Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application

For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.     

Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin

Staphylococcal α-hemolysin (Hla) is a principal small β-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, β-hexosaminidase. However, Hla more than doubled the releases of β-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130?mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.     

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells

Molecular and Cellular Biochemistry - Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of...     

Melanoderma boninense,a new species of Melanoderma (Polyporales,Agaricomycota) from the Bonin Islands in Japan

Melanoderma boninense is described and illustrated as a new species from the Bonin Islands, Japan, on the basis of morphological and phylogenetic investigations. This species is characterized by sessile basidiocarps with an ungulate pileus, a black crust on the pileus surface, pores 4–6/mm, a dimitic hyphal system comprising clamped generative hyphae and cyanophilous skeletal binding hyphae with or without a dextrinoid reaction, cylindrical basidiospores measuring 5.5–7.5 × 2–3 μm, and cystidioles on the sides of tubes and near the pore surface. Morphological examination of authentic specimens of other Melanoderma species revealed that the genus is variable in terms of the shape and size of cystidioles. A black crust on the pileus surface composed of palisade and highly agglutinated hyphae is a distinctive diagnostic morphological feature of Melanoderma that differentiates it from allied genera. Melanoderma boninense, which is currently known from a restricted area of the Bonin Islands, is potentially threatened by environmental reduction of the type locality due to the invasive tree Bischofia javanica.     

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.     

Adipocyte-specific Disruption of Fat-specific Protein 27 Causes Hepatosteatosis and Insulin Resistance in High-fat Diet-fed Mice

White adipose tissue (WAT) functions as an energy reservoir where excess circulating fatty acids are transported to WAT, converted to triglycerides, and stored as unilocular lipid droplets. Fat-specific protein 27 (FSP27, CIDEC in humans) is a lipid-coating protein highly expressed in mature white adipocytes that contributes to unilocular lipid droplet formation. However, the influence of FSP27 in adipose tissue on whole-body energy homeostasis remains unclear. Mice with adipocyte-specific disruption of the Fsp27 gene (Fsp27^ΔAd) were generated using an aP2-Cre transgene with the Cre/LoxP system. Upon high-fat diet feeding, Fsp27^ΔAd mice were resistant to weight gain. In the small WAT of these mice, small adipocytes containing multilocular lipid droplets were dispersed. The expression levels of the genes associated with mitochondrial abundance and brown adipocyte identity were increased, and basal lipolytic activities were significantly augmented in adipocytes isolated from Fsp27^ΔAd mice compared with the Fsp27^F/F counterparts. The impaired fat-storing function in Fsp27^ΔAd adipocytes and the resultant lipid overflow from WAT led to marked hepatosteatosis, dyslipidemia, and systemic insulin resistance in high-fat diet-treated Fsp27^ΔAd mice. These results demonstrate a critical role for FSP27 in the storage of excess fat in WAT with minimizing ectopic fat accumulation that causes insulin-resistant diabetes and non-alcoholic fatty liver disease. This mouse model may be useful for understanding the significance of fat-storing properties of white adipocytes and the role of local FSP27 in whole-body metabolism and estimating the pathogenesis of human partial lipodystrophy caused by CIDEC mutations.     

Preliminary evaluation of greenhouses employing positive-pressure forced ventilation to prevent invasion by insect pests

We conducted a preliminary comparison of greenhouses using positive-pressure forced ventilation (PFV) systems and natural ventilation (NV) systems, and assessed the effectiveness of both systems for preventing the invasion of greenhouses used to cultivate tomatoes by insect pests. In Trial 1 (August–December 2006), greenhouses using a PFV system and an insect-proof screen (mesh size 1.0 mm) had fewer sweetpotato whiteflies and Bemisia tabaci (Gennadius), and more onion thrips, Thrips tabaci (Lindeman), than greenhouses that employed an NV system fitted with the same screen. Tomato leafminers, Liriomyza sativae Blanchard, were not observed in the greenhouse using the PFV system, but some were observed in the greenhouse using the NV system. In Trial 2 (August–December 2007), the greenhouse using the PFV system combined with an insect-proof screen (mesh size 0.4 mm) had higher whitefly densities after late October compared to the greenhouse using the NV system and the same screen. However, there were more thrips in the greenhouse using the PFV system compared to the greenhouse using the NV system. In both trials, Tomato yellow leaf curl virus, which is transmitted by B. tabaci, was recorded in the greenhouse using the NV system but not in the greenhouse using the PFV system. The results showed that the PFV system was effective for preventing invasion by leafminers and partially effective for preventing invasion by whiteflies, but not effective for preventing invasion by thrips.     

Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase

The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.