一氧化氮合成酶抑制剂对宰后成熟过程中牦牛肉品质的影响

【目的】研究一氧化氮对宰后牦牛肉品质的影响,为牦牛肉品质改善提供理论依据。【方法】以3—5岁去势甘南牦牛肉为材料,取其背最长肌,剔除筋膜、脂肪等,切成大小均匀的薄片（8 cm×8 cm）,用20 G针均匀穿刺,分别采用去离子水（对照组）和1、10、100 mmol·L ^-1一氧化氮合成酶（NOS）抑制剂（N-硝基-L-精氨酸甲酯盐酸盐）在4℃条件下1﹕1（V/m）浸泡肉样1 d,然后在4℃条件下成熟,测定成熟期间（0、1、3、5和7 d）NOS活性与一氧化氮含量、肌原纤维小片化指数（MFI）、总巯基含量、羰基含量、pH、色度等指标。【结果】NOS抑制剂处理后的牦牛背最长肌NOS活性和一氧化氮含量显著降低,成熟第7天时,处理组的NOS活性分别比对照组低30.9%、43.6%、74.7%,一氧化氮含量分别比对照组低4.7%、12.5%、21.5%。处理后的牦牛肉pH显著降低,第7天时,处理组分别比对照组低0.8%、5.7%、15.2%,其中10和100 mmol·L ^-1 NOS抑制剂处理组显著低于对照组。处理显著降低了牦牛肉羰基含量,成熟第7天时,处理组分别比对照组低4.1%、19.0%、22.2%。此外,处理组MFI显著上升,成熟第7天时,处理组分别比对照组低31.1%、23.3%、9.6%。处理组牦牛肉总巯基含量显著上升,第7天时,对照组比NOS抑制剂处理组分别低3.2%、3.7%、2.7%。成熟过程中,NOS抑制剂处理使肉色a ^*值显著降低,肉色L ^*值显著升高,第7天时,处理组的肉色a ^*值分别比对照组低7.1%、40.2%、30.7%,肉色L ^*值分别比对照组高1.1%、2.0%、1.1%。【结论】一氧化氮促进宰后牦牛肉蛋白质氧化,抑制牦牛肉嫩化,使肉色L ^*值降低,a ^*值升高,对宰后牦牛肉的品质产生负面影响,而NOS抑制剂能降低肌肉中NOS活性。     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

紫斑牡丹种子浸提液对植物种子萌发和幼苗生长的影响

以紫斑牡丹种子为研究对象,研究不同质量浓度(0、0.025、0.050、0.075、0.100 g·mL-1)的种皮和胚乳水浸提液对白菜、小麦和绿豆种子萌发、幼苗生长和幼苗的抗氧化酶活性的影响,探究紫斑牡丹种子内源抑制物质的活性,为进一步研究紫斑牡丹种子休眠机理提供理论依据。结果表明:紫斑牡丹种皮和胚乳中均含有抑制受体植物种子萌发和幼苗生长的物质,且同一质量浓度下,胚乳浸提液的抑制作用强于种皮浸提液;种皮和胚乳浸提液对白菜幼苗生长的抑制作用高于小麦和绿豆;种皮和胚乳浸提液对3种受体植物幼苗根长的抑制作用大于苗高和鲜质量;种皮和胚乳浸提液对3种受体植物幼苗的SOD、CAT和POD活性均产生了一定的影响。其中,随着种皮浸提液质量浓度的提高,受体植物幼苗SOD、POD活性先上升后下降,而CAT活性持续下降;加入胚乳浸提液后,受体植物幼苗的CAT、POD活性与对照相比显著下降,SOD活性则依然随着胚乳浸提液质量浓度的升高而表现出先上升后下降的趋势。由结果可知,紫斑牡丹种皮和胚乳浸提液中均含有能抑制受体植物种子萌发和幼苗生长的物质,并且能够影响受体幼苗的抗氧化酶活性,但是紫斑牡丹种皮和胚乳内源抑制物质的活性存在一定的差异。     

Effects of Manipulation of Proteases on Myofibril Protein Degradation of Tilapia Muscle in vitro and in vivo

This study investigated the role of three proteases in myofibril degradation. Tilapia muscle was soaked in MDL-28170, E-64, and AC-DEVD-CHO, specific inhibitors of calpain, cathepsin, and caspase, respectively, for 14 days to determine the in vivo degradation of myofibril. Desmin and troponin T were significantly inhibited after treatment with inhibitors compared to the control (p < .05). For in vitro analyses, myofibril was incubated with recombinant active μ-calpain, cathepsin B, and caspase-3. Desmin was significantly reduced when incubated with recombinant proteases (p < .05). However, troponin T showed significant degradation only in the μ-calpain and cathepsin B treatment groups (p < .05). Our results reveal μ-calpain and cathespin B have similar degradation patterns, while caspase-3 has partly similar degradation for myofibril in vivo and in vitro. This suggests that μ-calpain and cathespin B are the main proteases responsible for post-mortem myofibril degradation, while caspase-3 is minor.     

