Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease

Background  

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall.     

Effect of nanoscale topography on fibronectin adsorption,focal adhesion size and matrix organisation

Phase separation of PLLA/PS (50/50, w/w) solutions during a spin-casting process gives rise to well-defined nanotopographies of 14, 29 and 45 nm deep pits depending on the concentration of the solution. Their influence on the biological activity of fibronectin (FN) was investigated. FN adsorption was quantified by radiolabelling the protein. The amount of adsorbed FN was higher on the 14 nm deep pit nanotopography than on the other two surfaces. FN distribution between valleys and peaks was investigated by AFM combined with image analysis. FN tends to adsorb preferentially on the valleys of the nanotopography only for the 14 nm system and when adsorbed from solutions of concentration lower than 10 μg/ml. Higher concentration of the FN solution leads to evenly distribution of the protein throughout the surface; moreover, there is no difference in the distribution of the protein between valleys and peaks for the other two systems (29 and 45 nm) irrespective of the concentration of the FN solution. The biological activity of the adsorbed protein layer was assessed by investigating MC3T3 osteoblast-like cells adhesion, FN reorganisation and late matrix formation on the different substrates. Even if initial cell adhesion is excellent for every substrate, the size of the focal adhesion plaques increases as the size of the pits in the nanotopography does. This is correlated to FN reorganisation, which only takes places on the 29 and 45 nm deep pits surfaces, where enhanced late matrix production was also found.     

Identification and characterization of a bacterial glutamic peptidase

Background  

Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized.     

The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1

Background  

Though Dnmt1 is considered the primary maintenance methyltransferase and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals, these three enzymes may work together in maintaining as well as establishing DNA methylation patterns. It has been proposed that Dnmt1 may carry out de novo methylation at sites in the genome with transient single-stranded regions, such as replication origins, and then spread methylation from these nucleation sites in vivo, even though such activity has not been reported.     

Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions

Background  

Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells.     

Development and validation of a repharsed phase- HPLC method for simultaneous determination of rosiglitazone and glimepiride in combined dosage forms and human plasma

Background  

Rosiglitazone (ROZ) and glimepiride (GLM) are antidiabetic agents used in the treatment of type 2 diabetes mellitus. A survey of the literature reveals that only one spectrophotometric method has been reported for the simultaneous determination of ROS and GLM in pharmaceutical preparations. However the reported method suffers from the low sensitivity, for this reason, our target was to develop a simple sensitive HPLC method for the simultaneous determination of ROZ and GLM in their combined dosage forms and plasma.     

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Background  

We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity.     

Nonnatural amino acid incorporation into the methionine 214 position of the metzincin <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> alkaline protease

Background  

The alkaline protease from Pseudomonas aeruginosa(AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region.     

Function,expression and localization of annexin A7 in platelets and red blood cells: Insights derived from an annexin A7 mutant mouse

Background  

Annexin A7 is a Ca²⁺- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca²⁺-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components.     

The <Emphasis Type="Italic">FTO</Emphasis> (fat mass and obesity associated) gene codes for a novel member of the non-heme dioxygenase superfamily

Background  

Genetic variants in the FTO (fat mass and obesity associated) gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO.     

Supramolecular assembled of hexameric water clusters into a 1D chain containing (H<Subscript>2</Subscript>O)<Subscript>6</Subscript> and [(H<Subscript>2</Subscript>O)<Subscript>4</Subscript>O<Subscript>2</Subscript>] stabilized by hydrogen bonding in a copper complex

Background  

Various water clusters including hexamers, heptamers, octamers, decamers and 1D or 2D infinite water chains in a number of organic and inorganic-organic hybrid hosts, have been reported.     

Exploring laccase-like multicopper oxidase genes from the ascomycete <Emphasis Type="Italic">Trichoderma reesei</Emphasis>: a functional,phylogenetic and evolutionary study

Background  

The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.     

Design,structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease

Abstract  

Serine protease activity of the NS3 protein of Dengue virus is an important target of antiviral agents that interfere with the viral polyprotein precursor processing catalyzed by the NS3 protease (NS3pro), which is important for the viral replication and maturation. Recent studies showed that substrate-based peptidomimetics carrying an electrophilic warhead inhibit the NS2B-NS3pro cofactor-protease complex with inhibition constants in the low micromolar concentration range when basic amino acid residues occupy P₁ and P₂ positions of the inhibitor, and an aldehyde warhead is attached to the P₁. We have used computer-assisted combinatorial techniques to design, focus using the NS2B-NS3pro receptor 3D structure, and in silico screen a virtual library of more than 9,200 peptidomimetic analogs targeted around the template inhibitor Bz-Nle-Lys-Arg-Arg-H (Bz—benzoyl) that are composed mainly of unusual amino acid residues in all positions P₁–P₄. The most promising virtual hits were analyzed in terms of computed enzyme-inhibitor interactions and Adsorption, Distribution, Metabolism and Excretion (ADME) related physico-chemical properties. Our study can direct the interest of medicinal chemists working on a next generation of antiviral chemotherapeutics against the Dengue Fever towards the explored subset of the chemical space that is predicted to contain peptide aldehydes with NS3pro inhibition potencies in nanomolar range which display ADME-related properties comparable to the training set inhibitors.     

