高糖高脂膳食诱导大鼠肝脏ChREBPmRNA表达

目的：观察高糖高脂膳食对大鼠肝组织碳水化合物反应元件结合蛋白（ChREBP）表达的影响。方法：16只雄性SD大鼠随机分为2组,对照组给予普通饲料,高糖高脂组给予高糖高脂饲料。6周后,检测血脂和肝组织中ChREBP表达水平。结果：高糖高脂膳食组ChREBPmRNA表达水平、血总甘油三酯、总胆固醇和高密度脂蛋白浓度均显著高于对照组（P〈0．05）;血低密度脂蛋白明显低于对照组（P〈0．05）。结论：高搪高脂膳食能引起肝脏组织中碳水化合物反应元件结合蛋白表达增加。     

Hepatic Gluconeogenesis and the Activity of PDH in Individual Tissues of GTG-Obese Mice Following Adrenalectomy

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 ± 0.7g; GTG: 44.1 ± 0.4g; p<0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 ± 1.6 mmol/L; GTG: 29.4 ± 1.9 mmol/L; p<0.05) and serum insulin levels (GTG ADX: 76 ± 10μ.U/mL; GTG: 470 ± 63μU/mL; p<0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 ± 10 nmol glucose/10⁶ cells; GTG: 403 ± 65 nmol glucose/10⁶ cells; p<0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 ± 27 μmol/liver; GTG: 614 ± 60 pmol/Iiver; p<0.05) and fatty acid content (GTG ADX: 101 ± 9 mg fatty acid/g liver; GTG: 404 ± 40 mg fatty acid/g liver; p<0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

荔枝胚败育过程中内源激素与蛋白质含量的变化

连续3年(1999-2001年)对典型的荔枝焦核品种桂味、糯米糍和大核品种黑叶、怀枝花后10-40d的幼胚和胚乳内源激素、多酚含量及蛋白质动态变化进行研究。结果表明,焦核品种幼胚及胚乳中的IAA、GAs和ABA含量低于大核品种;多酚类物质含量在胚中低于大核品种,胚乳中则高于大核品种;胚和胚乳中的蛋白质含量均低于大核品种。蛋白质电泳结果显示,22．5、28．5和45kD这3类蛋白质在怀枝和黑叶的胚蛋白质代谢过程中表现出较高的稳定性,桂味和糯米糍胚蛋白质中的28．5kD蛋白质也有相似的特性。     

乌骨鸡胚胎发育过程中卵内蛋白质脂肪的变化研究

对乌骨鸡胚胎发育过程中卵内各组成成分(蛋清、蛋黄、胚胎)在不同胚龄(即入孵的第0、3、6、9、12、15、18d和出壳)时蛋白质、脂肪含量变化进行测定与分析．结果表明：在不同胚龄时,蛋清中蛋白质含量显著高于蛋黄,蛋黄中脂肪含量始终高于蛋清;而胚胎中的蛋白质、脂肪逐渐增加。另外,胚胎发育过程中胚胎蛋白质的主要来源是蛋黄中的蛋白质,说明蛋黄是禽类胚胎发育过程中营养需要的主要来源。     

桃蚜高效氯氰菊酯抗性选育过程中酯酶含量和活性变化

采用生物测定和微量滴度酶标板法监测了桃蚜对高效氯氰菊酯抗性的发展趋势。结果表明 ,随着筛选次数增加 ,桃蚜对高效氯氰菊酯的抗药性逐渐增强 :筛选 3代后相对抗性倍数为 13 0 0 ,10代后为 4 9 86倍。随着抗性增强 ,桃蚜酯酶酶源蛋白含量从 35 86 32增加到 6 6 2 990 μg/mL ,酯酶活性从 33 6 76 0增加到 171 0 0 5 2μmol/ (mL·min)。比较抗性发展过程中酯酶酶源蛋白含量及其活性个体频率分布结果发现 ,随着选育次数的增多 ,更多的桃蚜个体分布在酯酶酶源蛋白含量和酯酶活性高的区域。相关分析结果表明 ,抗性筛选过程中的LD50 与酯酶含量及活性变化之间呈显著正相关 ,酯酶在桃蚜对高效氯氰菊酯抗药性发展中可能起着十分重要的作用。     

