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Biomedical subjects

M Nonaka

Publications and source records attributed to M Nonaka.

At least 253 records · Page 14Linked to original sources

Ir gene expression on T cells: effect of a monoclonal antibody directed against I region-controlled determinants on T cells.

A monoclonal antibody directed at an I region-controlled epitope uniquely expressed on T cells (Iat) was studied for its in vivo effect on the antibody response under the control of an Ir gene. The antibody was produced by a hybridoma made from A.TH spleen cells immune to A.TL (anti-Ik), that was selected for its reactivity with T but not B cells and macrophages, and thus was designated as anti-IatK. The injection of this anti-Iatk into H-2k, H-2b and H-2k X bF1 mice resulted in the suppression of antibody response to poly-L-(His,Glu)-poly-D,L-Ala--poly-L-Lys [(H,G)-A--L] in H-2k and F1 mice but not that to poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys [(T,G)-A--L] both in H-2b and F1 mice. The adoptive cell transfer of the combinations of anti-Iatk- or normal mouse serum-treated T and B cells into irradiated hosts demonstrated that anti-Iatk primarily affected (H,G)-A--L-specific helper T cells but not B cells and macrophages, resulting in the specific elimination of the antibody response. Suppressor T cells were not induced by the treatment with anti-Iatk. The antibody specifically eliminated the (H,G)-A--L-specific but not (T,G)-A--L-specific helper T cells in F1 spleen cells that had been primed with both (H,G)-A--L and (T,G)-A--L. The results indicated that anti-Iatk affected the H-2k-associated Ir gene function born by T cells but not by antigen-presenting cells, which was expressed on F1 helper T cells with apparent exclusion of the other allele, and implied that the Iat antigen on helper T cells is one of the sites of expression of Ir genes.

Animals↗

Identification of the 5'-flanking regulatory region responsible for the difference in transcriptional control between mouse complement C4 and Slp genes.

To elucidate the molecular basis underlying the difference in the mode of gene expression between mouse complement C4 (constitutive) and sex-limited protein (Slp) (testosterone-regulated), we compared nucleotide sequences and transcriptional regulatory activities of the 5'-flanking regions of these two genes. Although the two sequences showed a high degree of overall homology (95%) up to 1.9 kilobases (kb) upstream from the transcription initiation site, the Slp sequence lacked a 31-nucleotide segment containing ACACCC repeats and a 60-nucleotide segment containing ACAC repeats, which are present, respectively, 1.6 kb and 200 base pairs (bp) upstream from the transcription initiation site of the C4 gene. When assayed in human hepatoma-derived HepG2 cells, the 1.8-kb 5'-flanking DNA fragment of the C4 gene demonstrated strong transcriptional activity, whereas the corresponding DNA fragment of the Slp gene showed only negligible activity. By progressive deletion experiments, it was shown that the difference in the constitutive transcriptional activity of the C4 and Slp genes was accounted for by the presence or absence of the positive regulatory domain located between 1700 bp and 400 bp upstream of the transcription initiation site.

Acetyltransferases↗

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects.

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.

Amino Acid Sequence↗

Identification and characterization of a variant of the third component of complement (C3) in rainbow trout (Salmo gairdneri) serum.

A rainbow trout serum protein that is cross-reactive with the third complement component of rainbow trout (C3-1) was purified to homogeneity and its structural and functional properties compared with those of C3-1. This protein (termed C3-related protein: C3-2) bears a close structural resemblance to C3-1, although C3-2 apparently shows no hemolytic activity. Like C3-1, C3-2 consists of two disulfide-linked polypeptide chains (128,000 alpha and 72,000 beta) and retains the unique thiol ester site in the alpha-chain. C3-2 shares some antigenicity with C3-1, but it also displays distinctive antigenic determinants of its own. Comparison of tryptic peptide maps revealed that about 20% of the peptides was specific to either C3-1 or C3-2, and about 80% of the peptides were common to both proteins. Amino acid compositions of the alpha- and beta-chains of C3-2 were similar to those of C3-1. Furthermore, amino acid sequence analysis of the NH2 termini of the alpha- and beta-chains of C3-2 revealed a high degree of homology with those of C3-1, 24 of 26 residues in the alpha-chain and all 20 in the beta-chain of C3-2 were identical with those found in C3-1. Both C3-1 and C3-2 were detected in all the adult rainbow trout tested and in first generation offspring randomly bred from them.

