The Study of Enzymology Characterization of Ctenopharyngodon idellus LDH Isozyme

LDH-A_4,B_4,and C_4 were isolated and purified from Ctenopharyngodon idellus tissues by means ofaffinity chromatography.The purity,amino acid content and kinetics were analyzed.The anti-LDH-A_4,B_4,and C_4 antibodies were prepared from rabbit sensitized by LDH-A_4,B_4,and C_4respectively.The LDH patterns of some freshwater fish and marine fish were identified by theantigen-antibody precipitation reaction.The homology between LDH isozyme molecules fromdifferent sources and the immunological homology of different LDH isozymes were discussed.     

TLR4全长及其截断体重组腺病毒的制备和功能鉴定

制备脂多糖 (LPS)Toll样受体 4 (TLR4 )全长及其胞内段缺失的TLR4截断体 (ΔTLR4 )的绿色荧光蛋白重组腺病毒并鉴定其功能 .用PCR方法扩增TLR4及ΔTLR4基因片段 ,酶切后亚克隆至腺病毒穿梭质粒中 ,形成带有目的基因的穿梭载体pAdTrack TLR4和pAdTrack ΔTLR4 .用BJ5 1 83细菌同源重组法将目的基因重组于腺病毒骨架载体中 ;将重组腺病毒质粒用PacⅠ酶切线性化后 ,用脂质体法转染HEK 2 93细胞进行腺病毒的包装扩增 .将重组腺病毒感染CHO K1细胞 ,采用荧光毒酶报告基因方法检测其对LPS诱导NF κB激活的影响 .酶切及测序表明 ,TLR4全长及其截断体ΔTLR4的重组腺病毒载体构建正确 .荧光素酶报告基因检测结果表明 ,TLR4全长及其截断体的重组腺病毒感染细胞对LPS诱导的反应具有不同的影响 ,Ad ΔTLR4明显抑制了LPS引起的NF κB激活 (P <0 0 5 ) ,Ad TLR4则使LPS引起的NF κB活性增强 (P <0 0 5 ) .LPS对细胞的激活作用依赖于TLR4的结构完整性     

Fluorimetric Assay of the Activity of Extracellular Lipases of Pseudomonas fluorescens and Serratia marcescens

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonas fluorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone palmitate and 4-methylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The extracellular lipases were isolated and purified by ammonium sulphate precipitation. The assay was carried out by relating the fluorescent intensity emitted by two lipase concentrations on five substrates against four standard curves. These standard curves were prepared by estimating the intensity of fluorescence given by varying dilutions of 4-methylumbelliferone at the four pH levels. The results indicated that the oleic ester of 4-methylumbelliferone was a suitable substrate at pH 8.0 and pH 10.0. These pH values were also optimum for fluorescent intensity on substrates of 4-methylumbelliferone elaidate, 4-methylumbelliferone butyrate and 4-methylumbelliferone palmitate. However, on substrate 4-methylumbelliferone nonanoate, the optimum pH was 4.0.     

Microsatellite markers reveal chimeric origin of redesignated chromosome 4A of wheat from Triticum urartu and other species.

Although a new nomenclature has been adopted for wheat in which chromosome 4A (4AO) has been renamed 4B (4BN) and chromosome 4B (4BO) has been renamed 4A (4AN), their specific origin remains uncertain. The use of wheat microsatellite (WMS) markers mapped to chromosomes 4AN and 4BN in a set of polyploid wheats and diploid genome donors has unequivocally indicated that the entire short arm of 4AN, some part of 4ANL. and a segment of 4BNL were derived from Triticum urartu. The presence of a T. urartu-specific allele at locus gwm368 on 4BNL and of an Aegilops speltoides allele at locus gwm397 on 4ANL suggests the possibility of a reciprocal translocation between 4ANL and 4BNL. The subcentromeric and telomeric regions of 4ANL corresponding to heterochromatic C-bands were derived neither from diploid wheats nor from Ae. speltoides or Aegilops longissima.     

