Genetic characterization of <Emphasis Type="Italic">Perna viridis</Emphasis> L. in peninsular Malaysia using microsatellite markers

A total of 19 polymorphic microsatellite loci were used to analyse levels of genetic variation for 10 populations of Perna viridis L. collected from all over peninsular Malaysia. The populations involved in this study included Pulau Aman in Penang, Tanjung Rhu in Kedah, Bagan Tiang in Perak, Pulau Ketam in Selangor, Muar, Parit Jawa, Pantai Lido and Kampung Pasir Puteh in Johore, and Kuala Pontian and Nenasi in Pahang state. The number of alleles per locus ranged from two to seven, with an average of 3.1. Heterozygote deficiencies were observed across all the 10 populations. Characterization of the populations revealed that local populations of P. viridis in peninsular Malaysia were genetically similar enough to be used as a biomonitoring agent for heavy metal contamination in the Straits of Malacca. Cluster analysis grouped the P. viridis populations according to their geographical distributions with the exception of Parit Jawa. The analysis also revealed that P. viridis from the northern parts of peninsular Malaysia were found to be the most distant populations among the populations of mussels investigated and P. viridis from the eastern part of peninsular Malaysia were closer to the central and southern populations than to the northern populations.     

Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis,and consequently enhances Ras/ERK signalling

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF- mediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DeltaN11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation.     

Ultrastructural studies of developing goat oocytes in vitro

The structure and distribution of organelles within developing goat oocytes at various stages of incubation were studied. In oocytes with 5 or more layers of cumulus cells, at 0 h of incubation, the zona pellucida had developed although zonation was not evident. Lipid bodies were present but no mitochondria were observed. At 20 h, the zona pellucida had differentiated into thicker and thinner regions. Clusters of membrane-bound electron-transparent bodies were present in the perivitelline space. The mitochondria were fully developed, distributed evenly and usually in close proximity with dilated endoplasmic reticula. Cortical granules were distributed at the periphery. At 40 h of incubation, a number of mitochondria was hooded. In oocytes of 2 to 4 layers of cumulus cells at 0 h, the zona pellucida was penetrated by cumulus cell processes, and the mitochondria were not well developed. However, in 20-h incubated oocytes, fully developed mitochondria, many of which were hooded, could be observed. Clusters of membrane-bound electron-transparent bodies were also observed, while cortical granules were at the periphery. In cumulus-free oocytes, zonation within the zona pellucida was indistinct. Very few vesicles and lipid bodies were observed. At 20 h, mitochondria were sparsely distributed and were not well developed and lacked cristae. At 40 h, the zona pellucida was less compact, and the membrane-bound electron-transparent bodies were less numerous compared with those of the other groups. Endoplasmic reticula were not dilated, and cortical granules were few and had no definite pattern of distribution.     

The role of long chain omega-3 polyunsaturated fatty acids in reducing lipid peroxidation among elderly patients with mild cognitive impairment: a case-control study

The present work explores the effect of dietary omega-3 polyunsaturated fatty acids (PUFAs) intake on lipid peroxidation among mild cognitive impairment (MCI) patients. The plasma lipid hydroperoxide (LPO) levels in 67 MCI patients were compared to those of 134 healthy elderly controls. Omega-3 PUFA intake was assessed using an interviewer-administered food frequency questionnaire. Apolipoprotein E genotyping was performed using polymerase chain reaction and restriction enzyme digestion. The association between various confounders and lipid peroxidation was evaluated using regression analysis. The influence of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intake on LPO level was investigated. The results revealed that LPO levels were significantly higher in the MCI group than in the control group. Inverse correlations were found between DHA and EPA intake and LPO level among the MCI group. LPO levels decreased significantly with increasing DHA and EPA intake. In summary, the findings revealed that DHA and EPA can play a role in alleviating oxidative stress and reducing the risk of neurodegenerative diseases.     

Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.     

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.     

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 10⁷ K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.     

Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques

Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000-40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.     

Extracellular matrix as the early structural marker for <Emphasis Type="Italic">Centella asiatica</Emphasis> embryogenic tissues

Embryogenic and non-embryogenic calli were induced from the Centella asiatica leaf explants on Murashige and Skoog medium supplemented with kinetin and 2,4-dichlorophenophenoxyacetic acid. The extracellular matrix (ECM) layer was seen on the surface of embryogenic cells but not on the non-embryogenic cells. The ECM formed bridges with net-like material between the embryogenic cells. This network like structure was believed to play an important role in plant morphogenesis and can serve as an early structural marker of embryogenic competence in Centella asiatica calli culture.