IL-18 is redundant in T-cell responses and in joint inflammation in antigen-induced arthritis

IL-18 is an important cofactor in Th1 immune responses and it has additional roles in inflammation. Recent reports suggest the contribution of IL-18 to immune responses may vary between mouse strains and immune contexts. We investigated the contribution of IL-18 to T-cell activation and joint inflammation in Ag-induced arthritis (AIA) in C57Bl/6 mice. AIA and cutaneous delayed-type hypersensitivity (DTH) reactions were induced in wild-type (WT) and IL-18-/- C57Bl/6 mice, and Ag-specific T-cell proliferation and IFN-gamma and IL-4 production were measured. The humoral immune response was measured as serum antibody to the disease-initiating Ag, methylated BSA (mBSA). Splenocyte production of IL-6 was measured by ELISA. To confirm the dependence of this model on Th1-cell-mediated immunity, IL-12p40-/- mice were similarly studied. WT mice developed synovitis, joint effusion, cartilage destruction and bone damage associated with induction of DTH, and in vitro Ag-specific T-cell proliferation and IFN-gamma production. Unexpectedly, IL-18-/- mice developed AIA and indices of T-cell activation were similar to those of WT mice. In contrast, IL-12p40-/- mice did not develop AIA, DTH or T-cell activation. WT and IL-18-/- mice, but not IL-12p40-/- mice, developed significantly increased serum antibody to mBSA compared with naive controls. WT and IL-18-/- splenocytes produced high levels of IL-6, whereas IL-12p40-/- cells had significantly lower IL-6 production compared with both. In conclusion, IL-18 is redundant both as a Th1 response cofactor and inflammatory cytokine, whereas IL-12p40-/- is a key cytokine, in AIA in C57Bl/6 mice.     

Characterization of ATPases of plain synaptic vesicle and coated vesicle fractions isolated from rat brains.

The plain synaptic vesicle and the ocated vesicle fractions were isolated from rat brains, and the ATPase [EC 3.6.1.3] activities were characterized in terms of ionic effects, drug effects, and protein components. Coated vesicle fraction contained three times as much actomysin-like proteins as plain vesicle fraction, although both fractions had an identical ratio of actin-like protein to myosin-like protein. The ATPases of these two fractions were activated by both Mg2+ and Ca2+, and, in the presence of either of the cations, were inhibited by KCl. Reserpine activated plain vesicle ATPase only in the presence of Cl-. Colchicine and vinblastine inhibited coated vesicle ATPase only. The results are consistent with the view that actomyosin-like proteins are involved in the synaptic retrieval process.     

Characterization of Polypeptides that Accumulate in Cultured Nicotiana tabacum Cells

We characterized the polypeptides that accumulate in photoautotrophicallycultured cells of tobacco. Microsequencing of these polypeptidesaccumulated in large amounts revealed four NH₂-terminal aminoacid sequences that were highly homologous to those of the knownstress proteins, osmotin and chitinase. Further analyses ofour tobacco cell line grown with sucrose in light and in darkness,as well as analyses of newly established cultured cells andregenerating adventitious shoots, clearly showed that all thein vitro cultured cells accumulated these stress proteins. Theaccumulation of these proteins were also observed in old leaves,roots, and leaves infected with Tobacco Mosaic Virus, but notin young healthy leaves. (Received September 27, 1989; Accepted December 4, 1989)     

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.     

Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress

Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H(2)O(2) and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H(2)O(2). JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage.     

Species-specificity of the lytic activity of the cell wall in the genus Chlorella

Cell wall lytic activity was compared among strains IAM C-27, C-87, SAG 211-1c, -1d, -9a, -8b, -8c, -8l, -11f, -8k, -11g, and -11h/9 of the genus Chlorella . The optimal pH was alkaline in strains with glucosamine as the characteristic group of the rigid wall, and acidic in strains characterised by glucan groups. The lytic enzymes of strains in the former type of algae lyzed the cell wall mainly to soluble high molecular oligosaccharides. The lytic activity of the Chlorella cell wall thus appears species- and strain-speicific.     

Identification of RET autophosphorylation sites by mass spectrometry

The catalytic and signaling activities of RET, a receptor-type tyrosine kinase, are regulated by the autophosphorylation of several tyrosine residues in the cytoplasmic region of RET. Some studies have revealed a few possible autophosphorylation sites of RET by [(32)P]phosphopeptide mapping or by using specific anti-phosphotyrosine antibodies. To ultimately identify these and other autophosphorylation sites of RET, we performed mass spectrometry analysis of an originally prepared RET recombinant protein. Both the autophosphorylation and kinase activity of myelin basic protein as an external substrate of the recombinant RET protein were substantially elevated in the presence of ATP without stimulation by a glial cell line-derived neurotrophic factor, a natural ligand for RET. Mass spectrometric analysis revealed that RET Tyr(806), Tyr(809), Tyr(900), Tyr(905), Tyr(981), Tyr(1062), Tyr(1090), and Tyr(1096) were autophosphorylation sites. Levels of autophosphorylation and kinase activity of RET-MEN2A (multiple endocrine neoplasia 2A), a constitutively active form of RET with substitution of Tyr(900) by phenylalanine (Y900F), were comparable with those of original RET-MEN2A, whereas those of the mutant Y905F were greatly decreased. Interestingly, those of a double mutant, Y900F/Y905F, were completely abolished. Both the kinase activity and transforming activity were impaired in the mutants Y806F and Y809F. These results provide convincing evidence for both previously suggested and new tyrosine autophosphorylation sites of RET as well as for novel functions of Tyr(806), Tyr(809), and Tyr(900) phosphorylation in both catalytic kinase activities and cell growth. The significance of the identified autophosphorylation sites in various protein-tyrosine kinases registered in a data base is discussed in this paper.