Inhibitory potential of flavonoids on PtdIns(3,4,5)P3 binding with the phosphoinositide-dependent kinase 1 pleckstrin homology domain

Many membrane-associated proteins are involved in various signaling pathways, including the phosphoinositide 3-kinase (PI3K) pathway, which has key roles in diverse cellular processes. Disruption of the activities of these proteins is involved in the development of disease in humans, making these proteins promising targets for drug development. In most cases, the catalytic domain is targeted; however, it is also possible to target membrane associations in order to regulate protein activity. In this study, we established a novel method to study protein-lipid interactions and screened for flavonoid-derived antagonists of PtdIns(3,4,5)P₃ binding with the phosphoinositide-dependent kinase 1 (PDK1) pleckstrin homology (PH) domain. Using an enhanced green fluorescent protein (eGFP)-tagged PDK1 PH domain and 50% sucrose-loaded liposomes, the protein-lipid interaction could be efficiently evaluated using liposome pull-down assays coupled with fluorescence spectrophotometry, and a total of 32 flavonoids were screened as antagonists for PtdIns(3,4,5)P₃ binding with the PDK1 PH domain. From this analysis, we found that two adjunct hydroxyl groups in the C ring were responsible for the inhibitory effects of the flavonoids. Because the flavonoids shared structural similarities, the results were then subjected to quantitative structure-activity relationship (QSAR) analysis. The results were then further confirmed by in silico docking experiments. Taken together, our strategy presented herein to screen antagonists targeting lipid-protein interactions could be an alternative method for identification and characterization of drug candidates.     

Morphometric analysis of the genusHemerocallis L. (Liliaceae) in Korea

To better understand the patterns of variability and distributions ofHemerocallis in Korea, 53 locations were visited and measurements of 19 morphological and phenological characters were taken on plants directly from their natural habitats. For morphometric analysis, 10 plants from each of 34 populations and five herbarium specimens ofH. middendorffii were used and the data from 12 quantitative characters was analyzed using univariate analysis. Except the littoral populations of Cheju, Hong, Taehuksan, and Sohuksan Islands (H. hongdoensis M. Chung & S. Kang), three peninsular KoreanHemerocallis species can be recognized mainly in South Korea:H. hakuunensis Nakai (=H. micrantha Nakai, growing on southern, central, and northwestern Korea);H. thunbergii Baker (=H. coreana Nakai, found on southeastern and central Korea); andH. middendorffii Tr. et Mey. (central and northeastern Korea). Morphological and phenological features contributing to recognition of the three groups were; color of perianth, shape of roots, shape of inflorescence, flowering time, odor, length of inflorescence, width of the lowest bracts, length of perianth tube enclosing a ovary, width of the inner perianth lobes. Natural hybridization seems to be rare in KoreanHemerocallis. It appears that the KoreanHemerocallis species are relatively well characterized by their distribution patterns, phenology, and habitats compared with the JapaneseHemerocallis species.     

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra‐high‐density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l ‐asparagine content variation across populations, respectively. Metabolite?agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite?agronomic trait relationship and the corresponding genetic basis.     

Melanoma chondroitin sulfate proteoglycan regulates matrix metalloproteinase-dependent human melanoma invasion into type I collagen

Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes.     

Development and characterization of wheat-Psathyrostachys huashanica partial amphiploids for resistance to stripe rust

Two partial amphiploid lines, B113 (32 plants) and B21 (13 plants), derived from a wheat-Psathyrostachys huashanica intergeneric cross were characterized by Giemsa C-banding and SDS-PAGE and evaluated for stripe rust resistance. All 15 partial amphiploid plants were aneuploids with either 50 (8 plants), 51 (6 plants) or 54 (1 plant) chromosomes. Some showed regular meiosis and all the P. huashanica chromosomes were included, although not in a single plant. Of 45 plants 34 showed specific bands on SDS-PAGE representing high molecular weight glutenin subunit (HMW-GS) and 41 had bands representing P. huashanica low molecular weight glutenin subunit (LMW-GS), including two new subunits. All 45 plants were highly resistant (10) or immune (35) to stripe rust mixed races CYR-30, CYR-31, Shuiyuan 7 and Shuiyuan 14. These amphiploid plants could be useful germplasm for enhancing stripe rust resistance and might improve wheat grain quality.     

Genetic and clonal diversity in Korean populations ofVitex rotundifolia (Verbenaceae)

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (P _P), mean number of alleles per locus (A _P), and mean genetic diversity (He _P) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanG _ST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P<0.001). On the other hand, levels of genotypic diversity within and among populations were moderate. About 44% (18/41) of multilocus genotypes were “local genotypes” (genotypes occurring in only one population), whereas only one “widespread genotype” (genotypes occurring in more than 75% of the populations) were detected. The mean number of multilocus genotypes per population (G) and mean genotypic diversity index (D _G) were 8.4 and 0.74, respectively. Most common multilocus genotypes found in populations were homozygous for five polymorphic loci. The abundance of ramets of these genets is responsible for the low levels of expected heterozygosity within populations. The results indicate that clonal reproduction may act as an enhancer of genetic drift by reducing effective size of local populations ofV. rotundifolia.     

Activation of ERK5 in angiotensin II-induced hypertrophy of human aortic smooth muscle cells

Extracellular signal-regulated kinase 5 (ERK5), a recently discovered mitogen-activated protein kinase (MAPK), plays a key role in the development and pathogenesis of cardiovascular disease. In order to clarify the pathophysiological significance of ERK5 in vascular remodeling, we investigated ERK5 phosphorylation in hypertrophy of human aortic smooth muscle cells (HASMCs) induced by angiotensin II (Ang II). The AT1 receptor was involved in Ang II-induced ERK5 activity. Hypertrophy was detected by the measurement of protein synthesis with [³H]-Leu incorporation in cultured HASMCs. Ang II rapidly induced phosphorylation of ERK5 at Thr218/Tyr220 residues in a time- and dose-dependent manner. Activation of myocyte enhancer factor-2C (MEF2C) by ERK5 was inhibited by PD98059. Transfecting HASMCs with small interfering RNA (siRNA) to silence ERK5 inhibited Ang II-induced cell hypertrophy. Thus, ERK5 phosphorylation contributes to MEF2C activation and subsequent HASMC hypertrophy induced by Ang II, for a novel molecular mechanism in cardiovascular diseases induced by Ang II.     

Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10⁷ cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.     

FamPipe: An Automatic Analysis Pipeline for Analyzing Sequencing Data in Families for Disease Studies

In disease studies, family-based designs have become an attractive approach to analyzing next-generation sequencing (NGS) data for the identification of rare mutations enriched in families. Substantial research effort has been devoted to developing pipelines for automating sequence alignment, variant calling, and annotation. However, fewer pipelines have been designed specifically for disease studies. Most of the current analysis pipelines for family-based disease studies using NGS data focus on a specific function, such as identifying variants with Mendelian inheritance or identifying shared chromosomal regions among affected family members. Consequently, some other useful family-based analysis tools, such as imputation, linkage, and association tools, have yet to be integrated and automated. We developed FamPipe, a comprehensive analysis pipeline, which includes several family-specific analysis modules, including the identification of shared chromosomal regions among affected family members, prioritizing variants assuming a disease model, imputation of untyped variants, and linkage and association tests. We used simulation studies to compare properties of some modules implemented in FamPipe, and based on the results, we provided suggestions for the selection of modules to achieve an optimal analysis strategy. The pipeline is under the GNU GPL License and can be downloaded for free at http://fampipe.sourceforge.net.

This is a PLOS Computational Biology Software article.