Six papaya samples showing downward leaf curling were collected in Guangdong and Guangxi provinces, China. The result of TAS-ELISA showed they were all infected by geminiviruses. Comparison of partial DNA-A sequences reveals that these virus isolates can be classified into two groups. Group I includes isolates G2, G4, G5, G28 and G29 from Guangxi province, while isolate GD2 from Guangdong province belongs to Group II. The complete DNA-A sequence of G2 and GD2 were characterized. Sequence comparisons showed that the DNA-A of G2 and GD2 were most closely related to that of Ageratum yellow vein China virus- [Hn2] and Ageratum yellow vein virus , respectively, with 83.4 and 75.2% nucleotide sequence identity, while DNA-A sequence between G2 and GD2 had only 73.4% sequence identity. The molecular data suggests that G2 and GD2 are two distinct begomoviruses, for which the name Papaya leaf curl China virus (PaLCuCNV) for G2 and Papaya leaf curl Guangdong virus (PaLCuGDV) for GD2 are proposed. Comparison of individual encoded proteins showed the coat protein of G2 and GD2 shared highest amino acid sequence identity (97.7 and 94.2%, respectively) with that of Pepper leaf curl virus -[Malaysia] (PepLCV-[MY]), suggesting the CP of these viruses may have identical ancestor.
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.
A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China.
Bats are increasingly being recognized as important reservoir hosts for a large number of viruses, some of them can be highly virulent when they infect human and livestock animals. Among the new bat zoonotic viruses discovered in recent years, several reoviruses (respiratory enteric orphan viruses) were found to be able to cause acute respiratory infections in humans, which included Melaka and Kampar viruses discovered in Malaysia, all of them belong to the genus Orthoreovirus, family Reoviridae. In this report, we describe the isolation of a highly related virus from an adult patient who suffered acute respiratory illness in Malaysia. Although there was no direct evidence of bat origin, epidemiological study indicated the potential exposure of the patient to bats before the onset of disease. The current study further demonstrates that spillover events of different strains of related orthoreoviruses from bats to humans are occurring on a regular basis, which calls for more intensive and systematic surveillances to fully assess the true public health impact of these newly discovered bat-borne zoonotic reoviruses.
Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) play diverse roles in the cellular biology of many organisms, including signal transduction, secretion and vesicular trafficking, and regulation of cytoskeleton assembly. Discovery of the PIP5K gene in Eimeria tenella may shed light on its role in the biology of this avian protozoan, and afford further understanding of the cell-host interaction, particularly during the invasion process. In this study, we report the identification of the PIP5K coding region in the genome sequence of Eimeria tenella using in silico gene prediction approaches. Prediction of the PIP5K coding sequence was confirmed by mapping the full-length cDNA sequence, generated via the Rapid Amplification of cDNA Ends (RACE) method, to the genomic sequence. The putative PIP5K gene of Eimeria tenella is located on the complementary strand of the E1080B12.b1 contig, and comprises 12 exons. Further analysis showed that the coding region spans from exon 1 to exon 7, with all exons obeying the adopted 'gt...ag' splicing rule of intronic sequences. Consensus of the hexameric 5' donor-splice site was deduced as GTRDBB... and the consensus for the 3' acceptor-splice sites as ...BHDYAG. The gene encodes a 252-amino acid residue protein. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a PIP5K.
VCP (Valosin-Containing Protein), a member of the AAA (ATPases Associated to a variety of cellular Activities) family of proteins, possesses a duplicated highly conserved ATPase domain. An expressed sequence tag (EST), representing a clone from the Eimeria tenella merozoite cDNA library, was found to have high similarity to VCP genes from other organisms. A complete sequence derived from the corresponding clone (designated eth060) shows amino acid identity of 42-62% with other members of the VCP subfamily. Sequence analysis identified a putative ATPase domain in the eth060 sequence. This domain was PCR-amplified using gene-specific primers and cloned into a pBAD/Thio-TOPO expression vector. Expression in Escherichia coli demonstrated that the putative ATPase domain, which consists of 414 amino acid residues, produced a fusion protein of approximately 60 kDa in size.
Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
1. The receptors for relaxin in the rat atria and uterus were investigated and compared by use of a series of synthetic and native relaxin analogues. The assays used were the positive chronotropic and inotropic effects in rat spontaneously beating, isolated right atrium and electrically driven left atrium and the relaxation of K+ precontracted uterine smooth muscle. 2. Relaxin analogues with an intact A- and B-chain were active in producing powerful chronotropic and inotropic effects in the rat isolated atria at nanomolar concentrations. Single-chain analogues and structural homologues of relaxin such as human insulin and sheep insulin-like growth factor I had no agonist action and did not antagonize the effect of the B29 form of human gene 2 relaxin. 3. Shortening the B-chain carboxyl terminal of human gene 1 (B2-29) relaxin to B2-26 reduced the activity of the peptide and removal of another 2 amino acid residues (B2-24) abolished the activity. This suggests that the B-chain length may be important for determination of the activity of relaxin. More detailed studies are needed to determine the effect of progressive amino acid removal on the structure and the bioactivity of relaxin. 4. Porcine prorelaxin was as active as porcine relaxin on a molar basis, suggesting that the presence of the intact C-peptide did not affect the binding of the prorelaxin to the receptor to produce functional responses. 5. Relaxin caused relaxation of uterine longitudinal and circular smooth muscle precontracted with 40 mM K+. The pEC50 values for human gene 2 and porcine relaxins were lower than those in the atrial assay, but rat relaxin had similar pEC50 values in both atrial and uterine assays. Rat relaxin was significantly less potent than either human gene 2 or porcine relaxin in the atrial assay, but in the uterine assay they were equipotent. The results suggest that the relaxin receptor or the signalling pathway in rat atria may differ from that in the uterus.
