Digestive protease activities and free amino acids in white muscle as indicators for feed conversion efficiency and growth rate in Atlantic salmon (Salmo salar L.)

The aim of the present experiment was to screen several biochemical indices in fish and their interrelations in order to select variables for future studies of growth rate and feed conversion. Several parameters [trypsin activity, chymotrypsin activity, free amino acids (FAA) in plasma and white muscle, and RNA and RNA/protein ratio in the white muscle] were measured together with specific growth rate (SGR), feed intake and feed conversion efficiency (FCE) in four groups of diploid or triploid Atlantic salmon (Salmo salar L.) reared under different light regimes. SGR was measured on individually tagged fish, whereas feed intake and feed conversion was estimated on tank basis. A principal component analysis (PCA) explained 80.6% of the variance in the data, using all measured parameters, regardless of ploidy and light regime. Muscle free hydroxyproline showed the highest correlation, alone explaining 55% of SGR variability. The SGR also significantly correlated with trypsin activity (r=0.34), the activity ratio of trypsin to chymotrypsin (T/C) (r=0.39), plasma essential FAA (EAA) (r=0.39), plasma total FAA (TFAA) (r=0.37), the ratio of essential to non-essential FAA (EAA/NEAA) in the white muscle (r=–0.45), muscle RNA (r=–0.45) and RNA/protein ratio (r=–0.41). Tank FCE correlated positively (r=0.97) with SGR, T/C ratio and muscle free hydroxyproline, and negatively (r=–0.90) with muscle EAA/NEAA. The groups reared under continuous light (LL) regime showed significantly higher SGR than simulated natural photoperiod (SNP) groups, and with an apparently higher FCE. A higher growth rate was associated with either a higher consumption rate and/or a higher feed utilization. A negative correlation between muscle RNA concentration and SGR may indicate that increased growth rate under LL regime was not caused by an increased protein deposition rate.     

The effect of feeding three anabolic steroids in different combinations on the growth,food conversion efficiency and protein and nucleic acid levels of liver,kidney, brain and muscle of mirror carp (Cyprinus carpio)

A study on the effect of three different anabolic-androgenic steroids on the growth, food conversion efficiency and nucleic acid contents of liver, kidney, brain and muscle of carp,Cyprinus carpio was undertaken. The three steroids, methyltestosterone (MT), ethylestrenol (EE) and oxandrolone (ON), were fed in different combinations at final concentrations of 5 or 6 mg/kg diet for 60 days. No effect on the growth was observed in any of the experimental groups. A decrease in the specific growth rate (11–21%) and food conversion efficiency (20–29%) was noted. Feeding of drugs increased the cranio-somatic and reno-somatic index in all except one group. Hepatosomatic (ON+MT) and viscero-somatic (ON+MT; EE+MT+ON) indices decreased. Protein increased and RNA/DNA decreased in only one group while a decrease in protein/DNA was observed in the liver of all experimental groups. RNA/DNA increased and protein/RNA decreased only in one group while no effect was seen in protein and protein/DNA contents in any of the treated kidneys. Proteins, protein/RNA and protein/DNA decreased in certain groups in brain tissue. In muscle, no effect was seen in proteins or protein/DNA. Protein/RNA decreased in all but one group while RNA/DNA was higher only in the group fed all the three steroids together.     

A comparison of RNA concentrations and ornithine decarboxylase activity in Atlantic salmon (Salmo salar) muscle tissue,with respect to specific growth rates and diel variations

RNA concentrations and enzyme activities are often used as indices of recent growth in fish, but few studies have used both methods to assess the same fish. This study measured RNA concentrations and ornithine decarboxylase (ODC) activity in muscle tissue of juvenile Atlantic salmon (Salmo salar) to compare their usefulness for reflecting specific growth rates, and to determine whether either growth index was influenced by diel variations or time of feeding. Three groups (n = 54 in total) were fed 1.5% of body weight in commercial pellets in four feedings per day. One group was fed only in the morning (0830–1230h), one in the afternoon (1430–1830h), and one in the morning and afternoon (0830–1830h). At the end of ten days, fish were sampled at three times (0130h, 1030h, 1630h) over a single 24h period. Correlations to specific growth rate were slightly higher for RNA concentrations than for ODC activity, but both were highly significant. RNA and ODC activity were also correlated to each other. These results suggest that RNA concentration and ODC activity, taken together, can be used to monitor changes in both the numbers and activity of ribosomes. For RNA concentrations, there was no evidence of an effect of diel variations or the time of feeding. For ODC activity, a significant diel effect (all feed schedules combined) was detected if one non-growing fish was excluded from the analysis; activity of the enzyme was slightly higher in the sample taken at night (0130h) than in the two daytime samples. Contribution no. 8, Catamaran Brook Habitat Research Project     

基于RNA-Seq技术的鲮转录组分析

为满足标记辅助育种的要求,通过454测序平台首次开展了鲮Cirrhina molitorella全鱼转录组深度测序,并用 Newbler 等软件进行数据精细分析。结果表明：共获得了1297479条 reads,总碱基数为486586191 bp,组装后得到19962条contigs,平均长度为1269 bp, N50为1509 bp。基因功能注释研究共获取了10577个特异蛋白,根据特异蛋白注释结果进行GO分析,有7314条contigs有GO注释,包含5381个特异蛋白;采用GO 功能分类工具可将已注释转录物序列划分为分子功能、生物途径和细胞成分3类,为下一步开展生长等性状相关基因功能验证研究提供丰富的序列资源;共鉴定出5931个具有完整的ORF的全长cDNA序列,并且鉴定出2438个微卫星和5014个SNP位点。本研究中,还建立了鲮转录组数据库和网站,方便同行随时调取数据,这为深入开展鲮分子标记辅助的遗传育种、种群遗传学和资源评估等研究提供了丰富的标记资源。     

