Induction of IL-2 receptor expression and proliferation of T cell clones by a novel cytokine(s).

We found a unique T cell IL-2 receptor (IL-2R)3-inducing activity in the supernatant (SN) of the TH1 clone stimulated with antigen on spleen cells as antigen-presenting cells (APC). We have tentatively named this activity the IL-2R-inducing factor (IL-2RIF) and have characterized the activity. The SN induced IL-2R and proliferation of TH1 clones stimulated with B cell APC, which could not induce IL-2R in the absence of the SN. Other known cytokines were examined for a IL-2RIF activity; however, none of cytokines examined exerted a similar activity. Moreover, the neutralizing antibodies against the known cytokines tested did not block the IL-2RIF activity in the SN. When TH1 clones were stimulated with immobilized anti-CD3 or with fixed B cell APC in the presence of partially purified IL-2RIF, these clones expressed IL-2R and showed IL-2-dependent proliferation, whereas they induced neither IL-2R expression nor proliferation in the absence of IL-2RIF activity. These observations suggest that IL-2RIF activity is mediated by a novel cytokine(s) and the cytokine plays an important role as a second signal in the activation of the TH1 clone.     

Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies.

Five monoclonal antibodies (mAbs) directed against the flagellin of Clostridium chauvoei were used to analyse the structural and antigenic characteristics on the bacterial flagellar surface. Immune electron microscopy showed that three protective mAbs recognized the surfaced-exposed epitopes on the flagellar filament of this bacteria. In contrast, two non-protective mAbs recognized internal epitopes of the flagellar filament. These findings have been confirmed by ELISA using mAbs absorbed with whole cells of C. chauvoei possessing flagella. Competitive binding assays showed that protective mAbs indicated reciprocal competition, while each of the non-protective mAbs had topographically distinct epitopes. Moreover, immunoblotting analysis with cyanogen-bromide-cleaved flagellin showed that protective mAbs may preferentially recognize conformational epitopes, whilst one of the non-protective mAbs may recognize a linear and conformation-independent epitope in the flagellin of C. chauvoei.     

The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-beta-D-arabinofuranosylcytosine.

Dose-dependent induction of micronuclei with 1-beta-D-arabinofuranosylcytosine (ara-C) was clearly shown in CD-1 mouse peripheral blood reticulocytes (RETs) using an acridine orange (AO) supravital staining method, as well as in the conventional bone marrow assay. The maximum frequencies of micronucleated RETs (MNRETs) in peripheral blood and of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow were comparable, as shown in two laboratories independently. The maximum frequencies of MNRETs in peripheral blood lagged about 24 and 12 h behind those of MNPCEs in bone marrow in experiments with 24- and 12-h sampling intervals, respectively. The proportion of each type of RET was examined periodically after treatment with ara-C at doses ranging from 6.25 to 50.0 mg/kg. The proportion of type I RETs among total RETs decreased 24 or 48 h after treatment according to the dose level. This suggest that this ratio could be a good indicator of the bone marrow cell toxicity of test chemicals.     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Chronobiologic Evaluation of Drug Efficacy in Hypertension

Time-associated variations of blood pressure became accesible due to the modern development of pressure monitoring technology. The data collection and analysis should be standardized and formulated. Time-associated changes of pressure were evaluated not only by nonparametric and conventional but also by parametric and rhythmometric estimation. The reference data (chronodesms) are essential to define the deviant magnitude of pressure. This chronobiologic approach became feasible to quantitate the efficacy of antihypertensive agents in hypertensives.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

Possible involvement of arachidonic acid metabolites of cytochrome P450 monooxygenase pathway in vasopressin-stimulated glycogenolysis in isolated rat hepatocytes

Arachidonic acid (AA) is reported to be metabolized by three major pathways, i.e., cyclooxygenase (CO), lipoxygenase (LO), and NADPH-dependent cytochrome P450 monooxygenase (MO) pathways. Monooxygenase metabolites of AA have been proposed to play an important role in hormone action in various cells. Recently it was reported that the MO pathway may exist in rat liver. The present study was carried out to investigate the role of MO metabolites in vasopressin-induced glycogenolysis in isolated rat hepatocytes. The pretreatment of isolated rat hepatocytes with eicosatetraynoic acid (ETYA), an inhibitor of CO, LO, and MO pathways, and ketoconazole and SKF 525A, inhibitors of the MO pathway, dose-dependently reduced vasopressin-induced phosphorylase activation, while the pretreatment with indomethacin, an inhibitor of the CO pathway, had no effect. The increment of cytosolic calcium concentration in vasopressin-stimulated hepatocytes was also dose-dependently decreased by ETYA, ketoconazole, and SKF 525A. In vitro addition of epoxyeicosatrienoic acid (EET) dose-dependently increased both phosphorylase a activity and cytosolic calcium concentration. 14,15-EET was the most potent among four regioisomeric EETs. These results suggest that MO metabolites of AA, most likely EETs, may be involved in vasopressin-induced glycogenolysis probably via the activation of phosphorylase by increasing the cytosolic calcium concentration.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.