Induction of inflammatory response of mice exposed to diesel exhaust is modulated by CD4(+) and CD8(+) T cells.

Exposure to diesel exhaust (DE) increased airway inflammatory responses and airway responsiveness to allergen challenge. To clarify the roles of T cells in DE exposure-induced early inflammation, we studied the effect of CD4 and CD8 cells on the effect DE might have on allergic inflammation by using monoclonal antibody-mediated cellular depletion assays. In the bronchoalveolar lavage (BAL) fluid, the numbers of inflammatory cells from 3 mg/m(3) DE-exposed and ovalbumin (OVA)-immunized mice markedly increased. Depletion of CD4(+) cells resulted in reduced accumulation of inflammatory cells. DE exposure to OVA-immunized mice significantly increased interleukin (IL)-1 beta production but decreased IL-12 production. DE exposure significantly enhanced production of the macrophage inflammatory proteins (MIP)-1 alpha and MIP-2, but not monocyte chemoattractant protein (MCP)-1 and regulated upon activation normal T cells expressed and secreted (RANTES). Treatment with anti-CD4 and anti-CD8 mAbs abrogated the adverse effect of DE exposure. In CLN cells from OVA + DE-exposed mice, CD45R/B220-, CD3-, CD4-, and CD8-positive cells were significantly increased, but the OVA-stimulated cytokine production remained at the same levels with OVA-immunized mice. These findings suggest that the induction of early inflammatory responses by DE exposure may initially be related to the modulated function of lymphocyte subpopulations.

p21Cip-1/SDI-1/WAF-1 gene is involved in chondrogenic differentiation of ATDC5 cells in vitro.

Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. Here, to understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. Up-regulation of p21 and p27 mRNA was observed following a decrease in growth rate of prechondrocytes, and both transcripts subsequently accumulated during chondrogenic differentiation; p15, p18, and p19 mRNA, in contrast, did not change during differentiation. Only the up-regulation of p21 mRNA during differentiation was prevented by the continuous treatment of early chondrogenic inhibitor, parathyroid hormone, indicating a close correlation between differentiation and p21 induction in ATDC5 cells. Therefore, to examine the role of p21 during chondrogenesis, we established stable cell lines overexpressing full-length p21 antisense RNA in ATDC5. The reduction of endogenous p21 in these cell lines caused inhibition of early chondrogenic differentiation in ATDC5, indicating that p21 gene plays an important role in this process of the cells in vitro. Furthermore, the level of p21 protein and p21.CDK2 complexes transiently increased during differentiation, but not in undifferentiated cells, leading to a decrease in CDK2-associated kinase. However, differentiation-dependent expressed p21 protein was degraded by a proteasome-dependent pathway. Thus, the progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway.

Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice.

Through an imbalance in Th1 and Th2 cytokine profiles, diesel exhaust particles (DEP) are thought to induce Th2-dominated IgE and IgG1 production. However, the roles of CD4+ and CD8+ T-cell subtypes in the increased immune responses to antigen in mice exposed to DEP are unclear. In the present study, we investigated whether treatment with anti-CD4 or anti-CD8 mAb abrogated the adjuvant activity of DEP. On day -1 and day 1, each group of mice was injected intraperitoneally with anti-CD4, anti-CD8, or rat IgG (vehicle). On day 0, the mice were immunized with ovalbumin (OVA) or OVA plus DEP. After 3 weeks, each mouse was boosted with 10 microg of OVA alone. On day 7 after the first injection with OVA+DEP or OVA alone, the numbers of total, IA+, CD80+/IA+ and CD86+/IA+ cells in peritoneal exudate cells (PEC) were higher in OVA+DEP-immunized mice than in OVA-immunized mice. Depletion of CD8+ cells resulted in a modulation of the production of granulocyte-macrophage colony-stimulating factor, IL-12 and PGE(2) in peritoneal exudate fluid from OVA+DEP-immunized mice. On day 28, DEP injection markedly increased IL-4 production in the culture supernatants of spleen cells from CD4+ or CD8+-depleted mice. Depletion of CD8+ cells in OVA+DEP-immunized mice resulted in a decrease in IFN-gamma production compared with that in OVA-immunized mice. Adjuvant activity of DEP was observed in anti-OVA IgE, anti-OVA IgG1, anti-OVA IgG3, and total IgE production. Depletion of CD4+ T cells abrogated the adjuvant effect of DEP on anti-OVA IgE, and anti-OVA IgG1 production in plasma. However, depletion of CD8+ T cell inhibited the upregulated anti-OVA IgG3 production. These findings suggest that DEP injection may affect not only the function of CD4+ cells but also that of CD8+ T-cell subsets to modulate the synthesis of proinflammatory cytokine in PEC and type-1 and type-2 cytokine production in spleens.

