Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.     

双向BN/SDS-PAGE分离鉴定痢疾杆菌福氏5型M90T株毒力相关膜蛋白复合物

通过蓝色非变性凝胶电泳(BN-PAGE)比较痢疾杆菌福氏5型野生株M90T和大质粒缺失株M90T△T的膜蛋白复合物,发现一个野生株特有的复合物,其分子量的为290 kD,命名为M90T-290.通过第二向SDS-PAGE分离M90T-290得到6个蛋白亚基,质谱鉴定为:一个由大质粒编码的毒力蛋白Apyrase(ATP-二磷酸水解酶)和5个染色体编码的蛋白,这些蛋白可能以膜复合物的形式影响毒力蛋白IcsA的单极性分布和痢疾杆菌在细胞间的扩散.这个新发现的毒力相关膜复合物在痢疾杆菌的致病过程中可能发挥重要作用.     

Phylogeographic analysis reveals two cryptic species of the endangered fern <Emphasis Type="Italic">Ceratopteris</Emphasis><Emphasis Type="Italic">thalictroides</Emphasis> (L.) Brongn. (Parkeriaceae) in China

Ceratopteris thalictroides (L.) Brongn. (Parkeriaceae) is a difficult fern species to taxonomically classify. Three cryptic species were revealed in the previous studies, referred to as the north type, the south type, and the third type. Because much of the distribution range of C. thalictroides in China was not included in the sampling of the previous studies, the taxonomic complexity of C. thalictroides in China remained uncertain. In order to identify the uncharacterized cryptic species, we examined four chloroplast DNA (cpDNA) non-coding regions and compared sequence variation within this species complex. Sequence data were obtained from 143 individuals in 24 populations throughout the natural distribution of the species in China. Nineteen haplotypes were identified. Molecular systematic and phylogeographical analyses revealed two genetically distinct clusters of cpDNA haplotypes in China. One cluster included haplotypes associated with the north type, and another with the south type cryptic species. The N _ST value was significantly higher than the G _ST value (N _ST = 0.768 > G _ST = 0.434, P < 0.05), indicating the presence of a significant phylogeographical structure of C. thalictroides in China. The results of AMOVA analysis showed a significant inter-group differentiation (F _ST = 0.918; P < 0.001). Analyses based on different, but complementary methods suggest that in China, C. thalictroides contains only two of the cryptic species (the north and south types). Two haplotypes, H8 and H17, of the interior node in the minimum-spanning network (MSN) of cpDNA haplotypes are widespread. The origin of the widespread haplotypes in China may have resulted from long-distance dispersal to China.     

Identification of histone demethylases in Saccharomyces cerevisiae

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

Fe binding properties of two soybean (Glycine max L.) LEA4 proteins associated with antioxidant activity

Late embryogenesis abundant (LEA) group 4 (LEA4) proteins play an important role in the water stress tolerance of plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, the protective functions and mechanisms of LEA4 proteins are still not clear. In this study, the metal binding properties of two related soybean LEA4 proteins, GmPM1 and GmPM9, were tested using immobilized metal ion affinity chromatography (IMAC). The metal ions Fe(3+), Ni(2+), Cu(2+) and Zn(2+) were observed to bind these two proteins, while Ca(2+), Mg(2+) or Mn(2+) did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe(3+) was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe(3+)/H(2)O(2) system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions (100 mM NaCl), although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. These results suggest that GmPM1 and GmPM9 are metal-binding proteins which may function in reducing oxidative damage induced by abiotic stress in plants.     

Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse

Conventional knockout of the microsomal triglyceride transfer protein large subunit (lMTP) gene is embryonic lethal in the homozygous state in mice. We have produced a conditional lMTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal plasma lipids. Intravenous injection of an adenovirus harboring Cre recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of lMTP mRNA and immunoreactive protein in a liver-specific manner. There was also disappearance of plasma apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels. Wild-type mice showed no response, and heterozygous mice, an intermediate response, to AdCre1. Wild-type mice doubled their plasma cholesterol level following a high cholesterol diet. This hypercholesterolemia was abolished in AdCre1-treated lMTP-/- mice, the result of a complete absence of very low/intermediate/low density lipoproteins and a slight reduction in high density lipoprotein. Heterozygous mice showed an intermediate lipoprotein phenotype. The rate of accumulation of plasma triglyceride following Triton WR1339 treatment in lMTP-/- mice was <10% that in wild-type animals, indicating a failure of triglyceride-rich lipoprotein production. Pulse-chase experiments using hepatocytes isolated from wild-type and lMTP-/- mice revealed a failure of apoB secretion in lMTP-/- animals. Therefore, the liver-specific inactivation of the lMTP gene completely abrogates apoB-100 and very low/intermediate/low density lipoprotein production. These conditional knockout mice are a useful in vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins.     

Proteomic characterization of human milk whey proteins during a twelve-month lactation period

Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breast-fed infant.