Alleles↗

Purification of a major serum protein of rainbow trout (Salmo gairdneri) homologous to the third component of mammalian complement.

The present paper describes the purification and characterization of a major serum protein of the rainbow trout (Salmo gairdneri) required for complement-related functions (target cell lysis and opsonization). This protein, termed C3-1, was identified as the third component (C3) of rainbow trout complement. It is homologous to component C3 of human and other mammalian complements based on its structural and functional characteristics. Rainbow trout C3-1 is a beta-globulin of Mr = 190,000 composed of two polypeptide chains (Mr = 128,000 alpha-chain and Mr = 74,000 beta-chain) linked by disulfide bonds. The chain structure of C3-1 is, therefore, very similar to those of the third and fifth components of mammalian complement. C3-1 retains, on its alpha-chain, a unique hidden thiol site that can be exposed upon attachment of methylamine in the same way as human C3 and C4. The amino acid composition and NH2-terminal sequence of C3-1 show significant homology to mammalian C3 and to cobra venom factor (cobra C3).

Amino Acid Sequence↗

Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence.

Amino Acid Sequence↗

Is there multiplicity in I-J subregion products?

Recent studies in molecular genetics have revealed the striking fact that the previously known I-J subregion (or I-J gene) does not exist at the exactly prescribed position in the I-region of the murine major histocompatibility complex (MHC). How, then, can we comprehend the established serological and functional significance of I-J genes and I-J products? To answer this question we reexamined systematically the specificities and functional activities of monoclonal anti-I-J antibodies. A series of monoclonal anti-I-Jk antibodies were newly established by fusion of B10.A(3R) spleen cells immune to B10.A(5R) lymphoid cells with P3X63-Ag8-653. Their abilities to eliminate known functions of T cell subsets and to react with I-J+ T cell clones of defined functions were examined in an in vitro secondary antibody response and by fluorescence-activated cell sorter analysis. The monoclonal antibodies (mAb) were divided into three groups: (1) those reactive with suppressor inducer T cells (Tsi), Tsi hybridomas, and the antigen-specific T cell factor (TsF) derived from them; (2) those specific for suppressor effector T cells (Tse) and Tse clones; and (3) those reactive with some but not all helper T cells. The determinants detected by these three different groups of antibodies are apparently present on separate, nonoverlapping cell populations and functionally distinct clones. The above results indicate the multiplicity of I-J products expressed on different T cell subsets, and sharply contradict the notion, derived from molecular genetics, that not even a single gene can be accommodated in the I-J subregion. To resolve this dilemma, we compared the above results with those obtained with another set of mAb that also detected I-region-controlled determinants on augmenting and helper T cells. Although the specificities of these mAb clearly mapped to within the I-region, none of them reacted with conventional class II Ia antigens of B cells and macrophages. The common properties shared by these anti-Ik and anti-I-Jk antibodies are that (1) they react only with T cells and T cell clones with I-region-controlled functions; (2) they can block some of the Ia-restricted cell interactions, including those of helper and suppressor T cells; and (3) they inhibit the syngeneic and/or allogeneic mixed lymphocyte reaction (MLR) by blocking the responder but not the stimulator cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Human IgE synthesis in vitro by pokeweed mitogen-stimulated human lymphoid cells: verification with a reconfirmed epsilon-specific radioimmunoassay.