Over the past year, many reports have been published on a variety of clinical manifestations related to antiphospholipid antibodies. The low prevalence of anticardiolipin antibodies with the rare occurrence of thrombosis and a low rate of fetal loss in studies in Malaysia and China showed a potential role for local factors. A study of cross-reactive idiotype of the anticardiolipin antibody suggested that anticardiolipin antibodies are derived from a set of natural autoreactive clones. Regarding the pathogenic role of the antiphospholipid antibody, evidence has been presented that the epitopes formed between cardiolipin and beta 2 glycoprotein I are the targets of the antiphospholipid antibody. Complement activation, abnormalities of natural anticoagulants such as protein S deficiency, and genetic association with DR4, DR7, and DRw53 have also been studied.
Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3-hydroxy-4-methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro-organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV.
Tuberculosis (TB), an epidemic disease, affects the world with death rate of two million people every year. The bacterium Mycobacterium tuberculosis was found to be a more potent and disease-prolonged bacterium among the world due to multi-drug resistance. Emergence of new drug targets is needed to overcome the bacterial resistance that leads to control epidemic tuberculosis. The pathway thiamine biosynthesis was targeting M. tuberculosis due to its role in intracellular growth of the bacterium. The screening of enzymes involved in thiamin biosynthesis showed novel target thiazole synthase (ThiG) involved in catalysis of rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine. We carried out homology modeling for ThiG to understand the structure-function relationship, and the model was refined with MD simulations. The results showed that the model predicted with (α + β)8-fold of synthase family proteins. Molecular docking of ThiG model with substrate DXP showed binding mode and key residues ARG46, ASN69, THR41, and LYS96 involved in the catalysis. First-line anti-tuberculosis drugs were docked with ThiG to identify the inhibition. The report showed the anti-tuberculosis drugs interact well with ThiG which may lead to block thiamin biosynthesis pathway.
Infection with Plasmodium knowlesi, a zoonotic primate malaria, is a growing human health problem in Southeast Asia. P. knowlesi is being used in malaria vaccine studies, and a number of proteins are being considered as candidate malaria vaccine antigens, including the Apical Membrane Antigen 1 (AMA1). In order to determine genetic diversity of the ama1 gene and to identify epitopes of AMA1 under strongest immune selection, the ama1 gene of 52 P. knowlesi isolates derived from human infections was sequenced. Sequence analysis of isolates from two geographically isolated regions in Sarawak showed that polymorphism in the protein is low compared to that of AMA1 of the major human malaria parasites, P. falciparum and P. vivax. Although the number of haplotypes was 27, the frequency of mutations at the majority of the polymorphic positions was low, and only six positions had a variance frequency higher than 10%. Only two positions had more than one alternative amino acid. Interestingly, three of the high-frequency polymorphic sites correspond to invariant sites in PfAMA1 or PvAMA1. Statistically significant differences in the quantity of three of the six high frequency mutations were observed between the two regions. These analyses suggest that the pkama1 gene is not under balancing selection, as observed for pfama1 and pvama1, and that the PkAMA1 protein is not a primary target for protective humoral immune responses in their reservoir macaque hosts, unlike PfAMA1 and PvAMA1 in humans. The low level of polymorphism justifies the development of a single allele PkAMA1-based vaccine.
Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.
There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.
There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.
Phytol (PHY), a chlorophyll-derived diterpenoid, exhibits numerous pharmacological properties, including antioxidant, antimicrobial, and anticancer activities. This study evaluates the anti-diarrheal effect of phytol (PHY) along with its possible mechanism of action through in-vivo and in-silico models. The effect of PHY was investigated on castor oil-induced diarrhea in Swiss mice by using prazosin, propranolol, loperamide, and nifedipine as standards with or without PHY. PHY at 50 mg/kg (p.o.) and all other standards exhibit significant (p < 0.05) anti-diarrheal effect in mice. The effect was prominent in the loperamide and propranolol groups. PHY co-treated with prazosin and propranolol was found to increase in latent periods along with a significant reduction in diarrheal section during the observation period than other individual or combined groups. Furthermore, molecular docking studies also suggested that PHY showed better interactions with the α- and β-adrenergic receptors, especially with α-ADR1a and β-ADR1. In the former case, PHY showed interaction with hydroxyl group of Ser192 at a distance of 2.91Å, while in the latter it showed hydrogen bond interactions with Thr170 and Lys297 with a distance of 2.65 and 2.72Å, respectively. PHY exerted significant anti-diarrheal effect in Swiss mice, possibly through blocking α- and β-adrenergic receptors.
Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.
A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.
Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.
Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.