The Mitochondrial Protein Translocation Motor: Structural Conservation between the Human and Yeast Tim14/Pam18-Tim16/Pam16 co-Chaperones

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing Saccharomyces cerevisiae as a model system. Recently, several mammalian homologues of the mitochondrial motor proteins were identified. Of particular interest for us is the human Tim14/Pam18-Tim16/Pam16 complex. We chose a structural approach in order to examine the evolutionary conservation between yeast Tim14/Pam18-Tim16/Pam16 proteins and their human homologues. For this purpose, we examined the structural properties of the purified human proteins and their interaction with their yeast homologues, in vitro. Our results show that the soluble domains of the human Tim14/Pam18 and Tim16/Pam16 proteins interact with their yeast counterparts, forming heterodimeric complexes and that these complexes interact with yeast mtHsp70.     

发酵液中FDP的酶法分析

利用三种酶.即3-磷酸甘油脱氢酶、磷酸丙糖异构酶和醛缩酶,通过测定还原性辅酶1吸光值的变化来测定发酵液中FDP(1,6-二磷酸果糖)的含量。同时研究了pH、温度、pH对酶法测定结果的影响及最佳酶的加量。对FDP标准曲线进行了统计处理,结果表明:采用酶法测定FDP,其线性关系好.专一性强。对样品的重复测定表明,其相对测定相对偏差下超过0.6%,回收率101     

16％辛·氧乐乳油的毒性研究

叙述了辛硫磷．氧化乐果混合乳油的毒性,即混合乳油的LD50和LC50。     

Folding and Biogenesis of Mitochondrial Small Tim Proteins

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.     

Stereospecific synthesis of chiral 2,3-dihydro-1,4-benzodithiine and methyl-2,3-dihydro-1,4-benzodithiine derivatives and their toxic effects on Trypanosoma brucei

Preparation of chiral 2,3-dihydro-1,4-benzodithiine and methyl-2,3-dihydro-1,4-benzodithiine derivatives with known absolute configurations from the easily accessible chiral synthons benzyl 4-O-trifloxy-2,3-anhydro-beta-L-ribopyranoside and benzyl 4-O-trifloxy-2,3-anhydro-alpha-D-ribopyranoside is described. These compounds showed significant in vitro toxicity of the bloodstream form of Trypanosoma brucei with an IC50 of 11 microM. The parasites' energy metabolism and consumption of oxygen were found to be affected during incubation.     

应用细胞法代替小鼠法检测狂犬病毒毒力

目的应用细胞法代替小鼠法检测狂犬病毒毒力。方法将34批病毒样品采用小鼠法检测半数致死量LD50。同时,将病毒样品感染BSR细胞,检测病毒的荧光灶单位(FFU/ml)。结果用细胞法所得的logFFU值与对应的小鼠法所得LD50之间呈正相关性;分次测得同一狂犬病毒logFFU值在3·03~3·28之间,而logLD50值在2~3之间。结论细胞法代替小鼠法检测狂犬病毒的毒力是可行的。     

Discovery of Inhibitors of Trypanosoma brucei by Phenotypic Screening of a Focused Protein Kinase Library

A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm . Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H‐imidazo[4,5‐b]pyrazin‐2(3H)‐one scaffold that showed sub‐micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold‐hopping exercise led to the identification of a 1H‐pyrazolo[3,4‐b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H‐imidazo[4,5‐b]pyrazin‐2(3H)‐one series were found to be either static or growth‐slowing and not cidal. Compounds with the 1H‐pyrazolo[3,4‐b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.     

Antibodies against Mucin‐Based Glycopeptides Affect Trypanosoma cruzi Cell Invasion and Tumor Cell Viability

This study describes the synthesis of glycopeptides NHAc[βGal]‐(Thr)₂‐[αGalNAc]‐(Thr)₂‐[αGlcNAc]‐(Thr)₂Gly‐OVA ( 1 ‐OVA) and NHAc[βGal‐αGalNAc]‐(Thr)₃‐[αLacNAc]‐(Thr)₃‐Gly‐OVA ( 2 ‐OVA) as mimetics of both T. cruzi and tumor mucin glycoproteins. These glycopeptides were obtained by solid‐phase synthesis, which involved the prior preparation of the protected glycosyl amino acids αGlcNAc‐ThrOH ( 3 ), αGalNAc‐ThrOH ( 4 ), βGal‐ThrOH ( 5 ), αLacNAc‐ThrOH ( 6 ), and βGal‐αGalNAc‐ThrOH ( 7 ) through glycosylation reactions. Immunizations of mice with glycopeptides 1 ‐OVA and 2 ‐OVA induced high antibody titers (1:16 000), as verified by ELISA tests, whereas flow cytometry assays showed the capacity of the obtained anti‐glycopeptides 1 ‐OVA and 2 ‐OVA antibodies to recognize both T. cruzi and MCF‐7 tumor cells. In addition, antisera induced by glycopeptides 1 ‐OVA and 2 ‐OVA were also able to inhibit T. cruzi fibroblast cell invasion (70 %) and to induce antibody‐mediated cellular cytotoxicity (ADCC) against MCF‐7 cells, with 50 % reduction of cell viability.     

Design, synthesis and biological evaluation of Trypanosoma brucei trypanothione synthetase inhibitors

Trypanothione synthetase (TryS) is essential for the survival of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis. It is one of only a handful of chemically validated targets for T. brucei in vivo. To identify novel inhibitors of TbTryS we screened our in-house diverse compound library that contains 62,000 compounds. This resulted in the identification of six novel hit series of TbTryS inhibitors. Herein we describe the SAR exploration of these hit series, which gave rise to one common series with potency against the enzyme target. Cellular studies on these inhibitors confirmed on-target activity, and the compounds have proven to be very useful tools for further study of the trypanothione pathway in kinetoplastids.     

Synthesis, Cruzain docking, and in vitro studies of aryl-4-oxothiazolylhydrazones against Trypanosoma cruzi

Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a valid chemotherapeutic target. Herein we describe a small library of aryl-4-oxothiazolylhydrazones that have been tested in assays against T. cruzi cell cultures. The docking studies carried out suggest that these compounds are potential ligands for the TCC enzyme. The most promising compound of this series, N-(4-oxo-5-ethyl-2'-thiazolin-2-yl)-N'-phenylthio-(Z)-ethylidenehydrazone (6 f), was shown to be very active at non-cytotoxic concentrations in in vitro assays with mammalian cells and has a potency comparable with reference drugs such as nifurtimox (Nfx) and benznidazole (Bdz).