In silico characterization of a RNA binding protein of cattle filarial parasite Setaria digitata

Human lymphatic filariasis (HLF) is a neglected tropical disease which threatens nearly 1.4 billion people in 73 countries worldwide. Wuchereria bancrofti is the major causative agent of HLF and it closely resembles cattle filarial parasite Setaria digitata. Due to difficulties in procuring W. bancrofti parasite material, S. digitata cDNA library has been constructed to identify novel drug targets against HLF and many of the cDNA sequences are yet to be assigned structure and function. In this study, a 549 bp long cDNA (sdrbp) has been sequenced and characterized in silico. The shortest ORF of 249 bp from the isolated cDNA encodes a polypeptide of 82 amino acids and shows an amino acid identity of 54% with the RRM domain of human cleavage stimulation factor-64 kDa subunit (CstF-64). Structure of the protein (sdRBP) obtained by homology modelling using RRM of CstF-64 as template adopts classical RRM topology (β1α1β2β3α2β4). sdRBP model built was validated by superimposition tools and Ramachandran plot analysis. CstF-64 plays an important role in pre-mRNA polyadenylation by interacting with specific GU-rich downstream sequence element. Molecular docking studies of sdRBP with different RNA molecules revealed that sdRBP has greater binding affinity to GU-rich RNA and comparable results were obtained upon similar docking of RRM of CstF-64 with the same RNA molecules. Therefore, sdRBP is likely to perform homologous function in S. digitata. This study brings new dimensions to the functional analysis of RNA binding proteins of S. digitata and their evaluation as new drug targets against HLF.     

Terpenes of Dipterocarpus and Doona species

From the resins of Dipterocarpus hispidus, Dipterocarpus zeylanicus and Doona macrophylla, asiatic (2α,3β,23α-trihydroxyurs-12-en-28-oic) and 2α,3β-dihydroxyurs-12-en-28-oic acids have been isolated. The resin of Doona macrophylla contains ursolic acid and that of Doona congestiflora asiatic acid, 20β-hydroxy-3-oxo dammar-23-ene (Dipterocarpol) and a dihydroxyolean-12-en-28-oic acid. The bark of Dipterocarpus hispidus contains betulinic acid, dipterocarpol, and 3β,20β-dihydroxydammar-23-ene (dammarenediol 20S) whilst the timber contained dipterocarpol and asiatic acid.     

Cytosolic protein quality control machinery: Interactions of Hsp70 with a network of co-chaperones and substrates

The chaperone heat shock protein 70 (Hsp70) and its network of co-chaperones serve as a central hub of cellular protein quality control mechanisms. Domain organization in Hsp70 dictates ATPase activity, ATP dependent allosteric regulation, client/substrate binding and release, and interactions with co-chaperones. The protein quality control activities of Hsp70 are classified as foldase, holdase, and disaggregase activities. Co-chaperones directly assisting protein refolding included J domain proteins and nucleotide exchange factors. However, co-chaperones can also be grouped and explored based on which domain of Hsp70 they interact. Here we discuss how the network of cytosolic co-chaperones for Hsp70 contributes to the functions of Hsp70 while closely looking at their structural features. Comparison of domain organization and the structures of co-chaperones enables greater understanding of the interactions, mechanisms of action, and roles played in protein quality control.     

The synthesis of [14C]streptozotocin and its distribution and excretion in the rat

[(14)C]Streptozotocin was synthesized specifically labelled at three positions in the molecule. The biological activity of synthetic streptozotocin was characterised by studies in vivo of its diabetogenic activity and its dose-response curves. After this characterization the excretion pattern of all three labelled forms of streptozotocin was studied. With [1-(14)C]streptozotocin and [2'-(14)C]streptozotocin the injected radioactivity was excreted (approx. 70% and 80% respectively) mainly in the urine, the greater part of the excretion occurring in the first 6h period; small amounts (approx. 9% and 8% respectively) were found in the faeces. In contrast, with [3'-methyl-(14)C]streptozotocin a much smaller proportion (approx. 42%) of the injected radioactivity was excreted in the urine, the major proportion appearing in the first 6h, whereas approx. 53% of the injected radioactivity was retained in the carcasses. In whole-body radioautographic studies very rapid renal clearance and hepatic accumulation of the injected radioactivity was observed with all three labelled forms of the drug. There was some evidence for biliary and intestinal excretion. Major differences were apparent in the tissue-distribution studies, with each of the three labelled forms, particularly with [3'-methyl-(14)C]streptozotocin. There was no accumulation of [1-(14)C]streptozotocin in the pancreas for the 6h period after administration. However, with [3'-methyl-(14)C]streptozotocin (and also [2'-(14)C]streptozotocin) there was evidence of some pancreatic accumulation after 2h. The results indicate that streptozotocin is subjected to considerable metabolic transformation and to rapid renal clearance. The implication of these suggestions is evaluated with particular reference to the diabetogenic action of streptozotocin.     

A putative nuclear growth factor‐like globular nematode specific protein

Expressed sequence tags (ESTs) are an effective approach for discovery of novel genes. In the current study, approximately 250 ESTs of the cattle parasitic nematode Setaria digitata were examined and a cDNA clone identified whose coding sequence could not be functionally annotated by searching over publicly available genome, protein, EST and STS databases. Here, we report the extensive characterization of this ORF (UP) and its homologues using a bioinformatic approach. Uncharacterized protein (SDUP) of S. digitata consists of 204 amino acids with a predicted molecular weight and isoelectric point of 22.8KDa and 9.94, respectively. A search carried out using SDUP over nucleotide, EST and protein databases at NCBI, NEMBASE3 and Parasite Genome Database (PGD) identified homologous counterparts from the human parasitic nematodes Wuchereria bancrofti (WB), Brugia malayi (BM), Onchocerca volvulus (OV), the mouse filarial worm Litomosoides sigmodontis (LS), swine parasitic nematodes Ascaris suum (AS) and diverged counterparts from the plant parasitic nematode Meloidogyne hapla (MH) and free living nematodes Caenorhabditis elegans (CE) and Caenorhabditis briggsae (CB). Phylogenetic analyses revealed the UPs to be undergoing divergent evolution. A search of the ESTs at PGD showed that UP is expressed in all the stages of BM. Secondary structure analyses of multiply-aligned sequences of homologues using Jpred server indicated UPs to be rich in beta-pleated structures. TMMHH server and beta barrel finder programme indicated, UPs to be neither transmembrane or beta barrels proteins but are likely to be globular proteins. Further, the Motif discovery tool of MEME identified three novel potential motifs for UPS, of which only two are present in CE, CB & MH. Analyses of UPs using Signal IP, TargetP, Psort servers predicted this group of proteins to be devoid of signal peptide cleavage sites, are not mitochondrial targeting peptides but appear to be localized to the nucleus, respectively. Further analyses of the UPs using ScanProsite server for phosphorylation revealed potential sites for cAMP­ and cGMP­dependent protein kinase, Protein kinase C and Casein kinase II. Putative functional analysis using ProtFun 2.1 Server indicated UPs to be nonenzymatic, growth factor like protein. Finally, collating all the information derived from bioinformatic analyses, we conclude that the UPs of nematodes are most likely to be expressed at all stages in the life cycle, localized to the nucleus, regulated by phosphorylation, rich in beta­pleated strands and are growth factor like nematode specific proteins.     

Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti

Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.     

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae)

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus D?nitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.