不同日粮添加组合蛋白酶对育肥猪生长性能、营养物质表观消化率和氮利用率的影响

试验旨在探讨不同日粮添加组合蛋白酶对育肥猪生长性能、营养物质表观消化率和氮利用率的影响。选取120头体重约60 kg"杜×长×大"三元猪,按照公母各半的原则随机分为3组,每组4个重复,每个重复10头猪。各组猪分别饲喂玉米-豆粕型、玉米-杂粕型和小麦-杂粕型日粮。每组2个重复设为对照,不添加组合蛋白酶;2个重复设为试验,添加0.1%的组合蛋白酶。试验期40 d。结果显示,与对照猪相比,饲喂添加组合蛋白酶不同日粮的试验猪的末重和平均日增重极显著增加(P<0.01);平均日采食量增加,料重比降低,但差异不显著(P>0.05)。添加组合蛋白酶日粮的试验猪对干物质、有机物、粗蛋白、粗脂肪和粗纤维的表观消化率极显著提高(P<0.01),氮摄入量显著提高(P<0.05),粪便和尿中氮排放量极显著降低(P<0.01),氮利用量和氮利用率极显著提高(P<0.01)。玉米-豆粕型日粮的试验猪平均日增重显著低于玉米-杂粕型和小麦-杂粕型日粮(P<0.05)。研究表明,饲喂添加组合蛋白酶的不同的日粮可以提高育肥猪的生长性能,提高饲料中的营养物质表观消化率,降低粪尿中氮的排放量。

组合蛋白酶对肉仔鸡采食偏好性、血清生化指标和肠道组织形态的影响

本试验旨在探讨组合蛋白酶对肉仔鸡采食偏好性、血清生化指标和肠道组织形态的影响。试验1采用自由选择试验法,选择1日龄体况相近、健康的爱拔益加(AA)肉仔鸡40只,随机分为4个处理,每个处理10只鸡。4个处理的肉鸡同时饲喂基础饲粮(饲粮Ⅰ)以及在基础饲粮中分别添加4(饲粮Ⅱ)、8(饲粮Ⅲ)和12 U/g(饲粮Ⅳ)组合蛋白酶的饲粮,测定肉仔鸡对不同饲粮的采食偏好性。试验2采用单因素完全随机试验设计,选择1日龄体况相近、健康的AA肉仔鸡240只,随机分为4个组,每组6个重复,每个重复10只鸡。对照组(Ⅰ组)饲喂基础饲粮,试验组(Ⅱ~Ⅳ组)分别饲喂在基础饲粮中添加4、8和12 U/g组合蛋白酶的饲粮,测定肉仔鸡血清生化指标和肠道组织形态。试验鸡自由采食和饮水,2个试验期均为42 d。结果表明:1)各处理肉仔鸡对4种饲粮的耗料量、采食次数、总采食时间和每顿采食时间之间均无显著差异(P>0.05);但22~42日龄,肉仔鸡对饲粮Ⅱ的采食百分比显著高于其他3种饲粮(P<0.05)。2)1~21日龄,Ⅳ组肉仔鸡血清总蛋白(TP)、球蛋白(GLB)、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇显著高于Ⅰ组(P<0.05);22~42日龄,Ⅲ组肉仔鸡血清TP和GLB含量显著高于Ⅰ组(P<0.05),Ⅲ组血清甲状腺素含量显著低于Ⅰ组(P<0.05)。3)Ⅱ组肉仔鸡空肠和十二指肠隐窝深度显著低于Ⅲ组(P<0.05),空肠绒毛高度/隐窝深度(V/C)值显著高于Ⅲ组和Ⅳ组(P<0.05),十二指肠V/C值显著高于Ⅰ组和Ⅲ组(P<0.05);但与Ⅰ组相比,Ⅱ组回肠绒毛高度显著降低(P<0.05)。综上所述,肉仔鸡仅在其生长后期(22~42日龄)对添加4 U/g组合蛋白酶的饲粮表现出采食偏好性;饲粮添加12 U/g组合蛋白酶改善了21日龄肉仔鸡机体脂类合成,增强了免疫力;饲粮添加8 U/g组合蛋白酶显著提高了42日龄肉仔鸡血清TP和GLB含量;饲粮添加4 U/g组合蛋白酶可以改善或维持肉仔鸡肠道组织形态。综合考量,本试验中组合蛋白酶的适宜添加量为8 U/g。

