小分子免疫调节剂 L—2—(3—羟基—4—羟甲基苯基)—甘氨酸前体物 DL—2—(3—羟基—4—羟甲基苯基)—N—乙酰基甘氨酸的合成

本文通过对小分子免疲调节剂L—2—(3—羟基—4—羟甲基苯基)—甘氨酸(英文名:Forphenicinol)前体物DL—2—(3—羟基—4—羟甲基苯基)—N—乙酰基甘氨酸的合成方法研究,探索了终产物Forphenicinol 的合成路线及实验方法、并通过对各步产物之红外光谱分析,确证各步产物之主要特征吸收峰,为今后的合成工作打下良好之基础、     

DL—1—(2,6—二甲基苯氧基)—2——氨基丙烷氨基酸衍生物的研究

<正> DL—1—(2,6—二甲基苯氧基)—乙—氨基丙烷盐酸盐,商品名为慢心律(Mcxiletine)是七十年代进行临床研究的一个抗心律失常药物。目前国内外广泛用于临床,其特点是可以长期口服治疗和预防室性心律失常。根据国内外近十年的临床观察,若用量超过600—800mg/日,则有明显副作用如神经兴奋、噁心、复视、抽搐、振颤等。为了寻找更理想的抗心律失常药物,国内外已做过一些慢心律的类似物供药理筛选研究。本文是从前体药物的角度以慢心律自由碱DL—1—(2,6—甲基苯氧基)—2—氨基丙     

新型O—连接糖基化—O—GlcNAc

一种新型糖基化,单糖Ｎ－乙酰葡萄糖胺通过Ｏ－连接与蛋白质共价相连,随着研究的不断深入,这种新型糖基化受到愈来愈多的重视,本主要论述它的发现,存在部位及可能的功能。     

越南——黄连国家公园

说起越南,国人头脑里首先出现的是下龙湾、芽庄、岘港这些海边的旅游胜地,而相比之下西方人则更钟情于越南北部大山里的一个小县——沙坝（Sa Pa）。这个被誉为亚洲的＂欧洲小镇＂＂越南丽江＂的地方不仅有美丽的法式风格建筑和独特的越南少数民族的风情,更著名的名片则是秀色可餐的黄连国家公园。     

水中精灵——热带鱼

一说起热带鱼,很多人脑海中就浮现出色彩斑斓的小鱼欢快游动的情景,但是这情景很可能是朦胧的,因为我们大多数人对于热带鱼都没有很清晰的概念,也可以说,热带鱼本身就没有一个明确的定义。     

非典—SARS—冠状病毒

2002年11月,一种神秘不明的疾病出现在我国广东省继而肆虐全球,世界上有27个国家和地区相继报道出现这种疫情。由于这种疾病的症状不同于典型肺炎,故初期称之为“非典型肺炎”(简称“非典”)并沿用至今。其学名应称为“严重急性呼吸综合征”(Severe Acute Respiratory Syndrome,     

Non-homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human-like enzymes with novel activities

In antiviral and cancer therapy, deoxyribonucleoside kinases (dNKs) are often the rate-limiting step in activating nucleoside analog (NA) prodrugs into their cytotoxic, phosphorylated forms. We have constructed libraries of hybrid enzymes by non-homologous recombination of the pyrimidine-specific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster; their low sequence identity has precluded engineering by conventional, homology-dependent shuffling techniques. From these libraries, we identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2',3'-didehydro-3'-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, our results exposed a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity.     

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.     

Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates

The 2′-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2′-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level.     

Structural and mechanistic conservation in DNA ligases

DNA ligases are enzymes required for the repair, replication and recombination of DNA. DNA ligases catalyse the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA. Despite their occurrence in all organisms, DNA ligases show a wide diversity of amino acid sequences, molecular sizes and properties. The enzymes fall into two groups based on their cofactor specificity, those requiring NAD⁺ for activity and those requiring ATP. The eukaryotic, viral and archael bacteria encoded enzymes all require ATP. NAD⁺-requiring DNA ligases have only been found in prokaryotic organisms. Recently, the crystal structures of a number of DNA ligases have been reported. It is the purpose of this review to summarise the current knowledge of the structure and catalytic mechanism of DNA ligases.     

Enzymes from extremophiles

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.     

An early divergence of KhoeSan ancestors from those of other modern humans is supported by an ABC-based analysis of autosomal resequencing data

Sub-Saharan Africa has consistently been shown to be the most genetically diverse region in the world. Despite the fact that a substantial portion of this variation is partitioned between groups practicing a variety of subsistence strategies and speaking diverse languages, there is currently no consensus on the genetic relationships of sub-Saharan African populations. San (a subgroup of KhoeSan) and many Pygmy groups maintain hunter-gatherer lifestyles and cluster together in autosomal-based analysis, whereas non-Pygmy Niger-Kordofanian speakers (non-Pygmy NKs) predominantly practice agriculture and show substantial genetic homogeneity despite their wide geographic range throughout sub-Saharan Africa. However, KhoeSan, who speak a set of relatively unique click-based languages, have long been thought to be an early branch of anatomically modern humans based on phylogenetic analysis. To formally test models of divergence among the ancestors of modern African populations, we resequenced a sample of San, Eastern, and Western Pygmies and non-Pygmy NKs individuals at 40 nongenic (～2 kb) regions and then analyzed these data within an Approximate Bayesian Computation (ABC) framework. We find substantial support for a model of an early divergence of KhoeSan ancestors from a proto-Pygmy-non-Pygmy NKs group ～110 thousand years ago over a model incorporating a proto-KhoeSan-Pygmy hunter-gatherer divergence from the ancestors of non-Pygmy NKs. The results of our analyses are consistent with previously identified signals of a strong bottleneck in Mbuti Pygmies and a relatively recent expansion of non-Pygmy NKs. We also develop a number of methodologies that utilize "pseudo-observed" data sets to optimize our ABC-based inference. This approach is likely to prove to be an invaluable tool for demographic inference using genome-wide resequencing data.     

