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H Nakauchi

Publications and source records attributed to H Nakauchi.

At least 163 records · Page 9Linked to original sources
PubMed1988

Molecular cloning of the murine homologue of CD2. Homology of the molecule to its human counterpart T11.

In order to characterize the mouse homologue of the CD2 molecule, which has not yet been identified by immunologic means, cDNA clones putatively encoding mouse CD2 have been isolated by cross-hybridization with a cDNA probe for rat CD2. The predicted amino acid sequence of the putative mouse CD2 protein is consistent with that of a transmembrane glycoprotein, i.e., it consists of an N-terminal region of 186 amino acids bearing six potential N-glycosylation sites, a hydrophobic transmembrane segment of 25 residues, and a large cytoplasmic region of 116 amino acids rich in proline and basic residues. Comparison with the human and rat CD2 sequences clearly indicated the predicted mouse protein to be the mouse equivalent. Striking evolutionary conservations between mouse, rat, and human CD2 were found in their cytoplasmic region, suggesting a functional consequence of that segment for the physiologic role of CD2, such as signal transduction. RNA blot hybridization analysis demonstrated the expression of CD2 in T lymphocytes and in the NK cell lineage in mice. These data strongly suggest that the putative mouse CD2 molecule may perform some biologic functions in mouse T lymphocytes and NK cells as documented in humans.

Amino Acid Sequence↗
PubMed1988

Mouse immunoglobulin allotypes: multiple differences between the nucleic acid sequences of the IgEa and IgEb alleles.

To clarify the allotypic difference of the IgE antibody molecule, we determined the complete nucleotide sequence of the genes encoding the constant portion of mouse IgE of a (BALB/c) as well as b (B10.A) allotypes. A comparison of the sequences revealed that there were 12 single-base changes: 2 single-base changes in CH1 and CH2, 3 in CH3, and 7 in CH4. Five of them were silent changes, but seven resulted in amino acid substitutions. Although the silent changes are scattered through CH1 to CH4, the nonsilent substitutions were found only in CH3 (two substitutions) and CH4 (five). The allotypic determinant(s) that conventional antisera detect most likely reflects an amino acid difference(s) in CH3 and/or CH4.

Alleles↗
PubMed1988

Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique.

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.

Autoradiography↗
PubMed1987

Molecular evidence that SJL reticulum cell sarcomas are derived from pre-B cell. Clonal rearrangement of heavy chain but not of light chain immunoglobulin genes.

In order to investigate the clonal origin of SJL reticulum cell sarcoma (RCS), two-color cell membrane-staining and molecular biologic analyses were performed. Flow cytometric analysis revealed that the SJL RCS consists of about 50 to 60% Thy-1-positive and 20 to 30% B220-positive cells and that the majority of the Thy-1-positive cells are L3T4-positive, whereas a few are Lyt-2-positive. In spite of this pleomorphic nature of SJL RCS shown by cell membrane analysis, when the immunoglobulin heavy chain J segment (JH) was used to probe the DNA obtained from the tumor, we observed clonal rearrangements of the gene. Results of the cell-sorting experiment combined with Southern hybridization using the JH gene probe confirmed that those clonally expanding cells are Ia and B220 antigen-positive. Furthermore, all tumors derived from a given mouse showed the same rearrangement pattern. However, no clonal rearrangement was observed when the DNA was probed with the T cell receptor beta-chain gene or with immunoglobulin kappa- or lambda-light chain genes. From these data, we conclude that SJL RCS is a tumor of B cells and that the neoplastic event takes place at an immature B cell stage.

Animals↗
PubMed1987

Isolation and characterization of the gene for the murine T cell differentiation antigen and immunoglobulin-related molecule, Lyt-2.