Mutagenic activity of possible metabolites of 4-nitrobiphenyl ether

A series of possible metabolites--4-nitrosobiphenyl ether (4-NO), 4-hydroxylaminobiphenyl ether (4-NHOH), 4-aminobiphenyl ether (4-NH2), 4-hydroxyacetylaminobiphenyl ether (4-N(OH)Ac), 4-acetoxyacetylaminobiphenyl ether (4-N(OAc)Ac)involved in the toxic effects of 4-nitrobiphenyl ether (4-NO2) was synthesized and tested for mutagenic activity toward Salmonella typhimurium TA100 strain in the presence and the absence of liver homogenates of guinea pig treated with Kaneclor-500. 4-NO2, 4-NO and 4-NHOH showed direct-acting mutagenicity. 4-NO and 4-NHOH showed high mutagenic activity, while the mutagenic activity of 4-NO2 was very weak compared to 4-NO and 4-NHOH. 4-NO showed antimicrobial action at high concentrations. The other three compounds tested induced no mutation. Upon addition of NAD(P)H, the mutagenic activities of 4-NO and 4-NHOH were slightly enhanced, but no enhancement was observed by addition of NAD(P)+. Metabolic activation with guinea pig liver homogenates enhanced the mutagenic activities of 4-NO2 and 4-NO, and converted 4-NH2, 4-N(OH)Ac and 4-N(OAc)Ac to the product(s) responsible for the mutagenic activity. Addition of bis(p-nitrophenyl)phosphate, a deacetylase inhibitor, inhibited the mutagenic activities of 4-N(OH)Ac and 4-N(OAc)Ac by about 70% in the presence of NADPH and about 77% in the absence of NADPH. High performance liquid chromatography (HPLC) analysis of non-enzymatic conversion-products of 4-NHOH and 4-BO with and without NADPH indicated that 4-NHOH disappeared after 30 min of incubation and was converted completely to 4-NO without NADPH, while with NADPH, 4-NHOH disappeared very slowly and was detected even after 4 h of incubation. In the case of 4-NO, no decrease of 4-NO was observed without NADPH, while with NADPH 4-NO decreased quickly and a significant amount of 4-NHOH appeared. The mechanism of the NAD(P)H-dependent increase in mutagenicity is also discussed.     

Characterization of the 46,000-dalton subunit of eIF-4F

Three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F are required for the ATP-dependent binding of mRNA to the ribosome. To extend the characterization of the eIF-4A-like subunit of eIF-4F, a cDNA clone encoding eIF-4A has been isolated from a rabbit liver cDNA library and sequenced. The clone is almost full length for the coding region and complete for the 3' noncoding region. The sequence of the rabbit cDNA has been compared to the sequence of the two similar, but not identical, genes and cDNAs encoding mouse eIF-4A (termed eIF-4AI and eIF-4AII). The rabbit cDNA sequence is very similar to the mouse eIF-4AI genomic and liver cDNA sequence with 100% identity at the amino acid level and 90% identity at the nucleotide level within the protein coding region; however, there is very little similarity in the 3' noncoding region. Amino acid sequencing of purified rabbit reticulocyte eIF-4A protein indicates that it is eIF-4AI (encoded by the eIF-4AI gene and cDNA) and none of the amino acid residues sequenced are in disagreement with those predicted from the mouse liver or rabbit liver cDNA sequences. Subsequently, we have analyzed the p46 subunit of eIF-4F, a three subunit protein whose molecular weights have been estimated by sodium dodecyl sulfate gel electrophoresis to be 220,000, 46,000 and 24,000. The p46 subunit has physical properties similar to eIF-4A. This subunit was isolated from rabbit reticulocyte eIF-4F and sequenced chemically. Our results indicate that this peptide is a mixture of eIF-4AI and eIF-4AII in an approximate ratio of 4 to 1, respectively. No eIF-4AII was observed in our rabbit reticulocyte eIF-4A preparation. Therefore we have concluded that either the eIF-4AI and the eIF-4AII proteins were resolved from each other in the purification of rabbit reticulocyte eIF-4A or that eIF-4AII preferentially associates with the p220 and p24 subunits of eIF-4F. Evidence favoring the latter possibility is discussed.     