水解面筋蛋白高效制备谷氨酰胺结合肽的研究

采用食品级胃蛋白酶、胰蛋白酶、Protamex复合酶双酶组合对面筋蛋白进行水解制备Gln结合肽。先确定三种单酶各自对面筋蛋白适宜的初始pH、温度、酶浓度和底物浓度。并以双酶进行两两组合水解。在进行水解度控制实验时取样,分析水解液中氨基氮的含量,计算平均肽链长度,并用高效液相色谱分析水解液中Gln的得率。实验结果表明胰蛋白酶和胃蛋白酶联合水解的效果最好,条件为:胰蛋白酶在pH8.0,50℃,S%=12.8%(W/V),E%=9%(W/W)水解6h,再用胃蛋白酶pH2.0,40℃,E%=5%(W/W)条件下水解5h,可得到平均肽链长度为2.20个氨基酸残基的蛋白水解液,Gln得率最高,为60.43%。认为胰蛋白酶和胃蛋白酶组合是面筋蛋白中高效制备谷氨酰胺结合肽的最佳组合。

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner

AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.

Expression and purification of recombinant human hemangiopoietin in Escherichia coli

Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.

A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases

Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase(Pol B), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7 d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of Pol B and Pol D from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPf A1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both Pol B and Pol D were efficient in strand displacement. HPf A1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPf A1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

Effect of Lichong decoction on expression of Bcl-2 and Bcl-2-associated X protein mRNAs in hysteromyoma model rat

OBJECTIVE:To study on effects of Lichong decoction on expression of apoptosis-controlling genes,Bcl-2 and Bcl-2-associated X protein(Bax) mRNAs in hysteromyoma tissue of the hysteromyoma model rat.METHODS:Fifty Wistar female rats were randomly divided into a normal group,a model group,a Lichong decoction group,a Guizifuling capsule group and a Mifepristone group.The hysteromyoma rat model was established by intraperitoneal injection of exogenous estrin and progestogens.Pathological examination of uterine tissue,uterine coefficient and uterine transverse diameter were made under optic microscope and expressions of Bcl-2 and Bax mRNAs in uterine tissue in the groups were detected with real-time fluorescent quantitative polymerase chain reaction(PCR) technique.RESULTS:After treatment,under microscope it was found that in the Lichong decoction group myometrium thinned,muscle fiber slightly overgrowth or long and thin,regular arrangement,inserting phenomenon of inner circular muscle and external longitudinal muscle was occasionally or not seen in the Lichong decoction group.The uterine coefficient and the uterine transverse diameter significantly decreased(P<0.01),and Bcl-2 mRNA expression significantly decreased(P<0.01) and Bax mRNA expression significantly increased in hysteromyoma tissue(P<0.01) in the Lichong decoction group as compared with the model group.CONCLUSION:Therapeutic effects of Lichong decoction on hysteromyoma is related with decrease of Bcl-2 mRNA expression and increase of Bax mRNA expression.

