Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

Epinephrine-induced hyperpolarization of islet cells without KATP channels

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.     

N-4-methansulfonamidobenzyl-N'-2-substituted-4-tert-butyl-benzyl thioureas as potent vanilloid receptor antagonistic ligands

A series of N-4-methansulfonamidobenzyl-N'-2-substituted-4-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that there is a space for another hydrophobic binding interaction around 2-position in 4-tert-butylbenzyl region. Among the prepared derivatives, 6n show the highest antagonistic activity against the vanilloid receptor (IC(50)=15 nM).     

Design and synthesis of 3,7-diarylimidazopyridines as inhibitors of the VEGF-receptor KDR

3,7-Diarylsubstituted imidazopyridines were designed and developed as a new class of KDR kinase inhibitors. A variety of imidazopyridines were synthesized and potent inhibitors of KDR kinase activity were identified with good aqueous solubility.     

N-4-Substituted-benzyl-N'-tert-butylbenzyl thioureas as vanilloid receptor ligands: investigation on the role of methanesulfonamido group in antagonistic activity

A series of N-4-substituted-benzyl-N'-tert-butylbenzyl thioureas were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor in rat DRG neurons. Their structure-activity relationship reveals that not only the two oxygens and amide hydrogen of sulfonamido group, but also the optimal size of methyl in methanesulfonamido group play an integral role for the antagonistic activity on vanilloid receptor.     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

<Emphasis Type="Italic">Prorocentrum vietnamensis</Emphasis> sp. nov. (Prorocentraceae Dinophyta): A new armored dinoflagellate from Vietnam coastal waters

TheProrocentrum species, which are primarily marine, are distributed worldwide and occur in ocean, neritic, and littoral environments.Prorocentrum is either a planktonic or benthic genus, with some species possibly being both. Until now, about 35 species had been reported including two that are fresh-water. Some species contain toxic substances. As one of many currently active studies, we collectedP. vietnamensis sp. nov. from Hon Mé, Gulf of Tongkin, Vietnam. Based on its cell shape, smooth valve surface, arrangement of valves and marginal pores, plus characteristics of the central pyrenoid, we propose thatP. vietnamensis is a new species. Its attributes include straight cell sides that are parallel to each other, as well as anterior and posterior areas with rounded ends that form a short, rod-shaped cell that is unique to this species.     

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.