UV-sensitive photoreceptor protein OPN5 in humans and mice

A variety of animal species utilize the ultraviolet (UV) component of sunlight as their environmental cues, whereas physiological roles of UV photoreception in mammals, especially in human beings, remain open questions. Here we report that mouse neuropsin (OPN5) encoded by the Opn5 gene exhibited an absorption maximum (λmax) at 380 nm when reconstituted with 11-cis-retinal. Upon UV-light illumination, OPN5 was converted to a blue-absorbing photoproduct (λmax 470 nm), which was stable in the dark and reverted to the UV-absorbing state by the subsequent orange light illumination, indicating its bistable nature. Human OPN5 also had an absorption maximum at 380 nm with spectral properties similar to mouse OPN5, revealing that OPN5 is the first and hitherto unknown human opsin with peak sensitivity in the UV region. OPN5 was capable of activating heterotrimeric G protein Gi in a UV-dependent manner. Immuno-blotting analyses of mouse tissue extracts identified the retina, the brain and, unexpectedly, the outer ears as the major sites of OPN5 expression. In the tissue sections of mice, OPN5 immuno-reactivities were detected in a subset of non-rod/non-cone retinal neurons as well as in the epidermal and muscle cells of the outer ears. Most of these OPN5-immuno-reactivities in mice were co-localized with positive signals for the alpha-subunit of Gi. These results demonstrate the first example of UV photoreceptor in human beings and strongly suggest that OPN5 triggers a UV-sensitive Gi-mediated signaling pathway in the mammalian tissues.     

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.     

Volatile C6-aldehydes and Allo-ocimene activate defense genes and induce resistance against Botrytis cinerea in Arabidopsis thaliana

Green leafy volatiles or isoprenoids are produced after mechanical wounding or pathogen/herbivore attacks in higher plants. We monitored expression profiles of the genes involved in defense responses upon exposing Arabidopsis thaliana to the volatiles. Among the genes investigated, those known to be induced by mechanical wounding and/or jasmonate application, such as chalcone synthase (CHS), caffeic acid-O-methyltransferase (COMT), diacylglycerol kinase1 (DGK1), glutathione-S-transferase1 (GST1) and lipoxygenase2 (LOX2), were shown to be induced with (E)-2-hexenal, (Z)-3-hexenal, (Z)-3-hexenol or allo-ocimene (2,6-dimethyl-2,4,6-octatriene). A salicylic acid-responsive gene, pathogenesis-related protein2 (PR2), was not induced by the volatiles. Detailed analyses of the expression profiles showed that the manner of induction varied depending on either the gene monitored or the volatile used. A chemically inert compound, (Z)-3-hexenol, was also potent, which suggested that chemical reactivity was not the sole requisite for the inducing activity. With a jasmonate-insensitive mutant (jar1), the induction by the volatiles was mostly suppressed, however, that of LOX2 was unaltered. An ethylene-insensitive mutant (etr1) showed responses almost identical to the wild type, with minor exceptions. From these observations, it was suggested that both the jasmonate-dependent and -independent pathways were operative upon perception of the volatiles, while the ETR1-dependent pathway was not directly involved. When Botrytis cinerea was inoculated after the volatile treatment, retardation of disease development could be seen. It appears that volatile treatment could make the plants more resistant against the fungal disease.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Cardiac abnormalities cause early lethality of jumonji mutant mice

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.     

Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs

Although c-Jun NH(2)-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-kappaB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4 degrees C for 4 h followed by reperfusion at 37 degrees C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-kappaB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-alpha into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

Cytokine gene expression in Walker 256: a comparison of variants A (aggressive) and AR (regressive)

Two variants of this Walker 256 tumor have been previously reported as Walker 256 A and variant AR. The variant A has more aggressive property than variant AR and can induce systemic effects such as anorexia, sodium and water retention, followed by weight loss and death. The mechanisms involved in enhancing tumor regression and progression in this model are still incompletely understood. In the present study, serum and spleen mononuclear cells and tumor cells from animals inoculated with variants A and AR, were isolated to investigate the TGF-beta, IL-12, IFN-gamma and TNF-alpha and relationship with anemia, weight of animals, weight of spleen, volume of tumor and osmotic fragility compared with controls inoculated with Ringer Lactate. Results demonstrate that the group inoculated with variant A, compared to variant AR, shows high levels of TGF-beta gene expression in both tumor tissue and spleen cells, no expression of IFN-gamma and a progressive and higher levels of IL-12 in tumor tissue without inflammatory infiltrate visualized by optical microscopy. These results suggest that the aggressively of variant A is relate to cytokine modulation, facilitating the growth and escape of tumor cells. Furthermore, IL-12 seems to be constitutively expressed in both tumor lineage A and AR.