Deactivation of photosynthetic activities is triggered by loss of a small amount of water in a desiccation-tolerant cyanobacterium, Nostoc commune

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation-tolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm'-F)/Fm' values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss.     

Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.     

Efficient uptake and rapid degradation of plasmid DNA by murine dendritic cells via a specific mechanism

In spite of the important roles of dendritic cells in DNA-based therapies, the cellular uptake mechanism of plasmid DNA (pDNA) in dendritic cells is poorly understood. The present study was undertaken to investigate the binding and uptake of pDNA in vitro using a murine dendritic cell line, DC2.4 cells. A significant and time-dependent cellular association of [32P]pDNA with DC2.4 cells was observed at 37 degrees C and this fell markedly at 4 degrees C. The binding and uptake of [32P]pDNA were significantly inhibited by cold pDNA, polyinosinic acid (poly[I]), dextran sulfate, or heparin, but not by polycytidylic acid (poly[C]), dextran, or EDTA, suggesting that a specific mechanism mediated by a receptor like the macrophage scavenger receptor may be involved. The TCA precipitation experiments showed that DC2.4 cells rapidly endocytosed and degraded a significant amount of [32P]pDNA at 37 degrees C and released the degradation products into the medium. The pDNA degradation was also significantly inhibited by poly[I], but not poly[C]. The rate of pDNA degradation by DC2.4 cells was significantly higher than that by macrophages. A confocal microscopic study using fluorescein-labeled pDNA confirmed the rapid internalization and degradation of pDNA by the dendritic cells. Taken together, these results indicate that pDNA is efficiently taken up and rapidly digested by the dendritic cells via a specific mechanism. These findings may suggest the important role of the dendritic cells in the innate immune system for host defense.     

Muscular dystrophy: Centronucleation may reflect a compensatory activation of defective myonuclei

Muscular dystrophy has long been believed to be characterized by degeneration and abortive regeneration of muscle fibers (the muscle degeneration theory), but unfortunately its pathogenesis is still unclear and an effective treatment has yet to be developed. As a challenge to the theory, we have proposed an alternative muscle-defective-growth theory and a further bone muscle growth imbalance hypothesis supposing possible defects in bone-growth-dependent muscle growth based on our findings in hereditary dystrophic dy mice (C57BL/6Jdy/dy). This review presents some new insights into the pathogenesis of the disease along with our hypothesis, focusing on the physiological meaning of centronucleation, one of the major pathological changes commonly observed in dystrophic muscles of man and experimental animals.     

Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives

N,N'-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the sigma factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.     

Possible involvement of hydroxyl radical on the stimulation of tetrahydrobiopterin synthesis by hydrogen peroxide and peroxynitrite in vascular endothelial cells

We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.     

Effects of post low-dose X-ray irradiation on carbon tetrachloride-induced acatalasemic mice liver damage

The catalase activities in the blood and organs of the acatalasemic (C3H/AnLCsb-Csb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCSa-Csa) mouse. We examined the effects of post low-dose (0.5 Gy) X-ray irradiation which reduced the oxidative damage under carbon tetrachloride-induced hepatopathy in acatalasemic or normal mice. As a result, the 0.5 Gy irradiation after carbon tetrachloride administration decreased the glutamic oxaloacetic and glutamic pyruvic transaminase activity in the acatalasemic mouse blood to a level similar to that of the acatalasemic mouse blood not treated with carbon tetrachloride; this is in contrast to a high-dose (15 Gy) irradiation. In the same manner, pathological disorder was improved by 0.5 Gy irradiation. The fat degeneration in normal mice was quickly reduced, in contrast to acatalasemic mice. These findings suggest that low-dose irradiation after carbon tetrachloride administration accelerates the rate of recovery and that catalase plays an important role in the recovery from hepatopathy induced by carbon tetrachloride, in contrast to high-dose irradiation.     

Monitoring of active HHV-6 infection in bone marrow transplant recipients by real time PCR; comparison to detection of viral DNA in plasma by qualitative PCR

Twelve (46%) of the 26 patients had human herpesvirus 6 (HHV-6) viremia after bone marrow transplant (BMT). All isolates were recovered from the samples obtained at 2 weeks after BMT. The sensitivity and the specificity of detection of viral DNA in plasma by qualitative polymerase chain reaction (PCR) for monitoring active virus replication were 92% and 97% respectively. Moreover, the positive (85%) and negative (99%) predictive values were also high. The patients with HHV-6 viremia showed a clear peak in HHV-6 DNA in peripheral blood mononuclear cells (PBMCs) at 2 weeks after BMT, which was measured by real time PCR. The virus DNA level in PBMCs between the two groups (patients with viremia and patients without viremia) was statistically different at 2 weeks after BMT (P = 0.033). In patients with HHV-6 viremia, mean HHV-6 DNA copy number was higher in the samples collected at 2 weeks after BMT than the samples collected at any other time period.