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

表油菜素内酯对黄瓜子叶过氧化物酶活性和可溶性蛋白含量的影响

０．５ｍｇ／Ｌ的表油菜素内酯（ｅｐｉ－ＢＲ）能显著地促进黄瓜子叶叶绿素ａ、叶绿素ｂ的含量和叶绿素ａ／叶绿素ｂ比值的下降,表明ｅｎｉ－ＢＲ能促进子叶的衰老。从过氧化物酶（ＰＯＤ）的活性测定及同工酶谱发现ｅｐｉ－ＢＲ可提高其活性,暗示它可能通过提高子叶ＰＯＤ的活性而加速子叶叶绿素的降解。另一方面,ｅｐｉ－ＢＲ促进子时可溶性蛋白含量的下降,其中主要是ＲｕＢＰ羧化酶含量的下降,同时,ｅｐｉ－ＢＲ引起子叶游离氨基酸的累积。     

构树形成层活动中内源IAA的变化及其结合蛋白的研究

构树（Broussonetia papyrifera (L.)Vent.）形成层活动周期中用酶联免疫吸附法测定了形成层区域的内源IAA浓度的变化,并用自行发展的免疫荧光检测方法测定了由此处生活细胞制得的原生质体中IAA结合蛋白的分布。结果表明,形成层旺盛形成未成熟木质部和未成熟韧皮部时期,内源IAA急剧增加,当这些细胞分化成熟时IAA浓度降低,并维持在一定范围内。IAA结合蛋白主要分布于质膜、胞质、核膜及核质中。     

Regulation of Sucrose Metabolism in Higher Plants: Localization and Regulation of Activity of Key Enzymes

ABSTRACT

Sucrose (Sue) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Sue synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Sue degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Regulation of Sucrose Metabolism in Higher Plants: Localization and regulation of Activity of Key Enzymes

Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and ‘demand’ for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskel-eton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.     

Soluble Protein Content,Bioactive Compounds and the Antioxidant Activity in Seeds of Ten Rheum tanguticum Lines from Qinghai-Tibet Plateau

Rheum tanguticum (Rh. tanguticum) is a Chinese medicinal plant traditionally used in the treatment of constipation. As a byproduct, the seeds of this plant are rich in nutrients and phytochemicals. This study aimed to determine and assess seed germination ability, seed physical characteristics, soluble protein content, chemical constituents and antioxidant capacity from different breeding lines, to promote the development and utilization of seed resources. Significant differences were observed for the soluble protein content and antioxidant assays among the ten lines. The contents of aloe-emodin, rhein and catechins accumulated in seeds were extremely low and significantly different from those in roots. In contrast, emodin and chrysophanol were abundant in seeds, and significant differences were observed between seeds and roots. It was found that associations between gallic acid and catechins were not significant for either soluble protein or antioxidant capacity. There was a significantly positive correlation between the contents of four anthraquinones (aloe-emodin, rhein, emodin and chrysophanol) and soluble protein. Seeds have potent antioxidative capacity and relatively high levels of soluble protein content. The rich chemical composition of seeds can be widely used in the medical industry for further development.     

TRPV1 Agonist Monoacylglycerol Increases UCP1 Content in Brown Adipose Tissue and Suppresses Accumulation of Visceral Fat in Mice Fed a High-Fat and High-Sucrose Diet

The administration of such a transient receptor potential vanilloid 1 (TRPV1) agonist as capsaicin, which is a pungent ingredient of red pepper, promotes energy metabolism and suppresses visceral fat accumulation. We have recently identified monoacylglycerols (MGs) having an unsaturated long-chain fatty acid as the novel TRPV1 agonist in foods. We investigated in this present study the effects of dietary MGs on uncoupling protein 1 (UCP1) expression in interscapular brown adipose tissue (IBAT) and on fat accumulation in mice fed with a high-fat, high-sucrose diet. The MG30 diet that substituted 30% of all lipids for MGs (a mixture of 1-oleoylglycerol, 1-linoleoylglycerol and 1-linolenoylglycerol) significantly increased the UCP1 content of IBAT and decreased the weight of epididymal white adipose tissue, and the serum glucose, total cholesterol and free fatty acid levels. The diet containing only 1-oleoylglycerol as MG also increased UCP1 expression in IBAT. MGs that activated TRPV1 also therefore induced the expression of UCP 1 and prevented visceral fat accumulation as well as capsaicin.     