Bacterial metabolism of polychlorinated biphenyls

Microbial metabolism is responsible for the removal of persistent organic pollutants including PCBs from the environment. Anaerobic dehalogenation of highly chlorinated congeners in aquatic sediments is an important process, and recent evidence has indicated that Dehalococcoides and related organisms are predominantly responsible for this process. Such anaerobic dehalogenation generates lower chlorinated congeners which are easily degraded aerobically by enzymes of the biphenyl upper pathway (bph). Initial biphenyl 2,3-dioxygenases are generally considered the key enzymes of this pathway which determine substrate range and extent of PCB degradation. These enzymes have been subject to different protein evolution strategies, and subsequent enzymes have been considered as crucial for metabolism. Significant advances have been made regarding the mechanistic understanding of these enzymes, which has also included elucidation of the function of BphK glutathione transferase. So far, the genomes of two important PCB-metabolizing organisms, namely Burkholderia xenovorans strain LB400 and Rhodococcus sp. strain RHA1, have been sequenced, with the rational to better understand their overall physiology and evolution. Genomic and proteomic analysis also allowed a better evaluation of PCB toxicity. Like all bph gene clusters which have been characterized in detail, particularly in strains LB400 and RHA1, these genes were localized on mobile genetic elements endowing single strains and microbial communities with a high flexibility and adaptability. However, studies show that our knowledge on enzymes and genes involved in PCB metabolism is still rather fragmentary and that the diversity of bacterial strategies is highly underestimated. Overall, metabolism of biphenyl and PCBs should not be regarded as a simple linear pathway, but as a complex interplay between different catabolic gene modules.     

Filamentous fungi as cell factories for heterologous protein production

Filamentous fungi have been used as sources of metabolites and enzymes for centuries. For about two decades, molecular genetic tools have enabled us to use these organisms to express extra copies of both endogenous and exogenous genes. This review of current practice reveals that molecular tools have enabled several new developments. But it has been process development that has driven the final breakthrough to achieving commercially relevant quantities of protein. Recent research into gene expression in filamentous fungi has explored their wealth of genetic diversity with a view to exploiting them as expression hosts and as a source of new genes. Inevitably, the progress in the 'genomics' technology will further develop high-throughput technologies for these organisms.     

Mitochondrial and herpesvirus-specific deoxypyrimidine kinases.

To characterize and compare the thymidine (TdR) and deoxycytidine (CdR) kinase isozymes of uninfected and herpesvirus-infected cells: (i) the subcellular distribution of the isozymes has been studied; (ii) a specific assay for CdR kinase has been devised; (iii) the TdR kinase isozymes have been partially purified; and (iv) the purified enzymes have been analyzed by disc polyacrylamide gel electrophoresis, isoelectric focusing, and glycerol gradient centrifugation and by substrate competition and dCTP inhibition studies. The results indicate that there are interesting individual differences with respect to nucleoside acceptor specificity between the cytosol and mitochondrial pyrimidine deoxyribonucleoside kinases of uninfected cells and between the enzymes induced by different herpesviruses. In the cytosol of uninfected mouse, chicken, and owl monkey kidney cells, two different proteins, TdR kinase F and CdR kinase 2, catalyze the phosphorylations of TdR and CdR, respectively. TdR kinase F does not phosphorylate CdR, nor does CdR kinase 2 phosphorylate TdR. A second TdR kinase isozyme present in HeLa(BU25) mitochondria (TdR kinase B) also lacks CdR phosphorylating activity. In contrast, a genetically distinctive deoxypyrimidine kinase (TdR kinase A) of mouse, human, and chick mitochondria catalyzes the phosphorylation of both TdR and CdT. Three herpesviruses, marmoset herpesvirus and herpes simplex virus types 1 and 2, induce in the cytosol fraction of LM(TK-) mouse cells isozymes which share common properties with mitochondrial TdR kinase A, including the ability to catalyze the phosphorylation of both TdR and CdR. However, the herpesvirus-induced deoxypyrimidine kinases differ from mitochondrial TdR kinase A with respect to sedimentation coefficient, sensitivity to dCTP inhibition, and antigenic determinants. The herpesvirus-specific and the mitochondrial deoxypyrimidine kinases exhibit a preference for TdR over CdR as nucleoside acceptor. Pseudorabies virus and herpesvirus of turkeys induce cytosol TdR kinases resembling the other herpesvirus-induced TdR kinases in several properties, but like cellular TdR kinase F, the pseudorabies virus and herpesvirus of turkeys TdR kinases lack detectable CdR phosphorylating activities. Finally, a marmoset herpesvirus nutant resistant to bromodeoxyuridine, equine herpesvirus type 1, and Herpesvirus aotus induces neither TdR nor CdR phosphorylating enzymes during productive infections.     

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.