Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorff's perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.     

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.     

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.     

Population genetics of Haminoea (Haloa) japonica Pilsbry, 1895, a widespread non-indigenous sea slug (Mollusca: Opisthobranchia) in North America and Europe

Haminoea japonica is an opisthobranch mollusk with a large non-indigenous range. This species is a vector for a parasite that causes the human skin disease cercarial dermatitis, and may have negative effects on populations of native species. Molecular evidence from the mitochondrial cytochrome c oxidase I gene and the histone 3 nuclear gene indicates that previously published morphology-based hypotheses on the spread of H. japonica out of Japan are correct. The most likely explanation for the current range of the species, which includes Japan, Korea, France, Spain, Italy, Canada and the USA is a recent, human-mediated dispersal from Japanese populations. The highest levels of nucleotide and haplotype diversity are found in Japan. Non-indigenous populations have low levels of genetic diversity (indicating bottlenecking). Haplotypes that were detected in the non-indigenous range of H. japonica have only been found in two localities in the native range; these two localities are in north-eastern Japan. In addition, the haplotype network structure and Spatial Analysis of Molecular Variance results confirm the origins of non-indigenous populations most likely trace to north-eastern Japan, which is where most Pacific oyster exports to North America also originated. Because there are no major shipping ports in north-eastern Japan, ballast water is less likely to be the mechanism of dispersal. The results of this study provide important data for the development of policies and regulations aimed to prevent further spread of this species in non-indigenous ranges.     

Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.     

Influence of Aging and Gender Differences on Feeding Behavior and Ghrelin-Related Factors during Social Isolation in Mice

Psychological stress due to social isolation is known to cause abnormal feeding behaviors, but the influences of gender and aging on subchronic stress-induced changes in feeding behaviors are unknown. Thus, we examined the changes in body weight, food intake, and orexigenic ghrelin-related factors during 2 weeks of isolation stress in young and aged mice. Food intake increased significantly in young mice in the isolation group compared with the group-housed control throughout the experimental period. This isolation-induced increase in food intake was not observed in aged mice. In young mice, there were no significant differences in body weight between the isolated group and group-housed control up to 2 weeks. However, aged male mice exhibited significant weight loss at 2 weeks and a similar tendency was observed in aged female mice. Young male mice, but not female mice, had significantly increased (2.2-fold) plasma acylated ghrelin levels after 1 week of isolation compared with the group-housed control. A significant but lower increase (1.3-fold) was also observed in aged male mice. Hypothalamic preproghrelin gene expression decreased significantly with isolation in young male mice, whereas it increased significantly in female mice. The expression levels of NPY and AGRP in the hypothalamus, which are transmitted by elevated peripheral ghrelin signals, increased significantly in isolated young male mice, whereas the AGRP expression levels decreased significantly in young female mice. Isolation caused no significant differences in the expression levels of these genes in aged mice. In isolation, young female mice exhibited markedly increased dark- and light-phase locomotor activities compared with male mice, whereas male and female aged mice exhibited no obvious increases in activity immediately after the dark phase started. We conclude that the gender-specific homeostatic regulatory mechanisms required to maintain body weight operated during subchronic psychological stress in young mice but not in aged mice.