Inflammatory cytokines induce a reduction in E-cadherin expression and morphological changes in MDCK cells

Epithelial–mesenchymal transition (EMT) is a fundamental phenomenon in organisms that occurs during gastrulation, wound healing, and cancer metastasis. Various cytokines induce EMT processes through complex mechanisms. Inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), induce EMT in human cell lines. However, whether inflammatory cytokines can affect EMT processes in canine cell lines remains unclear. In this study, we investigated the role of transforming growth factor beta (TGF-β), TNF-α, and IL-6 in Madin-Darby canine kidney (MDCK) cells. We found that the localization of E-cadherin, a cell adhesion molecule, was shifted and that its expression was decreased. We also observed morphological changes in MDCK cells under persistent stimulation of inflammatory cytokines. Morphological changes in cells may occur during late stages of EMT processes; inflammatory cytokines may be important in these changes.     

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.     

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

CD47-SIRPα阻断剂在实体癌和血液癌中的研究现状

CD47属于免疫球蛋白家族成员之一,广泛存在于细胞膜表面,特别是在肿瘤细胞中高表达。肿瘤细胞表面的CD47可与巨噬细胞表面的抑制性免疫受体--信号调节蛋白α(SIRPα)相结合,形成CD47-SIRPα信号复合体,该复合体能抑制巨噬细胞对肿瘤细胞的吞噬,使肿瘤细胞逃避免疫监视,从而促进肿瘤的发展。目前免疫治疗主要是通过抗CD47抗体抑制CD47-SIRPα信号通路,激活先天性免疫反应和适应性免疫反应,提高巨噬细胞靶向杀伤肿瘤细胞的能力。CD47-SIRPα正逐渐成为肿瘤免疫治疗的新型有效免疫治疗靶标,论文对CD47-SIRPα阻断剂在实体癌和血液癌的研究现进行以综述,以期为实体癌和血液癌的研究提供参考。     

Once-yearly sampling for the detection of trends in biodiversity: The case of Willow Slough, California

The butterfly fauna at Willow Slough, Yolo County, California has been censused for 32 years as part of a participatory citizen-science project, the Fourth of July Butterfly Count. While the utility of a once-a-year census as a monitoring tool is potentially compromised by lack of standardization in counting protocols and variation in observer skill, at Willow Slough these issues have been minimized.We examined the Willow Slough count data for trends in both faunal diversity and the probability of presence of individual species. During the study, the number of species observed at a visit declined by 39%. Regressions of per-visit species counts against time did not detect a statistically significant decline until year 24. In contrast, Fisher’s α, a statistic designed to reduce sample-size bias, detected the decline as early as year 13. Twelve of the 24 species analyzed showed significant declines in probability of occurrence; a further nine exhibited negative but non-significant trends. Butterflies that overwinter as eggs or larvae were more likely to decline than those that overwinter as pupae or adults. Many species in decline at Willow Slough have also been observed less frequently at nearby sites which are monitored year-round, supporting the value of once-a-year monitoring. Although correlations with climatic data have been identified, they are too weak to account for the observed faunal decline. We suspect broader patterns of land use and habitat continuity are implicated in butterfly declines across the region.We conclude that once-a-year sampling, if properly and rigorously done, is in fact useful as a monitoring tool for butterfly faunas, and that Fisher’s α is well suited to early detection of trends in repeated diversity sampling.     

Short term pharmacological immobilization in macaque monkeys

ObjectiveTo develop a safe and effective immobilization protocol in rhesus monkeys, which is not based on dissociative anaesthetic agent.Study designProspective, randomised, experimental trial.AnimalsTwenty rhesus monkeys, weighing 2.6–8.0 kg, 1–3 years of age, of both sexes.MethodsThe monkeys received 50 μg kg^?1 medetomidine, 0.25 mg kg^?1 midazolam and 5 μg kg^?1 fentanyl with 150 IU hyaluronidase intramuscularly (IM). The animals were closely observed for behavioural changes and reaction to sound stimulus. Pulse rate and oxygen saturation of haemoglobin (SpO₂) were monitored every 5 minutes, for 20 minutes. After this period, 250 μg kg^?1 atipamezole or a placebo was administered IM and behavioural changes were closely observed.ResultsFull immobilization was observed after mean 269 ± SD 116 seconds. Ten minutes after injection mean arterial oxygen saturation of haemoglobin was 94 ± 4%, but did not fall significantly further. The median pulse rate was 116 beats minute^?1 5 minutes after the administration of the drug. This level further decreased to a median level of 108 beats minute^?1 20 minutes after the drug's administration. The median time to recover from immobilization was significantly shorter after atipamezole administration when compared to placebo (2.7 versus 55 minutes). All animals awoke smoothly and no side effects such as vomiting or agitation were observed.ConclusionsShort term and reversible pharmacological immobilization was achieved using combination of midazolam, medetomidine, and fentanyl.Clinical relevanceThe present study demonstrates that 20-minute pharmacological immobilization with a combination of midazolam, medetomidine, and fentanyl is feasible in rhesus monkeys with minimal effect on heart rate.     

