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991.
Proteoglycans (PGs) are among the most structurally complex biomacromolecules in nature. They are present in all animal cells and frequently exert their critical biological functions through interactions with protein ligands and receptors. PGs are comprised of a core protein to which one or multiple, heterogeneous, and polydisperse glycosaminoglycan (GAG) chains are attached. Proteins, including the protein core of PGs, are now routinely sequenced either directly using proteomics or indirectly using molecular biology through their encoding DNA. The sequencing of the GAG component of PGs poses a considerably more difficult challenge because of the relatively underdeveloped state of glycomics and because the control of their biosynthesis in the endoplasmic reticulum and the Golgi is poorly understood and not believed to be template driven. Recently, the GAG chain of the simplest PG has been suggested to have a defined sequence based on its top-down Fourier transform mass spectral sequencing. This review examines the advances made over the past decade in the sequencing of GAG chains and the challenges the field face in sequencing complex PGs having critical biological functions in developmental biology and pathogenesis. 相似文献
992.
Nguyen TK Arungundram S Tran VM Raman K Al-Mafraji K Venot A Boons GJ Kuberan B 《Molecular bioSystems》2012,8(2):609-614
Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation. 相似文献
993.
Dervillez X Gottimukkala C Kabbara KW Nguyen C Badakhshan T Kim SM Nesburn AB Wechsler SL Benmohamed L 《Future virology》2012,7(4):371-378
Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as "asymptomatic" protective epitopes") could boost local and systemic "natural" protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging "asymptomatic" T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease. 相似文献
994.
Background
Supertree methods combine trees on subsets of the full taxon set together to produce a tree on the entire set of taxa. Of the many supertree methods, the most popular is MRP (Matrix Representation with Parsimony), a method that operates by first encoding the input set of source trees by a large matrix (the "MRP matrix") over {0,1, ?}, and then running maximum parsimony heuristics on the MRP matrix. Experimental studies evaluating MRP in comparison to other supertree methods have established that for large datasets, MRP generally produces trees of equal or greater accuracy than other methods, and can run on larger datasets. A recent development in supertree methods is SuperFine+MRP, a method that combines MRP with a divide-and-conquer approach, and produces more accurate trees in less time than MRP. In this paper we consider a new approach for supertree estimation, called MRL (Matrix Representation with Likelihood). MRL begins with the same MRP matrix, but then analyzes the MRP matrix using heuristics (such as RAxML) for 2-state Maximum Likelihood.Results
We compared MRP and SuperFine+MRP with MRL and SuperFine+MRL on simulated and biological datasets. We examined the MRP and MRL scores of each method on a wide range of datasets, as well as the resulting topological accuracy of the trees. Our experimental results show that MRL, coupled with a very good ML heuristic such as RAxML, produced more accurate trees than MRP, and MRL scores were more strongly correlated with topological accuracy than MRP scores.Conclusions
SuperFine+MRP, when based upon a good MP heuristic, such as TNT, produces among the best scores for both MRP and MRL, and is generally faster and more topologically accurate than other supertree methods we tested. 相似文献995.
K Fritzsche P Scheib N Ko M Wirsching A Kuhnert J Hick G Schüßler W Wu S Yuan NH Cat S Vongphrachanh NT Linh NK Viet 《BioPsychoSocial medicine》2012,6(1):17
ABSTRACT: BACKGROUND: With the "ASIA-LINK" program, the European Community has supported the development and implementation of a curriculum of postgraduate psychosomatic training for medical doctors in China, Vietnam and Laos. Currently, these three countries are undergoing great social, economic and cultural changes. The associated psychosocial stress has led to increases in psychological and psychosomatic problems, as well as disorders for which no adequate medical or psychological care is available, even in cities. Health care in these three countries is characterized by the coexistence of Western medicine and traditional medicine. Psychological and psychosomatic disorders and problems are insufficiently recognized and treated, and there is a need for biopsychosocially orientated medical care. Little is known about the transferability of Western-oriented psychosomatic training programs in the Southeast Asian cultural context. METHODS: The curriculum was developed and implemented in three steps: 1) an experimental phase to build a future teacher group; 2) a joint training program for future teachers and German teachers; and 3) training by Asian trainers that was supervised by German teachers. The didactic elements included live patient interviews, lectures, communication skills training and Balint groups. The training was evaluated using questionnaires for the participants and interviews of the German teachers and the future teachers. RESULTS: Regional training centers were formed In China (Shanghai), Vietnam (Ho Chi Minh City and Hue) and Laos (Vientiane). A total of 200 physicians completed the training, and 30 physicians acquired the status of future teacher. The acceptance of the training was high, and feelings of competence increased during the courses. The interactive training methods were greatly appreciated, with the skills training and self-experience ranked as the most important topics. Adaptations to the cultural background of the participants were necessary for the topics of "breaking bad news," the handling of negative emotions, discontinuities in participation, the hierarchical doctor-patient relationship, culture-specific syndromes and language barriers. In addition to practical skills for daily clinical practice, the participants wanted to learn more about didactic teaching methods. Half a year after the completion of the training program, the participants stated that the program had a great impact on their daily medical practice. CONCLUSIONS: The training in psychosomatic medicine for postgraduate medical doctors resulted in a positive response and is an important step in addressing the barriers in providing psychosomatic primary care. The transferability of western concepts should be tested locally, and adaptations should be undertaken where necessary. The revised curriculum forms the basis of training in psychosomatic medicine and psychotherapy for medical students and postgraduate doctors in China, Vietnam and Laos. 相似文献
996.
Nicholas V Coleman Nga B Le Mai A Ly Hitoha E Ogawa Victoria McCarl Neil L Wilson Andrew J Holmes 《The ISME journal》2012,6(1):171-182
The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH4) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C2–C4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C2–C4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible. 相似文献
997.
David J. Leibly Trang Nhu Nguyen Louis T. Kao Stephen N. Hewitt Lynn K. Barrett Wesley C. Van Voorhis 《PloS one》2012,7(12)
Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl2, proline, xylitol, NDSB 201, CTAB and K2PO4) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher. 相似文献
998.
Christopher M. Clouthier Sébastien Morin Sophie M. C. Gobeil Nicolas Doucet Jonathan Blanchet Elisabeth Nguyen Stéphane M. Gagné Joelle N. Pelletier 《PloS one》2012,7(12)
Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions. 相似文献
999.
Bharat Bhusan Patnaik Dong Hyun Kim Seung Han Oh Yong-Su Song Nguyen Dang Minh Chanh Jong Sun Kim Woo-jin Jung Atul Kumar Saha Bharat Bhushan Bindroo Yeon Soo Han 《PloS one》2012,7(12)
Background
Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Methodology/Principal Findings
Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.Conclusions/Significance
The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba. 相似文献1000.
We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w(1118); iso-2; iso-3 strain and the reference y(1); cn(1) bw(1) sp(1) strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5'UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5' and 3' UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory. 相似文献