利用酵母双杂交系统筛选与草莓镶脉病毒移动蛋白P1互作的寄主因子

 构建感染草莓镶脉病毒(SVBV)森林草莓的酵母cDNA文库,利用酵母双杂交系统,筛选出与SVBV P1蛋白互作的15种寄主因子。生物信息学分析发现,这15种寄主因子参与茉莉酸途径、泛素化、光合作用、抗病抗逆、蛋白修饰、蛋白运输和氧化还原等多种生物过程。另外,这些寄主因子还具有其他分子功能,包括氧化还原酶活性、蛋白二硫化物异构酶活性和金属离子结合活性等。本研究初步探讨了P1与寄主因子的互作机理,为揭示SVBV侵染森林草莓以及SVBV在寄主中扩展的分子机制提供理论依据。     

MicroRNA-145 inhibits epithelial-mesenchymal transition in human renal cancer A-498 cells by ADAM28

AIM: To investigate the inhibitory effect of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) in renal cancer A-498 cells. METHODS: The A-498 cells were transfected with miR-145 mimics (M145) and mimic negative control(MNC), which served as M145 group and MNC group, respectively. Mock control (MC) group was set up using untreated A-498 cells. The expression level of miR-145 in each group was detected by RT-qPCR. Transwell assay was used to detect the invasion ability of the cells. The protein expression of vimentin, E-cadherin and ADAM28 was determined by Western blot. Bioinformatic method was used to predict the target genes of miR-145. Antagonistic effect of ADAM28 over-expression on the inhibition of EMT by miR-145 was detected by Western blot. The relationship between miR-145 and ADAM28 was analyzed by dual-luciferase reporter assay. RESULTS: The expression level of miR-145 in M145 group was significantly up-regulated than that in MC group (P<0.05). The number of invasive cells in M145 group was 12.78±3.37, which was significantly lower than that in MC group (P<0.05). ADAM28 may be the target gene of miR-145. Compared with MC group, the protein expression of vimentin and ADAM28 in M145 group was significantly decreased (P<0.05), while the protein expression of E-cadherin was significantly increased (P<0.05).After ADAM28 over-expression, the protein expression of vimentin in the A-498 cells of M145 group was significantly increased (P<0.05), and the protein expression of E-cadherin was significantly decreased (P<0.05). The results of dual-lucife-irasei reporter assay showed that ADAM28 was a downstream target gene of miR-145. CONCLUSION: miR-145 may inhibit the expression of EMT-related proteins through the downstream target gene ADAM28 and inhibit the EMT process of renal cancer A-498 cells.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

Partial fishmeal replacement by mussel meal or meat and bone meal in low‐fishmeal diets for juvenile Ussuri catfish (Pseudobagrus ussuriensis): Growth,digestibility, antioxidant capacity and IGF‐I gene expression

This study evaluated the effects of replacing fishmeal with mussel meal or meat and bone meal in low‐FM diet on growth performance, body composition, digestibility, antioxidant capacity and IGF‐I gene expression in juvenile Ussuri catfish (Pseudobagrus ussuriensis). The results showed that no significant changes in SGR, FE and PER were observed between MM₄₀ and LFM groups, but significantly reduced result was found in MBM₄₀ group. MM₄₀ group showed the higher ADC of lipid (93.30%) and lipase activity (95.00 U/gprot) than LFM group (90.97%; 70.18 U/gprot). MM₄₀ or MBM₄₀ diets led to significant reduction of SOD and CAT activities. MM₄₀ group showed significantly higher MDA level (5.84 nmol/mg) than LFM group (4.73 nmol/mg). The activities of LZM decreased significantly in MM₄₀ and MBM₄₀ groups compared with LFM group. MM₄₀ and MBM₄₀ groups showed no significant difference in hepatic IGF‐I gene expression levels compared with LFM group. The findings demonstrated that MM could substitute 400 g/kg of FM in low‐FM diet for Ussuri catfish without influencing the growth, but to some extent, spontaneous oxidative stress and immune damage could occur; when 400 g/kg of FM was replaced by MBM, significantly negative effects were observed on growth, antioxidant capacity and non‐specific immune response of Ussuri catfish.     

