Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro²⁴⁷, in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.     

The Silicon Layer Supports Acid Resistance of Bacillus cereus Spores

Silicon (Si) is considered to be a “quasiessential” element for most living organisms. However, silicate uptake in bacteria and its physiological functions have remained obscure. We observed that Si is deposited in a spore coat layer of nanometer-sized particles in Bacillus cereus and that the Si layer enhances acid resistance. The novel acid resistance of the spore mediated by Si encapsulation was also observed in other Bacillus strains, representing a general adaptation enhancing survival under acidic conditions.Silicon (Si), the second-most-abundant element in the earth''s crust, is an important mineral for living organisms; it acts as a component of the outer skeleton of diatomaceous protozoans (1), as a trace element to help animal bone and tooth development (5), and as an element in plants that enhances their tissue strength and disease resistance (8, 9). These organisms take up silicate from the environment and accumulate it as silica that is formed from highly concentrated silicate (27). In 1980, relatively high concentrations of Si were observed at the spore coat region of Bacillus cereus and Bacillus megaterium spores by an analysis using scanning transmission electron microscopy (STEM) (14, 23). However, due to the low resolution and relatively weak signal, the precise localization of Si was not determined. On the other hand, the Si contents of Bacillus coagulans and Bacillus subtilis spores were reported to be almost absent or under the detection limit (4, 24). Some bacteriologists familiar with these data consider the presence of Si an anomaly (17). The presence of Si in bacterial spores (specifically, the spores of Bacillus anthracis) again became the focus of attention when anthrax spores were mailed to U.S. senators in the fall of 2001 (17). The Senate anthrax spores could be easily dispersed as single spores when the container was opened. The investigators considered that coating spores with silica might be involved in preventing spores from sticking to each other (17). Thus, if silica is normally absent from spores, its presence in B. anthracis spores suggested that they had been weaponized (17). Subsequent analysis convinced the investigators that the Si was a natural occurrence (3). However, since silica-rich and -poor spores of the same bacterial strain have never been compared, any relationship between naturally accumulated silica and spore dispersion remained hypothetical.In the present study, we screened for the bacterium that takes up the largest amount of silicate from among a number of strains isolated from paddy field soil in order to study Si uptake, clarify the localization of Si, and reveal the roles of Si in bacteria. The effect of silica on spore dispersion was also discussed.     

Ultrastructural morphometric analysis of ameloblasts exposed to fluoride during tooth development

Since a considerable amount of the world population is exposed to high doses of fluoride, it is of special concern to investigate its action mechanisms during dental enamel development. In this study, the toxicity of fluoride in ameloblasts during enamel development was evaluated by means of ultrastructural morphometric analysis. A total of 18 male Wistar rats were distributed into three groups. In Group I, the animals received deionized drinking water ad libitum (negative control) and in Groups II and III, they received sodium fluorided (NaF) drinking water at doses of 7 and 100 ppm ad libitum, respectively, for 6 weeks. Morphometric data were expressed as volume density of the most significant organelles present in the secretory and maturation phases of amelogenesis such as RER, granules, lysosomes, phagic vacuoles, microfilaments and mitochondria. The results showed that the volume density of mitochondria in the 100 ppm experimental group was 29% (P < 0.05) higher than the control group in secretory ameloblasts. No remarkable differences were found in maturation ameloblasts for all organelles evaluated. Taken together, these data indicate that NaF at high doses is able to induce cellular damage in secretory ameloblasts, whereas no noxious effect was observed during maturation stage of amelogenesis as depicted by ultrastructural analysis.     

Carcinogenic role of tumor necrosis factor-alpha inducing protein of Helicobacter pylori in human stomach

Helicobacter pylori is the definitive carcinogen for stomach cancer and is known to induce proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1(IL-1) in the stomach. Based on our findings that TNF-alpha is an endogenous tumor promoter, we identified the TNFalpha inducing protein (Tipalpha) gene family, and confirmed Tipalpha and HP-MP1 as new carcinogenic proteins of H. pylori.Tipalpha protein is unique to H. pylori, and this paper shows the strong tumor promoting activity of Tipalpha gene family, in cooperation with Ras protein and its mechanisms of action in relation to NF-kappaB activation, and discusses the carcinogenic role of Tipalpha in stomach cancer. Our recent finding showing that penicillin-binding proteins of other bacteria are weak homologues of Tipalpha is also discussed.     

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Seasonal variation in gastric myoelectrical activity in young Japanese females

In order to test our hypothesis that there is seasonal variation in digestion and absorption of dietary carbohydrate from the intestine, we previously determined the amount of unabsorbed carbohydrate after breakfast by the breath hydrogen test in the four seasons. In pursuing our hypothesis further, we also recorded gastric myoelectrical activity before and after the breakfasts. In the current report, we describe the seasonality of gastric myoelectrical activity. Twenty-six Japanese female subjects were studied in winter, spring, summer and autumn. The cutaneous electrogastrogram was analysed by spectral analysis to compute the pre- and post-prandial dominant slow wave frequency (DF), and percentage of the 2 - 4 cpm gastric slow wave (Normal %). Two-factor ANOVA indicated that there was no significant seasonal variation in DF and Normal %. These results indicate that seasonal variations in digestion and absorption of dietary carbohydrate are caused by factors other than gastric and small intestinal motility.     

Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16)

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.