Study of the characterization and crystallization of 4-hydroxy-2-pyrrolidone

A systematic study of the characterization for racemic species of 4-hydroxy-2-pyrrolidone was undertaken. The melting point phase diagram of (R)- and (S)-4-hydroxy-2-pyrrolidone was determined by differential scanning calorimetry. The ternary phase diagram of (R)- and (S)-4-hydroxy-2-pyrrolidone with isopropanol was constructed at 15, 20, 25, and 35 degrees C. The crystalline nature of 4-hydroxy-2-pyrrolidone racemate was also characterized by means of comparison of solid-state FTIR spectra and powder X-ray diffraction patterns of the racemic mixture with those of one of the enantiomers. It is shown that (+/-)-4-hydroxy-2-pyrrolidone is a racemic conglomerate. The enthalpies of fusion of (R)-4-hydroxy-2-pyrrolidone and (+/-)-4-hydroxy-2-pyrrolidone and entropy of mixing of (R)- and (S)-4-hydroxy-2-pyrrolidone were calculated using the thermodynamic data. The solubility and supersolubility diagrams of (R)- and (S)-4-hydroxy-2-pyrrolidone in isopropanol were determined over a temperature range of 4-35 degrees C. The optical resolution of (+/-)-4-hydroxy-2-pyrrolidone was successfully achieved by preferential crystallization.     

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

培养基成分对杜仲愈伤组织生长及次生代谢产物含量的影响

以Bs＋0．5mg／L NAA＋0．5mg／L BA为基本培养基,研究了B5培养基中8种主要无机盐浓度对杜仲愈伤组织生长及绿原酸和总黄酮两种次生代谢产物含量的影响。结果表明：在1000～5000mg／L范围内增加培养基中KNO3的含量有利于愈伤组织生长,B5培养基中当KNO3的浓度达到2／3时,绿原酸和总黄酮含量及产量最高;(NH4)2SO4以4／3原浓度时对愈伤组织生长量、总黄酮含量及产量最高,对绿原酸的含量则是其为原浓度的1／3时最高;MgSO4以2／3浓度对生长量及1／3浓度对绿原酸、总黄酮积累最高;NaH2PO4、CaCl2和MnSO4以原浓度的愈伤组织生长和次生代谢产物合成最好;ZnSO4和FeSO4的原浓度愈伤组织的生长量最大,而1／3浓度的绿原酸和总黄酮含量最高。     

扬子鳄的CaSox4基因的分子克隆和进化分析

The completely identical HMG-box motif of CaSox4 gene from both male and female genomic DNA of the Chinese alligator (alligator sinensis) was cloned and sequenced by degenerate primer PCR.Compared with the human and mouse SRY,CaSox4 revealed 51% and 57% nucleotide homology respectively and 49% and 55% amino acid identity respectively,CaSox4 belongs to subgroup C of the Sox gene family.The GC content is 86% in the HMG-box region of the CaSox4 gene,Blast analysis showed that the CaSox4 gene shares 100^ amino acid identity with human Sox4.bird SoxLF4,turtle Sra4 and lizard CvSox4 genes.Casox4 may be orthologous with the human SOx4 gene.This indicates that CaSox4 gene shows the remarkable evolutionary conservation during the evolution of Alligator Sinensis,The extensive sequence conservation of the Sox4 gene between reptiles,mammals and birds suggests major functional constraints[Acta Zoologica Sinica 49(3):404-407,2003].     

Preparation of alpha and beta anomers of 1,2,3,6-tetra-O-acetyl-4-chloro-4-deoxy-D-galactopyranose based upon anomerization and kinetic acetylation

4-Chloro-4-deoxy-alpha-d-galactopyranose, 1,2,3,6-tetra-O-acetyl-4-chloro-4-deoxy-alpha-d-galactopyranose and 1,2,3,6-tetra-O-acetyl-4-chloro-4-deoxy-beta-d-galactopyranose were readily prepared from 1,4:3,6-dianhydro-beta-d-fructofuranosyl 4-chloro-4-deoxy-alpha-d-galactopyranoside. In the study, we found an interesting anomerization phenomenon of 4-chloro-4-deoxy-d-galactose. The molar ratio of alpha and beta anomers in solution is about 1:2 when the anomerization reaches a dynamic equilibrium, and the beta anomer could completely convert to the alpha anomer in the process of crystallization and precipitation. The acetylation of 4-chloro-4-deoxy-d-galactopyranose is kinetically controlled, and the configuration of the starting galactose determines the configuration of the resulting acetates. The influence of the chloro group at C-4 and the O-acetyl group at the anomeric carbon on the galactopyranose ring conformations is discussed, based upon the crystallographic data for the alpha and beta anomers of 1,2,3,6-tetra-O-acetyl-4-chloro-4-deoxy-d-galactopyranose.     