We present here the sequence of the 5310 base pair Hind III-cleaved genomic DNA segment that includes the gene for the Lyt-2, a murine differentiation antigen expressed on most immature T lymphocytes as well as the cytotoxic suppressor T cell subset. We also present the complete intron/exon structure of Lyt-2. There are five exons: a fused leader and immunoglobulin variable region like exon, a hinge region exon, a transmembrane exon and two alternatively spliced intracytoplasmic exons (alternative splicing of these exons yields the 38 kDa alpha and 34 kDa alpha' Lyt-2 polypeptides). The promotor region contains a "TATA" box and sequences homologous to the putative immunoglobulin transcriptional control elements cd/pd. S1 protection analysis reveals that thymocytes, T cells from lymph nodes, and a Lyt-2 transfectant obtained by introduction of total genomic DNA have the same initiation site. In the 3' region, there is a polyadenylation signal sequence after a 700 bp long 3' untranslated region.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of Lyt-3, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to immunoglobulin and T-cell receptor variable and joining regions.

Lyt-3 is a membrane glycoprotein expressed on thymocytes and class I major histocompatibility complex-restricted cytotoxic T cells. Lyt-3 is expressed as a heterodimer with Lyt-2, and this complex is considered to be a homologue of the human Leu-2/T8 (CD8) that has been postulated to be a receptor for the class I major histocompatibility complex. We have determined the complete primary structure of Lyt-3 from the nucleotide sequence of its cDNA clones. Analysis of the predicted amino acid sequence indicates that the Lyt-3 polypeptide has a 21-amino acid leader peptide, and the mature protein consists of an NH2-terminal region of 146 amino acids, a transmembrane region of 27 residues, and a C-terminal region of 19 amino acids. The NH2-terminal 110 residues show clear homology to the T-cell receptor and immunoglobulin variable region sequences. In addition, Lyt-3 has 11 residues that have strong homology to the joining region sequences of the T-cell receptor and the immunoglobulin heavy and light chains. The presence of immunoglobulin variable- as well as joining-region-related sequences in Lyt-3 further supports the idea that these molecules may be recognition molecules belonging to the immunoglobulin super gene family.

Amino Acid Sequence↗
PubMed1986

Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation.

We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.

Animals↗
PubMed1985

Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions.

The sequence of Lyt-2 cDNA shows that it is a new member of the immunoglobulin super gene family. Analysis of the predicted amino acid sequence indicates that the Lyt-2 polypeptide is synthesized with a 27-amino acid leader, and that the mature protein has an immunoglobulin variable region (Ig V)-related sequence of approximately 100 amino acids, an extracellular spacer of 43, a transmembrane region of 38, and an intracytoplasmic region of 27 amino acids. Lyt-2 and its human analogue Leu-2 are 56% homologous; analysis indicates that the Ig V-related domains of the two molecules have evolved away from each other faster than the carboxyl-terminal half of the proteins.

Amino Acid Sequence↗
PubMed1985

T-cell subsets in patients with gastric cancer.

Peripheral blood T-cell subsets of 70 patients with untreated gastric cancer and 55 age- and sex-matched normal controls were studied by using monoclonal antibodies and flow cytometry. The number of T cells (Leu 4+ cells) was significantly decreased in stage IV patients. Statistically significant decreases in the number of Leu 3a+ cells and in the Leu 3a+/Leu 2a+ ratio were also found in stage IV patients. Stage I-III patients showed no marked imbalance in T-cell subsets. Quantitative changes in T-cell subsets in the advanced stage may be one of the factors responsible for the immunosuppression in advanced gastric cancer.

Adult↗
PubMed1985

Production of interleukin 3 and gamma-interferon by an antigen-specific mouse suppressor T cell clone.

