Effect of cadmium on the cytoskeleton and morphology of gill chloride cells in parr and smolt Atlantic salmon (Salmo salar)

This study documented the effect of cadmium on salmon parr and smolt gill morphology. Cadmium-induced changes in chloride cell (CC) cytoskeletal elements were investigated, as well as the modifications of CC surface area and density. In cadmium-treated parr (10 µg Cd l^-1 for 2 days), immunofluorescent light microscopy revealed the appearance of an intense actin staining located in the CC apical part. Transmission electron microscopic observations revealed a change in the organization of the microfilaments at the CC apex, with the appearance of numerous aggregates of filamentous actin. Higher cadmium concentrations (30 and 50 µg l^-1) and prolonged treatment times (7 to 14 days) did not modify such reorganisation. Microtubules were not significantly affected by similar treatments. Further, scanning electron microscopic analysis revealed that cadmium induces a significant increase of parr CC surface area as early as the second day of exposure. After 2 days, mature CC density had also increased. In smolt, a rise in CC surface area was observed, although CC density did not significantly increase.     

Expression of pancreatic enzyme genes during the early larval stage of Japanese eel Anguilla japonica

ABSTRACT: To reveal the ontogeny of pancreatic exocrine function in the early larval stage of eel, cDNAs encoding major pancreatic enzymes, trypsinogen, amylase and lipase were identified from the Japanese eel Anguilla japonica and their expression pattern in larvae was analyzed. The cloned eel trypsinogen precursor consisted of 224 amino acids and showed 82.2% identity to trypsinogen-2 of winter flounder Pleuronectes americanus . The eel amylase precursor consisted of 512 amino acids and showed 77% identity to winter flounder amylase. Eel pancreatic lipase was composed of 470 amino acids and had 58.3% of identity to human pancreatic lipase. In the eel larvae, mRNA expression of trypsinogen and amylase was first detected at 6 days post-hatching (d.p.h.), and the expression level increased between 7 and 8 d.p.h. In contrast, mRNA expression of lipase was first detected at 8 d.p.h. Eel larvae start to feed actively at 8 d.p.h. Thus, it was indicated that eel pancreas starts to synthesize digestive enzymes at 6 d.p.h. and acquires full function by the onset of exogenous feeding at 8 d.p.h.     

Protective Effects of Marine Alkaloid Neolamellarin A Derivatives against Glutamate Induced PC12 Cell Apoptosis

Marine alkaloids obtained from sponges possess a variety of biological activities and potential medicinal value. The pyrrole-derived lamellarin-like alkaloids, especially their permethyl derivatives, show low cytotoxicity and potent MDR reversing activity. Neolamellarin A is a novel lamellarin-like alkaloid which was extracted from marine animal sponges. We reported the synthetic method of permethylated Neolamellarin A and its derivatives by a convergent strategy in 2015. In 2018, we reported the synthesis and the neuroprotective activity in PC12 cells of 3,4-bisaryl-N-alkylated permethylated Neolamellarin A derivatives. In this report, another series of 15 different 3,4-bisaryl-N-acylated permethylated Neolamellarin A derivatives were synthesized, and the outstanding protective effects of these compounds against glutamate induced PC12 cell apoptosis were presented and discussed. These Neolamellarin A derivatives which possessed low cytotoxicity and superior neuroprotective activity may have the potential to be developed into antagonists against glutamate induced nerve cell apoptosis.     

