从天然提取物或分离部位中以烯醇式丙酮酸转移酶为靶点的抗细菌活性筛选

目的是以烯醇式丙酮酸转移酶(EPT)为靶点筛选其抑制剂,以期寻找抗细菌活性样品。实验是在96％孔酶标板上对来源于169个科、560个属、916种动植物2490个提取物或分离部位样品在EPT模型上进行了批量筛选。结果表明在96.15μg/ml浓度下发现了来源于80个科、169个届、218个种的309个样品有活性,其中14个样品的IC50小于10．00μg/ml,40个样品的IC50在10．01-30．00μg/ml范围,83个样品的IC50在30．01—50．00μg/ml范围,172个样品的IC50在50．01—96.15μg/ml范围。通过以上工作我们认为以烯醇式丙酮酸转移酶为分子靶点的体外筛选方法稳定、方便、快速、微量、有效,特别适用于天然产物的抗细菌活性筛选。     

组织蛋白酶B的新型天然抑制剂

组织蛋白酶B是木瓜蛋白酶类半胱氨酸蛋白酶家族的重要成员,它与人类多种疾病相关,尤其是在恶性肿瘤的侵袭转移过程中扮演了重要角色.通过随机筛选,发现了五个对组织蛋白酶B具有较好抑制活性的天然化合物prodelphinidin B-23'-O-gallate(1),prodelphinidin B-2(2),ImJcyarddin B-2(3),puexin A(4)和(-)epigallocatechin-3-O-gallate(5),其IC50值分别为0.58,0.44,0.76,2.07和0.96umol/L.这五个抑制剂为黄烷醇类化合物,均为组织蛋白酶B的新型天然抑制剂.     

抗细菌药物默诺霉素的化学生物学研究进展

默诺霉素(moenomycins)家族类化合物主要是由链霉菌产生,属于磷酸糖脂类抗生素.该类化合物通过与细菌细胞壁肽聚糖糖基转移酶(peptidoglycan transferase,PGT)的活性位点结合,可以抑制众多革兰氏阳性细菌细胞壁的合成,具有很强的生物活性和重要的应用开发潜力.本文针对默诺霉素的化学结构、生物...     

几类天然产物对纤溶系统的作用

在初筛天然来源的小分子ＰＡＩ－１抑制剂过程中发现一些天然产物显示出活性苗头,进一步试验和选择性试验表明这些化合物都不同程度别对纤溶系统中存在的不同的酶具有抑制作用。化合物１（ｅｘｐｏｘｙｒｏｌｉｎ Ｂ）对纤溶酶有一定的抑制,化合物２（ｍｕｒｉｃａｔｉｎｃ）对ＰＡＩ－１具有一定的抑制作用且有较好的选择性。化合物４（ｃｈｅｒｉｍｏｌｉｎ－２）对纤溶酶较强的抑制。化合物５（ｍｏｒｉｎｄｏｎｅ）对凝血酶、     

植物来源的蛋白磷酸酶Cdc25C的新抑制剂

蛋白磷酸酶Cdc25C能够使有丝分裂激酶CDK1/cyclin B去磷酸化,从而促进细胞周期的进程.已经在一些肿瘤细胞中检测到Cdc25C的过量表达,这使得Cde25C成为肿瘤治疗中的潜在靶标.通过随机筛选,发现了八个CAe25C的天然新抑制剂(1-8),其IC50值在1.66～75.07umol/L之间.肿瘤细胞毒试验结果表明,其中四个化合物(化合物3,4,5,7)对十种肿瘤细胞株显示一定的细胞毒活性,其IC50值皆小于10ug/ml.     