温水浸种对杜仲种子萌发过程中保护酶活性及内源激素含量变化的影响

探讨杜仲（Eucommia ulmoides Oliver）种子萌发过程中保护酶活性及内源激素含量的变化，为其种子破眠及萌发不同时期采取有效处理措施，从而提高出苗率提供参考。利用室温（CK）、40℃、50℃、60℃和70℃不同水温，探讨温水浸种对杜仲种子萌发的影响。种子萌发进程中，利用常规生理生化方法测定种子内超氧化物歧化酶（SOD）、过氧化物酶（POD）活性和丙二醛（MDA）含量，以及酶联免疫法测定赤霉素（GA₃）、吲哚乙酸（IAA）、脱落酸（ABA）和玉米素核苷（ZR）含量。结果表明：①温水浸种处理能提高了杜仲种子的发芽率及发芽势，其中以50℃温水处理最为显著。②与室温浸种处理相比，50℃温水浸种处理提高了种子内SOD及POD活性，减轻了质膜氧化损伤程度，致使MDA含量的持续下降，从而促使种子朝向有利于萌发的方向发展。此外，50℃温水浸种处理在提高种子内促进生长内源GA₃、IAA及ZR含量的同时，并降低了抑制生长的内源ABA含量，致使GA₃/ABA、IAA/ABA和ZR/ABA值均明显高于室内浸种处理，最终促进了杜仲种子的萌发。     

百合小鳞茎离体发生过程中内源多胺水平和多胺氧化酶活性的变化

以百合小鳞茎离体发生为实验系统,研究了鳞片(外植体)内多胺和多胺氧化酶活性的变化与小鳞茎发生之间的相关性。随着细胞脱分化、愈伤组织形成、小鳞茎发生等过程,内源腐胺、亚精胺和精胺的含量均失上升后下降,变化曲线为“钟形”,其含量达最高峰的时间为肉眼可见小鳞茎出现之前。多胺氧化酶活性在小鳞茎发生初期逐渐下降,6d后开始上升,并在小鳞茎突起时达最大值。由于内源多胺水平和多胺氧化酶活性的变化与小鳞茎发生有密切关系,可作为这一形态发生过程的生化指示。     

德国鸢尾‘常春黄’花芽分化的形态观察及两种代谢产物的动态变化

鸢尾是世界著名观赏花卉,为研究其花芽分化期的形态和生理指标变化情况,我们以德国鸢尾两季花品种‘常春黄’（Iris germanica cv. Lovely Again）为材料,运用扫描电镜（SEM）观察了德国鸢尾‘常春黄’的花芽分化过程。结果表明：整个形态分化过程可分为6个阶段：花序原基分化期、外轮花被分化期、雄蕊分化期、内轮花被分化期、雌蕊分化期、髯毛形成期。结合上述形态分化过程,分别取其二次花花芽分化时期的顶芽、根茎和叶片部位,以蒽酮比色法测定可溶性糖,以考马斯亮蓝G-250法测定蛋白质含量。结果表明：可溶性糖在花序原基分化的初始阶段含量最高,且在3个部位的含量大小关系始终是：根茎﹥叶片﹥顶芽;蛋白质含量呈先上升后下降的趋势,蛋白质含量的峰值出现于花序伸展初期。     

小麦返白系返白阶段叶片蛋白质变化与叶绿素含量的关系

小麦返白系春天出现白色心叶。白叶中有脱辅基蛋白存在,但含量很低。白叶复绿时色素条带中ＣＰⅡ出现先于ＣＰＩ;叶绿素含量的增加与色素蛋白的出现呈正相关,同时影响蛋白含量,但２１、２５ｋＤ蛋白即使在全白叶中也有较高水平。白叶中ＲｕｂｉｓｃｏＬＳ含量极低,而ＳＳ仅略有降低。复绿初期蛋白质合成速率随叶绿素含量增加而显等提高,ＬＳ也迅速增加。     

Identification, Characterization, Immunocytochemical Localization, and Developmental Changes in the Activity of Calcium/Calmodulin-Dependent Protein Kinase II in the CNS of Bombyx mori During Postembryonic Development

Abstract: In the present investigation, in vitro phosphorylation of CNS proteins of the silkworm Bombyx mori during the postembryonic development have been studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of phosphorylated proteins revealed the presence of major phosphoproteins of 59/60 kDa. Based on molecular mass, calcium/calmodulin-dependent autophosphorylation, substrate specificity, KN-62 inhibition, apparent K _m for ATP and syntide-2, these proteins were identified as calcium/calmodulin-dependent protein kinase II (CaM kinase II). Anti-rat CaM kinase II monoclonal antibody showed immunoreactivity with Bombyx CaM kinase II isoforms. This kinase showed a high degree of autophosphorylation in neural tissue. During postembryonic development of Bombyx , two distinct peaks of enzyme activity could be noticed, one at the late-larval and another at the late-pupal stage, which were associated with an increase in amount of the enzyme. These results suggested that the expression of CaM kinase II in the CNS of Bombyx was developmentally regulated.