Mitogenomic analysis of the genus Panthera

The complete sequences of the mitochondrial DNA genomes of Panthera tigris,Panthera pardus,and Panthera uncia were determined using the polymerase chain reaction method.The lengths of the complete mitochondrial DNA sequences of the three species were 16990,16964,and 16773 bp,respectively.Each of the three mitochondrial DNA genomes included 13 protein-coding genes,22 tRNA,two rRNA,one O L R,and one control region.The structures of the genomes were highly similar to those of Felis catus,Acinonyx jubatus,and Neofelis nebulosa.The phylogenies of the genus Panthera were inferred from two combined mitochondrial sequence data sets and the complete mitochondrial genome sequences,by MP (maximum parsimony),ML (maximum likelihood),and Bayesian analysis.The results showed that Panthera was composed of Panthera leo,P.uncia,P.pardus,Panthera onca,P.tigris,and N.nebulosa,which was included as the most basal member.The phylogeny within Panthera genus was N.nebulosa (P.tigris (P.onca (P.pardus,(P.leo,P.uncia)))).The divergence times for Panthera genus were estimated based on the ML branch lengths and four well-established calibration points.The results showed that at about 11.3 MYA,the Panthera genus separated from other felid species and then evolved into the several species of the genus.In detail,N.nebulosa was estimated to be founded about 8.66 MYA,P.tigris about 6.55 MYA,P.uncia about 4.63 MYA,and P.pardus about 4.35 MYA.All these estimated times were older than those estimated from the fossil records.The divergence event,evolutionary process,speciation,and distribution pattern of P.uncia,a species endemic to the central Asia with core habitats on the Qinghai-Tibetan Plateau and surrounding highlands,mostly correlated with the geological tectonic events and intensive climate shifts that happened at 8,3.6,2.5,and 1.7 MYA on the plateau during the late Cenozoic period.

Enzyme/pH-sensitive dendritic polymer-DOX conjugate for cancer treatment

It is in a great demand to design a biodegradable, tumor microenvironment-sensitive drug delivery system to achieve safe and highly efficacious treatment of cancer.Herein, a novel pH/enzyme sensitive dendritic pdi HPMADOX conjugate was designed. di HPMA dendritic copolymer with GFLG segments in the branches which are sensitive to the intracellular enzyme of the tumor was prepared through RAFT polymerization. DOX was attached to dendritic di HPMA polymer through a pH-sensitive hydrazone bond. The dendritic pdi HPMA-DOX conjugate self-assembled into nanoparticles with an ideal spherical shape at a mean size of 103 nm. The DOX attached to the polymeric carrier was released in an acidic environment, and the GFLG linker for synthesizing the dendritic vehicle with a high molecular weight（M_W, 220 kDa） was cleaved to release low MWsegments（〈40 kDa） in the presence of cathepsin B. The dendritic polymeric conjugate was internalized via an endocytic pathway, and then released the anticancer drug, which led to significant cytotoxicity for tumors. The blood circulation time was profoundly prolonged, resulting in high accumulation of DOX into tumors. In vivo anti-tumor experiments with 4 T1 tumor bearing mice demonstrated that the conjugate had a better antitumor efficacy in comparison with free DOX. Additionally, body weight measurements and histological examinations indicated that the conjugate showed low toxicities to normal tissues. This dendritic polymeric drug carrier in a response to intracellular enzyme and acidic pH of tumor tissue or cells holds great promise in tumor-targeted therapy.

Activating transcription factor 5 regulates lipid metabolism in adipocytes

Activating transcription factor 5(ATF5) is a member of the activating transcription factor/cA MP response element binding protein(ATF/CREB) family, and is highly expressed in liver and adipose tissue. Previous reports have shown that ATF5 promoted 3T3-L1 preadipocytes differentiation. In this study, we found that ATF5 was highly expressed in mature adipocytes, suggesting a potential role of ATF5 in mature adipocytes, which has not been reported previously. To understand the function of ATF5 in mature adipocytes, we knocked down the expression of ATF5 in 3T3-L1 mature adipocytes and observed decreased lipid droplets. Consistent with the in vitro experiment, the knockdown of ATF5 in white adipose tissue led to less adipose tissue and smaller adipocytes size. Further research revealed that the inhibition of ATF5 diminished the adipocytes size via the inhibition of fatty acid synthetase, stearyl coenzyme A desaturation enzyme 1, and the induction of carnitine palmitoyl transferase 1, one key enzyme of lipid metabolism. In addition, ATF5 knockdown in inguinal white adipose tissue improved whole body insulin sensitivity.Our work provides a new understanding of ATF5 function in mature adipocytes and a potential therapeutic target of diabetes.