Purification and functional characterisation of Rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion

The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Functional and molecular characterization of Tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK, receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RTPCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin INK, receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK, receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK, receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

Characterization of a highly biodiverse floodplain meadow using hyperspectral remote sensing within a plant functional trait framework

We assessed the potential for using optical functional types as effective markers to monitor changes in vegetation in floodplain meadows associated with changes in their local environment. Floodplain meadows are challenging ecosystems for monitoring and conservation because of their highly biodiverse nature. Our aim was to understand and explain spectral differences among key members of floodplain meadows and also characterize differences with respect to functional traits. The study was conducted on a typical floodplain meadow in UK (MG4-type, mesotrophic grassland type 4, according to British National Vegetation Classification). We compared two approaches to characterize floodplain communities using field spectroscopy. The first approach was sub-community based, in which we collected spectral signatures for species groupings indicating two distinct eco-hydrological conditions (dry and wet soil indicator species). The other approach was “species-specific”, in which we focused on the spectral reflectance of three key species found on the meadow. One herb species is a typical member of the MG4 floodplain meadow community, while the other two species, sedge and rush, represent wetland vegetation. We also monitored vegetation biophysical and functional properties as well as soil nutrients and ground water levels. We found that the vegetation classes representing meadow sub-communities could not be spectrally distinguished from each other, whereas the individual herb species was found to have a distinctly different spectral signature from the sedge and rush species. The spectral differences between these three species could be explained by their observed differences in plant biophysical parameters, as corroborated through radiative transfer model simulations. These parameters, such as leaf area index, leaf dry matter content, leaf water content, and specific leaf area, along with other functional parameters, such as maximum carboxylation capacity and leaf nitrogen content, also helped explain the species’ differences in functional dynamics. Groundwater level and soil nitrogen availability, which are important factors governing plant nutrient status, were also found to be significantly different for the herb/wetland species’ locations. The study concludes that spectrally distinguishable species, typical for a highly biodiverse site such as a floodplain meadow, could potentially be used as target species to monitor vegetation dynamics under changing environmental conditions.

Synthesis, chemical, electrochemical characterization of oxomolybdenum(V) complexes of 2-(3,5-dimethyl pyrazol-1-yl) benzothiazole ligand: crystal structure of ligand and oxomolybdenum(VI) complex and density functional theory (DFT) calculation

This work reports the ligational behavior of the neutral bidentate chelating molecule 2-(3,5-dimethyl pyrazol-1-yl) benzothiazole towards the oxomolybdenum(V) center. Both mononuclear complexes of the type (MoOX3L)-O-V and binuclear complexes of the formula (Mo2O4X2L2)-O-V (where X = Cl, Br) are isolated in the solid state. The complexes are characterized by elemental analyses, various spectroscopic techniques (UV-Vis IR), magnetic susceptibility measurement at room temperature, and cyclic voltammetry for their redox behavior at a platinum electrode in CH3CN. The mononuclear complexes (MoOX3L)-O-V are found to be paramagnetic while the binuclear complexes Mo2O4X2L2 are diamagnetic. Crystal and molecular structure of the ligand and the dioxomolybdenum complex (MoO2Br2L)-O-VI (obtained from the complex MoOBr3L during crystallization) have been solved by single crystal X-ray diffraction technique. Relevant DFT calculations of the ligand and the complex (MoO2Br2L)-O-VI are also carried out.

Characterization of para-Nitrophenol-degrading bacterial communities in river water by using functional markers and stable isotope probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [13C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Proteornics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructurall protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Characterization of an escherichia coli elaC deletion mutant

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as 3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22 (2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPD we generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Micro array-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, likewise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaCl protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaCl) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaCl) proteins. (C) 2004 Elsevier Inc. All rights reserved.

Soluble redox-active polymetallic chains [{Ru-0(CO)(L)(bpy)}(m)](n) (bpy=2,2 '-bipyridine, L = PrCN, Cl-; m=0,-1): electrosynthesis and characterization

Electrochemical and spectroelectrochemical techniques were employed to study in detail the formation and so far unreported spectroscopic properties of soluble electroactive molecular chains with nonbridged metal-metal backbones, namely, [{Ru-0(CO)(PrCN)(bpy)}(m)](n) (m = 0, -1) and [{Ru-0(CO)(bpy)Cl}(m)](n) (m = -1, -2; bpy = 2,2'-bipyridine). The precursors cis-(Cl)-[Ru-II(CO)(MeCN)(bpy)Cl-2] (in PrCN) and mer-[Ru-II(CO)(bpy)Cl-3](-) (in tetrahydrofuran (THF) and PrCN) undergo one-electron reductions to reactive radicals [Ru-II(CO)(MeCN)(bpy(center dot-))Cl-2](-) and [Ru-II(CO)(bpy(center dot-))Cl-3](2-), respectively. Both [bpy(center dot-)]-containing species readily electropolymerize on concomitant dissociation of two chloride ligands and consumption of a second electron. Along this path, mer-to-fac isomerization of the bpy-reduced trichlorido complex (supported by density functional theory calculations) and a concentration-dependent oligomerization process contribute to the complex reactivity pattern. In situ spectroelectrochemistry (IR, UV/vis a has revealed that the charged polymer [{Ru-0(CO)(bpy)Cl}(-)](n) is stable in THF, but in PrCN it converts readily to [Ru-0(CO)(PrCN)(bpy)](n). An excess of chloride ions retards this substitution at low temperatures. Both polymetallic chains are completely soluble in the electrolyte solution and can be reduced reversibly to the corresponding [bpy(center dot-)]-containing species.

