Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.     

Mammary tumorigenesis in ApcMin/+ mice is enhanced by X irradiation with a characteristic age dependence

The Apc(Min/+) (Min) mouse is genetically predisposed to both intestinal and mammary tumorigenesis. We investigated age-related changes in the susceptibility of mice (before, during and after puberty) to radiation-induced mammary tumorigenesis using this model. Female Min and wild-type mice having the C57BL/6J background were irradiated with 2 Gy of X rays at 2, 5, 7 and 10 weeks and killed humanely at 18 weeks of age. Min mice irradiated at 7-10 weeks of age (after puberty) developed mammary tumors with squamous metaplasia, whereas their wild-type littermates did not. Interestingly, irradiation of Min mice at 2-5 weeks (before and during puberty, respectively) did not induce mammary tumors but rather cystic nodules with metaplasia. The mammary tumors exhibited increased nuclear beta-catenin protein and loss of the wild-type Apc allele. Our results show that susceptibility to radiation-induced mammary tumorigenesis increases after puberty in Min mice, suggesting that the tumorigenic effect of ionizing radiation targets the lobular-alveolar progenitor cells, which increase in number with age and are controlled by beta-catenin signaling.     

Vasodilative effect of perillaldehyde on isolated rat aorta

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.     

Synthesis and structure-activity relationships of a series of benzazepine derivatives as 5-HT2C receptor agonists

To identify potent and selective 5-HT(2C) receptor agonists, a series of novel benzazepine derivatives were synthesized, and their structure-activity relationships examined. The compounds were evaluated for their 5-HT(2C), 5-HT(2A), and 5-HT(2B) receptor binding affinity and intrinsic activity for the 5-HT(2C) and 5-HT(2A) receptors. Among these compounds, 6,7-dichloro-2,3,4,5-tetrahydro-1H-3-benzazepine (6) was effective in a rat penile erection model when administered po, which is a symptom of the serotonin syndrome reflecting 5-HT(2C) receptor activation. Moreover, compound 6 was characterized as a partial agonist of 5-HT(2A) receptors; therefore, it had little effect on the cardiovascular system.     

Computational search for over-represented 8-mers within the 5'-regulatory regions of 634 mouse testis-specific genes

Accumulation of microarray data has enabled the computational analysis of gene expressions in various tissues. Although the genes showing testis-specific expression are most abundant among the genes exhibiting tissue-specific expression, no systematic study has been conducted for over-represented motifs within their regulatory regions. We have identified 117 over-represented 8-mers that appeared 2648 times within the regulatory regions of 634 testis-specific genes. Of these, 64 over-represented 8-mers were significantly more frequent in the regulatory regions of testis-specific genes than in those of non-testis-specific genes. In this group of 8-mers, 4 8-mers differed from the canonical cAMP response element (CRE) 8-mer by 1 letter, but the canonical CRE was not included in this group. We consider that CRE-like 8-mers participate in the regulatory expression of testis-specific genes to a greater extent than the canonical CRE 8-mer.     

The effects of a herbal medicine (Mao-to) in patients with chronic hepatitis C after injection of IFN-beta.

We found that a herbal medicine (Mao-to) relieves the side effects of interferon (IFN)-beta and the combination therapy improves the biochemical response rate. However, the exact mechanism by which Mao-to is effective remains to be established. We conducted a controlled trial to clarify the effects of Mao-to. The study was carried out in 18 patients with chronic hepatitis C, and we examined subjective symptoms, body temperature and cytokines such as interleukin (IL)-beta, IL-1receptor antagonist (ra), IL-6 and TNF-alpha. Each patient received 6 million units of IFN-beta intravenously. Mao-to was given orally just before, just after, and 1 hour after IFN administration. The control study was carried out 6 months after the combination therapy of Mao-to and IFN-beta. The scores for general malaise, arthralgia and discomfort were significantly lower in the combination group than in control group. Body temperature did not significantly differ between the two groups. Plasma IL-6 level and IL-1ra were significantly elevated in the combination group compared to control (P = 0.0057 and 0.0003, respectively). Mao-to did not affect plasma concentrations of IL-1beta and TNF-alpha. We considered the increment of IL-1ra caused by Mao-to is to be one of the key factors involved in reducing the flu-like symptoms accompanying IFN-beta and improving the biochemical response rate.     

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana , and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants ( ole1 , ole2 , ole3 and ole4 ) and generated three double mutants ( ole1 ole2 , ole1 ole3 and ole2 ole3 ). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2 , which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants ( ole1 , ole2 and ole3 ), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.