Minor xanthones of Garcinia mangostana

Two new trioxygenated xanthones with a 3,3-dimethyl allyl side chain have been isolated from the fruit hulls of Garcinia mangostana. The structures were established from spectral and chemical data.     

拟南芥IQM3基因的表达分析及其突变体的鉴定

对拟南芥(Arabidopsis thaliana)IQM3基因的功能进行了分析.结果表明,推定IQM3的启动子中存在多种光、非生物胁迫和植物激素反应的顺式作用元件,可能参与植物对环境变化的反应.RT-PCR分析表明,IQM3在拟南芥莲座叶、花序叶、茎、花和根中的表达较强,但在荚果中的表达很弱;IQM3基因的T-DNA插入突变体iqm3-1和iqm3-2分别是功能缺失和超表达突变体,对这些突变体的表型分析表明,IQM3基因与种子萌发及幼苗子叶膨大有密切关系.     

Serine-inserting UAA suppression mediated by yeast tRNASer

The number of loci that give rise to serine-inserting UAA suppressors in the yeast Saccharomyces cerevisiae was determined by examining over 100 of the revertants that suppressed the two UAA markers his4-1176 and leu2-1: the his4-1176 marker is suppressed by serine-inserting but not by tyrosine- or leueine-inserting suppressors and the leu2-1 marker is suppressed by all UAA suppressors. The suppressors could be assigned to one or other of the four loci: SUP16 and SUP17. which were previously known to yield serine-inserting suppressors, and SUP19 and SUP22. The chromosomal map position of SUP19 suggested that it may be allelic to the previously reported suppressor SUP20, while the SUP22 suppressor has not been described. Representatives of all of the four suppressors were found to insert serine at the UAA site in iso-1-cytochrome c from suppressed cyc1-72 strains. The degree of suppression by the serine-inserting suppressors was SUP16 > SUP17 > SUP19 > SUP22. The efficiency of suppression of each of the four serine suppressors was increased by the chromosomal mutation sal and by the cytoplasmic determinant ψ⁺. Read-through of the synthetase gene of the RNA bacteriophage Qβ in a cell-free system was used to demonstrate that tRNA^Ser from SUP16, SUP17 and SUP19 strains can translate UAA codons. In contrast, tRNA^Ser or total tRNA from SUP22 strains had no suppressing activity. The results suggest that the three loci SUP16, SUP17 and SUP19 encode iso-accepting species of tRNA^Ser, and that the UAA suppression is mediated by mutationally altered tRNA molecules. The mechanism of SUP22 suppression remains unknown.     

嗜热菌Bacillus fordii 3-2海因酶的纯化及性质

对自行筛选的海因酶法L-苯丙氨酸生产菌株Bacillus fordii 3-2中的海因酶进行了分离纯化及相关性质研究.该海因酶协同L-氨甲酰水解酶是海因酶法生产L-氨基酸的关键酶.B.fordii 3-2菌悬液经压力破碎离心后取上清液为粗酶液,粗酶液通过硫酸铵分级沉淀、Phenyl FF(high sub) 疏水层析以及Source 15Q离子交换层析,经SDS-PAGE分析达到电泳纯,亚基相对分子质量为55×103,海因酶的纯化回收率为20.5%,纯化倍数为149.23.该海因酶在pH 8.0～10.0的范围内具有较高的活性,在45～70℃具有很高的酶活力, 最适反应pH和温度分别为10.0 ℃和65 ℃.纯酶易氧化失活,DTT对该酶有一定的保护作用.二价金属离子,Mn2 、Co2 及Fe2 对酶活性有显著的促进作用,而Ca2 、Cu2 等对酶活性有抑制作用.相关研究可为该菌株及海因酶的进一步开发与应用提供理论指导.     

Mutagenicity of BCNU and related chloroethylnitrosoureas in Drosophila.