This study was performed to address the controversy concerning human IgE biosynthesis in vitro induced by stimulation with pokeweed mitogen (PWM) or other agents. The controversy has focused on the specificity of reagents employed for quantitatively determining human IgE in culture supernatant fluids. Specifically, questions have been raised as to whether certain anti-human IgE antibody reagents possess anti-idiotypic reactivities, thereby resulting in reactions with Fab determinants of polyclonal immunoglobulins which would yield false-positive readings of IgE protein levels. We present a detailed analysis confirming that the goat anti-human IgE antibody designated GAHE(PS), which was initially isolated by affinity chromatography with the same IgE(PS) myeloma protein used for immunization, binds poorly, if at all, with IgG, IgA, or IgM immunoglobulins, even at excessive concentrations (100 micrograms/ml). Moreover, GAHE(PS) displayed no reactivity with Fab fragments of IgG or free L-chains prepared from pooled polyclonal IgG isolated from Cohn fraction II. A second GAHE reagent was prepared by purification by affinity chromatography on a second, completely unrelated IgE myeloma protein (DZA), which differed from IgE(PS) in light chain class, thereby resulting in a reagent, designated GAHE(DZA), which was completely devoid of any possible reactivity with L-chain or idiotypic determinants affiliated with IgE(PS). By utilizing both reagents, the studies presented here confirmed that PWM-stimulated human lymphoid cell cultures synthesize increased quantities of IgE, which can be detected in comparable amounts by both GAHE(DZA) and GAHE(PS) in supernatant fluids from such cultures. Because incorporation of the reversible protein synthesis inhibitor, cycloheximide, totally abolished the PWM-induced increases in IgE levels in such cultures, these results verify that such increases reflect de novo synthesis of human IgE as a result of PWM stimulation in vitro.

Animals↗

Purification of a lamprey complement protein homologous to the third component of the mammalian complement system.

A lamprey protein homologous to the third component of mammalian complement was isolated from lamprey plasma and was tentatively designated lamprey C3. Lamprey C3 is a major protein in lamprey serum with electrophoretic mobility of beta-globulin and with m.w. of 190,000. It consists of three polypeptide chains (84,000-alpha, 74,000-beta, and 32,000-gamma chains) linked by disulfide bonds. The protein retains a unique internal thiolester bond on the alpha-chain that is cleaved on methylamine treatment or on limited proteolysis with trypsin. Lamprey C3 by itself could not bind to zymosan or rabbit red cells, but it could covalently bind to these substances when activated by other factors present in lamprey serum. The binding of lamprey C3 to activating surfaces is mediated by covalent bonds and is accompanied by limited proteolysis of the alpha-chain. A fragment with an m.w. of 35,000 containing the internal thiolester site was isolated from methylamine-treated lamprey C3 bound to activated thiol-Sepharose by extensive tryptic digestion followed by dithiothreitol treatment. Lamprey C3 functions as the essential factor in phagocytosis of rabbit red cells by lamprey phagocytes. However, it is not involved in naturally occurring hemolytic activity in lamprey serum.

Animals↗

Monoclonal suppressor factor specific for lactate dehydrogenase B. I. Mechanism of interaction between the factor and its target cells.

Hybridomas secreting a monoclonal T suppressor-effector factor (TseF) were produced by fusion of a lactate dehydrogenase B (LDHB)-specific long-term T suppressor-effector (Tse) cell line with the BW5147 thymoma. A short exposure (4 h) to TseF completely suppresses the antigen-specific and A-restricted proliferation of LDHB-primed Lyt-1+2- [possibly helper (Th)] cells. The action of TseF on Th cells, as that of the Tse cells themselves, is antigen-specific and A-restricted. The interaction of TseF with Th cells involves two binding events, of which one occurs via antigen bridge, and the other represents the recognition of a factor-derived Ak-like moiety by the anti-Ak receptor of Th cells. The Ak-like moiety of the TseF carries the determinants that serve as restriction elements for antigen recognition by Th cells, and additional determinants demonstrable by T cell-specific monoclonal "anti-Ak" antibodies, however, it lacks serologically detectable determinants of the B cell-derived A alpha A beta class II Mhc molecules.

Animals↗

Electrophoretic study on the DNA-breaking actions of ascorbic acid and triose reductone in the presence of Cu2+.