Actions of synthetic leukotrienes on platelets and blood vessels in the anesthetised pig: the release of a platelet derived vasodilator

The actions of leukotrienes (LT's) C4, D4, E4 and F4 have been investigated in the perfused hind-limb of the anesthetized pig. In the blood perfused hind limb LTC4, D4 and E4 increased the perfusion pressure in a dose-dependent fashion whereas LTF4 decreased perfusion pressure. In the Tyrode perfused hind limb all LT's increased perfusion pressure (rank order potency LTC4 = LTD4 much greater than LTF4). The actions of LTF4 were not affected by a wide variety of pharmacological treatments, including indomethacin, methysergide and FPL-55712. The LT's aggregated porcine platelets (rank order potency LTC4 greater than LTF4 greater than LTD4) and induced the release of a platelet-derived vasodilatory mediator. The results provide pharmacological evidence of specific leukotriene receptors in vivo and that leukotrienes can independently modulate blood flow. These data suggest that important interactions may occur between platelets, the arachidonate lipoxygenase products and platelet-derived substances in response to inflammatory stimuli in the cardiovascular system.     

Prebiotic synthesis of imidazole-4-acetaldehyde and histidine

The prebiotic synthesis of imidazole-4-acetaldehyde and imidazole-4-glycol from erythrose and formamidine has been demonstrated as well as the prebiotic synthesis of imidazole-4-ethanol and imidazole-4-glycol from erythrose, formaldehyde and ammonia. The products were identified by TLC, HPLC, and LC-MS by comparison with authentic samples. The maximum yields of imidazole-4-acetaldehyde, imidazole-4-ethanol, and imidazole-4-glycol obtained in these reactions are 1.6, 5.4, 6.8% respectively, based on the erythrose. Imidazole-4-acetaldehyde would have been converted to histidine on the primitive earth by a Strecker synthesis, and several prebiotic reactions would convert imidazole-4-glycol and imidazole-4-ethanol to imidazole-4-acetaldehyde.     

Characteristics of formation and further metabolism of leukotrienes in the chopped human lung

The bronchoconstrictive leukotrienes (LTs) LTC4, LTD4 and LTE4 (cysteinyl-LTs) and the chemoattractant LTB4 were formed in chopped human lung stimulated by the calcium ionophore A23187, or supplied with the precursor LTA4. In contrast, challenge with anti-IgE exclusively induced release of cysteinyl-LTs, indicating that LTB4 is not released as a primary consequence of IgE-mediated reactions in the human lung. Furthermore, several differences were observed with respect to formation and further conversion of LTB4 and LTC4 in the chopped lung preparation. Thus, exogenous [1-14C]arachidonic acid was dose-dependently converted to radioactive LTB4, whereas the cysteinyl-LTs released were not radiolabeled and the amounts of LTC4, D4 and E4 were not influenced by addition of increasing concentrations of arachidonic acid. LTC4 was rapidly and completely converted into LTD4 and LTE4, with no further catabolism of LTE4 within 90 min. The metabolism of LTB4 was much slower than that of LTC4. Thus, following a 60 min incubation approx. 25% of the material remained as LTB4, whereas 35% was omega-oxidized and 40% eluted on RP-HPLC as two unidentified peaks.     

Oligosaccharide structure of human C4

The oligosaccharide structure of human C4 was studied by using C4 purified from plasma and C4 secreted by human hepatoma-derived cell line, HepG2. The alpha- and beta-chains of human C4 are glycosylated, whereas the gamma-chain is devoid of carbohydrate. The alpha-chain has three complex fucosylated oligosaccharides of the biantennary type, one each on the alpha 2, alpha 3, and alpha 4 fragments. The beta-chain has a single high mannose oligosaccharide primarily of the Man9GlcNAc2 type. The approximately 2000 Mr difference between the alpha-chains of the two C4 gene products (C4A and C4B) was localized to the alpha 2 fragment and is not due to carbohydrate. Sulfation of the C4 alpha-chain was localized to the alpha 4 fragment of the alpha-chain. Hence, the Mr difference between the two gene products is likely to reside in amino acid differences. The oligosaccharide structure of three incompletely processed C4 molecules was also analyzed. These molecules have the oligosaccharide composition of the appropriate individual subunits. Therefore, intracellular proteolytic processing to the multi-chain form of C4 is not required for proper oligosaccharide processing.     

