<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Melastoma malabathricum</Emphasis> and <Emphasis Type="Italic">Tibouchina semidecandra</Emphasis> with sense and antisense dihydroflavonol-4-reductase (DFR) genes

Genetic engineering of a wide variety of plant species has led to the improvement of plant traits. In this study, the genetic transformation of two potentially important flowering ornamentals, Melastoma malabathricum and Tibouchina semidecandra, with sense and antisense dihydroflavonol-4-reductase (DFR) genes using the Agrobacterium-mediated method was carried out. Plasmids pBETD10 and pBETD11, each harbouring the DFR gene at different orientations (sense and antisense) and selectable marker nptII for kanamycin resistance, were used to transform M. malabathricum and T. semidecandra under the optimized transformation protocol. Putative transformants were selected in the presence of kanamycin with their respective optimized concentration. The results indicated that approximately 4.0% of shoots and 6.7% of nodes for M. malabathricum regenerated after transforming with pBETD10, whereas only 3.7% (shoots) and 5.3% (nodes) regenerated with pBETD11 transformation. For the selection of T. semidecandra, 5.3% of shoots and 9.3% of nodes regenerated with pBETD10 transformation, while only 4.7% (shoots) and 8.3% (nodes) regenerated after being transformed with pBETD11. The presence and integration of the sense and antisense DFR genes into the genome of M. malabathricum and T. semidecandra were verified by polymerase chain reaction (PCR) and nucleotide sequence alignment and confirmed by southern analysis. The regenerated putative transformants were acclimatized to glasshouse conditions. Approximately 31.0% pBETD10-transformed and 23.1% pBETD11-transformed M. malabathricum survived in the glasshouse, whereas 69.4% pBETD10-transformed and 57.4% pBETD11-transformed T. semidecandra survived. The colour changes caused by transformation were observed at the budding stage of putative T. semidecandra transformants where greenish buds were produced by both T. semidecandra harbouring the sense and antisense DFR transgenes. Besides that, the production of four-petal flowers also indicated another morphological difference of putative T. semidecandra transformants from the wild type plants which produce five-petal flowers.     

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (β-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (β-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.     

Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1–4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Neoplastic transformation and growth require a massive increase in glucose uptake and glycolytic flux not only for energy production but also for the synthesis of nucleic acids, amino acids, and fatty acids. A central control point of glycolysis is the negative allosteric regulation of a rate-limiting enzyme, phosphofructokinase-1 (PFK-1),² by ATP (i.e. the Pasteur effect) (1, 2). When intracellular ATP production exceeds usage, ATP inhibits PFK-1 and glycolytic flux. Fructose 2,6-bisphosphate (Fru-2,6-BP) is a potent allosteric activator of PFK-1 that overrides this inhibitory influence of ATP on PFK-1, allowing forward flux of the entire pathway (3–5).The steady-state cellular concentration of Fru-2,6-BP is dependent on the activities of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four independent genes (PFKFB1–4) (6, 7). The PFKFB3 mRNA is distinguished by the presence of multiple copies of an AUUUA instability motif in its 3′-untranslated region and the PFKFB3 protein product has a high kinase:phosphatase activity ratio (740:1) (8). PFKFB3 mRNA is overexpressed by rapidly proliferating transformed cells and the PFKFB3 protein is highly expressed in solid tumors and leukemias (8–11). PFKFB3 expression is increased in response to several mitogenic stimuli, including progesterone, serum, and insulin (12–14). These studies indicate that the PFKFB3 enzyme may serve an essential function in the regulation of glucose metabolism during cell proliferation.The PFKFB3 mRNA is spliced into several variants that encode distinct carboxyl-terminal domains (9, 15). Importantly, the functional consequences of the disparate carboxyl-terminal variants of PFKFB3 are unknown. The mRNA splice variant 5 is the dominant PFKFB3 mRNA in human brain, several transformed cells, and colon adenocarcinoma tissues (9, 10). In the following series of experiments, we present data that the carboxyl-terminal domain of PFKFB3 variant 5 localizes the enzyme to the nucleus where its product, Fru-2,6-BP, increases the expression and activity of cyclin-dependent kinase-1. These data demonstrate a heretofore unidentified function of the PFKFB3 enzyme that is distinct from glycolysis, and provide a potential mechanism for the coupling of metabolism and proliferation.     

Complex inheritance of the plasmodial surface anion channel in a Plasmodium falciparum genetic cross

Human erythrocytes infected with the malaria parasite Plasmodium falciparum have increased permeabilities to many solutes. The plasmodial surface anion channel (PSAC) may mediate these changes. Despite good understanding of the biochemical and biophysical properties, the genetic basis of PSAC activity remains unknown. Functional polymorphisms in laboratory isolates and two mutants generated by in vitro selection implicate a parasite-encoded channel, although parasite-induced modifications of endogenous channels have not been formally excluded. Here, we identified stable differences in furosemide efficacy against PSAC activity induced by HB3 and 3D7A parasites. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl^- and sorbitol are transported by a single-channel type. Examination of 19 progeny from a genetic cross between HB3 and 3D7A revealed complex inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure.     

Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53

Background  

Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells.     

On GPU’s viability as a middleware accelerator

Today Graphics Processing Units (GPUs) are a largely underexploited resource on existing desktops and a possible cost-effective enhancement to high-performance systems. To date, most applications that exploit GPUs are specialized scientific applications. Little attention has been paid to harnessing these highly-parallel devices to support more generic functionality at the operating system or middleware level. This study starts from the hypothesis that generic middleware-level techniques that improve distributed system reliability or performance (such as content addressing, erasure coding, or data similarity detection) can be significantly accelerated using GPU support. We take a first step towards validating this hypothesis and we design StoreGPU, a library that accelerates a number of hashing-based middleware primitives popular in distributed storage system implementations. Our evaluation shows that StoreGPU enables up twenty five fold performance gains on synthetic benchmarks as well as on a high-level application: the online similarity detection between large data files.

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases—a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archeaons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.     

REFOLD: an analytical database of protein refolding methods

The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources--REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community.     

Sarcocystosis among Wild Captive and Zoo Animals in Malaysia

Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 × 24.5 µm and the thickness of the wall up to 2.5 µm. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.