A two disulfide bridge Kazal domain from <Emphasis Type="Italic">Phytophthora</Emphasis> exhibits stable inhibitory activity against serine proteases of the subtilisin family

Background  

Kazal-like serine protease inhibitors are defined by a conserved sequence motif. A typical Kazal domain contains six cysteine residues leading to three disulfide bonds with a 1–5/2–4/3–6 pattern. Most Kazal domains described so far belong to this class. However, a novel class of Kazal domains with two disulfide bridges resulting from the absence of the third and sixth cysteines have been found in biologically important molecules, such as human LEKTI, a 15-domain inhibitor associated with the severe congenital disease Netherton syndrome. These domains are referred to as atypical Kazal domains. Previously, EPI1, a Kazal-like protease inhibitor from the oomycete plant pathogen Phytophthora infestans, was shown to be a tight-binding inhibitor of subtilisin A. EPI1 also inhibits and interacts with the pathogenesis-related P69B subtilase of the host plant tomato, suggesting a role in virulence. EPI1 is composed of two Kazal domains, the four-cysteine atypical domain EPI1a and the typical domain EPI1b.     

A sharp J-band-like electronic absorption peak observed for the chromonic liquid crystal of bisazo dye Ponceau SS in water

Abstract  

The bisazo dye Ponceau SS forms an acute oblique (V-type) dimer in dilute aqueous solution. In concentrated aqueous solution, these V-type dimers are considered to stack to form V-type blocks of chromonic liquid-crystalline (LC) columns. The weak interactions among these LC columnar V-type blocks have been spectroscopically investigated. On increasing the concentration of Ponceau SS at 25 °C, the V-type LC columnar blocks form a rhombus-type LC columnar block at 2 wt% concentration. These rhombus-type LC columnar blocks are further aligned at an acute angle at 5 and 10 wt% concentrations. The rapidly cooled 10 wt% LC sample shows an unusually sharp J-band-like peak in the electronic absorption spectrum. The emergence of this J-band-like peak has been analyzed from the viewpoint of an exciton model, suggesting that many nearest neighbor unique molecules belonging to many different rhombus-type LC columnar blocks are aligned in a head-to-tail manner to give a giant quasi-linear head-to-tail-type exciton.     

A density functional theory investigation of the bromide oxidation mechanism by a vanadium bromoperoxidase model complex

Abstract  

Density functional theory has been used to study the mechanism of bromide oxidation by the oxo-peroxo complex K[VO(O₂)Hheida] (heida = N-(2-hydroxyethyl)iminodiacetic acid), which has the highest reported rate constant for bromide oxidation of any vanadium complex. Two possible mechanisms were explored, involving bromide attack on a protonated or unprotonated peroxo atom. The direct nucleophilic attack of bromide on a protonated peroxo begins the reaction, i.e. the protonated peroxo ligand is the active site of reaction. We examined five different transition states in the mechanism. Two transition states were found to have lower activation barriers. A reduction in the potential energy barriers, when calculated using the polarisable continuum model, indicates that with the involvement of acetonitrile as a solvent the transition states become more stable.     

Synthesis, characterization, and electron transfer reaction of some surfactant–cobalt(III) complex ions

Abstract  

The surfactant complex ion cis-[Co(tmd)₂(C₁₂H₂₅NH₂)₂]³⁺ (tmd = 1,3-propanediamine, C₁₂H₂₅NH₂ = dodecylamine) has been synthesized and characterized by elemental analysis and spectral data. In addition we have determined the critical micelle concentration of the surfactant–cobalt(III) complex and studied the kinetics and mechanism of the complex with ferrocyanide anion. The reaction is found to be second order, and the second-order rate constant increases with increasing initial concentration of the surfactant–cobalt(III) complex due to the presence of self-micelles formed by the complex itself. The thermodynamic parameters were determined. The results have been analyzed.     

<Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> porphobilinogen synthase,a high activity metal ion independent hexamer

Background  

The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus.     

Ecofriendly and efficient procedure for hetero-Michael addition reactions with an acidic ionic liquid as catalyst and reaction medium

Abstract  

1-Methylimidazolium trifluoroacetate ([Hmim]TFA) is reported as a cost-effective catalyst for a simple and environmentally benign hetero-Michael reaction. [Hmim]TFA works both as reaction medium and catalyst. The reaction is applicable to various aromatic sulfur and nitrogen nucleophiles. This method has advantages such as high yields, short reaction time, and simple workup. The catalyst could be recycled several times without any loss of activity.