Long non-coding RNA and diabetes mellitus

Long non-coding RNAs (lncRNAs) are a group of RNAs, which are longer than 200 nucleotides without containing functional open reading frame and cannot encode protein. The study of lncRNA will help to understand the multi-level expression regulatory network of the body, and is expected to provide the basis of prediction, diagnosis and treatment of complex diseases. Although the functions and mechanism of lncRNA remain unclear, some studies indicate that lncRNA is involved in the development of diabetes mellitus, and those lncRNAs may be new diagnostic markers and therapeutic targets.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effects of RNA interference targeting CDC25agene on proliferation of human liver cancer HepG2 cells

AIM: To investigate the effect of silencing cell division cycle 25a (CDC25a) gene on the proliferation of human hepatoma HepG2 cells. METHODS: CDC25agene in human hepatoma HepG2 cells was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS: The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P＜0.05). The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P＜0.05). The cell proliferation in silence group was lower than that in negative control group and normal control group (P＜0.05). The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase. CONCLUSION: Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effectively inhibits the CDC25agene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25agene may be a key target for the treatment of liver cancer.     

Attenuated Salmonella carrying pGRIM-19-si-survivin co-expression plasmid inhibits growth of prostate cancer subcutaneous xenografts in vivo

AIM：To explore the effects of pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella on prostate cancer subcutaneous xenograft growth in nude mice. METHODS：Prostate cancer xenograft model was established in nude mice. Co-expression plasmids carried by attenuated Salmonella were introduced by intraperitoneal injection. The xenograft volumes were monitored timely. Immunohistochemical staining, RT-PCR and TUNEL assay were applied to investigate the related mechanisms that pGRIM-19-si-survivin inhibited tumor growth in vivo. RESULTS：Compared with psi-survivin and pGRIM-19 carried by attenuated Salmonella (control groups), the tumor volumes were reduced markedly in pGRIM-19-si-survivin plasmid group. The mean shrinkage rates were 2.36 and 3.02 times. pGRIM-19-si-survivin co-expression plasmid carried by attenuated Salmonella inhibited survivin expression but strengthened GRIM-19 expression obviously (P<0.05). The mRNA expression of apoptosis-related proteins such as Bcl-xL, Stat3, cyclin D1 and c-Myc was inhibited, and the vascular endothelial growth factor (VEGF) mRNA and Ki67 protein were also inhibited, but the caspase-3 mRNA expression was up-regulated (P<0.05) with significant cell apoptosis. CONCLUSION： pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella inhibits the growth of prostate cancer subcutaneous xenografts in nude mice by promoting cell apoptosis and inhibiting prostatic cancer proliferation.     

Effect of small interfering RNA on expression of β2M in pre-differentiated bone marrow mesenchymal stem cells

AIM：To study the effect of small interfering RNA (siRNA) on the expression of beta 2-microglo-bulin (β2M) in pre-differentiated bone marrow mesenchymal stem cells (BMSCs). METHODS：The β2M siRNA was transfected into the pre-differentiated BMSCs with Lipofectamine 2000. BMSCs were divided into transfection group, blank control group and negative control group. The expression of β2M at mRNA and protein levels was determined by real-time qPCR, Western blotting and laser confocal microscopy. The productions of aggrecan and type II collagen in pre-differentiated BMSCs were determined by toluidine blue staining and type Ⅱ collagen immunofluorescence. RESULTS：The results of real-time qPCR, Western blotting and laser confocal microscopy showed that siRNA successfully inhibited the expression of β2M at mRNA and protein levels in the pre-differentiated BMSCs. The results of toluidine blue and type Ⅱ collagen immunofluorescence staining showed that siRNA does not affect the productions of aggrecan and type Ⅱ collagen in the pre-differentiated BMSCs. CONCLUSION：siRNA targeting β2M reduces the expression of β2M in the pre-differentiated BMSCs and does not affect the chondrocyte characteristics of pre-differentiated BMSCs.     

Influence of high-mobility group box 1 on proliferation of neural stem cell in pre-infarction cortex of focal cerebral ischemia/reperfusion model rats

AIM：To investigate the influence of high-mobility group box 1 (HMGB1) on the proliferation of neural stem cells in peri-infarction cortex of focal cerebral ischemia/reperfusion model rats. METHODS：Male SD rats (n=48) were randomly divided into sham group, ischemia/reperfusion (I/R) group, RNA interference group and negative interference group. The rat middle cerebral artery was blocked to establish focal cerebral I/R model (ischemia for 1 h and reperfusion for 7 d). Lentivirus vector of HMGB1 shRNA was used to suppress the HMGB1 protein expression in the rat brain. The effect of RNA interference was evaluated by the methods of double-immunofluorescence labeling of HMGB1/GFAP and Western blotting. The proliferation of neural stem cells in the peri-infarction cortex was assessed by double labeling of BrdU/nestin. RESULTS：The protein expression of HMGB1 in I/R group was much higher than those in sham group (P<0.05). RNA interference effectively inhibited the HMGB1 expression (P<0.05). Double labeled BrdU/nestin positive cells in I/R group were more than that in sham group (P<0.05). The double labeled BrdU/nestin positive cells were significantly decreased in RNA interference group (P<0.05). CONCLUSION：Focal cerebral ischemia/reperfusion injury promotes the proliferation of neural stem cells in peri-infarction cortex by increasing HMGB1 protein level.