Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

Transfer of Two Burkholderia and An Alcaligenes Species to Ralstonia Gen. Nov.

Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phylogenetic analysis of 16S rDNA nucleotide sequences and rRNA-DNA hybridization, Burkholderia pickettii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia, and Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb, nov., and R. eutropha (Davis 1969) comb. nov. are proposed. The type species of the new genus is R. pickettii. Type strain of R. pickettii is ATCC 27511^T, of R. solanacearum is ATCC 10696^T, and of R. eutropha is ATCC 17697^T.     

Roles of dilution rate and nitrogen concentration in competition between the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and the diatom <Emphasis Type="Italic">Cyclotella</Emphasis> sp. in eutrophic lakes

In Lake Tega, Japan, the shift of dominant algal species was caused as a result of discharging water from the adjacent river into the lake. The transition from cyanobacteria (mainly the genus Microcystis) to diatoms (mainly the genus Cyclotella) resulted in a disappearance of algal blooms. Although some environmental conditions such as flow rate, nutrient concentration, and transparency were changed by the project, the decisive factor for the transition has not been clarified yet. For the effective control of algal blooms by water discharge, this study aimed to elucidate the effects of daily renewal rate and nitrogen concentration on the interspecific competition between Microcystis aeruginosa and Cyclotella sp. Monoculture experiments were conducted to obtain growth characteristics for each species and mixed culture experiments were performed to examine their competitive abilities under various daily renewal rates of the culture medium (15 and 30 %) and nitrate concentrations (71.4, 178, and 357 μM). In addition to prepared medium, Lake Tega water was also used for mixed culture experiments. The results showed that the increase in a daily renewal rate contributed to the dominance of Cyclotella sp., while a nitrate concentration had little influence on the competition. We conclude that algal blooms composed of the genus Microcystis would be controlled by maintaining a daily renewal rate up to 30 % or more, which corresponded to the dilution rate of 0.36 day^?1, under a nitrate concentration of ≤357 μM. The study would include essential information for the management of lakes suffering from frequent occurrences of algal blooms.     

Enzymatic activity of metal-binding proteins in epidermal cells

Summary Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu²⁺ or Zn^2– chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu²⁺ chelate was greater than that with Zn²⁺ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu²⁺ chelate-binding proteins, but not in Zn²⁺ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, -galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu²⁺, but not by Zn²⁺. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.     

Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress

Salt stress causes oxidative damage and cell death in plants. Plants accumulate proline and glycinebetaine (betaine) to mitigate detrimental effects of salt stress. The aim of this study was to investigate the protective effects of proline and betaine on cell death in NaCl-unadapted tobacco (Nicotiana tabacum) Bright Yellow-2 suspension-cultured cells subjected to salt stress. Salt stress increased reactive oxygen species (ROS) accumulation, lipid peroxidation, nuclear deformation and degradation, chromatin condensation, apoptosis-like cell death and ATP contents. Neither proline nor betaine affected apoptosis-like cell death and G(1) phase population, and increased ATP contents in the 200mM NaCl-stressed cells. However, both of them effectively decreased ROS accumulation and lipid peroxidation, and suppressed nuclear deformation and chromatin condensation induced by severe salt stress. Evans Blue staining experiment showed that both proline and betaine significantly suppressed increment of membrane permeability induced by 200mM NaCl. Furthermore, among the ROS scavenging antioxidant defense genes studied here, mRNA levels of salicylic acid-binding (SAbind) catalase (CAT) and lignin-forming peroxidase (POX) were found to be increased by proline and betaine under salt stress. It is concluded that both proline and betaine provide a protection against NaCl-induced cell death via decreasing level of ROS accumulation and lipid peroxidation as well as improvement of membrane integrity.     

Molecular structure and interaction of dipalmitoyl phosphatidylcholine in multilayers. Comparative study with phosphatidylethanolamine

As a model of phospholipid bilayers in solid an oriented multilayer film (built-up film) of L-α-dipalmitoyl phosphatidylcholine (DPPC) was prepared from the monolayer by the dipping method. Structural analysis has been carried out by measuring infrared dichroism of the built-up film. The results were compared with those of the built-up film of L-α-dipalmitoyl phosphatidylethanolamine (DPPE). The tilting of the hydrocarbon chains is larger for DPPC than for DPPE. The orientation of the bisector of the two non-esterified PO bonds is closer to the film plane for DPPC than for DPPE. The strong hydrogen bonding interaction between the polar head groups was shown for DPPE, but not for DPPC. These features resemble the structural differences between dilauroyl phosphatidylethanolamine (DLPE) and dimyristoryl phosphatidylcholine (DMPC) in crystals. The hydrogen bonding interaction of DPPE found in solid remains even in the presence of water, namely, in the gel state. More closed packing of the hydrocarbon chains of solid DPPE than DPPC in solid was concluded on the basis of infrared and Raman spectra.     

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2?](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2?](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2?](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2?](cyt) oscillations.     

Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations

Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10⁻⁸) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (P _combined = 2.3×10⁻¹⁰, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10⁻¹⁰, OR = 1.52, and DPB1*0901: P = 2.0×10⁻⁷, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.     

Labeling of v-Src and BCR-ABL tyrosine kinases with [C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [¹⁴C]herbimycin A. p60^v-src and p2 10^BCR-ABL in immune complexes were labeled with [¹⁴C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [¹⁴C]herbimycin A-labeled p60^v-src with Staphylococcus taureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60^v-src, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60^v-src by [¹⁴]C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60^v-src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.