Induction of the imbalance of helper T-cell functions in mice exposed to diesel exhaust.

Administration of diesel exhaust particles (DEP) increases antigen-specific IgE production and IgE-secreting cells, and induces Th2-type cytokine profiles in the airway in mice and humans. To determine the early effects of diesel exhaust (DE) inhalation on the cytokine production profile, BALB/c mice were exposed to 0 (controls) and 1.0 mg/m3 DE inhalation for 4 weeks. Intraperitoneal sensitization with ovalbumin (OVA) was conducted immediately before DE inhalation. Mice were treated with anti-CD4 or anti-CD8 mAb 1 day before and after the sensitization. On day 21, these mice were boosted with OVA and blood; bronchoalveolar lavage (BAL) fluid, and spleens were collected on day 28. In BAL fluid, both TNFalpha and IL-10 production in DE-exposed and control mice remained basically the same. IL-6 production in the anti-CD4 treatment group of DE-exposed mice, however, significantly increased compared with that of the controls. In vitro antigen-stimulated interleukin-4 (IL-4) and -10 (IL-10) production in spleen cells of exposed mice were not affected by low-dose DE inhalation. In vitro interferon (IFN)-gamma production in the anti-CD4 treated group of exposed mice decreased markedly. Although anti-OVA IgE production in the plasma of sham-treated mice exposed to DE was the same level as for controls, anti-CD4 mAb treatment in DE-exposed mice significantly reduced IgE production compared to controls. In anti-OVA IgG1 production, anti-CD4 or anti-CD8 mAb treatment in DE-exposed groups also significantly reduced. Anti-OVA IgG2a production was reduced by treatment with anti-CD4 mAb, but increased by anti-CD8 mAb treatment in DE-exposed mice. Low dose DE inhalation is thus shown to adversely affect the cytokine and antibody production in mice by altering CD4+ and CD8+ T-cell functions.

[The countermeasure to pollinosis by a web site of Internet].

For the countermeasure to pollinosis, we opened "the web site of pollinosis by allergic group of otorhinolaryngology, Jikei Medical School" and provided the information of pollinosis for patients in the web site of internet from the spring of 1997. In the web site we kept to be informed of the pollen forecast, daily dispersed pollens, and medical information being renewed frequently of prevention and therapy for pollinosis. For the principle of therapy, we adopted the guideline for allergic rhinitis which was produced by Japan Allergic Societies and recommended visitors to get standard therapy for pollinosis. Consequently, the web site was accessed up to 160,000 times by the summer of 1999 and we received 204 medical questions by e-mail and answered to these all mails. We then made a questionnaire study after 3 each pollen seasons and received over 200 answers which showed that our fresh information was useful to decrease symptoms of pollinosis. These results show that information by web site seems to be useful for the countermeasure to pollinosis and will be more important to support medical treatment in hospitals in future.

Colorimetry of dehydroalanine residues preserved as 'lost side chains' in thyroglobulin.

