Genetic and Molecular Studies for Regulation of Bolting Time of Onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.)

The control of bolting time in onion is an important approach for bulb and seed production, as onion plants which bolt do not produce marketable bulbs and seed yields are dependent on floral induction. However, genetic and molecular studies about bolting time in onion plants have not been examined yet to date. In order to understand the regulation of bolting time in onion plants, we conducted the genetic crosses between late bolting-type cultivar (MOS8) and very early bolting-type cultivar (Guikum). Segregation ratio of late to very early in F₂ populations indicated that this lateness trait was determined by a dominant locus. We also analyzed protein profiles in onion plants with different bolting time by a proteomics approach. Interestingly, a protein spot with significant similarities to chromodomains of mammalian chromo-ATPase/helicase-DNA-binding 1 or heterochromatin protein 1, which is involved in the histone modifications, was identified. Histone methyltransferase activity was also observed in onion plants. Taken together, these results suggest that a genetic pathway may be involved in the modulation of bolting time in onion plants, though there is no direct evidence that this protein spot obtained by proteomics is relevant to vernalization.     

Comparative proteome analysis of TGF‐β1‐induced fibrosis processes in normal rat kidney interstitial fibroblast cells in response to ascofuranone

Transforming growth factor‐β1 (TGF‐β1) has a wide range of biological functions such as the regulation of cell growth, differentiation, and immunological response in various types of cells. Particularly, TGF‐β1 induces plasminogen activator inhibitor‐1 (PAI‐1) as a major target protein. PAI‐1 is associated with fibrosis, thrombosis, and metabolic disorders. In this study, to identify proteins potentially involved in TGF‐β1‐induced fibrosis processes, we performed a proteomic analysis of TGF‐β1‐induced normal rat kidney cells exposed to ascofuranone (AF). In these cells, we detected 1500 proteins, with 74 differentially expressed proteins identified by MALDI‐TOF and reference to the NCBI and Swiss‐Prot databases, including PAI‐1, peroxisome prdifesator‐activated receptor, prohibitin, glutamate formyltransferase, LIM domain protein 1, LASP‐1, porphobilinogen deaminase, and peroxiredoxin 2. We also found that AF suppresses expression of profibrotic factors induced by TGF‐β in renal fibroblasts, including matrix proteins and PAI‐1. AF was also shown to inhibit selectively phosphorylation of epidermal growth factor receptor, and downstream kinases such as extracellular signal‐regulated kinase 1/2 (ERK‐1/2). Further ongoing analysis of fibrosis‐related proteins will determine AF's potential for application in fibrotic diseases and therapeutics.     

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.     

Calpain inhibitory flavonoids isolated from Orostachys japonicus

The n-butanol (n-BuOH) fraction of Orostachys japonicus A. Berger (Crassulaceae) significantly inhibited calpain activity. Through the activity-guided isolation from the n-BuOH fraction, herbacetin 8-O-alpha-D-ribopyranoside (1), kaempferol (2), quercetin (3), afzelin (4), astragalin (5), isoquercetin (6) and quercitrin (7) were obtained. Their structures were determined by spectroscopic techniques. Among them, compound 3 and 5 had significant calpain inhibitory activities.     

Dimerization of Translationally Controlled Tumor Protein Is Essential For Its Cytokine-Like Activity

Background

Translationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn).

Methods and Findings

We found that only NH₂-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH₂-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH₂-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice.

Conclusions

Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.     

Dissecting the pleiotropic consequences of a quantitative trait nucleotide

The downstream consequences of a single quantitative trait polymorphism can provide important insight into the molecular basis of a trait. However, the molecular consequences of a polymorphism may be complex and only a subset of these may influence the trait of interest. In natural isolates of Saccharomyces cerevisiae , a nonsynonymous polymorphism in cystathione β-synthase ( CYS4 ) causes a deficiency in both cysteine and glutathione that results in rust-colored colonies and drug-dependent growth defects. Using a single-nucleotide allele replacement, we characterized the effects of this polymorphism on gene expression levels across the genome. To determine whether any of the differentially expressed genes are necessary for the production of rust-colored colonies, we screened the yeast deletion collection for genes that enhance or suppress rust coloration. We found that genes in the sulfur assimilation pathway are required for the production of rust color but not the drug-sensitivity phenotype. Our results show that a single quantitative trait polymorphism can generate a complex set of downstream changes, providing a molecular basis for pleiotropy.     

<Emphasis Type="Italic">ClustalXeed</Emphasis>: a GUI-based grid computation version for high performance and terabyte size multiple sequence alignment

Background  

There is an increasing demand to assemble and align large-scale biological sequence data sets. The commonly used multiple sequence alignment programs are still limited in their ability to handle very large amounts of sequences because the system lacks a scalable high-performance computing (HPC) environment with a greatly extended data storage capacity.     

Solubility engineering and crystallization of human apolipoprotein D

Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions--W99H, I118S, and L120S--was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 A, b = 75.1 A, c = 166.0 A, alpha = beta = gamma = 90 degrees), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.     

Genome-Wide Profiling of Structural Genomic Variations in Korean HapMap Individuals

Background

Structural genomic variation study, along with microarray technology development has provided many genomic resources related with architecture of human genome, and led to the fact that human genome structure is a lot more complicated than previously thought.

Methodology/Principal Findings

In the case of International HapMap Project, Epstein-Barr various immortalized cell lines were preferably used over blood in order to get a larger number of genomic DNA. However, genomic aberration stemming from immortalization process, biased representation of the donor tissue, and culture process may influence the accuracy of SNP genotypes. In order to identify chromosome aberrations including loss of heterozygosity (LOH), large-scale and small-scale copy number variations, we used Illumina HumanHap500 BeadChip (555,352 markers) on Korean HapMap individuals (n = 90) to obtain Log R ratio and B allele frequency information, and then utilized the data with various programs including Illumina ChromoZone, cnvParition and PennCNV. As a result, we identified 28 LOHs (>3 mb) and 35 large-scale CNVs (>1 mb), with 4 samples having completely duplicated chromosome. In addition, after checking the sample quality (standard deviation of log R ratio <0.30), we selected 79 samples and used both signal intensity and B allele frequency simultaneously for identification of small-scale CNVs (<1 mb) to discover 4,989 small-scale CNVs. Identified CNVs in this study were successfully validated using visual examination of the genoplot images, overlapping analysis with previously reported CNVs in DGV, and quantitative PCR.

Conclusion/Significance

In this study, we describe the result of the identified chromosome aberrations in Korean HapMap individuals, and expect that these findings will provide more meaningful information on the human genome.