Inactivation of cytochrome P450 and inhibition of ferrochelatase by analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine with 4-nonyl and 4-dodecyl substituents.

Cytochrome P450- and heme-destructive effects of the 4-nonyl and 4-dodecyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were determined using hepatic microsomal preparations obtained from untreated, beta-naphthoflavone-treated, and phenobarbital-treated chick embryos. The 4-nonyl analogue of DDC was less efficacious than 4-ethyl DDC and 4-hexyl DDC, but more efficacious than 4-dodecyl DDC with respect to cytochrome P450-destructive activity. In all hepatic microsomal preparations, cytochrome P450 destruction by 4-nonyl DDC was accompanied by loss of microsomal heme. In contrast, 4-dodecyl DDC caused loss of heme only in hepatic microsomal preparations obtained from phenobarbital-treated chick embryos. The ability of 4-nonyl DDC and 4-dodecyl DDC to lower ferrochelatase activity was compared with that of 4-ethyl DDC and 4-hexyl DDC in cultured chick embryo hepatocytes. As the length of the 4-alkyl group was increased, the ferrochelatase-lowering efficacy and potency of the DDC analogue decreased. The 4-dodecyl DDC analogue was unable to lower ferrochelatase activity, which accorded with the finding that the administration of 4-dodecyl DDC to phenobarbital-treated rats did not lead to the accumulation of an N-alkylprotoporphyrin. The ability of 4-nonyl DDC to lower ferrochelatase activity was attributed to the formation of N-nonylprotoporphyrin IX following the administration of 4-nonyl DDC to phenobarbital-treated rats.     

高油玉米优良自交系再生体系的建立

以供试的5个高油玉米优良自交系为材料,建立了一个高效的高油玉米幼胚再生体系.研究表明,高油玉米幼胚组织培养的最适幼胚长轴长度在0.5 mm～2.0 mm左右;MB培养基是最适的胚性愈伤组织诱导培养基;各材料胚性愈伤组织诱导率差异较大,以4K261和4K296的胚性愈伤诱导率较高;不同材料最适的继代培养条件存在差异,但基因型仍然是决定各自交系胚性愈伤组织的继代能力的主导因素,其中以4K261最佳.5个自交系均能分化出幼苗,但分化率差异较大,以4K059分化率最高,达82.0 %;其次是4K261和4K296,分别为63.2 %和59.0 %;4K060和4K061表现最差.所以4K059、4K261和4K296均可作为遗传转化的受体材料.该体系的建立为高油玉米的遗传转化奠定了基础.     

Kruppel样因子4对内毒素所致IL-6基因表达的调控及机制研究

探讨Kruppel样因子4(KLF4)对内毒素所致白介素(IL-6)的基因表达以及释放的影响,并对其调控机制做了初步研究．使用RT-PCR和Western blot检测KLF4 mRNA和蛋白质的表达．采用KLF4过表达的RAW264.7巨噬细胞株或反义寡核苷酸技术抑制内源性KLF4的表达,用RT-PCR和ELISA检测内毒素(LPS)刺激后IL-6 mRNA和蛋白质的表达．采用荧光素酶报告基因检测RAW264.7细胞中KLF4过表达对IL-6基因启动子报告基因转录活性的影响．使用EMSA法检测细胞中KLF4与IL-6基因启动子区KLF4元件的结合．结果表明：LPS可以诱导RAW264.7巨噬细胞KLF4的表达以及IL-6蛋白表达．KLF4过表达明显抑制IL-6的mRNA和蛋白质的表达,而KLF4缺失使这种作用消失．荧光素酶报告基因的结果显示,KLF4可以抑制LPS所致的IL-6基因启动子的转录活性．EMSA显示KLF4不能与IL-6启动子区的KLF4结合元件直接结合．结果表明,LPS可以促进RAW264.7小鼠巨噬细胞KLF4的表达和IL-6的释放．KLF4能抑制LPS诱导的IL-6表达和释放,其机制是抑制IL-6启动子的转录活性,但KLF4的抑制作用不是通过直接与IL-6基因的启动子区相结合而实现的．