Putative PIP1 genes isolated from apple: expression analyses during fruit development and under osmotic stress

To gain insight into the function of plasma membrane intrinsic protein (PIP) genes in apple, two genes, MdPIP1a and MdPIP1b, were isolated. MdPIP1 expression was in accordance with the volume increase during fruit development, which is a loading process of water and solutes. In addition, the expression of MdPIP1 was up-regulated in the stems by osmotic stress. These results indicate that MdPIP1 may play important roles not only in fruit expansion, but also in maintaining water homeostasis under stress conditions.     

Cutting edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by APCs through its chemokine domain

SR-PSOX and CXC chemokine ligand (CXCL)16, which were originally identified as a scavenger receptor and a transmembrane-type chemokine, respectively, are indicated to be identical. In this study, we demonstrate that membrane-bound SR-PSOX/CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria. Importantly, our prepared anti-SR-PSOX mAb, which suppressed chemotactic activity of SR-PSOX, significantly inhibited bacterial phagocytosis by human APCs including dendritic cells. Various scavenger receptor ligands inhibited the bacterial phagocytosis of SR-PSOX. In addition, the recognition specificity for bacteria was determined by only the chemokine domain of SR-PSOX/CXCL16. Thus, SR-PSOX/CXCL16 may play an important role in facilitating uptake of various pathogens and chemotaxis of T and NKT cells by APCs through its chemokine domain.     

Functional domains essential for Gs activity in prostaglandin EP2 and EP3 receptors

The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E(2) (PGE(2)) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3beta and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE(2) binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3beta, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs.     

Complementation of Escherichia coli ubiF mutation by Caenorhabditis elegans CLK-1, a product of the longevity gene of the nematode worm

Caenorhabditis elegans CLK-1 was identified from long-lived mutant worms, and is believed to be involved in ubiquinone biosynthesis. The protein belongs to the eukaryotic CLK-1/Coq7p family, which is also similar to the bacterial Coq7 family, that hydroxylates demethoxyubiquinone, resulting in the formation of hydroxyubiquinone, a precursor of ubiquinone. In Escherichia coli, the corresponding reaction is catalyzed by UbiF, a member of a distinct class of hydroxylase. Although previous studies suggested that the eukaryotic CLK-1/Coq7 family is a hydroxylase of demethoxyubiquinone, there was no direct evidence to show the enzymatic activity of the eukaryotic CLK-1/Coq7 family. Here we show that the plasmid encoding C. elegans CLK-1 supported aerobic respiration on a non-fermentable carbon source of E. coli ubiF mutant strain and rescued the ability to synthesize ubiquinone, suggesting that the eukaryotic CLK-1/Coq7p family could function as bacterial UbiF.     

Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.     

Antibacterial effect of Kampo herbal formulation Hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang) on Helicobacter pylori infection in mice

Because Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases in humans, the eradication of H. pylori using antibiotics is very effective for the treatment of gastroduodenal diseases. However, it has recently been reported that resistance to these antibiotics is developing. In the present study, the antibacterial effect of a Kampo (traditional Japanese medicine) herbal formulation, Hochu-ekki-to (RET; Formula repletionis animalis et supletionis medii), against H. pylori was examined in vitro and in vivo. HET inhibited the growth of antibiotic-resistant strains of H. pylori as well as antibiotic-sensitive strains at a dose of 2.5 mg/ml in vitro. When 1,000 mg/kg of HET was administered orally to C57BL/6 mice for 7 days before or after inoculation with H. pylori, H. pylori in the stomach was significantly reduced in the HET-pre-treatment group compared with the control group. Furthermore, HET in combination with antibiotics completely eradicated the bacteria in mice. The expression of interferon (IFN)-gamma was induced in the gastric mucosa of the mice pre-treated with HET. There were no significant differences between the colonization of H. pylori in the control and HET treatment groups in IFN-gamma gene-deficient mice. These results suggest that the antibacterial effect of HET may be partly due to IFN-gamma induction, and that HET may be clinically useful for treatment of H. pylori infection.     

Intraperitoneal fluid accumulation induced by Clostridium perfringens alpha-toxin (phospholipase C)

We report that the intraperitoneal injection of Clostridium perfringens alpha-toxin into mice induces ascites. This phenomenon was monitored by measuring fluid volume and analyzing hematologic data. The mouse toxicity test provides a simple and useful model for examining C. perfringens alpha-toxin-induced vascular permeability.     

Effects of systemic and central nervous system localized inflammation on the contributions of metabolic precursors to the L-kynurenine and quinolinic acid pools in brain

L-Kynurenine and quinolinic acid are neuroactive L-tryptophan-kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [(2)H(3) ]-L-kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS-localized immune-activated, and systemically immune-activated. The specific activity of L-kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of L-kynurenine in brain was derived via local synthesis from L-tryptophan/formylkynurenine versus 78% from L-kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue L-tryptophan/formylkynurenine (10%), blood L-kynurenine (35%), blood 3-hydroxykynurenine/3-hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune-activation, however, L-kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood L-kynurenine (52%), blood 3-hydroxykynurenine or 3-hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS-localized immune activation, > 98% of both L-kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which L-kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources.     

Neurotoxic mechanisms triggered by Alzheimer's disease-linked mutant M146L presenilin 1: involvement of NO synthase via a novel pertussis toxin target

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.     

Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.