The mode of DNA-breaking actions of ascorbic acid (AsA) and triose reductone (TR) in the presence of Cu2+ was studied by use of 0.8% agarose slab gel electrophoretic analysis. The DNA-breaking actions of the mixture of AsA or TR and Cu2+ were inhibited by N2 gas, hydroxyl radical scavengers and catalase, indicating that the oxidation of AsA or TR by Cu2+ and the hydrogen peroxide and hydroxyl radicals resulting from the oxidation are essential for the fragmentation of DNA by the mixture of AsA or TR and Cu2+. However, the DNA-breaking activity of these reductones varied with pH, while their oxidation rates were proportional to the increase in pH. The marked fragmentation of DNA occurred at pH 4, 7 or 8. The breakages of DNA by AsA or TR in the presence of Cu2+ were concluded not to be associated with the oxidation rates of reductones.

Animals↗

Function of Cu2+ on the DNA-breaking actions of ascorbic acid and triose reductone.

The role of Cu2+ on the DNA-breaking action of ascorbic acid (AsA) and triose reductone (TR) was studied with an agarose slab gel electrophoretic analysis. AsA and TR decomposed calf thymus DNA which had been pretreated with Cu2+, and their decomposing activity was proportional to the concentration of Cu2+ bound to the DNA. The DNA-Cu2+ complex had the ability to oxidize reductones. AsA and TR also decomposed the pretreated DNA with Cu2+ more markedly than that pretreated with Cu2+ and successively with EDTA. The DNA-breaking activity of AsA and TR showed no Cu2+-concentration dependency. The maximal fragmentation of DNA occurred at the concentration ratio of DNA and Cu2+ of 4:1. Excess concentration of Cu2+ decreased the activity of reductones. The present results indicate that the binding of Cu2+ to DNA molecules is also essential for the DNA-breaking action of AsA and TR in the presence of Cu2+ and that Cu2+ bound to DNA molecules has more effective promoting activity than free Cu2+.

Animals↗

lambda DNA-fragmenting actions of ascorbic acid and triose reductone in the presence of Cu2+.

Lambda DNA-fragmenting actions of ascorbic acid (AsA) and triose reductone (TR) in the presence of Cu2+ were studied. The mixture of AsA or TR and Cu2+ caused a marked fragmentation of lambda DNA (3.2 X 10(7) daltons) within the first 1 min of reaction. Further incubation resulted in accumulation of the most abundant species of fragmented lambda DNA having a molecular weight of 1.3 X 10(5) daltons. The mixture of AsA or TR and Cu2+ fragmented calf thymus DNA to produce the fragmented DNA of which 5'-OH terminal groups have a mixture of free OH groups and phosphodiester linkage. The mixture of AsA or TR and Cu2+ was also found to fragment lambda DNA to produce dCMP predominantly as 5'-OH terminal nucleotides.

Adenosine Triphosphate↗

Induction of T cell responses in nonresponder mice by abolishing suppression with monoclonal antibodies recognizing A region-controlled, T cell-specific determinants.

Mouse strains carrying the kappa allele at loci A beta, A alpha, E beta, and E alpha are nonresponders to lactate dehydrogenase B (LDHB) and to allotypic determinants of IgG2a myeloma proteins (for example, UPC10 used in this study). The nonresponsiveness to these antigens is caused by T suppressor (Ts) cells that prevent antigen-primed T helper (Th) cells from proliferating. We demonstrate here that monoclonal antibodies specific for an A region-controlled molecule selectively expressed on T cells (A-T) are capable of inducing anti-LDHB and anti-UPC10 responses of primed T cells from nonresponder strains. A monoclonal anti-J antibody that cross-reacts with the A-T molecule also induces responsiveness, whereas another J-specific antibody that lacks this cross-reactivity fails to do so. The mechanism of response induction is blocking of the interaction between the Ts cell or its factor (TsF) and the target of suppression, the antigen-specific Lyt-1+2- (Th) cell. The blocking occurs at the level of the Ts cell and the TsF. The data indicate that Ts cells and TsF carry a unique, A region-controlled molecule that is not only functionally analogous but also serologically similar to the J molecule.

Animals↗