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.     

Proteomic characterization of the sulfur-reducing hyperthermophilic archaeon Thermococcus onnurineus NA1 by 2-DE/MS–MS

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, was isolated from a deep-sea hydrothermal vent area in Papua New Guinea. The strain requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides, amino acids and sugars. Recently, genome sequencing of Thermococcus onnurineus NA1 was completed. In this study, 2-DE/MS–MS analysis of the cytosolic proteome was performed to elucidate the metabolic characterization of Thermococcus onnurineus NA1 at the protein level. Among the 1,136 visualized protein spots, 110 proteins were identified. Enzymes related to metabolic pathways of amino acids utilization, glycolysis, pyruvate conversion, ATP synthesis, and protein synthesis were identified as abundant proteins, highlighting the fact that these are major metabolic pathways in Thermococcus onnurineus NA1. Interestingly, multiple spots of phosphoenolpyruvate synthetase and elongation factor Tu were found on 2D gels generated by truncation at the N-terminus, implicating the cellular regulatory mechanism of this key enzyme by protease degradation. In addition to the proteins involved in metabolic systems, we also identified various proteases and stress-related proteins. The proteomic characterization of abundantly induced proteins using 2-DE/MS–MS enables a better understanding of Thermococcus onnurineus NA1 metabolism.     

Chrysin,a natural flavone,improves murine inflammatory bowel diseases

Chrysin (5,7-dihydroxyflavone) is a natural flavone commonly found in many plants. It has previously been shown to be an anti-tumor agent. In this study, we investigated whether chrysin could alleviate the symptoms of dextran sodium sulfate (DSS)-induced colitis in mice and whether chrysin has an inhibitory effect on nuclear factor (NF)-κB activation in vitro. A significant blunting of weight loss and clinical signs was observed in DSS-exposed, chrysin-treated mice when compared to vehicle-treated mice. This was associated with a remarkable amelioration of the disruption of the colonic architecture, a significant reduction in colonic myeloperoxidase (MPO) activity, and a decrease in the production of inflammatory mediators such as nitric oxide (NO), prostaglandin (PG) E₂, and pro-inflammatory cytokines. In addition, chrysin inhibited tumor necrosis factor (TNF)-α-induced activation of NF-κB in IEC-6 cells. These findings suggest that chrysin exerts potentially clinically useful anti-inflammatory effects mediated through the suppression of NF-κB activation.     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane

Although phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP₂ remains unknown. GFP containing PIP₂-binding-PH domain of phospholipase Cδ (GFP-PHδ) was used to monitor PIP₂. Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHδ, while the opposite was observed when syndecan-4 was knocked-down. PIP₂ levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHδ was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHδ from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP₂ by negatively regulating PLC-dependent PIP₂ degradation.     

Casares’ map function: no need for a ‘corrected’ Haldane’s map function

A genetic map function M(d) = RF provides a mapping from the additive genetic distance d to the non-additive recombination fraction RF between a given pair of loci, where the recombination fraction is the proportion of gametes that are recombinant between the two loci. Genetic map functions are needed because in most experiments all we can directly observe are the recombination events. However, since a recombination event is only observed if there are an odd number of crossovers between the two loci, recombination fractions are not additive. One of the most widely used map functions is Haldane’s map function, which is derived under the assumptions of no chiasma and no chromatid interference, and has been in widespread use since 1919. However, Casares recently proposed a ‘corrected’ Haldane’s map function – we show here that this ‘corrected’ map function is not correct due to faulty assumptions and mistakes in its derivation.     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.     

Accurate intraocular pressure prediction from applanation response data using genetic algorithm and neural networks

The fact that Goldmann applanation tonometry does not accurately account for individual corneal elastic stiffness often leads to inaccuracy in the measurement of intraocular pressure (IOP). IOP should account not only for the effect of central corneal thickness (CCT) but should also account for other corneal biomechanical factors. A computational method for accurate and reliable determination of IOP is investigated with a modified applanation tonometer in this paper. The proposed method uses a combined genetic algorithm/neural network procedure to match the clinically measured applanation force-displacement history with that obtained from a nonlinear finite element simulation of applanation. An additional advantage of the proposed method is that it also provides the ability to determine CCT and material properties of the cornea from the same applanation response data. The performance of the proposed method has been demonstrated through a parametric study and via comparison with a well known clinical case. The proposed method is also shown to be computationally efficient, which is an important practical consideration for clinical application.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.