Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.     

Resistance to metastatic disease in STAT6-deficient mice requires hemopoietic and nonhemopoietic cells and is IFN-gamma dependent

Mice deficient for the STAT6 gene (STAT6(-/-) mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8(+) T cells. Immunity in STAT6(-/-) mice is unusually effective in that 45-80% of STAT6(-/-) mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT(+/+) (BALB/c) mice. Surprisingly, STAT6(-/-) and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6(-/-) hemopoietic and nonhemopoietic components. Likewise, CD1(-/-) mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6(-/-) and CD1(-/-) reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6(-/-) and CD1(-/-) mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4(+)CD25(+) T cells in BALB/c mice does not increase survival, demonstrating that CD4(+)CD25(+) cells do not regulate immunity. Cytokine production and tumor challenges into STAT6(-/-)IFN-gamma(-/-) mice indicate that IFN-gamma is essential for immunity. Therefore, immunosurveillance in STAT6(-/-) mice facilitates survival against metastatic cancer via an IFN-gamma-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4(+)CD25(+) T cells.     

Evidence for a relationship between activity and the tetraprotomeric assembly of solubilized pig gastric H/K-ATPase

Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K0.5 = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K0.5 = 28 mM) and H/K-ATPase (K0.5 = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/K-ATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K+ in C12E8-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C12E8-solubilized gastric H/K-ATPase is a tetraprotomer.     

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.     

Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80(+), the gene encoding Ku80. Both pku70(+) and pku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.     

Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum

Novel cloning vectors for glutamic acid-producing bacteria have been constructed. Two cryptic plasmids, pAM330 from Brevibacterium lactofermentum and pHM1519 from Corynebacterium glutamicum, were used as precursors, and recombined with pBR325 or pUB110. Resultant composite plasmids were able to propagate and to express the CmR or KmR phenotype in B. lactofermentum and C. glutamicum. A smaller, high-copy-number plasmid, pAJ43, was also isolated following deletion of a part of the pAM330-pBR325 composite plasmid. Furthermore, a cosmid vector, which can be packaged and transduced through phage infection, has been developed using a cohesive-end fragment of the f1A phage and plasmid pAJ43. These plasmids are suitable for use as cloning vectors in the glutamic acid-producing bacteria.     

Detection of intranuclear forces by the use of laser optics during the recovery process of elongated interphase nuclei in centrifuged protenemal cells ofAdiantum capillus-veneris

For the direct investigation of intranuclear dynamics in living cells, extremely deformed nuclei of basipetally centrifuged protonemal cells of the fernAdiantum capillus-veneris were manipulated by the laser trap and the laser scalpel. Whereas the nucleolus was tightly fixed at the central position inside the non-centrifuged nucleus and proved to be immovable by the optical trap, it could easily be trapped and moved towards three directions inside the bubble-like terminal widening of the basal thread-like extension of centrifuged nuclei. Due to the connection of the nucleolus to the chromatin inside the nuclear thread (NT), moving was not possible against the direction of the nuclear apical main body. Nucleoli in recovered nuclei were again immovable, thus indicating the presence of a dynamic nucleolar anchoring system inside the nucleus. When the nucleolus in the bubble was arrested during the thread shortening process by the optical trap, the acropetal movement of the bubble continued. Probably due to dragging forces, some nucleoli became stretched, and a thick strand of a still unknown composition stretched between the nucleolus and the insertion site of the shortening NT. To assess whether the shrinking of the nuclear envelope (NE) and the shortening of the chromatin inside the NT were independent processes, the chromatin above the bubble was cut inside the NT by the laser scalpel. After severance, a gap between the nucleolus and the end of the chromatin strand in the NT indicated the shortening of the chromatin inside the NT. From these findings it was concluded that a shortening force was existing in the chromatin of the NT and that probably no physical link existed between the chromatin and the NE.     

Colon-specific delivery of R68070, a new thromboxane synthase inhibitor, using chitosan capsules: therapeutic effects against 2,4,6-trinitrobenzene sulfonic acid-induced ulcerative colitis in rats

The objective of this study was to estimate the therapeutic effects of R68070, a new thromboxane synthase inhibitor, on 2,4,6-trinitrobenzenesulfonic acid sodium salt (TNBS)-induced ulcerative colitis in rats. We also examined the acceleration of the healing effect of R68070 with chitosan capsules to achieve its colon-specific delivery. The colonic injury and inflammation were assessed by measuring the myeloperoxidase (MPO) activities, colon wet weight/body weight (C/B) ratio and the damage score, respectively. These markers were decreased by the oral administration of R68070 with chitosan capsules and carboxymethyl-cellulose (CMC) suspension. The therapeutic effects of R68070 against ulcerative colitis were observed in both dosage forms in a dose dependent manner. In addition, its therapeutic effects were increased by the use of chitosan capsules, compared with CMC suspension. These results suggest that chitosan capsule might be a very useful dosage form for the colon-specific delivery of R68070 as an anti-inflammatory drug and for the therapy of ulcerative colitis.