One-pot synthesis and characterization of soluble poly(aryl ether-ketone)s having pendant carboxyl groups

Aromatic poly(ether-ketone)s having pendant carboxyl groups have been obtained by direct, one-pot, Friedel-Crafts copolycondensation of 4,4'-diphenoxybenzophenone with a mixture of terephthaloyl chloride (TC) and trimellitic anhydride acid chloride (TAAC), over a wide range of TAAC/TC molar ratios, in the presence of anhydrous aluminum chloride. The syntheses were performed as precipitation-polycondensations, and the polymers were obtained in particulate form. Besides globular particles of polymer, small quantities of elongated, needlelike particles were observed when the mole ratio TAAC/TC was less than 1. Use of X-ray microdiffraction with synchrotron radiation has revealed that the needlelike material consists of a cyclic compound containing 10 phenylene units, i.e., the crystals are of a [2 + 2] macrocyclic dimer. The polymers obtained are soluble in strong acids and in mixtures of methanesulfonic acid or trifluoroacetic acid with chlorinated hydrocarbons. The molecular structures of the polymers were confirmed by H-1 and C-13 NMR spectroscopy. Reaction of TAAC with 4,4'-diphenoxybenzophenone produced mainly meta-orientation of the resulting ketone linkages. The size of the polymer particles, their molecular weights, and the melting behavior of the products obtained depend on the TAAC/TC ratio used. Ortho-keto acid residues, formed during reaction of anhydride groups of TAAC with 4,4'-diphenoxybenzophenone, exhibit ring-chain tautomerism. A carboxyl-containing aromatic polyketone derived from p-terphenyl, and thus having with no ether linkages in the main chain, was prepared by analogous chemistry, and functional derivatives of carboxy-substituted polyketones were also obtained and characterized.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions (a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SIDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to similar to 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

Characterization of wheat puroindoline proteins

Puroindoline proteins were purified from selected UK-grown hexaploid wheats. Their identities were confirmed on the basis of capillary electrophoresis mobilities, relative molecular mass and N-terminal amino acid sequencing. Only one form of puroindoline-a protein was found in those varieties, regardless of endosperm texture. Three allelic forms of puroindoline-b protein were identified. Nucleotide sequencing of cDNA produced by RT-PCR of isolated mRNA indicated that these were the 'wild-type', found in soft wheats, puroindoline-b containing a Gly -> Ser amino acid substitution (position 46) and puroindoline-b containing a Trp -> Arg substitution (position 44). The latter two were found in hard wheats. Microheterogeneity, due to short extensions and/or truncations at the N-terminus and C-terminus, was detected for both puroindoline-a and puroindoline-b. The type of microheterogeneity observed was more consistent for puroindoline-a than for puroindoline-b, and may arise through slightly different post-translational processing pathways. A puroindoline-b allele corresponding to a Leu -> Pro substitution (position 60) was identified from the cDNA sequence of the hard variety Chablis, but no mature puroindoline-b protein was found in this or two other European varieties known to possess this puroindoline-b allele. Wheats possessing the puroindoline-b proteins with point mutations appeared to contain lower amounts of puroindoline protein. Such wheats have a hard endosperm texture, as do wheats from which puroindoline-a or puroindoline-b are absent. Our results suggest that point mutations in puroindoline-b genes may confer hard endosperm texture through accumulation of allelic forms of puroindoline-b proteins with altered functional properties and/or through lower amounts of puroindoline proteins.

Neural activation and functional connectivity during motor imagery of bimanual everyday actions

Bimanual actions impose intermanual coordination demands not present during unimanual actions. We investigated the functional neuroanatomical correlates of these coordination demands in motor imagery (MI) of everyday actions using functional magnetic resonance imaging (fMRI). For this, 17 participants imagined unimanual actions with the left and right hand as well as bimanual actions while undergoing fMRI. A univariate fMRI analysis showed no reliable cortical activations specific to bimanual MI, indicating that intermanual coordination demands in MI are not associated with increased neural processing. A functional connectivity analysis based on psychophysiological interactions (PPI), however, revealed marked increases in connectivity between parietal and premotor areas within and between hemispheres. We conclude that in MI of everyday actions intermanual coordination demands are primarily met by changes in connectivity between areas and only moderately, if at all, by changes in the amount of neural activity. These results are the first characterization of the neuroanatomical correlates of bimanual coordination demands in MI. Our findings support the assumed equivalence of overt and imagined actions and highlight the differences between uni- and bimanual actions. The findings extent our understanding of the motor system and may aid the development of clinical neurorehabilitation approaches based on mental practice.