Differential miRNA expression between equine ovulatory and anovulatory follicles

Relatively little is known about the physiological roles of microRNAs (miRNAs) during follicular development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to gain insight on the involvement of these miRNAs during follicle maturation. Follicular fluid was aspirated from dominant follicles (>32 mm) during the ovulatory season (July to October) and the anovulatory season (January to March) in each of 5 mares, and the levels of steroids, IGF1, and miRNAs were analyzed by immunoassays and quantitative PCR. Levels of progesterone, testosterone, and IGF1 were lower (P ≤ 0.05) in anovulatory than in ovulatory follicles. Relative to ovulatory follicles, anovulatory follicles had higher (P < 0.05) mean levels of miR-21, miR-23b, miR-378, and miR-202 and tended to have higher (P = 0.06) levels of miR-145. Levels of miR-224 and miR-383 could not be detected in follicular fluid. These novel results indicate a physiological association between increases in follicular miRNA levels and seasonal anovulation in mares; further studies should elucidate the precise involvement of miR-21, miR-23b, miR-145, miR-378, and miR-202 in follicle maturation in the mare.     

Hepatic and intestinal CYP3A expression and activity in broilers

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT‐PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra‐acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.     

The expression of interferon-stimulated gene 15 in equine endometrium

Establishment of pregnancy is critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The up-regulation of the interferon-stimulated protein 15 kDa (ISG15) during pregnancy has been described in various species and has been hypothesized to be part of the molecular repertoire that makes the uterus receptive to conceptus development. In the current study, the expression of ISG15 and enzymes involved in ISG15ylation was examined at the mRNA and protein level in equine endometrium at Day 14 of the luteal phase and at Day 14 and 50 of pregnancy. ISG15 mRNA showed a 2.63-fold higher expression at Day 14 of pregnancy when compared to Day 14 of the cycle, while mRNA abundance at Day 50 of pregnancy was unchanged compared to Day 14 of the cycle. Upon Western blot analysis using anti-ISG15 antibody, several higher molecular weight bands could be observed, representing proteins conjugated to ISG15. No free ISG15 could be detected. The pattern of ISG15 reactive proteins differed from those observed in non-uterine samples. Upon immunohistochemistry, ISG15 reactive proteins located primarily to luminal and glandular epithelial cells, while stromal cells showed weaker staining. In conclusion, the expression of ISG15-conjugated proteins in equine endometrium did not differ between cyclic and pregnant 14 days after ovulation and Day 50 of pregnancy. It is hypothesized that the unique subset of ISG15ylated proteins expressed in endometrial tissue contributes to normal cellular function and that, unlike other species, the modification of ISG15-conjugated proteins is not an active contributor to conceptus-maternal interaction in the mare.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Differential expression of microRNAs in bovine papillomavirus type 1 transformed equine cells

Bovine papillomavirus (BPV) types 1 and 2 play an important role in the pathogenesis of equine sarcoids (ES), the most common cutaneous tumour affecting horses. MicroRNAs (miRNAs), small non‐coding RNAs that regulate essential biological and cellular processes, have been found dysregulated in a wide range of tumours. The aim of this study was to identify miRNAs associated with ES. Differential expression of miRNAs was assessed in control equine fibroblasts (EqPalFs) and EqPalFs transformed with the BPV‐1 genome (S6‐2 cells). Using a commercially available miRNA microarray, 492 mature miRNAs were interrogated. In total, 206 mature miRNAs were differentially expressed in EqPalFs compared with S6‐2 cells. Aberrant expression of these miRNAs in S6‐2 cells can be attributed to the presence of BPV‐1 genomes. Furthermore, we confirm the presence of 124 miRNAs previously computationally predicted in the horse. Our data supports the involvement of miRNAs in the pathogenesis of ES.     