BCNU and 10 related chloroethylnitrosoureas were tested for their ability to induce sex-linked recessive lethals in Drosophila spermatozoa. All chloroethylnitrosoureas tested were potent mutagens. Among the substances with one chloroethylnitrosourea group, chlorozotocin, BCNU and methanesulfonyloxyethyl chloroethylnitrosourea exhibited the strongest mutagenic effects. Two hydroxyalkyl chloroethylnitrosoureas behaved as potent mutagens too, although the mutation frequencies obtained were one order of magnitude lower relative to the other substances. Among the compounds with two chloroethylnitrosourea groups, bisCNU-ethane and bisCNU-diphenylmethane were most active. When the interconnecting polymethylene chain was elongated from 2 methylene groups (bisCNU-ethane) to 6 methylene groups (bisCNU-hexane), the mutagenic activity decreased by a factor of 2. The mutagenic activity of polymethylene bischloroethylnitrosoureas with connecting chains of intermediate length was not different from bisCNU-hexane. Differences in mutagenic activity were supposed to reflect different concentrations reaching the target cells, possibly in part as a result of differences in transportability of the substances.     

Isolation and identification of urinary metabolites of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) in rabbits

After oral administration of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) to rabbits, the two unique metabolites, M-I and M-II, were isolated from the urine. M-I, yellow needles of mp 117°, was identified as a new type metabolite of nitrofuran derivative, 2-(β-carboxypropionyl)-3-(5-methylthio-2-furyl) acrylamide by its mass, ir and nmr spectrometries. M-II, yellow solid, appears to be cis-trans isomer of M-I considering from its uv and mass spectral data, and the behavior on tlc.     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

Unique presence of 2-methylthio-ribosylzeatin in the transfer ribonucleic acid of the bacterium Pseudomonas aeruginosa

Analysis of ³⁵S labled nucleosides prepared from tRNA of Pseudomonas aeruginosa by phosphocellulose column chromatography, thin layer chromatography and Sephadex LH-20 column chromatography revealed the presence of 2-methylthioribosylzeatin in it.     

Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2(209-223) peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS-PAGE and LC/MS identified 14-3-3epsilon as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)(TG2+/+) cells but not from MEF(TG2-/-) cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.     

Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-μl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C₁₈) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 μM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 μM and 14% (n=6 days) at 0.3 μM NTBC in plasma. The quantitation limit was approximately 0.3 μM using 20 μl of plasma.     

On the use of the spin labeling technique in the study of erythrocyte membranes

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri V.G.; Wallach D.F.H.; Lin P.S. (1974) Proc. Natl. Acad. Sci. U. S. 71, 4797–4801) are reproduced, it is shown that at label-to-lipid ratios of 1 : 10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.     

云南野生稻离子吸收效率及其动力学特征研究

分析和检测4种,共6个不同类型的野生稻吸收H2PO4^-、K^＋、Mg^2＋、NO3^-的动力学特征以及培养液pH值变化的研究结果表明,景洪普通野生稻吸收K^＋较强;药用野生稻和元江普通野生稻次之。小粒野生稻吸收磷的能力最强。元江普通野生稻吸收Mg^2＋和NO3^-的能力强。药用野生稻吸收Mg^2＋较强;直立型景洪普通野生稻有较强的吸收NO3^-的能力。     

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor

β-methylaspartate ammonia-lyase, EC 4.3.1.2, (β-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b , respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)- 3a yielded the α,β-substituted β-lactone (3R,4R) 6 with a very high diastereoisomeric excess (>95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl β-(2R,3S)-3-methylmalate] 8 , prepared by an anionic ring opening polymerization, was highly isotactic as determined by ¹³C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7 , to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of β-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions. Chirality 10:727–733, 1998. © 1998 Wiley-Liss, Inc.     

Preparation of 2- and 3-substituted gibberellins A9 and A4 for bioassay

To test the effects of preventing enzymatic 2β- and 3β-hydroxylation on the biological activities of gibberellins, the preparation of the following compounds is described: 2β-methyl- and 2,2-dimethyl-gibberellins A₄ and A₉; 2α-fluoro-, 2β-fluoro- and 2β-methoxy-gibberellin A₉; and 3β-chloro-, 3β-fluoro-, 3β-methoxy- and 3-methylene A₉.     

Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.     

Superior prevention of calcium ionophore cataract by E64d

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64D, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 of E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.     

Yeast UAA suppressors effective in psi+ strains: leucine-inserting suppressors.

We have previously reported the isolation and characterization of UAA suppressors from a haploid strain of yeast Saccharomyces cerevisiae containing the ψ⁺ non-Mendelian determinant which increases the efficiency of action of certain suppressors (Ono et al., 1979). Most of the suppressors caused the insertion of either tyrosine or serine. In contrast, the pattern of suppression of nutritional markers suggested that the rare suppressor, SUP26, inserted in an amino acid other than tyrosine or serine. In this investigation we report the characterization of additional suppressors, similar to SUP26, that were isolated on a medium lacking uracil and containing canavanine; this medium is expected to exclude serine-inserting suppressors because they do not suppress the ura4-1 marker, and to exclude tyrosine-inserting suppressors because they suppress the can1-100 marker. The total of 155 revertants similar to the SUP26 suppressor were analyzed genetically and these could be assigned to one or another of the six distinct loci SUP26, SUP27, SUP28, SUP29, SUP32 and SUP33. The SUP26, SUP27 and SUP29 loci mapped on chromosomes XII, IV and X, respectively. The detailed map position of the SUP29 suppressor suggests that it may be allelic to the SUP30 suppressor reported by Hawthorne &; Mortimer (1968). These six suppressors had the same pattern of suppression of UAA nutritional markers and all of them had a similar low efficiency of action on the iso-1-cytochrome c mutation cyc1-72. The efficiency of each of these suppressors was increased by a chromosomal allo-suppressor, sal. Each of the six suppressors caused the insertion of leucine in iso-1-cytochrome c at the UAA site of the cyc1-72 mutation. It is suggested that the gene products of these suppressors are redundant forms of the same leucine transfer RNA.     

朗德鹅胆汁的化学成分及金属蛋白酶抑制活性研究

应用多种色谱技术进行分离纯化,从朗德鹅胆汁85％乙醇提取物中分离得到6个化合物。经理化性质和光谱数据分析鉴定为苯乙酸（1）、鹅去氧胆酸（2）、鹅去氧胆酸乙酯（3）、棕榈酸-α-单甘油酯（4）、顺-6-十八碳烯酸（5）、（4E）-2-[2＇-hydroxyhexadecanoylamino]-4-octadecane-1,3-diol（6）。化合物1、3、4和6为首次从该属动物胆汁中分得,其中化合物6为首次从陆生动物胆汁中分得的一种神经酰胺类成分。首次对化合物2、4和5进行抑制金属蛋白酶活性的实验,评价了三个化合物的生物活性。     

Characterization of ligands for thyroid receptor subtypes and their interactions with co-regulators

Thyroid hormone receptors (TRs) are nuclear receptors that are activated by thyroid hormone ligands and co-regulator proteins. Two receptor subtypes, TRα and TRβ, have been suggested to play a role in numerous physiological functions. However, specificity of receptor subtype function and co-regulator interaction is unclear due to the lack of TR subtype-specific ligands. Five TR ligands were evaluated for their selectivity and interaction with the TR subtypes. A multiplex assay was used to identify co-regulator peptide interaction, and biochemical assays were used to characterize ligand-receptor specificity. In the biochemical assay, rank order ligand potencies were similar in the presence of co-activator peptides, SRC1-2 and SRC3-2, and the co-repressor peptide, NCoR1-2, with T3 and Triac potencies greater in the presence of the co-repressor. The potency of Tetrac was similar regardless of the co-regulator used while T4 and rT3 demonstrated selectivity for TRα subtype. The rank order among TR ligands at either receptor subtype in the biochemical assay correlated with the multiplex assay. These assays can be used to identify new ligands that can provide further insight into TR biology.