Abstract:Whether germline stem cells exist in postnatal female mammal ovaries has been disputed for more than one hundred years and not conclusive yet. In 2004, researchers found and isolated female germline stem cells (FGSCs) from postnatal mice, which challenged the viewpoint of existing nearly half a century that the bank of oocytes in ovaries will be never renewed in postnatal mammal female. And then a lot of researches not only pointed out that the de novo oocytes derived from FGSCs, but also found that if the FGSCs were delivered back into adult ovaries, they were capable of generating functional eggs produced healthy offsprings. However, some groups repeated or designed experiments carefully, but they did not get the same or even opposite results. Recently, some researchers announced that they had purified FGSCs from ovaries of reproductive-age women, which could produce functional oocytes in vivo and in vitro, but the fertilization competency of these oocytes still need to be clarified. Therefore, this paper reviews the study history of mammalian FGSCs, the long-standing controversy as well as prospects for the future use of these FGSCs.

Abstract:Chromatin packages eukaryotic DNA, it can affect accessibility of DNA, thus chromatin has important roles in gene transcription regulation, DNA replication, DNA repair, and so on. The basic unit of chromatin is nucleosome consisting of ～147 DNA which sharply bends and tightly wraps on surface of octamer of histones. Between two nucleosomes is linker DNA. Histones can be modified by methylation and acetylation. Chromatin state (euchromatin and heterochromatin) is determined by the combinatorial effects of nucleosome positioning, histone modifications, and chromatin-binding proteins. Recently, by virtue of high throughput sequencing technique, the distributions of the chromatin marks were genome-widely determined in various cells. Results indicated that the distribution mode is specific at key sites of genome and is dynamically changed with both cell types and environment stimulus, showing a complex plot. In this paper, we detailedly reviewed the distribution modes of chromatin marks, biological meanings of the modes, interactions of the chromatin marks (nucleosome, DNA and histone modifications, histone variants, chromatin-binding proteins), techniques that were used to determine the marks, and the computational analysis for chromatin states. This work is important in understanding epigenetic regulation mechanism.

Abstract:Lewy bodies (LBs) in dopaminergic neurons in substantia nigra pars compacta (SNpc) are the cardinal pathological hallmark of Parkinson's disease (PD), while LBs and/ or Lewy neurites are also seen in non-dopaminergic neurons in other brain regions of patients with PD, such as noradrenergic neurons in locus coeruleus (LC), and cholinergic neurons in prefrontal cortex (PFC) and temporal cortex (TC). To explicit protein contents of LBs in PD, here is an article that demonstrates five aspects of protein constitution of LBs by analyzing protein bioinformatics datasets related to LBs in PD: (1) LBs is filled with 2 types of alpha-synuclein (α-SYN) positive fibrilar aggregates and/or 6 types of α-SYN positive non-fibrilar aggregates (described frequently in literature as oligomers); (2) modification forms of pathological α-SYN in LBs are often associated with 5 species of chemical modifiers; (3) 19 α-SYN-associated proteins rich in LBs are respectively colocalized with α-SYN; (4) 117 previously reported proteins in LBs in immunochemistry study are at least classified into 10 different groups of functional proteins with close relationship; (5) proteomics database as protein candidate resource for LBs contains 84, 124 and 120 proteins identified respectively from raw tissue samples of LC, SNpc and PFC regions, and 108 proteins identified from a cellular separation of analyte captured from TC region, and 29 proteins identified from a sub-cellular fraction of analyte isolated from TC region. It is suggested that the aforementioned aspects comprehensively and fundamentally reflect protein composition of LBs in PD.

Abstract:Quorum-sensing systems is critical regulator of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C₁₂-HSL) and N-butyryl-L-homoserine lactone (C₄-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C₁₂-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C₁₂-HSL to induce apoptosis in mice-derived macrophages (RAW264.7) and reduce phagocytosis of the cell. When RAW264.7 cells were incubated with synthetic 3-oxo-C₁₂-HSL, the significant loss of viability was observed in a concentration (6.25 to 100 μmol/L) and incubation time (2 to 24 h) dependent manner. The cytotoxic activity of 3-oxo-C₁₂-HSL was also observed in RAW264.7 cells. The cells treated with 3-oxo-C₁₂-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C₁₂-HSL-treated cells was confirmed by multiple criteria (caspases 3, 8 and 9, mitochondrial depolarization, phosphatidylserine expression). Phagocytosis of neutral red assay demonstrated that higher concentration of 3-oxo-C₁₂-HSL significantly reduced phagocytosis of RAW264.7 cells (P < 0.05). High concentration of 3-oxo-C₁₂-HSL also obviously lowered RAW264.7 cells for gobbling up of P. aeruginosa capability (P < 0.001). These data suggest that 3-oxo-C₁₂-HSL specifically promotes induction of apoptosis and reduces the phagocytosis of RAW264.7 cells, which may be associated with 3-oxo-C₁₂-HSL-induced cytotoxicity in RAW264.7 cells. Our data suggest that the quorum-sensing signal molecule 3-oxo-C₁₂-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors, but also in the modulation of host responses.

