Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in-vitro de-epoxidation in monogalactosyldiacylglycerol micelles

The dynamics of the xanthophyll cycle relative to non-photochemical quenching (NPQ) were examined in tobacco plants overexpressing violaxanthin de-epoxidase (VDE), PsbS and PsbS+VDE for effects on NPQ and violaxanthin (V) de-epoxidation over a range of light intensities. Induction of de-epoxidation and NPQ increased in overexpressed VDE and PsbS plants, respectively. Surprisingly, under low light, overexpressing PsbS enhanced de-epoxidation in addition to NPQ. The effect was hypothesized as due to PsbS binding zeaxanthin (Z) or inducing the binding of Z within the quenching complex, thus shifting the equilibrium toward higher de-epoxidation states. Studies in model systems show that Z can stereospecifically inhibit VDE activity against violaxanthin. This effect, observed under conditions of limiting lipid concentration, was interpreted as product feedback inhibition. These results support the hypothesis that the capacity of the thylakoid lipid phase for xanthophylls is limited and modulates xanthophyll-cycle activity, in conjunction with the release of V and binding of Z by pigment-binding proteins. These modulating factors are incorporated into a lipid-matrix model that has elements of a signal transduction system wherein the light-generated protons are the signal, VDE the signal receptor, Z the secondary messenger, the lipid phase the transduction network, and Z-binding proteins the targets.     

Molecular cloning of protamine-2 and expression with aging in Japanese monkey (<Emphasis Type="Italic">Macaca fuscata</Emphasis>)

In order to isolate genes relating spermatogenesis with postnatal development and aging, we have attempted to obtain genes showing differences in expression in testis of Japanese monkeys (Macaca fuscata) by means of differential display PCR, and we have cloned, sequenced and characterized protamine-2 (PRM2) of Japanese monkey. The predicted open reading frame encoded a protein of 103 amino acid residues, most of which are common to those of other mammals. Northern analysis revealed that the PRM2 gene is expressed at adult and aged stages, but not at the juvenile stage. In situ hybridization revealed that the PRM2 gene is expressed mainly in late spermatids and its expressional potential is decreased from adult to aged stages. It suggests that PRM2 in spermiogenesis is mediated by the age of the animal.     

Long-lasting smooth-muscle relaxation by a novel PACAP analogue in human bronchi

We compared the relaxant effect of original pituitary adenylate cyclase-activating peptide (PACAP)1-27 with that of a newly developed, synthetic PACAP1-27 analogue, [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2, in human bronchi in vitro (n=4-5 in each group). Using precontraction by carbachol (0.1 microM), cumulative administration of PACAP1-27 and salbutamol caused concentration-dependent smooth muscle relaxation with similar potencies and maximum relaxant effects. Non-cumulative administration of the PACAP1-27 analogue and the original PACAP1-27 caused concentration-dependent relaxation with a similar maximum relaxant effect and potency as well. However, the onset and offset of action was markedly slower for the PACAP1-27 analogue than for the original PACAP1-27 (>90% versus <10% of peak relaxation remaining 5 h after administration). Peptidase inhibition by captopril (10 microM) and phosphoramidon (1 microM) significantly increased the maximum relaxant effect and duration of action of PACAP1-27 but not of the PACAP1-27 analogue, during the 3 h of observation in the human bronchi. We conclude that [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2 produces significant concentration-dependent and sustained bronchial smooth muscle relaxation in vitro. The sustained relaxant effect is due, at least in part, to the synthetic PACAP1-27 analogue being less susceptible to cleavage by peptidases than the original peptide PACAP1-27.     

Generation of reactive oxygen species undergoing redox cycle of nostocine A: a cytotoxic violet pigment produced by freshwater cyanobacterium Nostoc spongiaeforme

Nostocine A (1) is an extracellular cytotoxic violet pigment produced by the freshwater cyanobacterium, Nostoc spongiaeforme TISTR 8169. Treatment with 1 was found to accelerate the generation of reactive oxygen species (ROS) in the green alga, Chlamydomonas reinhardtii, in the light. In vitro analysis revealed that 1 specifically eliminated superoxide radical anion (O(2)(-)) among several ROS tested. During the course of the reaction, oxygen (O(2)) was simultaneously synthesized and the O(2) synthesizing rate increased with the amount of 1 added. In contrast, O(2)(-) generation occurred when NADPH or NADH was added to a solution of 1 under aerobic condition. The reduction potential of 1 is very similar to that of O(2) indicating that 1 and O(2) can easily exchange electrons depending on the mass balance between their oxidized and reduced forms. Based on these results, the following hypothesis is formulated for the mechanism of intracellular ROS generation by treatment with 1: 1 taken into the target cells is reduced specifically by intracellular reductants such as NAD(P)H. When the O(2) level is sufficiently higher than that of 1, the reduced product of 1 is immediately oxidized by O(2). This is accompanied by the synthesis of O(2)(-) from O(2). The generation of O(2)(-) successively occurs, undergoing repeated redox cycles of 1, when the levels of the reductant and O(2) are still dominant to promote these reactions. This similar intracellular ROS generation mechanism to that of paraquat may cause the cytotoxicity.     

NHE3 inhibition activates duodenal bicarbonate secretion in the rat

We examined the effect of inhibition of Na+/H+ exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using CO2-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 microM; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with Na+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 microM indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis, Na+-HCO3- cotransporter activation, or intracellular HCO3- formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.