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991.
Paola Magotti Daniel Ricklin Hongchang Qu You‐Qiang Wu Yiannis N. Kaznessis John D. Lambris 《Journal of molecular recognition : JMR》2009,22(6):495-505
Compstatin is a 13‐residue peptide that inhibits activation of the complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264‐fold higher activity; one of these analogs is now in clinical trials for the treatment of age‐related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp‐4, Asp‐6, Ala‐9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co‐crystal structure of compstatin and C3c. The KD values for the panel of tested analogs ranged from 10?6 to 10?8 M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4‐fold range in kon values (2–10 × 105 M?1 s?1) and a koff variation of >35‐fold (1–37 × 10?2 s?1) being observed. The stability of the C3b‐compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a β‐turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
992.
Xian-Feng Ma Hui-Min Yu Cheng Wen Hui Luo Qiang Li Zhong-Yao Shen 《World journal of microbiology & biotechnology》2009,25(8):1353-1361
Fusion proteins of d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) with Vitreoscilla Hemoglobin (VHb) and (His)6-tag were constructed and expressed in recombinant Escherichia coli. A fusing-position effect was revealed that (His)6-tag’s N-terminal fusion with TvDAAO (HDAAO) reduced the specific activity by ~29%, while the C-terminal fusion (DAAOH) remained unreduced. The N-terminal
fusion of VHb with TvDAAO and DAAOH significantly improved their activity. As in a 5 l fermentor, the activity of the triple fusion VHb-TvDAAO-(His)6 (VDAAOH) reached 2.53 U/mg dry cell at 9 h, ~58% increase than that of DAAOH together with ~40% biomass increase, confirming
the positive effect of VHb expression on cell level. After purification, the UV–visible and fluorescence spectrum of DAAOH
and VDAAOH were characterized. Enzyme kinetics studies further indicated that VDAAOH behaved a higher K
cat, but a weaker substrate affinity of K
m relative to DAAOH, revealing two distinct impacts of VHb-coupling with TvDAAO on protein level. 相似文献
993.
Zhong Li Ruo-Wei Chen Heng-Yi Lei Zhong Shan Tao Bai Qiang Yu Hua-Liang Li 《World journal of microbiology & biotechnology》2009,25(5):745-752
We studied a novel bioflocculant, PX, that is produced from Bacillus Bacillus circulans X3, and has excellent flocculating activity with regard to its characterization and flocculating properties. The bioflocculant
was purified from supernatant by ethanol precipitation, dialysis and gel permeation chromatography (GPC). The major component
of PX was an acid polysaccharide including uronic (19.8%), pyruvic (6.5%) and acetic acids (0.7%). It consisted of galactose,
mannose, xylitol, rhamnose and galacturonic acid in an approximate molar ration of 5:4.1:3:2:1.2. The molecular weight of
PX was about 4.85 × 104 Da as determined by GPC. The infrared spectrum of the bioflocculant indicated the presence of carboxyl, hydroxyl, amino and
methoxyl groups. Studies of the flocculating properties revealed that it was stable at 60–100°C and pH 4–10. Moreover, it
could flocculate a kaolin suspension over a wide range of pH and temperature in the presence of CaCl2. 相似文献
994.
Improving enzymatic hydrolysis of wheat straw using ionic liquid 1-ethyl-3-methyl imidazolium diethyl phosphate pretreatment 总被引:1,自引:0,他引:1
Qiang Li Yu-Cai HeMo Xian Gao JunXin Xu Jian-Ming YangLiang-Zhi Li 《Bioresource technology》2009,100(14):3570-3575
This study aims to establish a cellulose pretreatment process using ionic liquids (ILs) for efficient enzymatic hydrolysis. The IL 1-ethyl-3-methyl imidazolium diethyl phosphate ([EMIM]DEP) was selected in view of its low viscous and the potential of accelerating enzymatic hydrolysis, and it could be recyclable. The yield of reducing sugars from wheat straw pretreated with this IL at 130 °C for 30 min reached 54.8% after being enzymatically hydrolyzed for 12 h. Wheat straw regenerated were hydrolyzed more easily than that treated with water. The fermentability of the hydrolyzates, obtained after enzymatic saccharification of the regenerated wheat straw, was evaluated using Saccharomyces cerevisiae. This microbe could ferment glucose efficiently, and the ethanol production was 0.43 g/g glucose within 26 h. In conclusion, the IL [EMIM]DEP shows promise as pretreatment solvent for wheat straw, although its cost should be reduced and in-depth exploration of this subject is needed. 相似文献
995.
Alexander Heim-Riether Steven J. Taylor Shuang Liang Donghong Amy Gao Zhaoming Xiong E. Michael August Brandon K. Collins Bennett T. Farmer II Kathleen Haverty Melissa Hill-Drzewi Hans-Dieter Junker S. Mariana Margarit Neil Moss Thomas Neumann John R. Proudfoot Lana Smith Keenan Renate Sekul Qiang Zhang Jun Li Neil A. Farrow 《Bioorganic & medicinal chemistry letters》2009,19(18):5321-5324
Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1′ pocket. 相似文献
996.