Karanj protein isolate prepared from karanj seed cake: Effect on growth,body composition and physiometabolic responses in Labeo rohita fingerlings

A protein isolate was prepared from karanj seed (KPI) with 921.2 g protein/kg seed, which contained a negligible amount of anti‐nutritional factors and a balanced amino acid composition, especially rich in methionine. For 60‐day feeding trial, five isonitrogenous (300 g/kg CP) and isocaloric (15 MJ DE/kg) diets were formulated by replacing soybean protein isolate (SPI) on protein equivalent basis, KPI‐0 (control, 0 g/kg KPI); KPI‐25 (replacing 250 g/kg SPI protein with KPI); KPI‐50 (replacing 500 g/kg SPI protein with KPI); KPI‐75 (replacing 750 g/kg SPI protein with KPI) and KPI‐100 (replacing 1,000 g/kg SPI protein with KPI) for the feeding of L. rohita. The weight gain percentage, specific growth rate, feed conversion ratio and protein efficiency ratio were not significantly (p > .05) varied among the KPI fed and control groups. A significantly higher hepatosomatic index was recorded in the control and KPI‐25 groups compared with other groups. The whole‐body compositions, except ether extract, did not differ significantly (p > .05) among the groups. Digestive (amylase, protease, lipase and alkaline phosphatase) and metabolic enzyme activities (hexokinase, transaminases and lactate dehydrogenase) and glycogen stores were not significantly affected, whereas intestinal alkaline phosphatase differed significantly (p < .05). The RNA–DNA ratio was significantly (p < .05) higher in the KPI‐75 group. Thus, the study revealed that KPI can completely replace SPI protein at 191 g/kg inclusion level in the diets of L. rohita fingerlings.     

Effects of dietary protein on growth performance,nutrient utilization,digestive enzymes and physiological status of grey mullet,Mugil cephalus L. fingerlings reared in inland saline water

A 60 days feeding trial was conducted to illustrate the effect of graded levels of protein on the growth and metabolic enzymes of grey mullet (Mugil cephalus L.) fingerlings reared in inland saline water (ISW). Six isoenergetic (16 MJ/kg) and isolipidic (60 g/kg) diets containing 240, 260, 280, 300, 320 and 340 g crude protein (CP)/kg diet were formulated and fed to triplicate. Weight gain %, specific growth rate, protein utilizing efficiency, feed efficiency and RNA:DNA ratio were significantly higher (p < .05) in 320 and 300 g CP/kg diets. Fish fed with 240 g CP/kg diet showed significantly higher (p < .05) feed intake, whole‐body lipid content, hepatosomatic index value and liver glycogen content. Transaminase enzymes and malate dehydrogenase activities were elevated in fish fed 340 g CP/kg diet. Protease activity increased with increasing dietary CP level, but amylase activities showed an inverse relationship. No significant (p > .05) variations were observed for lactate dehydrogenase, oxidative stress enzymes, blood parameters and serum osmolality among all the treatment groups, but red blood cell count increases with increasing dietary CP levels. Based on the results, feeding dietary protein level of 300 g CP/kg is economically viable for rearing of grey mullet in ISW.     

Nutritional evaluation of two marine microalgae as feedstock for aquafeed

Using microalgae for animal nutrition provides an economically viable route for microalgae‐based technological innovation, especially in combination with CO₂ fixation given current global warming. However, this technology still lacks sufficient evaluation for screening microalgae for specific animals; meanwhile, current studies show some prejudice regarding ‘essential’ or ‘non‐essential’ ingredients. The results show that Dunaliella salina and Nannochloropsis salina were able to accumulate high protein (30%–57%) and lipid (20%–46%) content without affecting the performance of CO₂ fixation, which reached 0.28 and 0.23 g L^?1 day^?1 respectively. Both species exhibited high quality of lipids and proteins for Penaeus monodon based on the profiling. The essential fatty acid indexes (EFAI) for N. salina and D. salina were 3.81 and 9.02 respectively. Butyric acid was found to be present in both D. salina (12.03%) and N. salina (4.87%) based on the total fatty acids (FAs). The essential amino acid indexes (EAAI) for D. salina and N. salina were 2.23 and 1.29 respectively. Arginine was the most abundant essential amino acid (EAA) in both D. salina (10.83%) and N. salina (13.35%) on the basis of total amino acids (AAs). This study comprehensively compares the nutritional quality of the two commercial marine microalgae of D. salina and N. salina with the potential to be used as sustainable sources of lipids and proteins to reduce or even replace the traditional fish oil and fish meal in aquafeeds.