Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Óleo de peixe (fonte de ácidos graxos n-3) atenua inflamação das vias aéreas e hiper-reatividade pulmonar induzida por alérgeno em camundongos

O óleo de peixe é rico em ácidos graxos poli-insaturados (AGPI) n-3 e vem sendo apontado como anti-inflamatório associado à melhora de diversas doenças de natureza inflamatória. No presente estudo, objetivou-se avaliar a influência do óleo de peixe sobre a inflamação pulmonar e hiper-reatividade em camundongos ativamente sensibilizados desafiados com ovoalbumina (OVA). Camundongos A/J machos foram alimentados com dieta standard-chow (SC) ou dieta rica em óleo de peixe (Px) durante 8 semanas. Após 4 semanas do início da dieta, cada grupo foi subdividido aleatoriamente para ser desafiado com salina (SC-SAL e PX-SAL) ou ovoalbumina (SC-OVA e PX-OVA). A função pulmonar (resistência e elastância) foi avaliada através de pletismografia invasiva, na condição de aerolização ou não com metacolina 24 horas após o último desafio antigênico. Foi realizado lavado broncoalveolar (LBA) para contagem de leucócitos e quantificação de eotaxina-2. A deposição de muco e de matriz peribronquiolar e o infiltrado de eosinófilos foram quantificados no tecido pulmonar. Foram avaliados interleucina (IL)-13 através de imunohistoquímica e NFκB, GATA-3 e PPARγ, por western-blotting. O desafio com OVA resultou em aumento da infiltração de eosinófilos, elevada produção de citocinas inflamatórias, remodelamento pulmonar, produção de muco e hiper-reatividade das vias aéreas. Detectou-se aumento na expressão dos fatores de transcrição NFκB e GATA-3 nos camundongos do grupo sensibilizado e desafiado com OVA em comparação aos controles. Todas essas alterações foram atenuadas nos camundongos que receberam dieta com óleo de peixe. Expressão elevada de PPARγ foi detectada nos pulmões dos camundongos dos grupos alimentados com óleo de peixe. Em conclusão, nossos resultados mostram que a ingestão de óleo de peixe atenuou as características clássicas do quadro asmático através da modulação da síntese de mediadores inflamatórios, via regulação negativa de NFκB e GATA-3 e regulação positiva de PPARγ. O óleo de peixe parece ser uma terapia alternativa para o controle e tratamento da asma.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

毛白杨（Populus tomentosa）木质素生物合成相关基因分离与木质素合成调节

木质素是植物体中具重要生物功能的次生代谢产物。然而纸浆生产主要是将原料中的木质素与用于造纸的纤维素分离，该工艺过程产生了造纸工业的主要污染废液，并且增加造纸成本。本研究目的在于利用反义RNA技术，在分子水平调节木质素的生物合成，降低中国特有造纸树种毛白杨的木质素含量，培育更适于我国造纸工业的原料树种。以下为本研究已取得的相关研究进展： 1.通过RT-PCR技术，从毛白杨中克隆了木质素生物合成的三个相关酶的cDNAs，它们分别为咖啡酸甲基转移酶(caffeic acid O-methyltransferase，COMT)、咖啡酰CoA甲基转移酶(caffeoyl Co-enzyme A O-methyltransferase，CCoAOMT)及香豆酸：辅酶A连接酶(4-coumarate: CoA ligase，4CL)。序列分析显示了毛白杨这三个基因与杨属中其它种的相应基因cDNA核苷酸序列高度同源。Northern点杂交分析表明，COMT、CCoAOMT及4CL基因在毛白杨正在生长的次生木质部中高水平表达，其表达高峰与树木的木质化进程同步;而在叶与叶柄中，这三个基因均不表达。COMT、CCoAOMT及4CL是木质素生物合成的相关酶，该表达特征与其基因功能相一致。本研究克隆的COMT、CCoAOMT及4CL基因的cDNAs已在GenBank注册登记，接受号分别为AF237777、AF240466、AF314180 (publish on Jan l,2002)。 2.通过一系列的DNA重组，构建了携带反义COMT、CCoAOMT或4CLcDNA的反义表达载体以及同时整合反义COMT与CCoAOMT cDNA的双价反义表达载体，PCR扩增与酶切检测确证构建无误。 3.以田间取材的速生三倍体毛白杨B19、B331及B304的茎尖、叶片与嫩茎为外殖体，首次获得了三倍体毛白杨的组培再生试管苗，并建立了速生三倍体毛白杨的组培再生系统，为通过基因工程改良其造纸性能奠定了基础。 4.农杆菌介导转化烟草，PCR与PCR-Southern检测表明我们获得了整合反义COMT、CCoAOMT cDNA及反义COMT及CCoAOMT cDNA共整合的转基因烟草。以Digoxigenin标记的对应于反义链的单链RNA为探针与转基因烟草的总RNA进行NoIthern点杂交，结果表明整合到其中的反义cDNA均已表达。转基因烟草的木质素分析将有助于对COMT及CCoAOMT两个甲基化酶功能的认识。 5.通过农杆菌介导，将反义CCoAOMT cDNA转入欧洲山杨与银白杨的杂交杨（P tremulaXP.alba）。经PCR，PCR-Southern及Southern检测，确认获得了转基因植株。以Digoxigenin标记的对应于CCoAOMT cDNA反义链的单链RNA为探针与转基因杂交杨总RNA进行Northern点杂交，结果表明整合到其中的反义cDNA已在转录水平表达。测定生长5-6个月的转基因杨树下部茎杆的Klason木质素含量，结果显示其中一个株系的Klason木质素含量比野生型对照下降17.9%，表明抑制杨树内源CCoAOMT基因表达可有效降低转基因植株的木质素含量。

