Quantitative analysis of serum IL-6 and its correlation with increased levels of serum IL-2R in HIV-induced diseases

We have devised a luminescence sandwich ELISA for the quantification of IL-6 in both sera and cell culture supernatants, which had a detection limit of 100 fg/ml of test sample. By using the luminescence sandwich-ELISA, low but measurable levels of IL-6 (9.5 pg/ml on average) were found in the sera from normal individuals. The serum levels of IL-6 were elevated in HIV-seropositive asymptomatic carriers (55.5 pg/ml on average), and the IL-6 levels were correlated with the degree of HIV-induced disease progression (AIDS-related complex 106.8 pg/ml on average and (AIDS 283 pg/ml). IL-6 immunoreactivity in the sera of AIDS patients eluted at a 25,000 m.w. major peak, which was biologically active and heat-stable, and a 500,000 m.w. minor peak in size-exclusion HPLC. Interestingly, a significant correlation was observed between the serum IL-6 levels and soluble IL-2R levels. In vitro, HIV infection of PHA-activated PBMC led to enhanced release of IL-6 into the culture supernatants. Moreover, soluble IL-2R release was markedly increased by adding exogenous IL-6, whereas it was decreased by adding the neutralizing anti-IL-6 mAb to the cultures. These results demonstrate that increased IL-6 levels are significantly associated with sIL-2R levels, and suggest a cause of the increased levels of this receptor in patients with HIV infection. Furthermore, both serum IL-6 and serum IL-2R levels in HIV infection reflect the stage of the HIV-induced disease.     

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

Differentiation of germ cells in seminiferous tubules transplanted to testes of germ cell-deficient mice of W/Wv and Sl/Sld genotypes

(WB X C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice and (WC X C57BL/6)F1-Sl/Sld (hereafter, WCB6F1-Sl/Sld) mice are sterile due to the deficient spermatogenesis in the testes. The cause of deficient spermatogenesis in WBB6F1-W/Wv mice is considered to be a defect in germ cells themselves, whereas that in WCB6F1-Sl/Sld mice is considered to be a defect in tissue environment necessary for differentiation of germ cells. Seminiferous tubules isolated from cryptorchid testes of C57BL/6- +/+ mice were transplanted into the testes of WBB6F1-W/Wv and WCB6F1-Sl/Sld mice to clarify that the extratubular environment of these mice was intact or not. Type A spermatogonia in the transplanted tubules normally differentiated into spermatids, suggesting that the extratubular environment is intact in both WBB6F1-W/Wv and WCB6F1-Sl/Sld mice.     

An ultrastructural study on gastric endocrine cells in the stomach gland patch of the koala Phascolarctos cinereus

The endocrine cells in the stomach gland patch of the koala (Phascolarctos cinereus) were studied ultrastructurally. They were classified into 3 types based on the ultrastructural profiles of their endocrine granules and tentatively categorized as type I, II, and III endocrine cells. Type I cells contained round granules that were for the most part larger than those observed in the other 2 cell types. The granules ranged from moderate to relatively high in electron density. Type II cells were angular in shape and characterized by the presence of granules that were polymorphous in profile. Contents of the endocrine granules in type II cells also showed a range of high to moderate electron density. Type III cells were oval or pyramidal in shape. They contained highly polymorphous granules that were round, oval, dumbbell-like or comma in shape and characterized by the presence of a clear space or halo separating the high to low electron-dense core from the limiting membrane of granules. Type III cells were observed most often whereas type I and II cells were a less frequent observation.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.