海洋抗肿瘤活性大环内酯类化合物化学成分研究近况

最近,人们已从多种海洋生物中发现许多具有应用价值的生理活性物质。其中,抗肿瘤活性物质的开发研究尤为盛行。目前,人们已经分离得到许多在体内试验中具有明显抗肿瘤活性的化合物,并且确立了它们的化学结构,其中包括类萜类化合物、生物碱,肽类化合物、大环内酯类化合物、类前列腺素化合物、聚醚类化合物等。其中,已有数种大环内酯类化合物很有可能作为抗癌药物进入临床。bryostatin 1则是它们的代表性化合物之一。目前,美国National Cancer Instituie已经开始对该化合物进行临床研究。它是由作者(釜野)研究小组在美国亚利桑拿州立大学癌研究所开发研究出的一种新型化合物,经过进一步研究又分离得到了bryostatin 2(24)～13(35),并确立了它们的化学结构。因此,本文将对bryostatin类化合物的化学性质以及其生理活性作详细地综述。另外,还将论述halichondrin类化合物(14～21)和Aplyronine(22)。它们是最近几年在日本发现的,在体内试验中具有很强的抗肿瘤活性的一系列新型化合物。再者,本文还将概述其它一些重要的、具有抗肿瘤活性的大环内酯类化合物,即aplysiatoxin(Ia～Ic), latrunculinA(2), acutiphycin(3),tedanolide(4), swinholide A(5), bistheonellde A(7a), amphicinolides(8～10), kabiramides(11a 11e), ulapualide(12a, 12b)以及patellazole B(13)。     

植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用

植物磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)是广泛存在的一种细胞质酶,催化磷酸烯醇式丙酮酸(PEP)和HCO3-生成草酰乙酸(OAA),后者可转化生成三羧酸循环的多种中间产物.PEPC在植物细胞中参与植物的光合碳同化等重要代谢途径,并且在不同组织中具有多种生理功能.PEPC同时也参与调控植物种子的营养物质合成与代谢过程,控制糖类物质流向脂肪酸合成或蛋白质合成途径.以下介绍了植物PEPC的种类、蛋白质结构特点及其在植物组织中的调控方式,并重点论述了PEPC在生物基因工程中的应用方面的进展,随着对其功能机制和应用研究的深入,将有助于植物PEPC在高产优质农作物育种、能源植物和工业微生物等的开发利用等方面得到更好的发展与应用.     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

产琥珀酸重组大肠杆菌的发酵性能研究

研究了重组大肠杆菌JM001(△ppc)/pTrc99a-pck发酵产琥珀酸的性能,结果表明厌氧条件下其耗糖能力和产酸能力分别为对照菌株JM001的4.2倍和15.3倍。进一步优化发酵条件表明：采用接入菌泥的发酵方式比按照10%接种量转接厌氧发酵的效果要好,琥珀酸的对葡萄糖的质量收率提高了约10%,且副产物乙酸的量进一步降低。初始葡萄糖浓度高于60g/L时会对菌株的生长和产酸产生抑制,且浓度越高,抑制作用越明显。7L发酵罐放大实验中,整个厌氧发酵阶段葡萄糖的消耗速率为0.42g/(L.h),琥珀酸对葡萄糖的质量收率为67.75%,琥珀酸的生产强度为0.28g/(L.h)。     

水分胁迫对露花叶片中PEP羧化酶活性及其调节特性的影响

水分胁迫期间露花叶片中PEP羧化酶的活力随胁迫时间的延长明显增加,复水后PEPC同工酶的活力下降。从露花叶片中分离到3个具有不同动力学和物理学特性的PEPC同工酶(PCⅠ,PCⅡ,PCⅢ),其中同工酶PCⅠ只存在于水分胁迫下露花叶片中,复水后消失。 这3个同工酶的K_m(PEP)值不相同;PCⅠ的K_m(PEP)值介于PCⅡ与PCⅢ之间,它在PAGE上的相对迁移率(Rm)比PCⅡ和PCⅢ大,对效应剂G—6—P及Mal的反应不敏感,分子量为PCⅡ之半;PCⅡ被G—6—P激活和被Mal抑制的程度介于PCⅠ与PCⅢ之间,它在PAGE上的相对迁移率和分子量与PCⅢ极相近。     

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRS_Mabs and LeuRS_Mtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.     

Expressed peptide tags: an additional layer of data for genome annotation

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here, we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six-frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well-annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed a high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well-annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller subdatabases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While approximately 76% of Phytophthora EPTs supported the current annotation, a portion of them (7.7% and 12.9% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.     

Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model

Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.     

