Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.     

Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta

IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Strand asymmetry of +1 frameshift mutagenesis at a homopolymeric run by DNA polymerase III holoenzyme of Escherichia coli

We have recently shown that single-base frameshifts were predominant among mutations induced within the rpsL target sequence upon oriC plasmid DNA replication in vitro. We found that the occurrence of +1 frameshifts at a run of 6 residues of dA/dT could be increased proportionally by increasing the concentration of dATP present in the in vitro replication. Using single-stranded circular DNA containing either the coding sequence of the rpsL gene or its complementary sequence, the +1 frameshift mutagenesis by DNA polymerase III holoenzyme of Escherichia coli was extensively examined. A(6) --> A(7) frameshifts occurred 30 to 90 times more frequently during DNA synthesis with the noncoding sequence (dT tract) template than with the coding sequence (dA tract). Excess dATP enhanced the occurrence of +1 frameshifts during DNA synthesis with the dT tract template, but no other dNTPs showed such an effect. In the presence of 0.1 mM dATP, the A(6) --> A(7) mutagenesis with the dT tract template was not inhibited by 1.5 mM dCTP, which is complementary to the residue immediately upstream of the dT tract. These results strongly suggested that the A(6) --> A(7) frameshift mutagenesis possesses an asymmetric strand nature and that slippage errors leading to the +1 frameshift are made during chain elongation within the tract rather than by misincorporation of nucleotides opposite residues next to the tract.     

ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1

By using a selective ROCK inhibitor Y-27632, the role of Rho-ROCK signaling in the function of hepatic stellate cells in culture was studied. Stellate cells maintained the "star-like" configuration of the quiescent stage in the presence of Y-27632, while the expression of smooth muscle alpha-actin and PDGF receptor beta was not affected by the agent. Serum-stimulated migration of the cells was significantly suppressed by Y-27632. The contraction of stellate cells induced by 5 nM endothelin-1 was attenuated by the agent in a dose-dependent manner. Formation of F-actin stress fibers and phosphorylation of myosin light chain was apparently reduced by Y-27632 even under the stimulation with endothelin-1. On the other hand, ex vivo liver perfusion experiment revealed that endothelin-1 (2 nM)-induced increase of portal vein constriction was almost completely inhibited by 20 microM Y-27632 with a concomitant improvement of hepatocyte degeneration. These results suggest that ROCK is one of the key regulators of stellate cell motility and that the clinical application of ROCK inhibitors such as Y-27632 should be considered in the reduction of portal hypertension in liver fibrosis and cirrhosis.     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: first genome wide comparative sequence analysis of wild and cultivated rice

We determined the complete nucleotide sequence of the chloroplast genome of wild rice, Oryza nivara and compared it with the corresponding published sequence of relative cultivated rice, Oryza sativa. The genome was 134,494 bp long with a large single-copy region of 80,544 bp, a small single-copy region of 12,346 bp and two inverted repeats of 20,802 bp each. The overall A+T content was 61.0%. The O. nivara chloroplast genome encoded identical functional genes to O. sativa in the same order along the genome. On the other hand, detailed analysis revealed 57 insertion, 61 deletion and 159 base substitution events in the entire chloroplast genome of O. nivara. Among substitutions, transversions were much higher than transitions with the former even more frequent than the latter in the coding region. Most of the insertions/deletions were single-base but a few large length mutations were also detected. The frequency of insertion/deletion events was more in the coding region within inverted repeats. In contrast, a very few substitution events were identified in the coding region. Polymorphism was observed among rice cultivars at loci of large insertion/deletion events. This is the first report describing comparative and genome wide chloroplast analysis between a wild and cultivated crop.