Diapause development and acclimation regulating enzymes associated with glycerol synthesis in the Shonai ecotype of the rice stem borer larva,Chilo suppressalis walker

Overwintering larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, enter diapause in early September and terminate diapause at the end of October. Cold acclimation at 0°C did not influence glycerol, trehalose or glycogen content in larvae collected on 22 September. Acclimation at 0°C increased the glycerol content and reduced the glycogen content significantly in larvae collected on 2 October and 22 November compared with acclimation at 15°C. These results indicate that overwintering larvae at different phases of diapause development respond differently to the low temperature stimulus for glycerol synthesis. Thus, we evaluated the metabolic rearrangements associated with glycerol synthesis during diapause development and after temperature acclimation. Larvae collected on 2 October were acclimated at 15°C for 15 and 60 days. Some of those acclimated at 15°C were then moved to 0°C for 15 days. The larvae acclimated at 15°C for 15 days were in deep diapause and accumulated little glycerol, while larvae acclimated at 15°C for 60 days were nearly ready to emerge from diapause and accumulated glycerol at 155.5 μmol/g. When larvae acclimated to 15°C for 15 days were transferred to 0°C, glycerol accumulation was stimulated to the same extent (ca 140 μmol/g) as it was in larvae that were acclimated to 15°C for 60 days and then transferred to 0°C. These results indicate that low temperature has a cumulative effect on glycerol production in larvae at different phases of diapause development. Glycerol accumulation was accomplished by activation of glycogen phosphorylase and inhibition of fructose-1,6-bisphosphatase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

黄秋葵组培快繁的研究

黄秋葵 (ＨｉｂｉｓｃｕｓｅｓｃｕｌｅｎｔｕｓＬ .)为锦葵科一年生草本植物 ,别名羊角豆、秋葵。原产非洲 ,欧美及东南亚热带地区广泛栽培 (马传先 ,1999;李正应 ,1993;翁文 ,1997;雪珍等 ,1999)。黄秋葵是一种营养保健型蔬菜 ,其花、叶、芽、果均可食用 ,种子富含油脂、蛋白质及钾、钙、铁、锌、锰等矿物质 ,晒干后既可用于提取油脂和蛋白质 ,还可作为咖啡的添加剂或代用品。花、种子和根均可入药 ,对恶疮、痛疖有疗效。黄秋葵的愈伤组织在一定条件下比较容易产生体细胞胚并再生成完整植株 ,故黄秋葵的组培快繁研究也可为胚胎学研究和人…     

Quantitative proteomic analysis of Myc oncoprotein function

This study applies a new quantitative proteomics technology to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICAT) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. Our results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells.     

c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis

Activation of the caspase cascade is a pivotal step in apoptosis and can occur via death adaptor-mediated homo-oligomerization of initiator procaspases. Here we show that c-FLIP(L), a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 death-inducing signaling complex (DISC) and potently promotes procaspase-8 activation through hetero-dimerization. c-FLIP(L) exerts its effect through its protease-like domain, which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. Ectopic expression of c-FLIP(L) at physiologically relevant levels enhances procaspase-8 processing in the CD95 DISC and promotes apoptosis, while a decrease of c-FLIP(L) expression results in inhibition of apoptosis. c-FLIP(L) acts as an apoptosis inhibitor only at high ectopic expression levels. Thus, c-FLIP(L) defines a novel type of caspase regulator, distinct from the death adaptors, that can either promote or inhibit apoptosis.     

Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).