997.
在不同的电压与载样量下,分别使用溴化乙锭(EB)与新型核酸染料GoldViewna II及GoldView对凝胶中的DNA进行染色,观察电泳条带扭曲程度,了解电压、载样量与核酸染料对琼脂糖凝胶电泳条带扭曲程度的影响.使用GoldViewna II染色,当电压≥10V/cm时,DNA条带的扭曲程度随载样量增大而加剧;当DNA载样量≥10ng时,DNA条带的扭曲程度随电压升高而加剧. 使用EB染色,仅当载样量≥50ng时,条带出现轻微扭曲,扭曲程度与电压无明显关系.结果表明使用GoldView染色,当电压不超过20V/cm或载样量不超过100ng时,DNA条带不扭曲.在琼脂糖凝胶电泳中,若要避免DNA条带扭曲干扰实验结果,使用GoldViewna II染色,若DNA载样量≥10ng,应控制电压≤5V/cm;使用EB染色,载样量应≤50ng;使用GoldView染色,可以使用20V/cm的电压缩短实验时间. 相似文献
998.
999.
Nayana Prabhu Mookkan Prabakaran Hui-Ting Ho Sumathy Velumani Jia Qiang Michael Goutama Jimmy Kwang 《Journal of virology》2009,83(6):2553-2562
The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.Highly pathogenic avian influenza (HPAI) virus H5N1 strains are currently causing major morbidity and mortality in poultry populations across Asia, Europe, and Africa and have caused 385 confirmed human infections, with a fatality rate of 63.11% (37, 39). Preventive and therapeutic measures against circulating H5N1 strains have received a lot of interest and effort globally to prevent another pandemic outbreak. Influenza A virus poses a challenge because it rapidly alters its appearance to the immune system by antigenic drift (mutating) and antigenic shift (exchanging its components) (5). The current strategies to combat influenza include vaccination and antiviral drug treatment, with vaccination being the preferred option. The annual influenza vaccine aims to stimulate the generation of anti-hemagglutinin (anti-HA) neutralizing antibodies, which confer protection against homologous strains. Current vaccines have met with various degrees of success (31). The facts that these strategies target the highly variable HA determinant and that predicting the major HA types that pose the next epidemic threat is difficult are significant limitations to the current antiviral strategy. In the absence of an effective vaccine, therapy is the mainstay of control of influenza virus infection.Therefore, therapeutic measures against influenza will play a major role in case a pandemic arises due to H5N1 strains. Currently licensed antiviral drugs include the M2 ion-channel inhibitors (rimantidine and amantidine) and the neuraminidase inhibitors (oseltamivir and zanamivir). The H5N1 viruses are known to be resistant to the M2 ion-channel inhibitors (2, 3). Newer strains of H5N1 viruses are being isolated which are also resistant to the neuraminidase inhibitors (oseltamivir and zanamivir) (5, 17). The neuraminidase inhibitors also require high doses and prolonged treatment (5, 40), increasing the likelihood of unwanted side effects. Hence, alternative strategies for treatment of influenza are warranted.Recently, passive immunotherapy using monoclonal antibodies (MAbs) has been viewed as a viable option for treatment (26). The HA gene is the most variable gene of the influenza virus and also the most promising target for generating antibodies. It is synthesized as a precursor polypeptide, HA0, which is posttranslationally cleaved to two polypeptides, HA1 and HA2, linked by a disulfide bond. MAbs against the HA1 glycopolypeptide (gp) are known to neutralize the infectivity of the virus and hence provide good protection against infection (12). However, they are less efficient against heterologous or mutant strains, which are continuously arising due to antigenic shift and, to an extent, drift. Recent strategies for alternative therapy explore the more conserved epitopes of the influenza virus antigens (18, 33), which not only have the potential to stimulate a protective immune response but are also conserved among different subtypes, so as to offer protection against a broader range of viruses.The HA2 polypeptide represents a highly conserved region of HA across influenza A virus strains. The HA2 gp is responsible for the fusion of the virus and the host endosomal membrane during the entry of the virus into the cell (16). Previously, anti-HA MAbs that lacked HA inhibition activity were studied and were found to reduce the infectivity of non-H5 influenza virus subtypes by inhibition of fusion during viral replication (14). They are known to block fusion of the virus to the cell membrane at the postbinding and prefusion stage, thereby inhibiting viral replication. Furthermore, in vivo studies show that anti-HA2 MAbs that exhibit fusion inhibition activity contribute to protection and recovery from H3N2 influenza A virus infection (8). It is interesting that although the HA2 gp is generally conserved, the fusion peptide represents the most conserved region of the HA protein. So far, there have been no studies on the possible therapeutic effects of MAbs, specifically against the fusion peptide of HA, on lethal HPAI H5N1 infections.Previous studies have suggested that HA2 could contain a potential epitope responsible for the induction of antibody-mediated protective immunity (9). In the present study, a panel of MAbs against HA2 gp was characterized for their respective epitopes by epitope mapping. The therapeutic and prophylactic efficacies of these MAbs were evaluated in mice challenged with HPAI H5N1 virus infection. 相似文献
1000.