Effects of estrogen and estrogen receptor α on neuronal injury induced by glutamate

AIM：To observe the effects of estrogen and estrogen receptor α (ERα) on the neuronal damage induced by glutamate (Glu). METHODS：Primarily cultured mouse neurons were used in the study. The model of neuronal injury was established by Glu induction. The neuron-specific enolase (NSE) was identified by the immunohistochemical method to determine the purity of the neurons. The recombinant lentiviral vector containing ERα (V-ERα-RFP-flag) was constructed. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of ERα in the neurons. The neurons were divided into 3 groups: (1)control group: empty lentivirus (V-RFP-flag) was used to infect the injured cells; (2)estrogen group: estrogen intervention was applied to the injured cells;(3) lentivirus group: V-ERα-RFP-flag was used to infect the injured cells. The apoptosis of the neurons was detected by flow cytometry. The changes of N-methyl-D-aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter protein 1 (VGLUT1) were determined by the methods of real-time quantitative PCR and immunofluorescence. RESULTS：The neurons were primary cultured with the purity of over 90%. The injury model of the neuron was successfully induced by Glu. V-RFP-flag and V-ERα-RFP-flag (carrying red fluorescence) at MOI=7 were used to infect the neurons. After 72 h, the red fluorescence was observed significantly under a fluorescence microscope. Compared with the control lentivirus, V-ERα-RFP-flag significantly enhanced the mRNA and protein levels of ERα. Compared with control group, the apoptotic rates of the neurons in estrogen group and lentivirus group were decreased, and the mRNA expression of NMDAR1 and VGLUT1 in estrogen group and lentivirus group was reduced. The numbers of NMDAR1 and VGLUT1 positive cells were also decreased. CONCLUSION：Estrogen and ERα reduce Glu-induced damage in the neurons by inhibiting the expression of NMDAR1 and VGLUT1.     

中国大麦地方品种的遗传多样性及α-淀粉酶活性的 全基因组关联分析

【目的】了解中国大麦地方品种的遗传多样性,为大麦α-淀粉酶活性基因寻找有效的分子标记。【方法】利用覆盖全基因组的41对简单重复序列（SSR）标记引物,对257份中国大麦地方品种进行PCR扩增;采用Nei’s遗传距离和邻接（neighbour-joining）法进行聚类分析;在对群体结构和连锁不平衡分析的基础上,进行基于全基因组的表型与基因型的关联分析。【结果】共鉴定出709个等位变异,平均每个位点的等位变异数为17个。41个SSR标记位点的多态性信息指数（PI）变化范围为0.23（Bmag 0385）—0.94（Bmac0032）,平均为0.6385。257个中国大麦地方品种聚合成9个不同的结构类群,发现5个与大麦α-淀粉酶活性显著关联的标记位点。【结论】中国大麦地方品种中蕴藏着丰富的遗传等位变异;各类群的特性和品种来源符合“遗传关系密切、表型特征特性相同、地理生态相近”的同类群聚集规律。在5个关联位点中,位于7H染色体上的Bmag0385位点,其等位变异A215的酶活增强效应最大;此外,7H上Bmac0273的等位变异A141的增效作用较大,可用于啤酒大麦育种的分子标记辅助选择。     

Inhibition of TNF-α-mediated NF-κB activation by 4-hydroxynonenal contributes to liver injury in alcoholic liver disease

AIM: To study the role of 4-hydroxynonenal (4-HNE) in hepatocyte death induced by tumor necrosis factor α (TNF-α).METHODS: Human liver cell line HepG2 and primary mouse hepatocytes were used to establish the cell model. The effect of 4-HNE on TNF-α-induced cell death was determined by lactate dehydrogenase (LDH) release and MTT assays. The intracellular levels of 4-HNE-protein adducts were determined by Western blotting. The intranuclear NF-κB (p65) and its DNA binding activity were detected by Western blotting and ELISA, respectively. Long-term intake of alcohol in C57BL/6 mice was performed to establish the animal model. The histological changes of mouse hepatic tissues and the apoptosis of hepatocytes were observed by HE staining and TUNEL assay, respectively. The hepatic levels of triglyceride (TG), TNF-α and 4-HNE-protein adducts, and the plasma activity of alanine aminotransferase (ALT) were also detected.RESULTS: (1) 4-HNE significantly increased the sensitivity of HepG2 cells and primary mouse hepatocytes to the killing effect of TNF-α. (2) 4-HNE significantly increased the intracellular levels of 4-HNE-protein adducts. (3) 4-HNE inhibited TNF-α-mediated NF-κB (p65) activation in HepG2 cells. (4) Long-term intake of alcohol in mice resulted in high hepatic levels of 4-HNE and TNF-α, accompanied with the increases in hepatic TG content, plasma ALT activity and hepatocyte death.CONCLUSION: Long-term intake of alcohol induces oxidative stress and produces 4-HNE as a hepatocyte-sensitizing factor, which inhibits TNF-α-mediated NF-κB anti-apoptotic signaling pathway in hepatocytes, thus inducing alcoholic liver damage.