Interaction between LPS-induced NO production and IDO activity in mouse peritoneal cells in the presence of activated Vα14 NKT cells

In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Vα14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity.     

Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.     

Disturbances in nitric oxide/cyclic guanosine monophosphate system in SHR/NDmcr-cp rats, a model of metabolic syndrome

Metabolic syndrome is a cluster of metabolic abnormalities, including hypertension, hyperlipidemia, hyperinsulinemia, glucose intolerance and obesity. In such lifestyle-related diseases, impairment of nitric oxide (NO) production or bioactivity has been reported to lead to the development of atherogenic vascular diseases. Therefore, in the present study we investigated changes in the NO/cyclic guanosine monophosphate (cGMP) system in aortas of SHR/NDmcr-cp (cp/cp) rats (SHR-cp), a model of the metabolic syndrome. In aortas of SHR-cp, endothelium-dependent relaxations induced by acetylcholine and endothelium-independent relaxations induced by sodium nitroprusside were significantly impaired in comparison with Wistar-Kyoto rats. Furthermore, protein levels of soluble guanylyl cyclase and cGMP levels induced by sodium nitroprusside were significantly decreased. In contrast, protein levels of endothelium NO synthase and cGMP levels induced by acetylcholine were significantly increased, and plasma NO2 plus NO3 levels were also increased. The levels of lipid peroxide in plasma and the contents of 3-nitrotyrosine, a biomarker of peroxynitrite, in aortas were markedly increased. These findings indicate that in the aortas of SHR-cp, NO production from the endothelium is augmented, although the NO-induced relaxation response is impaired. Enhanced NO production may be a compensatory response to a variety of factors, including increases in oxidative stress.     

Hyaluronan fragments: an information-rich system

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-D-glucuronic acid-beta1,3-N-acetyl-D-glucosamine-beta1,4-]n. Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell-cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2 x 10(4) kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous "danger signals", while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.     

Smell intensity monitoring using metal oxide semiconductor odor sensors during intravenous olfaction test

The intravenous olfaction test using prosultiamine (PST) solution is simple to perform and has been used clinically in Japan. We monitored intranasal intensity of smell continuously in real time under various conditions of administration using metal oxide semiconductor odor sensors and established an optimal PST injection procedure. In this study, we found that 1) although there was fluctuation in the pattern of intensity of increase in smell in the PST original solution test, the pattern of increase in intranasal smell intensity could be stabilized by prolonging the injection time to 40 s and 2) dilution of PST with physiological saline was effective in preventing angialgia during intravenous injection. It appears that PST administration is best performed by adding 10 ml of saline to 10 mg (2 ml) of PST and injecting the resulting 12-ml solution (6x dilution) and that the best respiratory cycle for testing is once in every 2 s.     

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae)

During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.     

Detection of quantitative trait loci controlling pre-harvest sprouting resistance by using backcrossed populations of japonica rice cultivars

Backcrossed inbred lines (BILs) and a set of reciprocal chromosome segment substitution lines (CSSLs) derived from crosses between japonica rice cultivars Nipponbare and Koshihikari were used to detect quantitative trait loci (QTLs) for pre-harvest sprouting resistance. In the BILs, we detected one QTL on chromosome 3 and one QTL on chromosome 12. The QTL on the short arm of chromosome 3 accounted for 45.0% of the phenotypic variance and the Nipponbare allele of the QTL increased germination percentage by 21.3%. In the CSSLs, we detected seven QTLs, which were located on chromosomes 2, 3 (two), 5, 8 and 11 (two). All Nipponbare alleles of the QTLs were associated with an increased rate of germination. The major QTL for pre-harvest sprouting resistance on the short arm of chromosome 3 was localized to a 474-kbp region in the Nipponbare genome by the SSR markers RM14240 and RM14275 by using 11 substitution lines to replace the different short chromosome segments on chromosome 3. This QTL co-localized with the low-temperature germinability gene qLTG3-1. The level of germinability under low temperature strongly correlated with the level of pre-harvest sprouting resistance in the substitution lines. Sequence analyses revealed a novel functional allele of qLTG3-1 in Nipponbare and a loss-of-function allele in Koshihikari. The allelic difference in qLTG3-1 between Nipponbare and Koshihikari is likely to be associated with differences in both pre-harvest sprouting resistance and low-temperature germinability.     

Rev-derived peptides inhibit HIV-1 replication by antagonism of Rev and a co-receptor,CXCR4

Rev, a viral regulatory protein of HIV-1, binds through its arginine-rich domain to the Rev-responsive element (RRE), a secondary structure in transcribed HIV-1 RNA. Binding of Rev to RRE mediates export of singly spliced or unspliced mRNAs from the nucleus to the cytoplasm. It has been previously shown that a certain arginine-rich peptide exhibits not only RRE-binding ability but also cell permeability and antagonism of CXCR4, one of the major coreceptors of HIV-1. Here we designed and synthesized arginine-rich peptides derived from the RNA-binding domain of Rev (Rev_34-50) and evaluated their anti-HIV-1 activities. Rev_34-50-A₄C, comprising Rev_34-50 with AAAAC at the C-terminus to increase the α-helicity, inhibited HIV-1 entry by CXCR4 antagonism and virus production in persistently HIV-1-infected PM1-CCR5 cells. Interestingly, similar motif of human lymphotropic virus type I Rex (Rex_1-21) also exerted moderate anti-HIV-1 activity. These results indicate that arginine-rich peptide, Rev_34-50-A₄C exerts dual antagonism against CXCR4 and Rev.     

Rice-<Emphasis Type="Italic">Arabidopsis</Emphasis> FOX line screening with FT-NIR-based fingerprinting for GC-TOF/MS-based metabolite profiling

The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice-Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice-Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism.