Synthesis,Stability and Direct Antiproliferative Effect of New Cysteine Modified GnRH Analogs

International Journal of Peptide Research and Therapeutics - It is demonstrated that gonadotropin-releasing hormone (GnRH) analogs can directly inhibit the proliferation of reproductive tissue...     

Late-acting self-incompatibility in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The self-incompatibility of tea plant (Camellia sinensis (L.) O. Kuntze) was studied with the methods of aniline blue fluorescence assay and paraffin sections. The characteristics of pollen tube elongation after hand pollination was analyzed in 4 tea cultivars, including ‘Keemenzhong’, ‘Longjing-changye’, ‘Fuding-dabaicha’ and ‘Yabukita’, under self-pollination and cross-pollination, respectively. Although there were some difference among cultivars, pollen tubes elongated through the style and reach the ovary successfully at 48 h after pollination for both cross- and self-pollen tubes in all the four cultivars of tea. Pollen tubes entered into the ovule micropyles, however, only for cross-pollination, but not for self-pollination. Pollen tubes of selfing plants, failed in fertilizing, seemed have some difficulties to enter the ovule. All of which indicated that the self-incompatibility of tea plant is a late-acting self-incompatibility system (LSI) or an ovarian sterility (OS), in which the self incompatibility was due to none self pollen tube penetrating into the ovule and no fertilization.     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

Molecular dynamics simulations on the oligomer-formation process of the GNNQQNY peptide from yeast prion protein Sup35

Oligomeric intermediates are possible cytotoxic species in diseases associated with amyloid deposits. Understanding the early steps of fibril formation at atomic details may provide useful information for the rational therapeutic design. In this study, using the heptapeptide GNNQQNY from the yeast prion-like protein Sup35 as a model system, for which a detailed atomic structure of the fibril formed has been determined by x-ray microcrystallography, we investigated its oligomer-formation process from monomer to tetramer at the atomistic level by means of a molecular dynamics simulation with explicit water. Although the number of simulations was limited, the qualitative statistical data gave some interesting results, which indicated that the oligomer formation might start from antiparallel beta-sheet-like dimers. When a new single peptide strand was added to the preformed dimers to form trimers and then tetramers, the transition time from disorder aggregates to regular ones for the parallel alignment was found to be obviously much less than for the antiparallel one. Moreover, the parallel pattern also statistically stayed longer, providing more chances for oligomer extending, although the number of parallel stack events was almost equal to antiparallel ones. Therefore, our simulations showed that new strands might prefer to extend in a parallel arrangement to form oligomers, which agrees with the microcrystal structure of the amyloid fibril formed by this peptide. In addition, analysis of the pi-pi stacking of aromatic residues showed that this type of interaction did not play an important role in giving directionality for beta-strand alignment but played a great influence on stabilizing the structures formed in the oligomer-formation process.     

猪冠状动脉前降支结扎位点和左室功能的线性回归

目的探讨猪冠状动脉前降支(LAD)结扎百分位点和心梗体积、左室射血分数的关系,以期指导研究者能够根据急性心肌梗死模型的心功能要求选择合适的LAD结扎百分位点。方法将47只小型猪开胸结扎心脏LAD中远段约30%~75%的不同百分位点,分别于术前、术后1 h心脏超声检查左室射血分数(LVEF),术后3 d进行常规冠状动脉造影,4周处死测量前降支结扎位点和梗死体积,最后用简单直线回归模型分析LAD结扎百分位点和心梗体积、左室射血分数回归方程和相关系数。结果47例动物手术过程中死亡8只,剩余39只存活动物冠状动脉造影均显示LAD中远段结扎部位处完全闭塞,表明手术成功。LAD结扎百分位点和术后1 h LVEF、术后1 hLVEF下降值、梗死心肌体积均明显相关(相关系数r分别为0.87、0.78和0.90,P均<0.001),其回归方程分别为:术后LVEF(%)=65.88-0.55x结扎百分位点;术后LVEF下降值(%)=0.12 0.59x结扎百分位点;心肌梗死体积(%)=0.53x结扎百分位点-5.43。结论猪LAD结扎百分位点和术后左室功能、梗死心肌体积均存在显著的相关性,可根据实验目的和对心功能的要求选择合适的结扎百分位点。     

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis

Applied Microbiology and Biotechnology - Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain...     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.