巨噬细胞产生NO.和O_2~-自由基的分子机理

建立了用顺磁共振（ＥＳＲ）和化学发光技术测定巨噬细胞产生ＮＯ和氧自由基的方法．捕捉到了巨噬细胞受佛波酯刺激产生的ＮＯ．和Ｏ－２自由基．测定了在不同浓度Ｌ－精氨酸存在时佛波酯刺激后巨噬细胞产生的ＮＯ自由基．研究了巨噬细胞产生的ＮＯ和氧自由基的分子机理．结果表明巨噬细胞不仅产生氧自由基而且产生ＮＯ自由基．ＮＡＤＰＨ氧化酶产生氧自由基的部位位于巨噬细胞膜的外侧．ＮＯ合成酶活化产生ＮＯ自由基比ＮＡＤＰＨ氧化酶活化产生氧自由基晚几分钟．     

Pol β associated complex and base excision repair factors in mouse fibroblasts

During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an ‘affinity-capture’ procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3′-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3′-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3′-blocked intermediate.     

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA^M53I/A354V. Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.     

The posttranslational modification of HDAC4 in cell biology: Mechanisms and potential targets

Histone deacetylase 4 (HDAC4) is a member of the HDACs family, its expression is closely related to the cell development. The cell is an independent living entity that undergoes proliferation, differentiation, senescence, apoptosis, and pathology, and each process has a strict and complex regulatory system. With deepening of its research, the expression of HDAC4 is critical in the life process. This review focuses on the posttranslational modification of HDAC4 in cell biology, providing an important target for future disease treatment.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

Design and synthesis of highly selective pyruvate dehydrogenase complex E1 inhibitors as bactericides

In order to obtain PDHc-E1 inhibitors with high selectivity and efficacy, four series (7, 12, 15, and 19) of 35 novel 4-aminopyrimidine derivatives were rationally designed and synthesized based on the binding site of ThDP in E. coli PDHc-E1. 12, 15, and 19 were confirmed to be potent inhibitors against E. coli PDHc-E1. Selected compounds 12g, 12i, 15f, and 19a showed negligible inhibition against porcine PDHc-E1. To understand their selectivity, the interaction of inhibitor and E. coli PDHc-E1 or porcine PDHc-E1 was studied by molecular docking. The newly introduced acylhydrazone and N-phenylbenzamide moieties could form stronger interaction by hydrogen bond at the active site of E. coli PDHc-E1 compared with that of porcine PDHc-E1. A part of title compounds as potent PDHc-E1 inhibitors also exhibited notable antibacterial activity. In particular, 12e, 12f, 12g, 12o, and 19a exhibited 72–92% inhibition against Xanthomonas oryzae pv. Oryzae and Ralstonia solanacearum at 100?μg/mL, which was better than thiodiazole-copper (34 and 29%, respectively) and bismerthiazol (56 and 55%, respectively). The results proved that we could obtain effective bactericidal compounds as highly selective PDHc inhibitors by rational molecular design utilizing the binding model of active site of E. coli PDHc-E1.     

SAXSDom: Modeling multidomain protein structures using small-angle X-ray scattering data

Many proteins are composed of several domains that pack together into a complex tertiary structure. Multidomain proteins can be challenging for protein structure modeling, particularly those for which templates can be found for individual domains but not for the entire sequence. In such cases, homology modeling can generate high quality models of the domains but not for the orientations between domains. Small-angle X-ray scattering (SAXS) reports the structural properties of entire proteins and has the potential for guiding homology modeling of multidomain proteins. In this article, we describe a novel multidomain protein assembly modeling method, SAXSDom that integrates experimental knowledge from SAXS with probabilistic Input-Output Hidden Markov model to assemble the structures of individual domains together. Four SAXS-based scoring functions were developed and tested, and the method was evaluated on multidomain proteins from two public datasets. Incorporation of SAXS information improved the accuracy of domain assembly for 40 out of 46 critical assessment of protein structure prediction multidomain protein targets and 45 out of 73 multidomain protein targets from the ab initio domain assembly dataset. The results demonstrate that SAXS data can provide useful information to improve the accuracy of domain-domain assembly. The source code and tool packages are available at https://github.com/jianlin-cheng/SAXSDom .     

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20–80 min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA–primaquine conjugate (TriGalNAc-PGA–PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250 ng per million cells of the conjugate bound to one million rat hepatocytes at 0 °C, and approximately 2 μg per million cells of the conjugate was taken up over 7 h incubation at 37 °C. Furthermore, our results suggested that TriGalNAc-PGA–PQ was almost completely degraded over 24 h, and small degradation products were secreted into cell culture medium.The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.