低温等离子活化水对猕猴桃溃疡病菌抗菌活性及机制

为了探究低温等离子体活化水 (cold plasma activated water, PAW) 对丁香假单胞杆菌猕猴桃致病变种 (Pseudomonas syringae pv. actinidiae, PSA) 的抗菌活性和潜在机制。该研究通过介质阻挡放电(dielectric barrier discharge, DBD) 低温等离子体发生装置制备不同激活时间的PAW,考察放电时间与杀菌效果之间的关系。此外,通过评估PSA形态特征,细胞粒径、DNA、细胞膜损伤情况和胞内活性氧积累 (reactive oxygen species,ROS) 情况探究PAW对PSA的杀菌机制。结果表明：PAW对PSA的杀灭效果与PAW激活时间呈依赖性,与对照相比PAW处理120 s后,PSA显著 (P<0.05) 减少了4.38 lg (CFU/mL)。扫描电子显微镜 (field emission scanning electron microscope, FESEM) 结果清楚地表明,由于PAW处理,PSA细胞发生了明显的质壁分离。荧光染色结果显示,PSA细胞DNA、膜渗透屏障被破坏程度、内容物泄漏量和ROS积累量与PAW激活时间表现为正相关关系。因此,推测PAW由于自身酸性、较高的氧化还原电位 (oxidation-reduction potential, ORP),以及水性活性物质导致细胞的氧化损伤,而且这可能是杀灭PSA的主要原因。研究结果可为PAW控制猕猴桃细菌性溃疡病提供参考。     

鸭坦布苏病毒NS1蛋白单克隆抗体的研制及亚细胞定位

以鸭坦布苏病毒(duck Tembusu virus, DTMUV)为免疫原,以DTMUV NS1原核表达蛋白为筛选抗原,获得2株针对DTMUV NS1蛋白的单克隆抗体,并进行NS1蛋白的亚细胞定位。首先,采用DTMUV脑内接种方式免疫BALB/c小鼠3～4次,取BALB/c小鼠脾细胞与SP2/0细胞融合,通过间接ELISA法筛选NS1蛋白的单克隆抗体,并通过Western blot进行鉴定。其次,DTMUV感染BHK-21细胞24 h,加入获得的NS1蛋白单抗,结合TMUV感染细胞过程中产生的NS1蛋白,加入FITC标记山羊抗小鼠IgG反应,DAPI核染色,通过激光共聚焦显微镜观察绿色荧光的位置,确定NS1蛋白的亚细胞定位。结果显示,间接ELISA法筛选到2株针对NS1蛋白的杂交瘤细胞株,细胞上清和小鼠腹水抗体滴度高,敏感性强。Western blot鉴定2株杂交瘤分泌的单克隆抗体特异性强。激光共聚焦显微镜观察显示,代表NS1蛋白的绿色荧光主要位于BHK21的细胞质中,这与软件预测结果一致。结果表明,TMUV NS1蛋白亚细胞定位的明确,为进一步理解和揭示该蛋白的结构与功能,蛋白质之...     

湖羊PGR基因对卵泡颗粒细胞体外增殖与凋亡的影响

旨在探究孕酮受体(progesterone receptor, PGR)基因对湖羊卵泡颗粒细胞体外增殖与凋亡的影响。利用RT-PCR技术扩增和克隆获得PGR基因编码序列(CDS),通过生物信息学软件对其氨基酸序列及同源性进行比对;用所获得序列构建过表达载体和干扰siRNA,分别转染湖羊颗粒细胞,并用CCK8技术检测颗粒细胞的细胞活力;采用RT-qPCR和Western blot技术,检测细胞周期和凋亡的相关基因或蛋白表达水平。结果显示,羊PGR基因的CDS区全长2 736 bp,编码911个氨基酸;与其他物种氨基酸序列的同源性为39.66%～95.44%。干扰PGR基因通过下调CDK4、Bcl-2和上调Caspas3、Caspase8、BAX基因mRNA的表达(P<0.05),进而抑制颗粒细胞的增殖,但对CyclinD1未产生影响(P>0.05);过表达PGR基因通过上调CDK4、CyclinD1、Bcl-2和下调Caspase3、Caspase8、BAX基因mRNA的表达(P<0.05),进而促进颗粒细胞的增殖;BAX蛋白表达变化与对应mRNA的表达趋势一致(P&l...     