Abstract:Multiple sclerosis (MS), an autoimmune disease of the CNS with prominent demyelination and axonal degeneration, is more prevalent in women than men. EAE is a widely used animal model of MS for preclinical therapy development. In this report, a 29 days observation of the EAE model induced by MOG_33-35 in C57BL/6 mice was performed. From the clinical course, we found no significant difference between male and female mice in the incidence and onset time, but males had a more serious disease score than that in females. And histological examination of spinal cord was performed at day 21 post-immunization. Compared with male mice, female mice had a dramatic increase of leukocyte infiltration in the spinal cord. Luxol fast blue staining also revealed enhanced demyelination in male mice compared to female mice. The CNS leukocyte infiltration was also quantified by flow cytometry analysis at day 19 post-immunization. The results again confirmed that the CD4 T cells accumulated in the CNS of EAE mice were increased in male mice. We further quantified the number of T_H-1 and T_H-17 cells in the CNS infiltrates and found that the absolute number of both T_H-17 and T_H-1 cells were significantly increased in male mice. Taken together, these data indicate that sex difference exists in EAE induced by MOG_33-35 in C57BL/6 mice, and this will provided useful indications for further studies on the selection of animals in the EAE model.

Abstract:Fragile X syndrome (FXS) is a genetic mental retardation disease, with incidence second only to trisomy 21 syndrome. Fragile X mental retardation protein(FMRP), is the causative factor of FXS and encoded by the Fragile X mental retardation1(FMR1)gene, which is widely expressed in cells of the nerve, muscle, and testes. Fragile X related protein 1 (FXR1P) is encoded by a homologous gene to FMR1——Fragile X related gene 1 (FXR1) and can interact with proteins and RNAs. Many illnesses were involved in the altered expression of FXR1. To understand the biological effect of the interaction between FXR1P and CMAS, we constructed a FXR1 overexpression vector and investigated its expression in PC12 (the rat pheochromocytoma) cells and VSMC (vascular smooth muscle cell) and the effect of the overexpression on cell morphology and several cell processes related to CMP-N-acetylneuraminic acid synthetase (CMAS) activity. We demonstrate that the overexpression of FXR1 gene can increase activity of CMAS in PC12 cells and provide a certain degree growth protection for that cells. Thus, it suggests FXR1P is a tissue-specific regulator to alter the concentration of GM1 in PC12 cells, but not in VSMC.

Abstract:Better effective therapy for highly pathogenic influenza virus infection should consist of combinations of an effective antiviral agent and immunomodulatory agents to control viral replication and pulmonary injury, respectively. Here we evaluated the virus titres in different cells (A549 cells and MDCK cells) infected with influenza virus H5N1 by plaque assay with the various concentrations of Geldanamycin at different time points. The levels of proinflammatory cytokines from A549 cell infected with influenza virus H5N1were detected by ELISA in the absence or presence of Geldanamycin. Our findings showed that Geldanamycin significantly inhibited influenza virus growth in MDCK cells and A549 cells(P < 0.01). However, influenza virus growth were not inhibited with Geldanamycin on 36 h and 48 h(P > 0.05)after infection with influenza virus H5N1. Analysis of proinflammatory cytokines in Geldanamycin-treated A549 cells revealed significant reductions in IFN-α，TNF-α or IL-6 on 12 h and 24 h after infection with influenza virus H5N1(P < 0.05). All the results indicated that Geldanamycin have the dual effect of combine antivirus with anti-inflammation, which provided the science data for the potential use of Geldanamycin as a novel, highly effective anti-influenza virus drug.

Abstract:The transcription of plant pathogenesis related (PR) genes were known to be induced by pathogen invasion. Immerging evidences revealed that the transcription of PR genes also up-regulated under abiotic stresses, however, limited data for PR proteins expression was reported. To characterize the expression of rice PR proteins under stresses, we examined the expressions of eight PR proteins under cold, hot, drought, submerge and salt stresses at seedling stage by Western blotting (WB) via antibody-based proteomics strategy. The results showed that the expressions of PR8 was up-regulated under cold stress, the expressions of PR1a, PR3, PR5 and PR16 were down-regulated under hot stress. The expressions of PR1a, PR2 and PR8 were up-regulated while PR5 and PR16 were down-regulated under drought stress, the expressions of PR1, PR2 and PR15 were up-regulated and PR1a, PR3, PR5 and PR8 were down-regulated under submerge stress. The expressions of PR2 and PR3 were up-regulated while PR1a, PR5, PR8 and PR16 were down-regulated under salt stress. In addition, a number of stress responsive cis-elements, such as the ABRE, TC-rich repeats and HSE, were identified in the promoter region of PR genes. These data supported that PR proteins play important and specific roles in the process of stress tolerance.

Abstract:In E. coli, FabA, a bifunctional enzyme, is the key enzyme of the classic anaerobic pathway of unsaturated fatty acid synthesis and introduces the cis double bond into a 10-carbon intermediate. This intermediate is then elongated by FabB (one of long chain 3-ketoacyl-ACP synthases) to form the unsaturated fatty acids found in the membrane phospholipids. Sequence alignments indicated that Sinorhizobium meliloti SmFabA and SmFabB are 60.6% and 61.1% identical to E. coli FabA and FabB, respectively. Further analysis showed that the conservative active-site histidine residue in EcFabA and the Cys-His-His catalytic triads in EcFabB, are also found in SmFabA and SmFabB. The genetic complementary revealed that SmfabA is able to restore the growth and the fatty acid synthesis of the E. coli temperature sensitive mutant CY57 at nonpermissive temperature under addition low concentration triclosan to inhibit enoyl-acyl carrier protein reductases. Moreover, SmfabB is able to complement temperature sensitive mutant CY242. In vitro assay identifies that SmfabA, like E. coli FabA, is able to introduce the cis double bond into a 10-carbon intermediate, and SmfabB, like FabB, is able to condense the acyl-ACPs with malonyl-ACP to long-chain acyl-ACPs. However, we also attempted to inactivate the fabA and fabB genes by allelic replacement but none of the fabA and fabB deletion mutant was obtained, and it seemed likely that fabA and fabB are essential genes in S. meliloti. These results demonstrated that SmFabA and SmFabB are key enzymes in unsaturated fatty acid synthesis in S. meliloti.

Abstract:Dry bacteriorhodopsin (BR) film was fabricated into sandwich photocell and has differential photocurrent response to rectangular light pulse. And rectification was observed in the dry photocell of indium-tin oxide/BR film/Parafilm/stainless steel, but not of the steel/BR film/Parafilm/ ITO. This is an evidence of electrode-mediated rectification. Measurement of equilibrium potential suggests that the working electrode/BR film interface have different property from that of its counter electrode/Parafilm/BR film interface. The interfacial effect of the electrodes may dominate over that of the orientation of BR. Polarities of acid or base induced transient current confirmed the presence of rectification behavior of the electrode. The results will help to understand the mechanism of differential response of BR film.

Abstract:The 4-coumarate∶CoA ligase (4CL) is one of the most important enzymes of the plant specific phenylpropanoid pathway. It catalyzes the syntheses of coumaroyl-CoA thioesters, the precursors of lignin and other important phenylpropanoids, from corresponding coumarate compounds such as 4-coumaric acid, caffeic acid, and 3-methoxy-4-coumaric acid, ATP, and coenzyme A, in two-step reactions that involves the formation of coumarate-AMP anhydride as intermediates and subsequent formation of the the coumaroyl-CoA thioesters through the nucleophilic substitution of the AMP group by CoA. Because of the important roles played by 4CL in the biosynthesis of lignin, this protein has become the target of bioengineering aimed at improving the quality of plant products. We have solved the crystal structure of Populus tomentosa 4CL1 (Pt4CL1) complexed with its enzymatic intermediate 4-coumarate-AMP anhydride. The Pt4CL1 was found to adopt the conformation of the second step reaction based on comparisons with other members from the same superfamily. Structural analysis revealed that the residue His-234, which forms a hydrogen bond to the phosphate group of the 4-coumarate-AMP anhydride through its side chain imidazole group, plays essential roles in the enzymatic mechanisms of Pt4CL1, including facilitating both steps of the reactions by forming a hydrogen bond to, and reducing the negative charge of, the phosphate group; de-protonating the thiol group of CoA; and regulating the access of the active site to CoA through the conformational changes of its side chain. Enzymatic assays on Pt4CL1 and relevant mutant verified that His-234 is an essential residue of this enzyme.