MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.     

Intra‐specific pollen polymorphism in Mimosa pigra (Mimosaceae)

Five different types of pollen tetrads were detected from the short and long stamens of the male and hermaphrodite flowers of the androdioecious species, Mimosa pigra L. Light (LM) and scanning electron microscopes (SEM) were used in this study to redescribe the pollen forms and to identify the various pollen types encountered. An identification key and relative percentages of these pollen types were also presented. The study showed that the long stamens of the male flowers and those of the hermaphrodite ones have five (I, II, III, IV and V) and two (I and V) pollen types respectively, whereas the short stamens of both male and hermaphrodite flowers have only one pollen type (type I). We report here the first incidence of intra‐specific pollen polymorphism in androdioecious species.     

Chemical Conversion of Some Ribonucleosides into the Corresponding β-D-Arabinofuranosyl Derivatives1

Abstract

Five 3′,5′-di-O-acylribonucleosides were converted into the corresponding β-D-arabinofuranosyl derivatives through DMSO-oxidation followed by NaBH₄-reduction and deacylation with NaOMe-MeOH.     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.     

Plasma free amino acid profiling of five types of cancer patients and its application for early detection

Background

Recently, rapid advances have been made in metabolomics-based, easy-to-use early cancer detection methods using blood samples. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. Furthermore, PFAA profiles are known to be influenced by specific diseases, including cancers. Therefore, the purpose of the present study was to determine the characteristics of the PFAA profiles in cancer patients and the possibility of using this information for early detection.

Methods and Findings

Plasma samples were collected from approximately 200 patients from multiple institutes, each diagnosed with one of the following five types of cancer: lung, gastric, colorectal, breast, or prostate cancer. Patients were compared to gender- and age- matched controls also used in this study. The PFAA levels were measured using high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS). Univariate analysis revealed significant differences in the PFAA profiles between the controls and the patients with any of the five types of cancer listed above, even those with asymptomatic early-stage disease. Furthermore, multivariate analysis clearly discriminated the cancer patients from the controls in terms of the area under the receiver-operator characteristics curve (AUC of ROC >0.75 for each cancer), regardless of cancer stage. Because this study was designed as case-control study, further investigations, including model construction and validation using cohorts with larger sample sizes, are necessary to determine the usefulness of PFAA profiling.

Conclusions

These findings suggest that PFAA profiling has great potential for improving cancer screening and diagnosis and understanding disease pathogenesis. PFAA profiles can also be used to determine various disease diagnoses from a single blood sample, which involves a relatively simple plasma assay and imposes a lower physical burden on subjects when compared to existing screening methods.     

Reconstitution mechanism of nucleosome core particles mediated by poly(l-glutamic acid)

Poly(l-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5000–5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(l-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(l-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(l-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo ‘nucleosome assembly factors’ is also discussed.     

Human immunodeficiency virus type 1 Vpr induces G2 checkpoint activation by interacting with the splicing factor SAP145

Vpr, the viral protein R of human immunodeficiency virus type 1, induces G(2) cell cycle arrest and apoptosis in mammalian cells via ATR (for "ataxia-telangiectasia-mediated and Rad3-related") checkpoint activation. The expression of Vpr induces the formation of the gamma-histone 2A variant X (H2AX) and breast cancer susceptibility protein 1 (BRCA1) nuclear foci, and a C-terminal domain is required for Vpr-induced ATR activation and its nuclear localization. However, the cellular target of Vpr, as well as the mechanism of G(2) checkpoint activation, was unknown. Here we report that Vpr induces checkpoint activation and G(2) arrest by binding to the CUS1 domain of SAP145 and interfering with the functions of the SAP145 and SAP49 proteins, two subunits of the multimeric splicing factor 3b (SF3b). Vpr interacts with and colocalizes with SAP145 through its C-terminal domain in a speckled distribution. The depletion of either SAP145 or SAP49 leads to checkpoint-mediated G(2) cell cycle arrest through the induction of nuclear foci containing gamma-H2AX and BRCA1. In addition, the expression of Vpr excludes SAP49 from the nuclear speckles and inhibits the formation of the SAP145-SAP49 complex. To conclude, these results point out the unexpected roles of the SAP145-SAP49 splicing factors in cell cycle progression and suggest that cellular expression of Vpr induces checkpoint activation and G(2) arrest by interfering with the function of SAP145-SAP49 complex in host cells.     

Free radical scavenging activity of propolis

We investigated the radical scavenging activity of propolis by ESR spectroscopy using spin trapping method. In addition, we examined the influence of a diet of 2% propolis on mice under oxidative stress. At low concentrations, the methanolic extract of propolis exhibited strong scavenging activity in vitro towards both the superoxide anion radical, generated by the hypoxanthine-xanthine oxidase reaction, and the NO radical, generated from the mixture of NOC-7 (NO generator) and carboxy-PTIO (spin trapping agent). An inhibitory effect of propolis on lipid peroxidation in vivo was observed, as determined by measurement of thiobarbituric acid-reactive substances in mouse liver homogenate. The level of vitamin C in the brain of mice under oxidative stress significantly increased compared with control mice under atmosphere, which was not observed in the mice given 2% propolis. The level of alpha-tocopherol in the brain of mice given 2% propolis significantly increased compared with control mice under atmosphere, which was not observed in mice under oxidative stress. SOD activity in the brain and plasma of mice given 2% propolis significantly decreased under atmosphere and oxidative stress compared with control mice. These results suggest that propolis possesses potent antioxidant activity in vitro and in vivo.