牛CRH基因克隆及其在生殖轴中的表达研究

为了探讨促肾上腺皮质激素释放激素（corticotropin-releasing hormone，CRH）基因在牦牛繁殖中的调控作用，试验分别采集5头成年母牦牛和5头成年母黄牛的下丘脑、脑垂体前叶、输卵管、卵巢和子宫等组织，通过RT-PCR技术及生物信息学软件对牦牛CRH基因编码区进行扩增、克隆及序列分析，并构建系统进化树；采用实时荧光定量PCR法检测CRH基因的组织表达。结果表明，牦牛CRH基因编码区全长573 bp，编码190个氨基酸。牦牛与黄牛、猫、鸡、人、小鼠、大鼠和猪的CRH基因核苷酸同源性分别为99.8%、43.3%、36.1%、46.2%、39.0%、38.5%和56.4%。系统进化树表明，牦牛与黄牛亲缘关系最近，并与其他物种类聚，符合物种进化规律。CRH蛋白分子式为C₉₂₀H₁₅₀₃N₂₇₉O₂₆₃S₃，分子质量为20776.95 u，理论等电点为10.95，其氨基酸组成中亮氨酸（L）、脯氨酸（P）、丙氨酸（A）和精氨酸（R）所占比例较高，分别为15.8%、12.6%、12.1%和10.5%。CRH蛋白为不稳定亲水蛋白，存在信号肽和跨膜结构。CRH蛋白二级结构中α-螺旋、β-转角、延伸链和无规则卷曲分别占41.05%、1.05%、8.42%和49.48%，三级结构分析结果与其相一致。CRH基因mRNA在牦牛下丘脑中表达量最高，显著高于其他组织（P<0.05），在子宫中表达量最低。牦牛脑垂体前叶CRH基因mRNA表达量极显著高于黄牛（P<0.01），在卵巢、输卵管中表达量显著高于黄牛（P<0.05），而两个品种在下丘脑、子宫中的表达量差异不显著（P>0.05）。提示CRH基因在牦牛繁殖调控中具有重要作用。     

Pilot study of the ultrasonographic examination of the intact and transected medial glenohumeral ligament in dogs

Medial glenohumeral ligament injury is commonly reported during medial shoulder joint instability in dogs. Arthroscopy is considered the gold standard procedure, but it is invasive and requires distension of the joint. Ultrasonographic examination of the medial glenohumeral ligament has been studied as a possible, less invasive alternative to arthroscopy however it has not been considered a useful method of assessment due to the interference of the probe with the pectoral muscles. The aims of this prospective analytical randomized pilot study were to develop a standardized ultrasound protocol for visualizing the canine medial glenohumeral ligament and to compare goniometry and ultrasound findings in cadaver dogs with versus without transection of the medial glenohumeral ligament. Nine adult Beagle cadavers (18 shoulders) were used. The first six shoulders were used in a preliminary study to describe an ultrasound technique to identify the medial glenohumeral ligament. Arthroscopy was performed on the remaining 12 shoulders, with six randomly selected medial glenohumeral ligaments from these shoulders, transected during the procedure. Ultrasound examination was performed after each arthroscopic procedure by an ultrasonographer blinded to the patient group. Four medial glenohumeral ligaments (67%) were correctly identified during the preliminary study. Ultrasonographic examination failed to diagnose the transection of all six medial glenohumeral ligaments in the second part of the study. No difference was observed in the ligament thickness between the dogs with and without a transected medial glenohumeral ligament. Dogs with a transected medial glenohumeral ligament had a wider articular space compared to dogs without a transected ligament (P < 0.001), and an articular space wider than 8.2 mm was discriminatory of a transected medial glenohumeral ligament in all the shoulders. In conclusion, the medial glenohumeral ligament could be identified with a medial ultrasonographic approach of the shoulder and a wider articular space can be a sign of a medial shoulder joint instability. Further studies are needed to confirm these preliminary findings in living dogs, with and without shoulder instability.     

Nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed

Pomegranate peel is a nutritive-rich byproduct whose amounts are extensively growing due to the exponential increase in the production of pomegranate juice and "ready to eat" arils. Pomegranate peel is a rich source for antioxidants and thus may serve in the prevention of cattle diseases and in the improvement of beef products, making it an attractive component in beef cattle diets. The present study aims to evaluate the effect of commonly used storage practices on the nutritive and antioxidative properties of pomegranate peel. In general, storage conditions preserved most antioxidant capacity. Ensiling ambivalently affected the nutritive values of the peel and promoted increased levels of antioxidative components. In addition to polyphenols, nonphenolic components, such as alpha- and gamma-tocopherols, contributed to the total antioxidative capacity, and several minerals found in the peel added to its nutritional value. Dietary supplementation with fresh peels promoted significant increases in feed intake and alpha-tocopherol concentration in the plasma, with positive tendency toward increased weight gain of bull calves. All in all, the nutritive value and the antioxidant capacity of pomegranate peel turn it into a favorable health-promoting constituent of feedlot beef cattle diet.     

