Adaptive significance of death feigning posture as a specialized inducible defence against gape-limited predators

Death feigning is fairly common in a number of taxa, but the adaptive significance of this behaviour is still unclear and has seldom been tested. To date, all proposed hypotheses have assumed that prey manage to escape predation by sending a death-mimicking signal, although death-feigning postures are markedly different from those of dead animals. Moreover, the efficacy of this technique may largely depend on the foraging mode of the predator; death feigning seldom works with sit-and-wait predators that make the decision to attack and consume prey within a very brief time. We examined whether death feigning in the pygmy grasshopper Criotettix japonicus Haan was an inducible defence behaviour against the frog Rana nigromaculata, a sit-and-wait, gape-limited predator. The characteristic posture assumed by the grasshopper during death feigning enlarges its functional body size by stretching each of three body parts (pronotum, hind legs and lateral spines) in three different directions, thereby making it difficult for the predator to swallow the prey. Our result is the first consistent explanation for why death-mimicking animals do not always mimic the posture of dead animals.     

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.     

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid β-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.Peroxisomes are single membrane-bound organelles harboring at least one H₂O₂-generating oxidase and one H₂O₂-decomposing catalase, and they are present in virtually all eukaryotic cells, from yeast to mammals. The most conserved activity of peroxisomal metabolism is the β-oxidation of fatty acids (27).Peroxisome assembly requires more than 20 PEX gene products, termed peroxins, in any given organism (5). The impairment of peroxisomal protein transport caused by mutations in PEX genes causes fatal human peroxisome biogenesis disorders (PBDs) (34). In the cells of such PBD patients, essential enzymes normally localized to peroxisomes are found mostly in the cytosol. Mammalian cell lines harboring mutations in peroxins (including fibroblasts from PBD patients) grow well in cell culture. On the other hand, pex mutants of the methylotrophic yeast Pichia pastoris can grow normally on glucose but not oleate or methanol (37).Peroxisomal metabolic pathways can generate a high level of reactive oxygen species (ROS) (32). Therefore, peroxisomal disorders have been studied with a focus on the generation of ROS. However, the relationship between PBDs and the intracellular redox state is unclear (13, 32).Peroxisomes have long been thought to be in a more highly oxidized state than the cytosol due to this generation of ROS. However, there is no reported experimental evidence supporting this notion. We previously identified a 20-kDa peroxisomal membrane protein, named Pmp20, in methanol-induced peroxisomes of methylotrophic yeasts. Pmp20 had a glutathione (GSH) peroxidase activity, suggesting the presence of glutathione within the peroxisomes (9). However, we and other groups of investigators have been unable to determine the levels of the reduced and oxidized forms of glutathione due to technical difficulties and therefore have been unable to assess the redox state within peroxisomes by conventional biochemical methods.In general, the intracellular redox state is determined by the levels of redox-related metabolites that are generated by multiple metabolic pathways. (We herein refer to the “redox state” as an intracellular environment at steady state, which is distinct from oxidative stress or ROS, which functions as a signal for further intracellular events such as apoptosis.) Therefore, the redox state is considered to reflect the overall metabolic status. While the standard redox potential (E₀′) is a general index used to express the redox state of a compound, it cannot be used to describe the intracellular redox state because it does not take into account various physiological considerations, such as the cytosol, where many compounds coexist in a mixture of various redox states (14). Therefore, the equilibrium redox state in living cells has been estimated from indices such as the ratio of oxidized and reduced forms of glutathione, from indirect indices of the redox state, such as the NAD(P)H ratio (12, 40), or from the level of the expression of antioxidant enzymes. However, the measurement of these indices often yields contradictory results, making it difficult to evaluate the physiological redox state using any single index. This situation might have led to misunderstanding the redox state in cells from patients with PBDs. Reductive conditions could occur during conditions of oxidative stress, when the ROS defense system is functioning normally.With the aim of determining the intracellular redox state directly, we developed a fluorescent redox probe, Redoxfluor, with a novel sensing mechanism. Several green fluorescent protein (GFP) variants that report the in vivo redox state (roGFP [4, 7], rxYFP [18, 24, 25]) or H₂O₂ level (HyPer [3]) have been developed since the start of our research. However, none of these reporters have been used to visualize the redox state in mammalian cytosol, and differences in the redox potential between normal and pathological states have not been reported.In the present work, we developed a Redoxfluor that discriminates the redox state of peroxisome assembly mutant cell lines (34) from that of the normal cell line. Our findings shed light on how to tackle problems with monitoring the spatiotemporal dynamics of the redox state within living mammalian cells and also should pave the way for the development of a screen for drugs that can affect various metabolic disorders with abnormal redox state.     

