Functional partition of cells in the gill epithelium of the Japanese eel, Anguilla japonica, and the role of hormones

Viable chloride and pavement cells were isolated from the gill epithelium of Japanese eel, Anguilla japonica, by a 3-step percoll gradient centrifugation at low speed. Viability of the isolated cells were tested by the trypan blue exclusion test, rhodamine-123, or pre-labelling the cells with fluo-3 Ca²⁺ dye and examined by laser confocal microscopy. Isolated chloride cells responded to ionomycin with a rapid increase in Ca²⁺ fluorescence, which was abolished by chelating external Ca²⁺ with EGTA. Peptide hormones, including arginine vasotocin, isotocin, insulin-like growth factor I and II, and urotensin I increased Ca²⁺ entry, urotensin II had no effect, and eel corpuscles of Stannius extract reduced residual Ca²⁺ fluorescence. Isolated chloride cells and pavement cells from eels were analyzed for their enzymatic activities involved in intermediary and nitrogen metabolism. Chloride cells had high levels of glutaminase I, glutamate dehydrogenase, glutamate oxalacetate transaminase, HCO₃-ATPase and carbamoyl phosphate synthetase II. Pavement cells had highly active glucose-6-phosphate dehydrogenase and AMP deaminase. Both had high levels of lactate dehydrogenase compared with other tissues.     

细胞松弛素B对水牛体细胞核移植效果的影响

以水牛耳皮成纤维细胞为供体细胞,采用电融合方法,探讨细胞松弛素B(CB)对水牛体细胞核移植效果的影响.体外成熟培养22～24 h的水牛卵母细胞去核后.将经0.1 mg/L Aphidicolin(APD)+0.5%FBS培养2～9 d的水牛耳皮成纤维细胞注射到卵周隙中再经电融合(100 V/mm,15μs,电脉冲3次)构建核移植重构胚.重构胚经化学激活后(5 μmol/L)离子霉素5 min,2 mmol/L 6-DMAP 3 h)培养,7～9 d评定其胚胎发育能力.结果显示,在含CB(3 mg/L)的融合液中进行电融合后,核移植的融合率、重组胚的存活率、卵裂率和囊胚率与对照组(不含CB)相比均无显著差异(P>0.05);核移植重组胚激活前用含CB(6 mg/L)的培养液培养1 h,其激活后的存活率(97.52%)和体外囊胚发育率(22.09%)均显著地高于未经CB处理的重组胚的存活率(93.87%)和囊胚率(13.25%,P<0.05);重组胚经离子霉素激活5 min后,在6-DMAP+CB中培养3 h的分裂率明显低于放在6-DMAP中培养3 h的分裂率(65.37% vs 78.92%,P<0.05),但囊胚发育率无显著差异(11.19% vs 10.96%,P>0.05).这表明水牛体细胞核移植电融合时,融合液中不添加CB,而核移植重组胚激活前经CB培养处理后,有利于胚胎的进一步发育,但激活后用CB培养处理会降低胚胎的发育率.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

Immunoproteomics analysis of whole cell bacterial proteins of Riemerella anatipestifer

Riemerella antipestifer is one of the most important duck pathogens. It has worldwide distribution, and the lack of the information on bacteria-host interactions and an effective vaccine are limitations on the control of this infection. In this study, an immunoproteomic assay was used to identify immunogenic proteins among the whole cell bacterial proteins of R. anatipestifer virulent strain Th4. Duck antiserum against R. anatipestifer Th4 recognized 64 protein spots which were transferred from two-dimensional electrophoresis (2-DE) gel of the whole cell bacterial proteins onto polyvinylidene fluoride (PVDF) membrane. Immunogenic proteins on a duplicate gel were excised and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprinting (PMF), a total of 34 immunogenic proteins were found. With the exception of OmpA and GroEL, the other 32 proteins were newly recognized immunogenic antigens of R. anatipestifer. In addition, TonB-dependent outer membrane receptor was found to be a cross immunogenic antigen among serotypes 1, 2 and 10 of R. anatipestifer. Bioinformatics analysis showed that most of the immunogenic proteins were located in the outer membrane and cytoplasm, and were involved in cellular processes and metabolism. The newly identified immunogenic proteins of R. anatipestifer may help us to uncover the pathogenesis of the bacteria, develop novel vaccine candidates and serological diagnosis marker.