Endotoxin-induced changes in expression of cyclooxygenase isoforms in the lamellar tissue of extracorporeally haemoperfused equine limbs

Angiogenesis and sepsis-related equine laminitis have several features in common. Both events can be induced by endotoxin (lipopolysaccharide— LPS) and both are associated with increased expression of the enzyme cyclooxygenase (COX), of which two isoforms (COX-1 and COX-2) exist. To examine the causal relationship between LPS exposure and COX expression and to investigate the tissue distribution of COX in the LPS-exposed tissue, the technique of extracorporeal haemoperfusion of isolated equine forelimbs was utilized. Perfusion was performed for 10 hr under physiological conditions (control-perfused limbs, n = 5) and with addition of 80 ng/L of endotoxin (LPS-perfused limbs; n = 5). After perfusion, samples of lamellar tissue were collected from the dorsal aspect of the hoof wall. Additional control samples were collected from three non-perfused limbs. Immunohistochemical analysis was performed using antibodies against COX-1 and COX-2, and intensity of immunohistochemical staining was scored for each isoform. In the lamellar tissue of control- and LPS-perfused limbs, there was no significant difference in COX-1 staining intensity and distribution, whereas COX-2 expression was significantly increased in LPS-perfused limbs (especially in endothelial cells, fibroblasts and intravasal leucocytes as well as in epidermal basal cells at the base of the primary epidermal lamellae). These results suggest that COX-2 and its metabolites are involved in the initiation of pathological changes seen in sepsis-associated events such as sepsis-related laminitis. In such cases, COX-2 could therefore be an important therapeutic target; however, early therapy may be required as increase in COX-2 expression occurs within 10 hr after LPS exposure.     

Effect of breed and gender on bovine liver cytochrome P450 3A (CYP3A) expression and inter-species comparison with other domestic ruminants

The cytochrome P450 (P450) superfamily represents a group of relevant enzymes in the field of drug metabolism and several exogenous or constitutional factors contribute to regulate its expression. Cattle represent an important source of animal-derived food-products and studies concerning the P450 expression are needed for the extrapolation of pharmacotoxicological data from one species to another and for the evaluation of the consumer's risk associated with the consumption of harmful residues found in foodstuffs. In the present study, possible breed-, gender- and species-differences in P4503A (the P450 subfamily more expressed in the human liver) expression were studied in vitro in Piedmontese (PDM) and Limousin (LIM) meat cattle breeds of both sexes and in domestic Ruminants (cattle, sheep and goats). Cytochrome P450 and P4503A contents as well as CYP3A-dependent drug metabolising enzymes (DME) were measured in liver microsomes. Significant lower levels of P450 (P < 0.001) and P4503A (P < 0.05) contents were observed in PDM vs. LIM of both sexes; the P4503A-dependent DME activities were significantly (P values ranging from 0.05 up to 0.001) higher in PDM cattle, particularly in males. A gender-effect in DME activities was noticed (P < 0.05) only in PDM male cattle. With regards to the species, the expression of both P4503A apoprotein and some of the related DME activities were more pronounced in sheep (P < 0.01 vs. cattle) and in goats (P < 0.05 vs. sheep; P < 0.01 vs. cattle) than in cattle. The significant differences in P4503A expression observed in LIM and PDM cattle are consistent with previously published data on strain- and breed-differences pointed out in rats and men. As far as a possible sex-effect is concerned, no clear-cut evidence is likely to be drawn. Finally, P4503A expression was more relevant in small ruminants.     

盐生植物小花碱茅CYP86A基因的RNAi载体构建

拒盐型盐生植物小花碱茅根系具有高度木栓化的内皮层可能是其抵御盐胁迫的重要策略之一。本研究以盐胁迫下小花碱茅幼苗的根为材料,采用RT-PCR方法扩增到木栓质生物合成关键基因CYP86A的RNAi靶片段,以中间载体pHANNIBAL和植物表达载体pART27为基础,采用酶切连接结合In-Fusion的克隆方法成功构建了以35S启动子驱动,含PtCYP86A基因片段反向重复序列的RNAi植物表达载体pARC,为利用RNAi技术深入探究根系木栓化在小花碱茅拒盐中的功能奠定基础。     

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1β or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.     