An interleukin 2 (IL 2)-dependent, keyhole limpet hemocyanin (KLH)-specific, mouse suppressor T cell clone, 3D10, was found to produce interleukin 3 (IL 3) and gamma-interferon (IFN-gamma) in response to T cell mitogens Con A and PHA. Different from KLH-specific suppressor factor (TsF) that was spontaneously released into the medium when cultured in IL 2-containing conditioned medium, the production of IL 3 and IFN-gamma was induced by mitogenic stimuli. IL 3, IFN-gamma, and TsF were separable by gel filtration through a Sephadex G-100 column, being recovered in fractions of m.w. 25,000 to 30,000, 45,000 to 50,000 and 60,000 to 70,000, respectively. On the other hand, minimum size of IL 3 and IFN-gamma were shown to be about 25,000 and 20,000, respectively, by determining the lymphokine activities contained in the extracts from slices of SDS gels. These results indicate that IFN-gamma was present as a homodimer or hetero-complex with another carrier protein(s), whereas IL 3 was present as a monomeric form. A highly positive correlation (a correlation coefficient r = 0.96) between the titers of IL 3 and IFN-gamma produced by seven subclones derived from 3D10 was obtained, suggesting that IL 3 and IFN-gamma are induced by a process with a common mechanism. 3D10 also produced IL 3 and IFN-gamma when cultured with its specific antigen, KLH, in the presence of antigen-presenting cells. When Con A-stimulated 3D10 cells were labeled with L-[35S]methionine, we found that at least three proteins, with m.w. of 35,000, 25,000, and 20,000, were specifically released into medium by the stimulation. The latter two may be IL 3 and IFN-gamma described above, respectively, because of the similarities in m.w.

Animals↗
PubMed1985

The role of peripheral blood T and B lymphocytes in mitogen responses during human pregnancy.

Studies on the role of T and B lymphocytes of pregnant and post-partum women were performed in a pokeweed mitogen (PWM) stimulated culture system. The responses were assessed by immunofluorescent staining for intracytoplasmic immunoglobulins and solid-phase radioimmunoassay for Ig secretion into culture supernatants. As compared with non-pregnant control subjects, a slight decrease of helper T cell function and accelerated suppressor activity of T cells in pregnant and post-partum women in B cell differentiation promoted by PWM were demonstrated. Conversely, B lymphocytes in pregnant and post-partum women were hyperreactive in the presence of T lymphocytes from non-pregnant control subjects in the mitogen stimulated culture system.

Antibodies, Monoclonal↗
PubMed1984

Establishment and functional analysis of a cloned, antigen-specific suppressor effector T cell line.

A long-term cultured suppressor T cell line (3D10) was established from the spleen cells of KLH hyperimmunized C3H mice. The cells of 3D10 suppressed the secondary antibody response against dinitrophenylated KLH both in vitro and in vivo. The 3D10 cells were capable of directly suppressing the antibody response mounted by B cells and Lyt-1+2-helper T cells without participation of Lyt-1+2+ cells, which indicates that 3D10 is an effector rather than an inducer-type suppressor T cell. The cell line was unable to suppress the response against DNP-FGG even in the presence of free KLH. By analysis with a fluorescence-activated cell sorter, 3D10 was found to bear determinants detectable by conventional anti-Ia as well as rabbit anti-VH. The Ia locus was mapped in the I-J subregion and was detected by either conventional or monoclonal anti-I-J antibodies. The I-J determinants detected by monoclonal antibodies were different from those expressed on inducer-type suppressor T cells. The cell line also bears an antigenic determinant detected by a monoclonal SJL anti-SJA/9 antibody (HA16) that recognizes an allotypic determinant on T cells linked to the immunoglobulin heavy chain locus. The results indicate that the cell line 3D10 represents a suppressor effector T cell that acts directly on carrier-specific helper T cells.

Animals↗
PubMed1984

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line.

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.

Animals↗
PubMed1983

A quantitative analysis of cell surface glycosphingolipid with a fluorescence activated cell sorter.

The fluorescence activated cell sorter (FACS) was used with an indirect membrane immunofluorescence technique to detect antibody against the Forssman antigen, a glycosphingolipid. Sheep erythrocytes, which contain Forssman antigen as a major membrane glycosphingolipid, were used as the target antigen. Detection of the anti-Forssman antibody on the sheep erythrocytes was done with specific fluorescein-conjugated second antibody and analyzed on a FACS. Compared to other available methods, analysis with the FACS was simple, sensitive, reproducible and quantitative. More than 250 pg of antibody could be detected. In addition, as little as 1 ng of Forssman antigen could be estimated by a binding inhibition experiment.

Animals↗