A novel α-amylase was isolated successfully from Glaciozyma antarctica PI12 using DNA walking and reverse transcription-polymerase chain reaction (RT-PCR) methods. The structure of this psychrophilic α-amylase (AmyPI12) from G. antarctica PI12 has yet to be studied in detail. A 3D model of AmyPI12 was built using a homology modelling approach to search for a suitable template and to generate an optimum target-template alignment, followed by model building using MODELLER9.9. Analysis of the AmyPI12 model revealed the presence of binding sites for a conserved calcium ion (CaI), non-conserved calcium ions (CaII and CaIII) and a sodium ion (Na). Compared with its template-the thermostable α-amylase from Bacillus stearothermophilus (BSTA)-the binding of CaII, CaIII and Na ions in AmyPI12 was observed to be looser, which suggests that the low stability of AmyPI12 allows the protein to work at different temperature scales. The AmyPI12 amino acid sequence and model were compared with thermophilic α-amylases from Bacillus species that provided the highest structural similarities with AmyPI12. These comparative studies will enable identification of possible determinants of cold adaptation.
Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized.
A method to map the specific site on dengue virus envelope protein (E) that interacts with cells and a neutralizing antibody is developed using serially truncated dengue virus type 2 (DENV-2) E displayed on M13 phages as recombinant E-g3p fusion proteins. Recombinant phages displaying the truncated E consisting of amino acids 297-423 (EB2) and amino acids 379-423 (EB4) were neutralized by DENV-2 patient sera and the DENV-2 E-specific 3H5-1 monoclonal antibodies suggesting that the phages retained the DENV-2 E antigenic properties. The EB4 followed by EB2 recombinant phages bound the most to human monocytes (THP-1), African green monkey kidney (Vero) cells, mosquito (C6/36) cells, ScFv specific against E and C6/36 cell proteins. Two potential cell attachment sites were mapped to loop I (amino acids 297 to 312) and loop II (amino acids 379-385) of the DENV-2 E using the phage-displayed truncated DENV-2 E fragments and by the analysis of the E structure. Loop II was present only in EB4 recombinant phages. There was no competition for binding to C6/36 cell proteins between EB2 and EB4 phages. Loop I and loop II are similar to the sub-complex specific and type-specific neutralizing monoclonal antibody binding sites, respectively. Hence, it is proposed that binding and entry of DENV involves the interaction of loop I to cell surface glycosaminoglycan-motif and a subsequent highly specific interaction involving loop II with other cell proteins. The phage displayed truncated DENV-2 E is a powerful and useful method for the direct determination of DENV-2 E cell binding sites.
Various works have been carried out in developing therapeutics against dengue. However, to date, no effective vaccine or anti-dengue agent has yet been discovered. The development of protease inhibitors is considered as a promising option, but most previous works have involved competitive inhibition. In this study, we focused on rational discovery of potential anti-dengue agents based on non-competitive inhibition of DEN-2 NS2B/NS3 protease. A homology model of the DEN-2 NS2B/NS3 protease (using West Nile Virus NS2B/NS3 protease complex, 2FP7, as the template) was used as the target, and pinostrobin, a flavanone, was used as the standard ligand. Virtual screening was performed involving a total of 13 341 small compounds, with the backbone structures of chalcone, flavanone, and flavone, available in the ZINC database. Ranking of the resulting compounds yielded compounds with higher binding affinities compared with the standard ligand. Inhibition assay of the selected top-ranking compounds against DEN-2 NS2B/NS3 proteolytic activity resulted in significantly better inhibition compared with the standard and correlated well with in silico results. In conclusion, via this rational discovery technique, better inhibitors were identified. This method can be used in further work to discover lead compounds for anti-dengue agents.
Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.
Development of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) has been considered as major health burden, globally. In order to develop novel, potential molecules against drug resistant TB, twenty two (22) new 3-substituted-7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a-k) and 3-substituted-7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a-k) derivatives were designed and synthesized by using appropriate synthetic protocols. Pantothenate synthetase (PS) was considered as the target for the molecular docking studies and evaluated the binding pattern at active site, as PS plays a significant role in the biosynthesis of pantothenate in Mycobacterium tuberculosis (MTB). The preliminary in vitro antibacterial screening of test compounds was carried out against two strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The antimycobacterial screening was performed against MTB H37Rv and an isoniazid-resistant clinical isolate of MTB. The compounds 6b, 6c, 6d, 6k, 7b, 7c, 7d and 7k exhibited promising antibacterial activity MIC in the range of 15-73 μM against all bacterial strains used and compounds 6d and 7b showed antimycobacterial activity (IC50 <340 μM in LRP assay) and (MIC <9 μM in broth microdilution method).
The glycoprotein (G) of Nipah virus (NiV) is important for virus infectivity and induction of the protective immunity. In this study, the extra-cellular domain of NiV G protein was fused with hexahistidine residues at its N-terminal end and expressed in Escherichia coli. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 8 M urea and the protein was purified to homogeneity under denaturing conditions using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea. Light scattering analysis of the purified protein showed primarily monodispersity. The purified protein showed significant reactivity with the antibodies present in the sera of NiV-infected swine, as demonstrated in Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Taken together, the data indicate the potential usefulness of the purified G protein for structural or functional studies and the development of immunoassay for detection of the NiV antibodies.
Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.
Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT2 and α1 adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5-HT2 and adrenergic α1 receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT2 and α1 receptors with (R)-isolaureline showing the greatest potency (pKb = 8.14 at the 5-HT2C receptor). Both (R)- and (S)-glaucine also antagonized α1 receptors, but they behaved very differently to the other compounds at 5-HT2 receptors: (S)-glaucine acted as a partial agonist at all three 5-HT2 receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT2A receptor.
A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen>human fibrinogen>dog fibrinogen>goat fibrinogen>rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.