Implication of Echinochrome A in the Plasticity and Damage of Intestinal Epithelium

The diverse therapeutic feasibility of the sea urchin-derived naphthoquinone pigment, Echinochrome A (Ech A), has been studied. Simple and noninvasive administration routes should be explored, to obtain the feasibility. Although the therapeutic potential has been proven through several preclinical studies, the biosafety of orally administered Ech A and its direct influence on intestinal cells have not been evaluated. To estimate the bioavailability of Ech A as an oral administration drug, small intestinal and colonic epithelial organoids were developed from mice and humans. The morphology and cellular composition of intestinal organoids were evaluated after Ech A treatment. Ech A treatment significantly increased the expression of LGR5 (~2.38-fold change, p = 0.009) and MUC2 (~1.85-fold change, p = 0.08). Notably, in the presence of oxidative stress, Ech A attenuated oxidative stress up to 1.8-fold (p = 0.04), with a restored gene expression of LGR5 (~4.11-fold change, p = 0.0004), as well as an increased expression of Ly6a (~3.51-fold change, p = 0.005) and CLU (~2.5-fold change, p = 0.01), markers of revival stem cells. In conclusion, Ech A is harmless to intestinal tissues; rather, it promotes the maintenance and regeneration of the intestinal epithelium, suggesting possible beneficial effects on the intestine when used as an oral medication.     

Effects of IL-6 and IL-11 on differentiation of cord blood CD34+ cells towards megakaryocytes

AIM: To investigate the effects of interleukin-6 (IL-6) and interleukin-11 (IL-11) on differentiation of cord blood CD34+ cells towards megakaryocytes and platelet production in vitro.METHODS: The CD34+ cells from fresh umbilical cord blood samples were cultured in serum-free culture medium with thrombopoietin (TPO) 50 μg/L,IL-3 10 μg/L,stem cell factor (SCF) 50 μg/L as control groups,then 10 μg/L IL-6 or IL-11 or IL-6＋IL-11 respectively was added as treatment groups.Mononuclear cells (MNCs) in cultured cells were detected by cell counter,megakaryocytes (CD41+ cells) and platelets were measured by flow cytometry,respectively.Platelet agglutination after thrombin induced was observed by microscopy and flow cytometry.RESULTS: Compared with the control group,the number of MNCs was not significantly different（P>0.05）,but the numbers of CD41+ cells and platelets were increased significantly (P<0.05) in treatment groups.There were more platelet particles in treatment groups than those in control group by microscopy and the results also showed that the cytoplasmic fragments from the cultures responded to thrombin induction.CONCLUSION: It is concluded that both IL-6 and IL-11 induce the cord blood CD34+ cells to differentiate towards megakaryocytes and produce platelets.     

Biological characteristics of uNK and pNK cells in pregnancy

AIM: To study the biological characteristics of uterine natural killer cells (uNK cells) and peripheral natural killer cells (pNK cells) in early gestation and their difference on physiological functions. METHODS: MTT assay was used to detect cytotoxicity and proliferation of the uNK and pNK cells. RT-PCR technique was applied to examine the altered gene expression of the chemotaxis, angiopoiesis and other biological activity in uNK and pNK cells. RESULTS: Compared with pNK group, uNK cells showed a higher cytotoxicity against K562 and a lower proliferation activity. The expression of some chemokine receptor genes and angiogenic growth factor genes in uNK cells and pNK cells were detected, such as CCR1, CCR5, CCR7, CXCR2, CXCR4 and CX3CR1 expressed by uNK cell and high contents of CXCR4 and CX3CR1 mRNA were found. The ligand genes of the chemokine receptor were expressed by decidual tissue or trophoblast tissue in early pregnancy. PIGF and AngⅡ mRNA were only found in uNK cells. CONCLUSION: Compared with pNK cells, uNK cells have peculiarities of the behaviour that might contribute to uterine unique microenvironment during pregnancy.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.