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom

Rong Gao, Yun Zhang, Qing-Xiong Meng, Wen-Hui Lee, Dong-Sheng Li, Yu-liang Xiong and Wan-Yu Wang. Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejneger ) venom. Toxicon 36, 457-467, 1998.-From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-l, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatograghy (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mel. wt of -50 000, 31 000 and 32 000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnfibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA. and stejnobin, stejnefibrase-l, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially BE-chain while stejnefibrase-l and -3 cleaved concomitantly Ax and B beta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-l. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases. (C) 1998 Elsevier Science Ltd. All rights reserved.

The first study on the effects of microcystin-RR on gene expression profiles of antioxidant enzymes and heat shock protein-70 in Synechocystis sp PCC6803

Microcystins are heptapeptide toxins produced by cyanobacteria. Microcystin-RR(MC-RR) is a common variant among the 80 variants identified so far. There have been many investigations documenting the toxic effects of microcystins on animals and higher plants, but little is known on the toxic effects of microcystins on algae, especially at molecular level. We studied the effects of MC-RR on gene expression profile of a few antioxidant enzymes and heat shock protein-70 (Hsp70) in Synechocystis sp. PCC6803. After two days post-exposure, a high dose toxin (5 mg/l, about 4.8 x 10(-3) mM) significantly increased expression levels of the genes gpx1, sodB, katG, acnB, gamma-TMTand dnaK2, while a relatively low dose toxin (1 mg/l, about 9.63 x 10(-4) mM) induced a moderate and slow increase of gene expression. Our results indicate that MC-RR could induce the oxidative stress in Synechocystis sp. PCC6803 and the increase in gene expression of antioxidant enzymes and Hsp70 might protect the organism from the oxidative damage. in addition, cell aggregation was observed during the early period of exposure, which might be a specific oxidative stress reaction to MC-RR. (C) 2008 Elsevier Ltd. All rights reserved.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

Influence of dioxin and metal-contaminated sediment on phase I and II biotransformation enzymes in silver crucian carp

Several biochemical responses were measured in silver crucian carp (Carassius auratus gibelio) after exposure to sediments obtained from contaminated Ya-Er Lake, No, 1 pond, and an unpolluted reference site, Honglian Lake. After 1 week of exposure, a significant induction of the phase I biotransformation enzyme (ethoxylresorufin-o-deethylase, EROD) was found (83-fold of control), whereas the phase II biotransformation enzyme (glutathione S-transferase, GST) exhibited a slight, but significant induction (1,4-fold of control) after 4 weeks of exposure. The level of cellular glutathione in the liver was also slightly elevated after 4 weeks of exposure. The delayed response of GST to the contaminants indicates that the phase I and phase II biotransformation enzymes are regulated differently in fish. The results suggest that EROD is a sensitive bioindicator to assess the toxicity of dioxin-contamined sediment in the laboratory, (C) 1998 Academic Press.

An ambient proof and wide tuning range LC VCO with automatic amplitude control for tuner applications

This paper represents a LC VCO with AAC (Auto Amplitude Control), in which PMOS FETs are used as active components, and the varactors are directly connected to ground to widen Kvco linear range. The AAC circuitry adds little noise to the VCO and provides it with robust performance over a wide temperature and carrier frequency range. The VCO is fabricated in 50-GHz 0.35-mu m SiGe BiCMOS process. The measurement results show that it has -127.27-dBc/Hz phase noise at 1-MHz offset and a linear gain of 32.4-MHz/V between 990-MHz and 1.14-GHz. The whole circuit draws 6.6-mA current from 5.0-V supply.

A Smart Frequency Presetting Technique for Fast Lock-in LC-PLL Frequency Synthesizer

This paper proposes a smart frequency presetting technique for fast lock-in LC-PLL frequency synthesizer. The technique accurately presets the frequency of VCO with small initial frequency error and greatly reduces the lock-in time. It can automatically compensate preset frequency variation with process and temperature. A 2.4GHz synthesizer with 1MHz reference input was implemented in 0.35 mu m CMOS process. The chip core area is 0.4mm(2). Output frequency of VCO ranges from 2390 to 2600MHz. The measured results show that the typical lock-in time is 3 mu s. The phase noise is -112dBc/Hz at 600KHz offset from center frequency. The test chip consumes current of 22mA that includes the consumption of the I/O buffers.

Fabrication of 1.3 mu m Si-based MEMS tunable optical filter

In this paper we report the fabrication of 1.3 mum Si-based MEMS tunable optical filter, by surface micromaching. Through wet etching of polyimide sacrificial layer, a tunable Fabry-Perot filter was successfully fabricated. We make the capacitance measurement of the prototype device, compare the experimental curve with the theoretical one, and explain the difference between them.