Abscisic acid-induced hydrogen peroxide is required for anthocyanin accumulation in leaves of rice seedlings

The role of hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced anthocyanin accumulation in detached and intact leaves of rice seedlings was investigated. Treatment with ABA resulted in an accumulation of anthocyanins in detached rice leaves. Dimethylthiourea, a chemical trap for H(2)O(2), was observed to be effective in inhibiting ABA-induced accumulation of anthocyanins. Inhibitors of NADPH oxidase (diphenyleneiodonium chloride and imidazole), phosphatidylinositol 3-kinase (wortmannin and LY 294002), and a donor of nitric oxide (N-tert-butyl-alpha-phenylnitrone), which have previously been shown to prevent ABA-induced H(2)O(2) accumulation in detached rice leaves, inhibited ABA-induced anthocyanin increase. Exogenous application of H(2)O(2), however, was found to increase the anthocyanin content of detached rice leaves. In terms of H(2)O(2) accumulation, intact (attached) leaves of rice seedlings of cultivar Taichung Native 1 (TN1) are ABA sensitive and those of cultivar Tainung 67 (TNG67) are ABA insensitive. Upon treatment with ABA, H(2)O(2) and anthocyanins accumulated in leaves of TN1 seedlings but not in leaves of TNG67. Our results, obtained from detached and intact leaves of rice seedlings, suggest that H(2)O(2) is involved in ABA-induced anthocyanin accumulation in this species.     

Heptad repeats regulate protein phosphatase 2a recruitment to I-kappaB kinase gamma/NF-kappaB essential modulator and are targeted by human T-lymphotropic virus type 1 tax

The switching on-and-off of I-kappaB kinase (IKK) and NF-kappaB occurs rapidly after signaling. How activated IKK becomes down-regulated is not well understood. Here we show that following tumor necrosis factor-alpha stimulation, protein phosphatase 2A (PP2A) association with IKK is increased. A heptad repeat in IKKgamma, helix 2 (HLX2), mediates PP2A recruitment. Two other heptad repeats downstream of HLX2, termed coiled-coil region 2 (CCR2) and leucine zipper (LZ), bind HLX2 and negatively regulate HLX2 interaction with PP2A. HTLV-1 transactivator Tax also binds HLX2, and this interaction is enhanced by CCR2 but reduced by LZ. In the presence of Tax, PP2A-IKKgamma binding is greatly strengthened. Interestingly, peptides spanning CCR2 and/or LZ disrupt IKKgamma-Tax and IKKgamma-PP2A interactions and potently inhibit NF-kappaB activation by Tax and tumor necrosis factor-alpha. We propose that when IKK is resting, HLX2, CCR2, and LZ form a helical bundle in which HLX2 is sequestered. The HLX2-CCR2-LZ bundle becomes unfolded by signal-induced modifications of IKKgamma or after Tax binding. In this conformation, IKK becomes activated. IKKgamma then recruits PP2A via the exposed HLX2 domain for rapid down-regulation of IKK. Tax-PP2A interaction, however, renders PP2A inactive, thus maintaining Tax-PP2A-IKK in an active state. Finally, CCR2 and LZ possibly inhibit IKK activation by stabilizing the HLX2-CCR2-LZ bundle.     

No Increased Risk of Herpes Zoster Found in Cirrhotic Patients: A Nationwide Population-Based Study in Taiwan

Background

The association between liver cirrhosis (LC) and herpes zoster has rarely been studied. We investigated the hypothesis that LC, known as an immunodeficiency disease, may increase the risk of herpes zoster using a national health insurance database in Taiwan.

Materials and Methods

The study cohort included cirrhotic patients between 1998 and 2005 (n = 4667), and a ratio of 1∶5 randomly sampled age- and gender-matched control patients (n = 23,335). All subjects were followed up for 5 years from the date of cohort entry to identify whether or not they had developed herpes zoster. Cox proportional-hazard regressions were performed to evaluate 5-year herpes zoster-free survival rates.

Results

Of all patients, 523 patients developed herpes zoster during the 5-year follow-up period, among whom 82 were LC patients and 441 were in the comparison cohort. The adjusted hazard ratio (AHR) of herpes zoster in patients with LC was not higher (AHR: 0.77, 95% confidence interval: 0.59–1.01, p = 0.06) than that of the controls during the 5-year follow-up. No increased risk of herpes zoster was found in LC patients after stratification by age, gender, urbanization level, income, geographic region, and all comorbidities.

Conclusions

This large nationwide population-based cohort study suggests that there is no increased risk for herpes zoster among people who have LC compared to a matching population.     

Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficile-associated disease is recognized. In this study, we identified Fbp68, one of the colonization factors that bind to fibronectin (Fn), as a manganese-binding protein (K(D) = 52.70 ± 1.97 nM). Furthermore, the conformation of Fbp68 changed dramatically upon manganese binding. Manganese binding can also stabilize the structure of Fbp68 as evidenced by the increased T(m) measured by thermodenatured circular dichroism and differential scanning calorimetry (CD, T(m) = 58-65 °C; differential scanning calorimetry, T(m) = 59-66 °C). In addition, enhanced tolerance to protease K also suggests greatly improved stability of Fbp68 through manganese binding. Fn binding activity was found to be dependent on manganese due to the lack of binding by manganese-free Fbp68 to Fn. The C-terminal 194 amino acid residues of Fbp68 (Fbp68C) were discovered to bind to the N-terminal domain of Fn (Fbp68C-NTD, K(D) = 233 ± 10 nM, obtained from isothermal titration calorimetry). Moreover, adhesion of C. difficile to Caco-2 cells can be partially blocked if cells are pretreated with Fbp68C, and the binding of Fbp68C on Fn siRNA-transfected cells was significantly reduced. These results raise the possibility that Fbp68 plays a key role in C. difficile adherence on host cells to initiate infection.     

Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.