Screening of Human CYP1A2 and CYP3A4 Inhibitors from Seaweed In Silico and In Vitro

Phenolic compounds and carotenoids are potential inhibitors of cytochrome P450s. Sixteen known compounds, phenolic compounds and carotenoids from seaweed were examined for potential inhibitory capacity against CYP1A2 and CYP3A4 in silico and in vitro. Morin, quercetin, and fucoxanthin inhibited the enzyme activity of CYP1A2 and CYP3A4 in a dose-dependent manner. The IC₅₀ values of morin, quercetin, and fucoxanthin were 41.8, 22.5, and 30.3 μM for CYP1A2 and 86.6, 16.1, and 24.4 μM for CYP3A4, respectively. Siphonaxanthin and hesperidin did not show any significant effect on CYP1A2, but they slightly inhibited CYP3A4 activity at high concentrations. In silico modeling of CYP’s binding site revealed that the potential inhibitors bound in the cavity located above the distal surface of the heme prosthetic group through the 2a or 2f channel of CYPs. This study presents an approach for quickly predicting CYP inhibitory activity and shows the potential interactions of compounds and CYPs through in silico modeling.     

泛素激活酶抑制剂对猪精子获能状态、膜重构及体外受精的影响

【目的】评估泛素激活酶（E1）对猪精子获能、膜重构和体外受精的影响。【方法】选取5头长白猪采集精液，以硫醇酯法评估泛素激活酶（ubiquitin activating enzyme，E1）抑制剂（TAK-243）对精子中泛素（ubiquitin，Ub）与E1结合的影响；将精子分为鲜精组（Fresh）、DMSO组（DMSO）、获能组（Capacitated）及2.4、4.8和7.2 nmol/L TAK-243组，鲜精组用2 mL PBS重悬精子沉淀；获能组用2 mL获能液重悬精子沉淀；DMSO组及2.4、4.8和7.2 nmol/L TAK-243组分别用2 mL含0.07% DMSO及2.4、4.8和7.2 nmol/L TAK-243的获能液重悬精子沉淀，使用微生物动（静）态图像检测系统检测精子的动力学参数；以Western blotting法检测精子的酪氨酸磷酸化水平；Zn²⁺荧光探针检测精子的Zn²⁺含量；花生凝集素染色检测精子的顶体膜重构；免疫荧光法检测顶体表面精子黏附蛋白（AQN-1、AWN）的表达；体外受精法检测TAK-243对精卵结合和卵裂率的影响。【结果】硫醇酯分析结果表明，TAK-243处理组精子未形成Ub-E1复合物，说明TAK-243抑制了E1对Ub的激活作用。与鲜精组相比，获能组精子的曲线速度（VCL）和平均鞭打频率显著升高（P<0.05）。3个浓度TAK-243组与获能组精子的各项动力学参数差异均不显著（P>0.05）。与获能组相比，3个浓度TAK-243组酪氨酸磷酸化水平均显著降低（P<0.05），且随着TAK-243浓度的增加，精子蛋白的酪氨酸磷酸化水平逐渐降低。后续试验使用7.2 nmol/L TAK-243进行精子中Zn²⁺水平和顶体膜重构的研究，其结果表明，与获能组相比，7.2 nmol/L TAK-243组Zn²⁺水平、顶体膜完整性及AQN-1和AWN蛋白的表达水平均显著增加（P<0.05），精子与卵母细胞的黏附率和卵裂率均显著降低（P<0.05）。【结论】7.2 nmol/L TAK-243显著降低了精子获能期间蛋白酪氨酸磷酸化水平、精卵结合率和卵裂率，显著增加了Zn²⁺含量、顶体膜完整率及顶体表面蛋白AWN和AQN-1的表达，说明泛素-蛋白酶体信号通路参与了精子的体外获能。     

中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析

Kazal型丝氨酸蛋白酶抑制剂可以通过精确调控丝氨酸蛋白酶的活力,在生物体的防御应答等众多生物过程中发挥重要作用。以前期克隆的中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal, GenBank注册号为DQ318856)为基础,对其功能结构域进行序列比对和进化分析;组织表达分析结果表明,该基因在血细胞、鳃和淋巴器官等组织中高水平表达,而在眼柄、神经和肌肉中无表达;利用原核表达系统对该基因成熟肽区域成功进行了重组表达,纯化后的目的蛋白最终得率为0.4 g/L培养液;活性分析结果显示,复性后的rFc-Kazal对鳗弧菌、金黄色葡萄球菌、杀鲑气单胞菌、苏云金芽孢杆菌有明显的抑菌作用。     

Alteration of glutathione S-transferase properties during the development of <Emphasis Type="Italic">Micromelalopha troglodyta</Emphasis> larvae (Lepidoptera: Notodontidae)

Micromelalopha troglodyta (Graeser) is an important pest of poplar in China. Glutathione S-transferases (GSTs) are known to be responsible for adaptation mechanisms of M. troglodyta. The activities and kinetic constants of glutathione S-transferases in M. troglodyta were studied. Significant differences in glutathione S-transferase activity and kinetic characteristics were observed among five instars of M. troglodyta larvae. Furthermore, the inhibition of glutathione S-transferase activity in five instars by 24 inhibitors was conducted. The results show the inhibition of GST activity of different instars by 24 inhibitors was different. For GST activity in the 1st instar, chlorpyrifos, lambda-cyhalothrin, endosulfan, abamectin, fipronil and pyridaben were the best inhibitors tested, and for GST activity in the 2nd instar, tannic acid and quercetin were the most potent inhibitors tested, and for GST activity in the 3rd instar, the inhibitory effects of quercetin, chlorpyrifos and lambda-cyhalothrin were the highest, and for GST activity in the 4th instar, quercetin and lambda-cyhalothrin were the best inhibitors, and the inhibitory effect of phoxim was the highest for GST activity in the 5th instar. Our results show that glutathione S-transferases in different instars are qualitatively different in isozyme composition and thus different in sensitivity to inhibitors.