EPT昆虫群落分布与环境因子的相关性

为了研究EPT昆虫与水环境因子的相关性,在2006年6-7月和9-10月分季在丹江口水库两条主水源河流上分设5个采样点,对EPT昆虫和水质采样检测。结果共检测到EPT昆虫780头,为12科16种(或种团),水质理化指标7项。简单相关分析与复相关分析都表明,氮浓度、磷浓度、生化需氧量与EPT昆虫密度呈极显著正相关（P<0.01）,与EPT昆虫的种数呈极显著负相关（P<0.01）。典型相关分析表明,EPT昆虫组与环境因子组有极显著的正相关（P<0.0001）,EPT昆虫组主要目为蜉蝣目和襀翅目,环境因子组主要因子为氮浓度、磷浓度、生化需氧量。由此得出：即使在低污染、低营养程度的水环境下,EPT昆虫与环境因子也表现出群体的显著相关性,环境因子对EPT昆虫分布有重要影响。     

Inhibition of Cottonseed Choline- and Ethanolaminephosphotransferases by Calcium during Postgerminative Growth

Activities of choline- and ethanolaminephosphotransferase (CPT and EPT) were reproducibly high in microsomes from imbibed seeds of cotton (Gossypium hirsutum, L.). Initial studies showed that both activities dramatically declined during postgerminative growth when demand for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis was high. Addition of CaCl₂ (0.1 millimolar) or aliquots of supernatant fractions (150,000g, 60 minutes) from cotyledons of 48-hour-old seedlings to imbibed-seed microsomes reduced the CPT and EPT activities to levels approximating those found in 48-hour microsomes. Inhibition by supernatants was completely reversed by adding EGTA (1.0 millimolar), but not by boiling the supernatants. EGTA (1.0 or 5.0 millimolar) relieved inhibition in cellular fractions whether it was added to the homogenization media or the assay reaction mixtures. A time course of CPT and EPT activities in cellular fractions prepared with 1.0 millimolar EGTA showed that activities were well developed in imbibed seeds, doubled coincidentally to a peak at 36 hours, then declined during the next 12 hours to levels approximating those in imbibed seeds. Greater than 90% of the CPT and EPT activities were pelletable (150,000g, 60 minutes) at all ages examined. Calcium apparently was artificially released upon homogenization, to a progressively greater extent in older cotyledons, and severely inhibited CPT and EPT activities. This is the only time course of CPT and EPT activities reported for cotyledons of any oilseed; it is substantially different from that in oil-storing endosperm.     

Epithelial to mesenchymal transition of a primary prostate cell line with switches of cell adhesion modules but without malignant transformation

Background

Epithelial to mesenchymal transition (EMT) has been connected with cancer progression in vivo and the generation of more aggressive cancer cell lines in vitro. EMT has been induced in prostate cancer cell lines, but has previously not been shown in primary prostate cells. The role of EMT in malignant transformation has not been clarified.

Methodology/Principal Findings

In a transformation experiment when selecting for cells with loss of contact inhibition, the immortalized prostate primary epithelial cell line, EP156T, was observed to undergo EMT accompanied by loss of contact inhibition after about 12 weeks in continuous culture. The changed new cells were named EPT1. EMT of EPT1 was characterized by striking morphological changes and increased invasion and migration compared with the original EP156T cells. Gene expression profiling showed extensively decreased epithelial markers and increased mesenchymal markers in EPT1 cells, as well as pronounced switches of gene expression modules involved in cell adhesion and attachment. Transformation assays showed that EPT1 cells were sensitive to serum or growth factor withdrawal. Most importantly, EPT1 cells were not able to grow in an anchorage-independent way in soft agar, which is considered a critical feature of malignant transformation.

Conclusions/Significance

This work for the first time established an EMT model from primary prostate cells. The results show that EMT can be activated as a coordinated gene expression program in association with early steps of transformation. The model allows a clearer identification of the molecular mechanisms of EMT and its potential role in malignant transformation.     

