Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.     

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.     

Characterization of histopathology and gene-expression profiles of synovitis in early rheumatoid arthritis using targeted biopsy specimens

The disease category of early rheumatoid arthritis (RA) has been limited with respect to clinical criteria. Pathological manifestations of synovitis in patients whose disease is clinically classified as early RA seem to be heterogeneous, with regular variations. To clarify the relation between the molecular and histopathological features of the synovitis, we analyzed gene-expression profiles in the synovial lining tissues to correlate them with histopathological features. Synovial tissues were obtained from knee joints of 12 patients with early RA by targeted biopsy under arthroscopy. Surgical specimens of long-standing RA (from four patients) were examined as positive controls. Each histopathological parameter characteristic of rheumatoid synovitis in synovial tissues was scored under light microscopy. Total RNAs from synovial lining tissues were obtained from the specimens selected by laser capture microdissection and the mRNAs were amplified by bacteriophage T7 RNA polymerase. Their cDNAs were analyzed in a cDNA microarray with 23,040 cDNAs, and the levels of gene expression in multilayered lining tissues, compared with those of normal-like lining tissues in specimens from the same person, were determined to estimate gene-expression profiles characteristic of the synovial proliferative lesions in each case. Based on cluster analysis of all cases, gene-expression profiles in the lesions in early RA fell into two groups. The groups had different expression levels of genes critical for proliferative inflammation, including those encoding cytokines, adhesion molecules, and extracellular matrices. One group resembled synovitis in long-standing RA and had high scores for some histopathological features – involving accumulations of lymphocytes and plasma cells – but not for other features. Possible differences in the histopathogenesis and prognosis of synovitis between the two groups are discussed in relation to the candidate genes and histopathology.     

ER-60 domains responsible for interaction with calnexin and calreticulin

ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT). In this study, we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule. ER-60 was shown to be composed of at least four domains, named a, b, b', and a', by limited proteolysis. Recombinant fragments of ER-60, a, b', and a'c, were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient. These fragments each gave the circular dichroism (CD) spectrum of the folded protein. On the other hand, fragment b, which did not undergo the cooperative unfolding transition along a urea gradient gel, did not show any sign of the folded structure on the CD measurement. However, subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb'. Both a and a'c, which have a catalytic center CGHC motif, showed activity almost equivalent to half of that of wild-type ER-60. Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity, suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60. The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.     

Distribution of tetrodotoxin in the xanthid crab (Atergatis floridus) collected in the coastal waters of Kanagawa and Wakayama Prefectures

The objective of this investigation was to study the distribution of tetrodotoxin (TTX) in the xanthid crab (Atergatis floridus) found in the coastal waters of Kanagawa and Wakayama Prefectures of Japan using mouse assay methods. We used 32 crab samples (18 males and 14 females) and toxicity was analyzed on 13 parts of the body of each sample. The muscle of chelipeds was found to be toxic in all the samples with a wide range of toxicity (5–237 MU/g), whereas the toxicity in the muscle of the cephalothorax was found to be non-toxic (below detectable limit) in all the samples [Narita, H., Watanabe, K., Baba, K., Ohgami, H., Ai, T.K., Igarashi, Y., Nara, M., Noguchi, T., Hashimoto, K., 1987. The toxicity of digestive gland of trampet shells inhabiting the coast of Shizuoka Prefecture. J. Food Hyg. Soc. Jpn. 28, 115–118.]. Further investigation of different parts of the chelipeds indicated that the muscle of the palm and carpus are usually toxic and that of merus and ischium are almost non-toxic. Toxicity of the muscles of palm ranged between 7 and 52 MU/g, whereas toxicity of the muscle of ischium was below detectable limit. Results from our study indicate clear contrast in the distribution of tetrodotoxin in muscles of different parts of the xanthid crabs, plausibly due to some inherent physiological mechanism. Further investigation is necessary to understand the mechanism responsible for such contrast.     

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.     

Adenosine A2A receptor is involved in cell surface expression of A2B receptor

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.