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Biomedical subjects

K Inaba

Publications and source records attributed to K Inaba.

At least 73 records · Page 4Linked to original sources
PubMed1997

Structural and functional roles of modules in hemoglobin. Substitution of module M4 in hemoglobin subunits.

The alpha- and beta-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric alphaalphabeta- and betabetaalpha-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric alphaalphabeta-subunit drastically collapsed, while the chimeric betabetaalpha-subunit conserved the native globin structure (Wakasugi, K., Ishimori, K., Imai, K., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric alphaalphabeta-subunit. Based on tryptophan fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp14 and Tyr125 would be induced in the chimeric alphaalphabeta-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure. On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the betabetaalpha-subunit. The different stabilities of these module-substituted globins imply that modules would not always be stable "structural" units, and interactions between modules are crucial to construct stable globin subunits.

Circular Dichroism↗
PubMed1997

Ursodeoxycholic acid enhances glucocorticoid-induced tyrosine aminotransferase-gene expression in cultured rat hepatocytes.

Ursodeoxycholic acid (UDCA) is an effective treatment for immune-mediated liver diseases, suggesting that UDCA is functionally similar to glucocorticoids (GCs). We investigated the effects of UDCA on the enzyme activity and the mRNA levels of tyrosine aminotransferase (TAT), a hepatocyte-specific marker of GC action, in primary cultured rat hepatocytes. Addition of UDCA resulted in a significant increase in TAT activity in the presence of dexamethasone (DEX), compared with DEX alone, and this increase was completely suppressed by sphingosine, a protein kinase C (PKC) inhibitor, or actinomycin D, a transcriptional inhibitor. UDCA could not induce TAT activity in the absence of DEX. UDCA increased the TAT mRNA levels in the presence of DEX. In conclusion, UDCA enhances the GC-induced TAT-gene expression in hepatocytes, and UDCA-activated PKC may play a role in this upregulation.

Animals↗
PubMed1997

Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

Animals↗
PubMed1997

High levels of a major histocompatibility complex II-self peptide complex on dendritic cells from the T cell areas of lymph nodes.

T lymphocytes recirculate continually through the T cell areas of peripheral lymph nodes. During each passage, the T cells survey the surface of large dendritic cells (DCs), also known as interdigitating cells. However, these DCs have been difficult to release from the lymph node. By emphasizing the use of calcium-free media, as shown by Vremec et al. (Vremec, D., M. Zorbas, R. Scollay, D.J. Saunders, C.F. Ardavin, L. Wu, and K. Shortman. 1992. J. Exp. Med. 176:47-58.), we have been able to release and enrich DCs from the T cell areas. The DCs express the CD11c leukocyte integrin, the DEC-205 multilectin receptor for antigen presentation, the intracellular granule antigens which are recognized by monoclonal antibodies M342, 2A1, and MIDC-8, very high levels of MHC I and MHC II, and abundant accessory molecules such as CD40, CD54, and CD86. When examined with the Y-Ae monoclonal which recognizes complexes formed between I-Ab and a peptide derived from I-Ealpha, the T cell area DCs expressed the highest levels. The enriched DCs also stimulated a T-T hybridoma specific for this MHC II-peptide complex, and the hybridoma underwent apoptosis. Therefore DCs within the T cell areas can be isolated. Because they present very high levels of self peptides, these DCs should be considered in the regulation of self reactivity in the periphery.

Animals↗
PubMed1997

Developmental regulation of MHC class II transport in mouse dendritic cells.

Dendritic cells (DCs) have the unique capacity to initiate primary and secondary immune responses. They acquire antigens in peripheral tissues and migrate to lymphoid organs where they present processed peptides to T cells. DCs must therefore exist in distinct functional states, an idea that is supported by observations that they downregulate endocytosis and upregulate surface molecules of the class II major histocompatibility complex (MHC) upon maturation. Here we investigate the features of DC maturation by reconstituting the terminal differentiation of mouse DCs in vitro and in situ. We find that early DCs, corresponding to those found in peripheral tissues, exhibit a phenotype in which most class II molecules are intracellular and localized to lysosomes. Upon maturation, these cells give rise to a new intermediate phenotype in which intracellular class II molecules are found in peripheral non-lysosomal vesicles, similar to the specialized CIIV population seen in B cells. The intermediate cells then differentiate into late DCs which express almost all of their class II molecules on the plasma membrane. These variations in class II compartmentalization are accompanied by dramatic alterations in the intracellular transport of the new class II molecules and in antigen presentation. We found that although early DCs could not present antigen immediately after uptake, efficient presentation of the previously internalized antigen occurred after maturation, 24-48 hours later. By regulating class II transport and compartmentalization, DCs are able to delay antigen display, a property crucial to their role in immune surveillance.

