Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid,Acyrthosiphon pisum

In addition to the essential intracellular symbiotic bacterium Buchnera, several facultative endosymbiotic bacteria called collectively secondary symbionts (S-symbionts) have been identified from the pea aphid Acyrthosiphon pisum. We conducted an extensive and systematic survey of S-symbionts in Japanese local populations of A. pisum using a specific PCR detection technique. Five S-symbionts of A. pisum, PASS, PAUS, PABS, Rickettsia and Spiroplasma, and two facultative endosymbionts universally found in various insects, Wolbachia and Arsenophonus, were targeted. Of 119 isofemale strains originating from 81 localities, 66.4% of the strains possessed either of four S-symbionts: PASS (38.7%); PAUS (16.0%); Rickettsia (8.4%); and Spiroplasma (3.4%), while 33.6% of the strains contained only Buchnera. PABS, Wolbachia and Arsenophonus were not detected from the Japanese strains of A. pisum. In order to understand intra- and interpopulational diversity of S-symbiont microbiota in detail, 858 insects collected from 43 localities were examined for infection with the four S-symbionts. It was demonstrated that different S-symbionts coexist commonly in the same local populations, but double infections with two S-symbionts were rarely detected. Notably, the S-symbionts exhibited characteristic geographical distribution patterns: PASS at high frequencies all over Japan; PAUS at high frequencies mainly in the northeastern part of Japan; and Rickettsia and Spiroplasma at low frequencies sporadically in the southwestern part of Japan. These results indicate that the geographical distribution and infection frequency of the S-symbionts, in particular PAUS, might be affected by environmental and/or historical factors. Statistical analyses suggested that the distribution of PAUS infection might be related to host plant species, temperature and precipitation.     

Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise

Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.     

Bioassay-based screening of microorganisms that degrade dioxin using substrate-immobilized microtubes

In the current study, we attempted to develop a method for bioassay-based screening of microorganisms that degrade dioxin. However, a crucial problem encountered was that the standard dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to bacterial medium immediately disappeared from the liquid phase due to its adsorption onto polypropylene (PP) tubes. Among other aromatic hydrocarbons, adsorption onto PP tubes was also observed in beta-naphthoflavone but not in benzo[a]pyrene. Adsorption of TCDD was observed not only onto PP tubes but also onto polystyrene, glass, and PP tubes with low affinity for DNA or protein. Silanization was not effective at preventing adsorption of TCDD. TCDD immobilized onto PP tubes was recovered by organic solvents, including ethanol, methanol, and dimethyl sulfoxide (DMSO). The elution efficiency of the immobilized TCDD by DMSO was approximately 85%. Based on these findings, screening of bacteria that degrade dioxin was attempted as follows. First, TCDD was immobilized onto PP tubes. Second, bacterial suspension was added to the tubes and incubated for biodegradation of TCDD. Third, remaining, immobilized TCDD was eluted by DMSO and subjected to a reporter bioassay to evaluate the level of TCDD. Using this method, we demonstrated successful screening of bacteria that have the potential for degradation of dioxin.     

Staphylococcal enterotoxin A modulates intracellular Ca2+ signal pathway in human intestinal epithelial cells

We demonstrate here that staphylococcal enterotoxin A (SEA) induces an increase in intracellular calcium ([Ca2+]i) in human intestinal epithelial cells and the [Ca2+]i is released from intracellular stores. SEA-induced increase of [Ca2+]i was clearly inhibited by treatment with a nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine and guanidine. Intestinal epithelial cells express endothelial NOS in resting cell condition, and express inducible NOS after stimulating with tumor necrosis factor (TNF)-alpha. TNF-alpha-pretreated cells showed a significant increase in [Ca2+]i that was also inhibited by the NOS inhibitor. These results suggest that SEA modulated [Ca2+]i signal is dependent on NOS expression in human intestinal epithelial cells.     

Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state

We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.     

Roles of MAP kinase cascades in Caenorhabditis elegans

Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that are activated by diverse stimuli such as growth factors, cytokines, neurotransmitters and various cellular stresses. MAPK cascades are generally present as three-component modules, consisting of MAPKKK, MAPKK and MAPK. The precise molecular mechanisms by which these MAPK cascades transmit signals is an area of intense research, and our evolving understanding of these signal cascades has been facilitated in great part by genetic analyses in model organisms. One organism that has been commonly used for genetic manipulation and physiological characterization is the nematode Caenorhabditis elegans. Genes sequenced in the C. elegans genome project have furthered the identification of components involved in several MAPK pathways. Genetic and biochemical studies on these components have shed light on the physiological roles of MAPK cascades in the control of cell fate decision, neuronal function and immunity in C. elegans.     

Parasitism affects worker size in the Neotropical swarm-founding social wasp, <Emphasis Type="Italic">Polybia paulista</Emphasis> (Hymenoptera,Vespidae)

Summary We discovered two kinds of parasites, i.e., a strepsipteran, possibly Xenos myrapetrus (Trois) and an undescribed gregarine in the Neotropical swarm-founding paper wasp, Polybia paulista (Ihering). Although proportions of workers that were infected by these parasites varied greatly among colonies analyzed, prevalence of infected workers was recognized. Five external characters were measured and compared among uninfected workers, stylopized (i.e., infected by Strepsiptera) workers and workers that were infected by gregarines. Uninfected workers were significantly larger than stylopized workers, while smaller than workers that were infected by gregarines. Nutrients of stylopized workers may be plundered during their growth period, and consequently their body size may be reduced. However, the gregarines may manipulate host larvae to solicit more food from adults or increase development time of larvae longer, and therefore produce more parasites from a larger host.Received 1 July 2003; revised 1 December 2003; accepted 4 December 2003.     

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.