Characterization of β -Carotene Ketolases, CrtW, from Marine Bacteria by Complementation Analysis in Escherichia coli

A complementation analysis was performed in Escherichia coli to evaluate the efficiency of β-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25ΔcrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into α-Proteobacteria.     

2,3-Diphenylpropionic acids as potent VLA-4 antagonists

The discovery and SAR of 2,3-diphenylpropionic acid derivatives as highly potent VLA-4 antagonists are described. One representative compound, 9cc has inhibited intercellular adhesion by a VCAM-1/VLA-4 interaction with an IC(50) of 1.7 nM, and has good pharmacokinetics and oral bioavailability.     

Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM^SA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM^WT/SA) were indistinguishable from WT (VIM^WT/WT) mice. In VIM^SA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM^SA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM^SA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.     

Imidocarb,a potent anti-protozoan drug,up-regulates interleukin-10 production by murine macrophages

Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses. To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7. Imidocarb, (3,3'-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified. Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner. It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component. In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-alpha production by peritoneal macrophages. In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-alpha levels. Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.     

Decreased daytime light intensity at nonwindow hospital beds: Comparisons with light intensity at window hospital beds and light exposure in nonhospitalized elderly individuals

Light is crucial for the synchronization of internal biological rhythms with environmental rhythms. Hospitalization causes a range of unfavorable medical conditions, including delirium, sleep disturbances, depressed mood, and increased fall, especially in elderly people. The hospital room environment contributes significantly to patients’ circadian physiology and behavior; however, few studies have evaluated light intensity in hospital settings. In this study, bedside light intensity during the daytime (6:00–21:00) was measured at 1-min intervals using a light meter on 4869 bed-days at the Inabe General Hospital in Mie, Japan (latitude 35°N), for approximately 1 month in each season. Daytime light exposure in home settings was measured in nonhospitalized elderly individuals (n = 1113) for two consecutive days at 1-min intervals using a wrist light meter. Median daytime light intensities at window and nonwindow hospital beds were 327.9 lux [interquartile range (IQR), 261.5–378.4] and 118.4 lux (IQR, 100.6–142.9), respectively, and daytime light intensity measured in nonhospitalized elderly individuals was 337.3 lux (IQR, 165.5–722.7). Compared with data in nonhospitalized elderly individuals, nonwindow beds were exposed to significantly lower daytime light intensity (p < 0.001), whereas window beds were exposed to similar daytime light intensity to that of home settings (p = 1.00). These results were consistent regardless of seasons (spring, summer, fall, and winter) or room directions (north vs. south facing). The lowest median daytime light intensity was observed at nonwindow beds in north-facing rooms during the winter (84.8 lux; IQR, 76.0–95.8). Further studies evaluating the incidence of in-hospital outcomes between patients hospitalized in window and nonwindow beds are needed.     

Translocation of Bim to the endoplasmic reticulum (ER) mediates ER stress signaling for activation of caspase-12 during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress activates caspase-12 in murine cells, triggering the ER stress-specific cascade for implementation of apoptosis. In C2C12 murine myoblast cells, activation of the cascade occurs without release of cytochrome c from mitochondria, suggesting that the cascade is independent of mitochondrial damage. Stable overexpression of Bcl-xL in C2C12 cells suppressed activation of caspase-12 and apoptosis. In ER-stressed cells, but not in normal cells, Bcl-xL was co-immunoprecipitated with Bim, a pro-apoptotic member of the Bcl-2 family, suggesting that Bcl-xL sequesters Bim, thereby inhibiting the apoptotic signaling. Fractionation of C2C12 cells revealed that ER stress led to translocation of Bim from a dynein-rich compartment to the ER, while stable overexpression of Bcl-xL suppressed accumulation of Bim on the ER. Although the toxic effect of Bim had been previously observed only at the mitochondrial outer membrane, overexpression of a Bim derivative, Bim(ER), targeted at the surface of the ER led to apoptosis. A C2C12 transfectant overexpressing the caspase-12 suppressor protein was resistant to Bim(ER), suggesting that the toxic effect of Bim on the ER is dependent on activation of caspase-12. Knockdown of Bim by RNA interference provided cells resistant to ER stress. These results suggest that translocation of Bim to the ER in response to ER stress is an important step toward activation of caspase-12 and initiation of the ER stress-specific caspase cascade.     

Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.