Amino Acid Sequence↗
PubMed1997

The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex.

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

Amino Acid Sequence↗
PubMed1997

Presence of B cell progenitors in the thymus.

We characterize B cell progenitors in the thymus. Although mature thymic B cells are surface Ig+, B220(high), but CD43-, B cell progenitors in the thymus were found to be slg-, B220(med), and CD43+. Most progenitors showed rearranged Ig D-J and V-D-J patterns when DNA from sorted B cell progenitors was amplified by PCR, blotted, and probed. When B cell progenitors were purified from the thymus and injected intrathymically into Ig heavy chain (Igh) allotype-disparate recipients, B cells bearing donor-type Igh6 were detected in the thymus but not in the periphery. Thymic B cells generated from these progenitors were CD5+ cells, although there was a broad range of expression of CD5 molecules. Furthermore, B cells purified from thymus that had been inoculated with B cell progenitors were able to differentiate into Ab-forming cells under the influence of CD40 ligand plus IL-10, and secreted IgM with donor Igh6 allotype. We thus clarified the existence of B cell progenitors in the thymus and also characterized the surface markers and their developmental functions.

Animals↗
PubMed1997

Pluripotent hemopoietic stem cells are c-kit<low.

Pluripotent hemopoietic stem cells (P-HSCs) were thought to be c-kit+, but recent reports indicate that they are c-kit(low). In the present report, we provide evidence using Ly5 congenic mice that P-HSCs are c-kit(<low). Lineage-negative (Lin-)/CD71- cells among bone marrow cells (BMCs) from C57BL/6 Ly5.1 mice were separated into major histocompatibility complex class I(high) (class I(high))/c-kit(low) and class I(high)/ c-kit(<low) populations. Each population (500 cells) was transplanted into lethally (9.0 Gy) irradiated C57BL/6 Ly5.2 congenic mice along with Ly5.2 (2 x 10(5)) compromised cells. Donor-derived Ly5.1+ cells were detected 6 months after transplantation in primary recipients reconstituted with either class I(high)/c-kit(low) or class I(high)/c-kit(<low) cells. BMCs (1 x 10(6)) from the primary recipients were further transplanted into secondary recipients (Ly5.2 mice) to assess their long term repopulating activity. Six months after bone marrow transplantation, Ly5.1+ cells in all lineages were detected only in secondary recipients that had been given BMCs from the primary recipients reconstituted with class I(high)/c-kit(<low) cells but not in cells that were class I(high)/c-kit(low). When the BMCs (1 x 10(6)) of these secondary recipients were further transplanted into tertiary recipients, all tertiary recipients that had been given BMCs from the secondary recipients originally reconstituted with Lin-/CD71-/class I(high)/c-kit(low) cells died within 10 days whereas all six tertiary recipients originally reconstituted with Lin-/CD71-/class I(high)/c-kit(<low) cells showed donor (Ly5.1+)-derived cells in their peripheral blood. In the single tertiary recipient that was killed, donor-derived T cells, B cells, macrophages, and granulocytes also were detected in several major hematolymphoid organs. The remaining five mice continue to survive more than 6 months after the tertiary bone marrow transplantation.

Animals↗
PubMed1997

Contrasting effects of TGF-beta 1 and TNF-alpha on the development of dendritic cells from progenitors in mouse bone marrow.