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.     

Coproantigen survey for Echinococcus multilocularis prevalence of red foxes in Hokkaido, Japan

An epidemiological survey was conducted on the seasonal variation of Echinococcus multilocularis prevalence in red foxes from 1997 to 1998, using a monoclonal antibody-based detection of the tapeworm coproantigen. Thirty-six breeding dens of reproductive fox families were identified in the endemic area of Koshimizu, eastern Hokkaido, Japan. Fecal samples from each site were examined by coproantigen detection assay and fecal egg examination. Whereas the prevalence of coproantigen positive feces showed no seasonal fluctuation (51.6-66.7%), variation was found in the prevalence of egg positive feces in which a higher prevalence was observed in the summer and winter (31.1 and 38.7%) than spring and autumn (13.3 and 13.5%). Significant differences were observed between juveniles and adult foxes in both examinations. Samples from juvenile foxes gave higher coproantigen positive results and taeniid egg intensity. Those results suggest more juveniles infected with the cestode than adults in the same period. The practical use of coproantigen assay as a survey tool and factors which affect the prevalence and host age-related difference are discussed.     

Hyperglycemia impairs the insulin signaling step between PI 3-kinase and Akt/PKB activations in ZDF rat liver

Akt/PKB activation is reportedly essential for insulin-induced glucose metabolism in the liver. During the hypoinsulinemic and hyperglycemic phase in the Zucker diabetic fatty (ZDF) rat liver, insulin-induced phosphorylations of the insulin receptor (IR) and insulin receptor substrate (IRS)-1/2 were significantly enhanced. Similarly, phosphatidylinositol (PI) 3-kinase activities associated with IRS-1/2 were markedly increased in ZDF rat liver compared with those in the control lean rat liver. However, interestingly, insulin-induced phosphorylation and kinase activation of Akt/PKB were severely suppressed. The restoration of normoglycemia by sodium-dependent glucose transporter (SGLT) inhibitor to ZDF rats normalized elevated PI 3-kinase activation and phosphorylation of IR and IRS-1/2 to lean control rat levels. In addition, impaired insulin-induced Akt/PKB activation was also normalized. These results suggest that chronic hyperglycemia reduces the efficiency of the activation step from PI 3-kinase to Akt/PKB kinase and that this impairment is the molecular mechanism underlying hyperglycemia-induced insulin resistance in the liver.     

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.     

Susceptibility to kainate-induced seizures under dietary zinc deficiency

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.     

alpha-Mannosidase-catalyzed synthesis of a (1-->2)-alpha-D-rhamnodisaccharide derivative

Enzymatic transglycosylation using p-nitrophenyl alpha-D-rhamnopyranoside as the glycosyl donor and 6equiv of ethyl 1-thio-alpha-D-rhamnopyranoside as the glycosyl acceptor yielded a D-rhamnooligosaccharide derivative. The reaction was catalyzed by jack bean alpha-mannosidase in a 1:1 (v/v) mixture of 0.1 M sodium citrate buffer (pH4.5)-MeCN at 25 degrees C. The enzyme exhibited high catalytic activity for the reaction, to afford in 32.1% isolated yield (based on donor substrate) ethyl alpha-D-rhamnopyranosyl-(1-->2)-1-thio-alpha-D-rhamnopyranoside, which is a derivative of the common oligosaccharide unit of the antigenic lipopolysaccharides from Pseudomonas.