We developed a new assay method for dehydroalanine residues in thyroglobulin, which had been proposed to be the 'lost side chains' during thyroid hormonogenesis. Thyroglobulin preparations were labeled with 4-aminothiophenol at 30 degrees C for 10 days. Under the conditions, the reagent reacted only with dehydroalanine and cysteine residues. The 4-aminothiophenol bound to cysteine was eliminated by reductive cleavage. The 4-aminothiophenol-labeled dehydroalanine (4-aminophenylcysteine) residues were liberated by acidic hydrolysis, converted to a colored derivative by the Bratton-Marshall reaction and quantified colorimetrically. The number of dehydroalanine residues was the same as that of hormone residues in each thyroglobulin preparation. The results indicate that when one hormone residue is produced by the coupling of two iodotyrosine residues, the 'lost side chain' is preserved as one dehydroalanine residue in the thyroglobulin molecule.

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.

Properties of thyroglobulins from normal thyroid and thyroid tumor on a concanavalin A-sepharose column.

Affinity chromatography on a concanavalin A (con A)-Sepharose column is a potentially useful for the isolation of whole thyroglobulin (Tg) at least from normal thyroid tissue. In addition to being a simple procedure for the isolation of Tg, large amounts of Tg can be applied to the column and recovered in good yield with a buffer containing MeG. In gradient elution with buffer containing increasing amounts of MeG, a single but broad peak was obtained, without separation into subfractions. However, a hemagglutination-inhibition test showed that the Tg preparation eluted early from the column had less affinity for con A than the Tg preparation eluted later, suggesting a heterogeneous distribution of carbohydrate moieties among Tg preparations. When human Tg from thyroid tumor was applied to the column, tumor Tg partly passed through the column without being adsorbed. This unadsorbed Tg showed a very low affinity for lectins, con A and wheat germ agglutinin (WGA), as determined by a double diffusion reaction in agar gel. In contrast to this fraction, the Tg adsorbed on the con A-gel column showed a very strong affinity for WGA, differing from Tg of normal thyroid tissue. Therefore, tumor Tg preparation appears to have an abnormally modified carbohydrate structure, at least in part. The higher affinity for WGA (with a specificity for N-acetylglucosamine) seen in adsorbed Tg could be due to a larger amount of GlcNAc residues which bind irregularly in the carbohydrate moiety of tumor Tg.

Electron microscopy of monoclonal antibody-linked chains of thyroglobulin molecules.

Negative staining electron microscopy visualized immune complexes of hog thyroglobulin produced by 3 monoclonal antibodies, each towards a different conformation-dependent antigenic structure of the thyroglobulin. Equimolar mixtures of the thyroglobulin and any one of the 3 antibodies formed peculiar unbranched chains of 'ovoid' thyroglobulin molecules linked by the antibody molecules in an 'end to end' fashion. A mixture of the thyroglobulin and the 2 different antibodies produced branched chains. A set of 3 conformation-dependent antigenic structures was thus located near each end of the elongated thyroglobulin molecule. The results also indicated a topological similarity between the 2 subunits of thyroglobulin.

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase.

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.

Isoelectric points of erabutoxins and monoacyl derivatives of erabutoxin b. Estimation of the pK values of amino groups in erabutoxins by using isoelectric-focusing data.

The isoelectric points of erabutoxins a, b and c, neurotoxic proteins of a sea snake, Laticauda semifasciata, were determined by density-gradient isoelectric focusing. The same measurement was also made with monoacyl derivatives of erabutoxin b, in which each one of all amino groups had been either acetylated or propionylated. Erabutoxins a and b showed the same isoelectric point at pH 9.68. The values for ]1-N alpha-acetyl-arginine]-, [15-N6-acetyl-lysine]-, [27-N6-acetyl-lysine]-, [47-N6-propionyl-lysine]- and [51-N6-acetyl-lysine]-erabutoxin b were at pH 9.52, 9.31, 9.45, 9.22 and 9.09 respectively, being definitely different from each other and lower than the value for the unmodified molecule. The isoelectric point of erabutoxin c, which is [51-asparagine]-erabutoxin b, was the same as that of [51-N6-acetyl-lysine]erabutoxin b. Assuming that no change in pK occurs on monoacylation, the pK values of amino groups in erabutoxin b were calculated from the isoelectric-point data. It is indicated that the pK values of zeta-amino groups differ markedly from each other and that the value of alpha-amino group is anomalously high.

Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver.

Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89,000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154,000 and 149,000, respectively. They each consist of 4 polypeptide chains of identical size. Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.

Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. Macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) was purified to a homogeneous state from the water extracts of Koji cultures of Aspergillus niger var. macrosporus. The molecular weight of the enzyme was determined to e 136 000 by sedimentation equilibrium method. The denatured specimen of the enzyme exhibited a molecular weight of 60 000 in the sedimentation equilibrium in 6 M guanidinium chloride, suggesting that the native enzyme is composed of two identical subunits. However, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the enzyme showed an anomalous Ferguson plot, which may account for the inconsistent values of apparent molecular weights obtained by this method. The acid carboxypeptidase was found to be an acidic glycoprotein (pI, 4.1), composed of 955 amino acid, 140 mannose, 14 galactose and 30 glucosamine residues/molecule.

Electrophoretic analysis of thyroid iodoproteins in highly cross-linked polyacrylamide gels.

The electrophoretic behavior of thyroglobulin and larger thyroglobulin-like iodoproteins prepared from the hog thyroid was studied in polyacrylamide gels over a wide range of cross-linking degrees (C%). When examined at a constant total acrylamide monomer concentration (T%) of 5%, the mobility of 19S thyroglobulin decreased with increase in C below C = 5%, whereas above C = 5%, it increased markedly with increase in C, giving a minimum mobility at C = 5%. Similar biphasic mobility curves were obtained with 27S and 37S thyroid iodoproteins. Furthermore, in a region above C = 15%, at least two additional larger components which escaped detection in the gels with lower degrees of cross-linking appeared as separate bands. Ferguson plots constructed for thyroid iodoproteins at a higher constant C of 20% gave straight lines intersecting at a common point at T = 0%. From the calculated slopes of the Ferguson plots, it has been established that the thyroid contains a series of multimers of 19S thyroglobulin as constituent iodoproteins. Structural parameters of highly cross-linked gels were estimated under the assumption that the gels would be predominantly composed of a random meshwork of gel beads.

Properties of mitochondria and peroxisomal enoyl-CoA hydratases from rat liver.

Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.

Purification and properties of acyl-CoA oxidase from rat liver.

Acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was purified from rat liver. The final preparation was judged to be nearly homogeneous from the results of sedimentation analysis. Ultrogel AcA-34 column chromatography, and phosphocellulose column chromatography. The molecular weight of the enzyme was determined to be 139,000 by the sedimentation equilibrium method and Ultrogel AcA-34 column chromatography. The S020,W of the enzyme was 7.85S. Three protein components, A, B, and C, were found in the enzyme preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights, 75,500, 50,100, and 19,000) and high-pressure liquid chromatrography in the presence of 6 M guanidine . HCl (molecular weights, 71,900, 51,700, and 20,500). It was concluded that components B and C were formed from component A, probably by proteolytic cleavage, based on the result of amino acid analysis of each component. The pI of the enzyme was 9.2. The enzyme contained FAD as a prosthetic group, and exhibited absorption maxima at 278, 378, and 450 nm. The FAD content was 1.22 mol/mol of enzyme. When palmitoyl-CoA was added to the enzyme solution under anaerobic conditions, the bound FAD was reduced. The Km values were lower for C14 to C18 acyl-CoA's than for others tested, whereas Vmax values were roughly the same for C8 to C18 acyl-CoA's. The Km value for O2 was 5 microM. The optimal pH was 8. 3-Ketohexadecanoyl-CoA inhibited the enzyme (Ki=0.47 microM), forming a charge-transfer complex with the enzyme.