Molecular modeling on human CCR5 receptors and complex with CD4 antigens and HIV-1 envelope glycoprotein gp120

AIM: To investigate the interaction between human CCR5 receptors (CCR5) and HIV-1 envelope glycoprotein gp120 (HIV-1 gp120) and HIV-1 receptor CD4 antigens (CD4). METHODS: The structurally con served regions (SCR) of human CCR5 was built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of bacteriorhodopsin (bR) as the template. The coordinates for amino-ter minal residue sequence, and carboxyl-terminal residue sequence, extracellular and cytoplasmic loops were generated using LOOP SEARCH algorithm. Subsequently the structural model was merged into the complex with HIV-1 gp120 and CD4. RESULTS: Human CCR5 interacted with both an HIV-1 gp120 and CD4. The N-terminal residues (especially Met1 and Gln4) of human CCR5, contacted with CD4 residues, mainly 7Nith one span (56 - 59) of CD4 in electrostatic interaction and hydrogen-bonds. The binding sites of human CCR5 were buried in a hydrophobic center surrounded by a highly basic periphery. On the other hand, direct interatomic contacts were made between ? CCR5 residues and 6 gp120 amino-acid residues, which included van der Waals contacts, hydrophobic interaction, and hydrogen bonds. CONCLUSION: The interaction model should be helpful for rational design of novel anti-HIV drugs.

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 6 1 -residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop 11, and the cation groups of all three arginines face out of the molecular surface of NT1 This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3. (C) 2002 Elsevier Science B,V. All rights reserved.

Interpreting the input admittance of violins and guitars

The most widespread vibration measurement on musical instrument bodies is of the point mobility at the bridge. Analysis of such measurements is presented, with a view to assessing what range of information could feasibly be extracted from the corpus of data. Analysis approaches include (1) pole-residue extraction; (2) damping trend analysis based on time decay information; (3) statistical estimates based on SEA power-balance and variance theory. Comparative results are shown for some key quantities. Damping trends with frequency are shown to have unexpectedly different forms for violins and for guitars. Linear averaging to estimate the "direct field" component gives a simple and clear visualisation of any local resonance behaviour near the bridge, such as the "bridge hill", and reveals some violins that show a double hill, while viols show only weak hills, and guitars none at all. © S. Hirzel Verlag · EAA.

Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages.

Nanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118-131 and 137-140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation.

Simulations of thermal upgrading methods for oil shale reservoirs

This paper analyzes reaction and thermal front development in porous reservoirs with reacting flows, such as those encountered in shale oil extraction. A set of dimensionless parameters and a 3D code are developed in order to investigate the important physical and chemical variables of such reservoirs when heated by in situ methods. This contribution builds on a 1D model developed for the precursor study to this work. Theory necessary for this study is presented, namely shale decomposition chemical mechanisms, governing equations for multiphase flow in porous media and necessary closure models. Plotting the ratio of the thermal wave speed to the fluid speed allows one to infer that the reaction wave front ends where this ratio is at a minimum. The reaction front follows the thermal front closely, thus allowing assumptions to be made about the extent of decomposition solely by looking at thermal wave progression. Furthermore, this sensitivity analysis showed that a certain minimum permeability is required in order to ensure the formation of a traveling thermal wave. It was found that by studying the non-dimensional governing parameters of the system one can ascribe characteristic values for these parameters for given initial and boundary conditions. This allows one to roughly predict the performance of a particular method on a particular reservoir given approximate values for initial and boundary conditions. Channelling and flow blockage due to carbon residue buildup impeded each method's performance. Blockage was found to be a result of imbalanced heating. Copyright 2012, Society of Petroleum Engineers.

Nonlinear analysis of cardiac rhythm fluctuations using DFA method

An approach by which the detrented fluctuation analysis (DFA) method can be used to help diagnose heart failure was demonstrated. DFA was applied to patients suffering from congestive heart failure (CHF) to check correlations between DFA indices and CHF, and determine a correlation between DFA indices and mortality, with a particular attention to the residue parameter, which is a measure of the departure of the DFA from its power law approximation. DFA parameters proved to be useful as a complement to the physiological parameters weber and FE to sort out the patients into three prognostic group.

Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages

Nanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118-131 and 137-140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation. © 2013 Elsevier Ltd.

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Monitoring of organochlorine pesticides using PFU csystems in Yunnan lakes and rivers, China

Polyurethane foam unit (PFU) systems were collected from 11 lakes and three rivers in the Yunnan Plateau, China and, the PFU extrusion liquids, were analyzed for organochlorine pesticides (OCPs) by gas chromatography with electron capture detection (GCECD). The concentrations of pp'-DDE, HCB and HCHs were undetectable to 1.86 mu g l(-1) (mean 0.27 mu g l(-1)), undetectable to 0.72 mu g l(-1) (mean 0.11 mu g l(-1)), and 0.24-21.95 mu g l(-1) (mean 7.39 mu g l(-1)) respectively in lakes; and those in rivers were undetectable to 0.23 mu g l(-1) (mean 0.08 mu g l(-1)), 0.68-2.93 mu g l(-1) (mean 1.70 mu g l(-1)), and 2.71-37.56 mu g l(-1) (mean 17.01 mu g l(-1)) respectively. Notably, some residue levels of OCPs exceeded the US National Recommended Water Quality Criteria, implying Yunnan has levels of OCPs potentially harmful to human health. Further, the contamination by OCPs showed an obvious spatial distribution pattern. Amongst the lakes, Dianchi, Xingyun, Lugu and Yangzonghai had the highest OCP levels dominated by beta-HCH, whereas among rivers, Nujiang and Lancang Rivers had the highest contents of OCPs dominated by alpha-HCH. This demonstrates that HCHs are the predominant contaminants and some point sources of HCHs may still exist in Yunnan. The pollution levels in Yunnan were compared with other studies, suggesting the PFU method is suitable for long-term on-line monitoring of trace OCPs in aquatic ecosystems. Therefore, continuous studies monitoring OCPs in lakes and rivers are needed to further understand the future trend of contamination. (c) 2006 Elsevier Ltd. All rights reserved.

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus

The cDNAs and genes of two different types of leucine- rich repeat-containing proteins from grass carp ( Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced aminoacid sequence similarities with human glycoprotein A repetitions predominant precursor ( GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine- rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL ( x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod ( Sinergasilus major)- infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host - pathogen interactions.

Characterization and phylogenetic analysis of the chitinase gene from the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

A putative chitinase gene was identified within the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV, also called HaSNPIV) genome. The open reading frame (ORF) contains 1713 nucleotides (nt) and encodes a protein of 570 amino acids (aa) with a predicted molecular weight of 63.6 kDa. Transcription started at about 18 h post infection (p.i.) and the protein was first detected at 20 h p.i. The times of transcription and expression are characteristic of a late baculovirus gene. 5' and 3' RACE indicated that transcription was initiated from the adenine residue located at -246 nt upstream from the ATG start site and the poly (A) tail was added at 267 nt downstream from the stop codon. This is the first report on the molecular characterization of a chitinase from a single nucleocapsid NPV. The phylogeny of baculoviral chitinase genes were extensively examined in comparison with chitinases derived from bacteria, fungi, nematode, actinomycetes, viruses, insects and mammals. Neighbor-joining and most parsimony analyses showed that the baculoviral chitinases were clustered exclusively within gamma-proteobacteria. Our results strongly suggest that baculoviruses acquired their chitinase genes from bacteria. (C) 2004 Elsevier B.V. All rights reserved.

Rapid bioassay as indicator of potentially harmful effects for dioxin-like compounds in sample of Ya-Er Lake, China: Requirements for clean-up and comparison to chemical analysis

A rapid bioassay was established measuring the extracts of wildlife samples which were taken from Ya-Er Lake area, China. In extracts of these samples containing PCDD/Fs and PCBs, bioassay and chemically derived TCDD-equivalents (TEQs) were nearly identical. Our results indicate this bioassay is an excellent complement to chemical residue analysis and a useful tool in understanding the complex interactions of halogenated hydrocarbons. However, it must be mentioned that the proper prior clean-up method is very important for using the bioassay.