Effects of dietary docosahexaenoic acid (22:6n‐3) and arachidonic acid (20:4n‐6) on the growth,survival, stress resistance and fatty acid composition in black sea bass Centropristis striata (Linnaeus 1758) larvae

The objectives of this study were to determine the effects of the dietary docosahexaenoic acid (DHA) to arachidonic acid (ARA) ratio on the survival, growth, hypersaline stress resistance and tissue composition of black sea bass larvae raised from first feeding to metamorphic stages. Larvae were fed enriched rotifers Brachionus rotundiformis and Artemia nauplii containing two levels of DHA (0% and 10% total fatty acids=TFA) in conjunction with three levels of ARA (0%, 3% and 6% TFA). On d24ph, larvae fed the 10:6 (DHA:ARA) treatment showed significantly (P<0.05) higher survival (62.3%) than larvae fed 0:0 (DHA:ARA) (27.4%). Notochord length and dry weight were also significantly (P<0.05) greater in the 10:6 (DHA:ARA) treatment (8.65 mm, 2.14 mg) than in the 0:0 (DHA:ARA) (7.7 mm, 1.65 mg) treatment. During hypersaline (65 g L⁻¹) challenge, no significant differences (P>0.05) were observed in the median survival time (ST50) between larvae fed 10% DHA (ST50=25.6 min) and larvae fed 0% DHA (ST50=18.2 min). The results suggested that black sea bass larvae fed prey containing 10% DHA with increasing ARA within the range of 0–6% showed improved growth and survival from first feeding through metamorphic stages.     

牦牛FSHR基因克隆及其在生殖轴中的表达研究

本研究旨在阐明牦牛促卵泡素受体(follicle stimulating hormone receptor,FSHR)基因CDS序列及其在牦牛生殖轴中表达的特点,为探讨其在牦牛繁殖活动中的调控作用奠定基础。试验采集卵泡期的牦牛与黄牛下丘脑、脑垂体前叶、卵巢、输卵管及子宫组织,通过RT-PCR技术对牦牛FSHR基因cDNA进行扩增、克隆与序列分析;采用实时荧光定量PCR法检测FSHR基因在牦牛与黄牛中的组织表达差异。结果显示,牦牛FSHR基因编码区全长2 088bp,编码695个氨基酸,蛋白质分子式为C6378H10670N2088O2637S576,分子质量为177 263.85u,理论等电点(pI)为4.88,与黄牛、绵羊、山羊和猪氨基酸序列同源性较高(91.50%～99.38%)。FSHR蛋白为酸性不稳定疏水蛋白,存在信号肽与8个跨膜结构;二级结构由延伸链(15.54%)、α-螺旋(42.30%)、β-转角(1.44%)和无规则卷曲(40.72%)组成;系统进化树表明,牦牛与黄牛亲缘关系最近;实时荧光定量PCR结果显示,FSHR基因在黄牛、牦牛检测组织中均有表达,牦牛子宫中表达量显著或极显著高于除卵巢外的其他组织(P<0.05;P<0.01),而黄牛卵巢中表达量显著或极显著高于其他组织(P<0.05;P<0.01);黄牛卵巢中表达量极显著高于牦牛(P<0.01)。说明FSHR基因在动物进化中较为保守,其在牦牛卵巢中表达量低可能影响到牦牛繁殖机能。     

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping.

This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds.     

The Pho regulon and the pathogenesis of Escherichia coli

During the course of infection, bacteria must coordinately regulate gene expression in response to environmental stimuli. The phosphate (Pho) regulon is controlled by the two component-regulatory system PhoBR. PhoBR is activated during starvation and regulates genes involved in phosphate homeostasis. Several studies have highlighted the importance of the Pho regulon in bacterial pathogenesis, showing how induction of PhoBR, in addition to regulating genes participating in phosphate metabolism, leads to modulation of many cellular processes. The pleiotropic effects of Pho regulon activation include attenuated virulence and alteration of many virulence traits, including adhesion to host cells and resistance to cationic antimicrobial peptides, acidity and oxidative stresses. This review provides an overview of the relationship between the Pho regulon and virulence in Escherichia coli and illustrates that, in addition to regulating phosphate homeostasis, the Pho regulon plays a key role in regulating stress responses and virulence.     

Parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> and role of the gelatinous matrix

Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca²⁺-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.     

Changes in Protein Kinase A Activity Accompany Sclerotial Development in Sclerotinia sclerotiorum

ABSTRACT Sclerotia of Sclerotinia sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Sclerotial formation has been shown to be affected by increased intracellular cAMP levels. Cyclic AMP (cAMP) is a key modulator of cAMP-dependent protein kinase A (PKA) and the latter may prove to play a significant role in sclerotial development. Therefore, we monitored changes in relative PKA activity levels during sclerotial development. To do so, we first developed conditions for near-synchronous sclerotial development in culture, based on hyphal maceration and filtering. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in nonsclerotium-producing mutants. Furthermore, applying caffeine, an inducer of PKA activity, resulted in increased relative PKA activity levels and was correlated with the formation of sclerotial initial-like aggregates in cultures of the non-sclerotium-producing mutants. In addition, low PKA activities were found in an antisense smk1 strain, which exhibits low extracellular-signal-regulated kinase (ERK)-type mitogen-activated protein kinase (MAPK) activity, and does not produce sclerotia. The changes in PKA activity, as well as the abundance of phosphorylated MAPKs (ERK-like as well as p38-like) that accompany sclerotial development in a distinct developmental phase manner represent a potential target for antifungal intervention.     

Internally generated reactivation of single neurons in human hippocampus during free recall

The emergence of memory, a trace of things past, into human consciousness is one of the greatest mysteries of the human mind. Whereas the neuronal basis of recognition memory can be probed experimentally in human and nonhuman primates, the study of free recall requires that the mind declare the occurrence of a recalled memory (an event intrinsic to the organism and invisible to an observer). Here, we report the activity of single neurons in the human hippocampus and surrounding areas when subjects first view cinematic episodes consisting of audiovisual sequences and again later when they freely recall these episodes. A subset of these neurons exhibited selective firing, which often persisted throughout and following specific episodes for as long as 12 seconds. Verbal reports of memories of these specific episodes at the time of free recall were preceded by selective reactivation of the same hippocampal and entorhinal cortex neurons. We suggest that this reactivation is an internally generated neuronal correlate for the subjective experience of spontaneous emergence of human recollection.