Searching for aging-related proteins in human dermal microvascular endothelial cells treated with anti-aging agents

Endothelial cells constitute an interface between blood and tissue and act as a medium for active interaction between plasma and the intracellular environment for homeostasis. Aging of endothelial cells plays a significant role in the pathophysiology of age-related vascular diseases; however, precise mechanisms for senescence have not been elucidated. Proteomics allows identification of protein structures, functions, and characteristics, and can be applied to the study of aging processes. Using cultured human dermal microvascular endothelial cells and two-dimensional proteomic mapping, we studied the effects of kinetin, epigallocatechin-3-gallate, all-trans-retinoic acid, and selenium on their senescence and searched for the aging-related proteins. The treatments resulted in 68 qualitative changes and 172 quantitative changes, and we were able to identify 46 spots among them. All of the agents indicated above induced changes in the expression of moesin, rho guanosine-5'-diphosphate-dissociation inhibitor, and actin, confirmed by immunoblotting and confocal laser microscopy. As these proteins were associated with cell cycle and cytoskeleton, immunoblotting of the proteins related to cell cycle was performed. Although practical significance remains to be confirmed by in vivo research, this fundamental discovery may provide a basis for understanding the mechanism of aging and age-related diseases.     

Expression of EuNOD-ARP1 encoding auxin-repressed protein homolog is upregulated by auxin and localized to the fixation zone in root nodules of Elaeagnus umbellata

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.     

Development of a target component extraction method from GC-MS data with an in-house program for metabolite profiling

After gas chromatography-mass spectrometry (GC-MS) analysis, data processing, including retention time correction, spectral deconvolution, peak alignment, and normalization prior to statistical analysis, is an important step in metabolomics. Several commercial or free software packages have been introduced for data processing, but most of them are vendor dependent. To design a simple method for Agilent GC/MS data processing, we developed an in-house program, "CompExtractor", using Microsoft Visual Basic. We tailored the macro modules of an Agilent Chemstation and implanted them in the program. To verify the performance of CompExtractor processing, 30 samples from the three species of the genus Papaver were analyzed with Agilent 5973 MSD GC-MS. The results of CompExtractor processing were compared with those of AMDIS-SpectConnect processing by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The two methods showed good classification according to their species in HCA. The PC1+PC2 scores were 54.32-63.62% for AMDIS-SpectConnect and 56.65-85.92% for CompExtractor in PCA. Although the CompExtractor processing method is an Agilent GC-MS-specific application and the target compounds must be selected first, it can extract the target compounds more precisely in the raw data file with batch mode and simultaneously assemble the matrix text file.     

The impact of the stopping rule on sex ratio of last births in Vietnam

This study examines the hypothesis that the stopping rule - a traditional postnatal sex selection method where couples decide to cease childbearing once they bear a son - plays a role in high sex ratio of last births (SRLB). The study develops a theoretical framework to demonstrate the operation of the stopping rule in a context of son preference. This framework was used to demonstrate the impact of the stopping rule on the SRLB in Vietnam, using data from the Population Change Survey 2006. The SRLB of Vietnam was high at the level of 130 in the period 1970-2006, and particularly in the period 1986-1995, when sex-selective abortion was not available. Women were 21% more likely to stop childbearing after a male birth compared with a female birth. The SRLB was highest at parity 2 (138.7), particularly in rural areas (153.5), and extremely high (181.9) when the previous birth was female. Given the declining fertility, the stopping rule has a potential synergistic effect with sex-selective abortion to accentuate a trend of one-son families in the population.     

Functional characterization of ObgC in ribosome biogenesis during chloroplast development

The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT.     

The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.     

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.