高脂诱导非酒精性脂肪肝对CYP2A5表达的影响

为了研究高脂诱导脂肪肝对CYP2A5表达的影响,选取雄性C57BL/6J小鼠18只,随机分成对照组(control)、高脂组(NAFLD)和阳性对照组(PYR),每组6只,分别饲喂基础饲料和高脂饲料,阳性对照组饲喂基础饲料在处死前一天给予吡唑诱导,连续8周后小鼠断颈处死,取肝脏测定mRNA,CYP2A5的蛋白表达和酶活性。其结果阳性对照组CYP2A5的mRNA、蛋白表达和酶活性与对照组相比分别增加229%、80%、151%差异极显著(P0.01)。高脂诱导组与对照组相比CYP2A5的mRNA、蛋白表达和酶活性分别增加69%、34%、43%(P0.01)。表明高脂诱导非酒精性脂肪肝可增加CYP2A5的mRNA、蛋白表达水平和酶活力。     

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis

Reasons for performing study: There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis‐related laminitis than the black walnut extract (BWE) model. Objectives: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature >38.9°C (DEV group, n = 8) or at onset of Obel grade 1 lameness (OG1 group, n = 8). Control horses (CON group, n = 8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time‐quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. Results: Increased mRNA concentrations (P<0.05) for IL‐1β, IL‐6, IL‐12p35, COX‐2, E‐selectin and ICAM‐1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL‐2, IL‐4, IL‐10, TNF‐α, IFN‐γ or COX‐1 at either the DEV or OG1 time points. Conclusions: There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. Potential relevance: The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.     

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury.     

Effect of strenuous exercise and ex vivo TLR3 and TLR4 stimulation on inflammatory gene expression in equine pulmonary leukocytes

The effects of strenuous exercise and ex vivo stimulation of TLR3 and TLR4 pathways on the expression of six inflammatory genes in equine pulmonary leukocytes were investigated. The genes tested were interferon-beta (IFN-β), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interferon gamma-induced protein 10 (IP-10), chemokine (c-c motif) ligand 5 (RANTES) and tumor necrosis factor-alpha (TNF-α). We hypothesized that strenuous exercise would modulate basal gene expression on one hand and modulate the response to bacterial lipopolysaccharide (LPS) and to polyinosinic:polycytidylic acid (Poly IC) on the other hand. Eight young Thoroughbred mares were selected for the experiment. Bronchoalveolar lavages were performed on horses 48 h before and 24h after the completion of treadmill exercise until fatigue. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was used to quantify cytokine expression in pulmonary leukocytes. Target gene expression was normalized to the expression of three housekeeping genes (HKG). There were no significant differences in the mRNA expression of the six cytokines between pre-exercise and post-exercise cells. LPS and Poly IC induced respectively significant increases of TNF-α, IFN-β, IL-6, IL-1β, and TNF-α, IFN-β, IP-10 and RANTES, both before and after exercise. However, exercise induced a significant decrease of the genes response to LPS and Poly IC. These findings may suggest that strenuous treadmill exercise exerts a deleterious effect on part of the pulmonary immune response in horses 24h following an intense physical activity.     

小型猪肝脏细胞色素P450基因CYP3A29克隆与序列分析

设计引物通过RT-PCR扩增巴马香猪和贵州小型猪香猪药物代谢关键P450基因CYP3A29基因的全长编码区,纯化并克隆入pMD 18-T载体,通过序列测定和比对分析其序列变异情况。17头小型猪共发现CYP3A29基因的5个单核苷酸变异位点,均表现为同义替换,对编码的氨基酸序列无影响;猪CYP3A29的单核苷酸变异和品系相关,在不同品系中可表现出品系特有的单核苷酸变异;贵州小型香猪G3 a2902发现1个可变剪接,缺失89 bp并产生移码,改变编码蛋白。本研究克隆的巴马香猪和贵州小型香猪CYP3A29基因及其序列变异情况,可为其作为药物代谢模型的应用提供技术资料。     

马属动物cyclinT1基因的扩增与真核表达载体的构建

在幔病毒复制过程中cyclinT1(CycT1)是重要的辅助因子.为了揭示慢病毒的复制机理,试验使用RT-PCR方法扩增马和驴的cyclinT1基因,并进行克隆和序列分析,然后用DNASTAR(MegAlign)软件将测得的序列和国外发表的马属动物CycT1序列(GenBank中登录号为AF137509和AF190905)进行同源性比较.结果表明:马属动物CycT1大小为2 184 bp,编码727个氨基酸;4种CycT1基因氨基酸同源性在98%以上;将扩增出的马CycT1基因克隆至真核表达载体pcDNA3.1(+)中,经酶切及测序鉴定证实成功构建了pcDNA3.1(+)/eCycT1重组质粒.