Influence of morphohydraulic habitat structure on invertebrate communities (Ephemeroptera,Plecoptera and Trichoptera)

Fluvial geomorphology proposes the methodology of cognition and assessment of the riverine landscape and points to the possibilities of exploitation of its results in hydrobiological research. Habitat structure of two reaches of the Drietomica brook (Biele Karpaty Mts, Slovakia) was assesed at level of morphological and morphohydraulic units in the sense of the River Morphology Hierarchical Classification (RMHC)). Physical habitats were described by flow hydraulics and substrate properties as directly measured variables (current velocity, depth, substrate size) and related variables (flow type, Froude and Reynolds numbers). According to the shear stress (expressed by Fr and Re), the morphological units were divided into two main groups — with low shear stress — pools, glides, edgewaters, bar nooks and bars; with high shear stress — riffles, runs, rapids and scours; characterized also by different Ephemeroptera, Plecoptera and Trichoptera (EPT) communities. The EPT communities were analyzed in relation to the morphological, hydraulic and substrate characteristics of the stream channel. The main environmental gradient responsible for the variation in EPT fauna was found using Principal Component Analysis and was related to gradient of flow in term of current velocity and other hydraulic attributes covered by Fr and Re numbers. The EPT communities (by means of abundance, feeding types, current, microhabitat and zonation preferences) showed preferences for different morphological units, flow type and current velocity. Depth and substrate grain size showed only weak relation to EPT communities.     

The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae. Isolation of mutants and cloning of the EPT1 gene

A colony autoradiographic assay was used to identify nine Saccharomyces cerevisiae mutants defective in in situ ethanolaminephosphotransferase activity (ept mutants). Genetic analysis revealed five complementation groups. The EPT1 gene was cloned by complementation of ept1 using a yeast genomic library and was localized to a 2.1-kilobase region of DNA. An ept1 deletional mutant was constructed and introduced into the chromosome by integrative transformation. The ethanolaminephosphotransferase activities in membranes prepared from ept1 and ept2 mutants were reduced 30- to 90-fold and 2- to 3-fold compared with wild-type activity, respectively; the other ept mutants had activities similar to wild type. In strains transformed with a multicopy EPT1-bearing plasmid, a 22- to 33-fold overproduction of ethanolaminephosphotransferase activity was observed. The sn-1,2-diacylglycerol cholinephosphotransferase activities in membranes prepared from ept1 mutants were reduced 3.5- to 7-fold. In contrast to the residual CMP-sensitive cholinephosphotransferase activity observed in cpt1 mutants (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), the residual cholinephosphotransferase activity of ept1 mutants was CMP-insensitive. The cholinephosphotransferase activities in strains bearing the EPT1 gene on multicopy plasmids were elevated 13- to 23-fold and were CMP-sensitive. The data indicate that 1) the cloned EPT1 gene most likely represents the structural gene for the yeast ethanolaminephosphotransferase, 2) the EPT1 gene product possesses both ethanolamine- and cholinephosphotransferase activities, and 3) the EPT1 gene is nonessential for growth.     

Habituation to Experimentally Induced Electrical Pain during Voluntary-Breathing Controlled Electrical Stimulation (BreEStim)

Objective

Painful peripheral electrical stimulation to acupuncture points was found to cause sensitization if delivered randomly (EStim), but induced habituation if triggered by voluntary breathing (BreEStim). The objective was to systematically compare the effectiveness of BreEStim and EStim and to investigate the possible mechanisms mediating the habituation effect of BreEStim.

Methods

Eleven pain-free, healthy subjects (6 males, 5 females) participated in the study. Each subject received the BreEStim and EStim treatments in a random order at least three days apart. Both treatments consisted of 120 painful but tolerable stimuli to the ulnar nerve at the elbow on the dominant arm. BreEStim was triggered by voluntary breathing while EStim was delivered randomly. Electrical sensation threshold (EST) and electrical pain threshold (EPT) were measured from the thenar and hypothenar eminences on both hands at pre-intervention and 10-minutes post-intervention.

Results

There was no difference in the pre-intervention baseline measurement of EST and EPT between BreEStim and EStim. BreEStim increased EPT in all tested sites on both hands, while EStim increased EPT in the dominant hypothenar eminence distal to the stimulating site and had no effect on EPT in other sites. There was no difference in the intensity of electrical stimulation between EStim and BreEStim.

Conclusion

Our findings support the important role human voluntary breathing plays in the systemic habituation effect of BreEStim to peripheral painful electrical stimulation.