Gene Expression of Neuropilin-1 and Its Receptors,VEGF/Semaphorin 3a,in Normal and Cancer Cells

Extracellular domains of the transmembrane glycoprotein, neuropilin-1 (Np1), specifically bind an array of factors and co-receptors including class-3 semaphorins (Sema3a), vascular endothelial growth factor (VEGF), hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-β 1 (TGF-β1), and fibroblast growth factor2 (FGF2). Np1 may have a role in immune response, tumor cell growth, and angiogenesis, but its relative expression in comparison to its co-primary receptors, VEGF and Sema3a, is not known. In this study we determined the mRNA expression of Np1 and its co-receptors, VEGF and Sema3a, and the ratio of VEGF/Sema3a in different human and rodent cell lines. Expression of Np1, VEGF and Sema3a is very low in cells derived from normal tissues, but these proteins are highly expressed in tumor-derived cells. Furthermore, the ratio of VEGF/Sema3a is highly variable in different tumor cells. The elevated mRNA expression of Np1 and its putative receptors in tumor cells suggests a role for these proteins in tumor cell migration and angiogenesis. As different tumor cells exhibit varying VEGF/Sema3a ratios, it appears that cancer cells show differential response to angiogenic factors. These results bring to light the individual variation among the cancer-related genes, Np1, VEGF, and Sema3a, and provide an important impetus for the possible personalized therapeutic approaches for cancer patients.     

ABCG2: A potential marker of stem cells and novel target in stem cell and cancer therapy

ABCG2 is a member of the ATP binding cassette (ABC) transporters, which can pump a wide variety of endogenous and exogenous compounds out of cells. Widely expressed in stem cells, ABCG2 is also found to confer the side population phenotype and is recognized as a universal marker of stem cells. Although the precise physiological role of ABCG2 in stem cells is still unclear, existing data strongly suggest that ABCG2 plays an important role in promoting stem cell proliferation and the maintenance of the stem cell phenotype. In addition, ABCG2 is also found to be expressed in a number of cancer cells and appears to be a marker of cancer stem cells. Moreover, ABCG2 expression in tumors may contribute to their formation and progression. Thus, ABCG2 has potential applications in stem cell and tumor therapy.     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

Methyl lucidenate F isolated from the ethanol-soluble-acidic components of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> is a novel tyrosinase inhibitor

Tyrosinase is a key enzyme in the biosynthesis of melanin, and the use of inhibitors against tyrosinase can prevent hyperpigmentation by inhibiting enzymatic oxidation. However, the current use of tyrosine inhibitors is limited by their low activities and high toxicities. The aim of the present research was to develop novel whitening agents, or tyrosinase-targeted medicine, from a submerged culture of the fungus Ganoderma lucidum. Methyl lucidenate F was isolated from the ethanol-soluble-acidic components (ESACs) of G. lucidum, with the structure of ESACs elucidated via UV, LC-MS, and ¹³C-NMR spectral analysis. The tyrosinase inhibitory activity was measured using catechol as a substrate. Methyl lucidenate F displayed uncompetitive inhibition of the potato tyrosinase activity, for which Lineweaver-Burk plots revealed a maximum reaction rate (V _max) of 0.4367/min, Michaelis constant (K _m) of 6.765 mM and uncompetitive inhibition constant (K _i) of 19.22 μM. Meanwhile, methyl lucidenate F (tetra cyclic triterpenoid) exhibited high tyrosinase inhibitory activity, with an IC₅₀ of 32.23 μM. These results suggest that methyl lucidenate F may serve as a potential candidate for skin-whitening agents.     

Molecular phylogenetic relationships of China Seas groupers based on cytochrome <Emphasis Type="Italic">b</Emphasis> gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and T _S/T _V ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus.     

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.     

Allozyme variation in the diploid (A genome) populations ofScilla scilloides (Hyacinthaceae)

Allozyme variation at eleven loci encoding seven enzyme systems were examined in 20 populations of diploid (genome AA, 2n = 16)Scilla scilloides in China. In comparison with the average species of seed plants studied, populations of this species display a high amount of genetic variation (A = 2.0, P = 58.6%, H_o = 0.172, and H_e = 0.185). Allozyme variation pattern revealed predominant outcrossing within populations and considerable differentiation (F_ST = 0.314) among populations as well as between the subtropic and temperate regions. The wide distribution, long existence and outcrossing are presumably the main factors responsible for the high genetic diversity within populations. But the gravity dispersal of seeds and pollination by small insects set limits to the increase of genetic variation within populations and promote differentiation between populations and regions. In addition, allozyme variation does not distinguishS. scilloides var.albo-viridis and suggests that subtropic populations may be considered as a genetic entity.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Assessing indica-japonica differentiation of improved rice varieties using microsatellite markers

To assess the indica-japonica differentiation of improved rice varieties, a total of 512 modem varieties including 301 indica and 211 japonica accessions were analyzed using 36 microsatellites. The Fst coefficients ranged from 0.002 to 0.730 among the loci with an average of 0.315. Significant differentiation was detected at 94.4% of the loci studied （P 〈 0.05, pairwise Fst tests）, indicating that there was a high level of indica-japonica differentiation within the improved varieties. At 18 loci, about 74%-98% of the alleles of indica and japonica accessions were distributed in two ranges of amplicon length. Linkage disequilibrium analysis showed that the distribution trends were significantly nonrandomly associated. Using the differentiation trends at the 18 loci, microsatellite index （MI） was proposed for discrimination of the two subspecies. When rice accessions with MI value greater than zero were classified as indica, and those with MI value smaller than zero were classified as japonica, about 96.1% of the accessions could be classified. This result agrees with the classification based on morphological-physiological characters, indicating that this method is feasible and effective.     

Profilin‐1 overexpression inhibits proliferation of MDA‐MB‐231 breast cancer cells partly through p27kip1 upregulation

Profilin‐1 (Pfn1), a ubiquitously expressed actin‐binding protein, has gained interest in epithelial‐derived cancer because of its downregulation in expression in various adenocarcinoma. Pfn1 overexpression impairs tumorigenic ability of human breast cancer xenografts thus suggesting that Pfn1 could be a tumor‐suppressor protein. The objective of the present study was to determine how Pfn1 overexpression affects cell‐cycle progression of breast cancer cells. We show that Pfn1 overexpression in MDA‐MB‐231 breast cancer cells causes cell‐cycle arrest in G1 phase and dramatically reduced proliferation in culture. Pfn1 overexpression results in increased protein stability of p27^kip1 (p27—a major cyclin‐dependent kinase inhibitor) and marked elevation in the overall cellular level of p27. Proliferation defect of Pfn1 overexpressers can be partly rescued by silencing p27 expression thus suggesting a critical role of p27 in Pfn1‐induced growth inhibition of MDA‐MB‐231 cells. Finally, Pfn1 overexpression was found to sensitize MDA‐MB‐231 cells to apoptosis in response to cytotoxic stimulus thus suggesting for the first time that survival of breast cancer cells can also be negatively influenced by Pfn1 upregulation. These findings may provide novel insights underlying Pfn1's tumor‐suppressive action. J. Cell. Physiol. 223:623–629, 2010. © 2010 Wiley‐Liss, Inc.