Dendritic cells (DC) are a distinct population of leukocytes and specialized antigen-presenting cells for T cell responses. Prior work has shown that GM-CSF can induce the development of large numbers of DC from proliferating progenitors in mouse bone marrow. We have monitored the effects of potentially enhancing and suppressive cytokines in these cultures. In this system, many immature DC develop from proliferating precursors during the first six days of culture, and between days 6-8 maturation of typical nonadherent and nonreplicating DC takes place. The maturation is accompanied by a large increase in the expression of major histocompatibilities complex class II (MHC II) and B7-2/CD86, and in mixed leukocyte reaction stimulating activity. Tumor necrosis factor-alpha (TNF-alpha), previously shown to be required for development of human DC, was found to enhance the maturation of mouse DC in the last two days of culture. Transforming growth factor-beta 1 (TGF-beta 1), on the other hand, almost totally blocked DC maturation, but it had to be given in the first six days of culture when the DC were actively proliferating. TGF-beta 1 did not block the production of immature, MHC II-positive but B7-2/CD86-negative DC. Maturation would take place between days 6-8 as long as the cultures were depleted of Fc-receptor-bearing cells, or if TNF-alpha were added. In both instances, maturation was not blocked even when TGF-beta 1 remained in the culture. We conclude that the development of DC, in response to GM-CSF, can be modified by other cytokines. TGF-beta 1 is suppressive but only indirectly via Fc-receptor-bearing suppressive cells, presumably suppressive macrophages, while TNF-alpha enhances the final maturation of DC.

Animals↗
PubMed1997

Degradation of vitellogenins by 170 kDa trypsin-like protease in the plasma of the tilapia, Oreochromis niloticus.

Proteolytic degradation of plasma vitellogenins during purification procedure has been noted in several teleost fishes. We have characterized here a trypsin-like serine protease in the plasma of the tilapia, Oreochromis niloticus, which degrades vitellogenins. The molecular mass of the protease was estimated as 230 kDa by gel filtration and as 170 kDa both by nondenaturing and by SDS-polyacrylamide gel electrophoresis. The protease efficiently hydrolyzed the synthetic peptide substrates for trypsin-like proteases but not the substrates for chymotrypsin-like proteases nor aminopeptidases. Hydrolysis of the peptide substrates was strongly inhibited by leupeptin, aprotinin and N-tosyl-L-lysine chloromethyl ketone and to certain extent by chymostatin, 3,4-dichloroisocoumarin, phenylmethanesulfonyl fluoride, and soybean trypsin inhibitor. Leupeptin and aprotinin also inhibited the degradation of a vitellogenin in the plasma. Although the physiological functions of the 170 kDa protease in vivo have not been elucidated, the results on exzymatic properties of this protease will be useful for the isolation and characterization of vitellogenin not only in tilapia but also in other organisms.

Amino Acid Sequence↗
PubMed1997

Dendritic cells as antigen-presenting cells in vivo.

Antigen-presenting cells are of central importance for the generation and regulation of T cell-mediated immune responses. The specialized features of dendritic cells as antigen-presenting cells in selecting rare clones of antigen-specific T cells and activating them in vivo are described.

Animals↗
PubMed1997

Dendritic cells in the T-cell areas of lymphoid organs.

Substantial numbers of dendritic cells (DCs) are found in the T-cell areas of peripheral lymphoid organs such as the spleen, lymph node and Peyer's patch. By electron microscopy these DCs (also called interdigitating cells) form a network through which T-cells continually recirculate. The cytological features of DCs in the T-cell areas, as well as a number of markers detected with monoclonal antibodies, are similar to mature DCs that develop from other sites such as skin and bone marrow. Some markers that are expressed in abundance are: MHC II and the associated invariant chain, accessory molecules such as CD40 and CD86, a multilectin receptor for antigen presentation called DEC-205, the integrin CD11c, several antigens within the endocytic system that are detected by monoclonal antibodies but are as yet uncharacterized at the molecular level, and, in the human system, molecules termed S100b, CD83 and p55. DCs in the periphery can pick up antigens and migrate to the T-cell areas to initiate immunity. However, there are new observations that DCs within the T-cell areas also express high levels of self-antigens and functional fas-ligand capable of inducing CD4+ T-cell death. We speculate that there are at least 2 sets of DCs in the T-cell areas, a migratory myeloid pathway that brings in antigens from the periphery and induces immunity, and a more resident lymphoid pathway that presents self-antigens and maintains tolerance.

Animals↗
PubMed1997

Na+(Li+)/H+ antiporter in Pseudomonas aeruginosa and effect of Li+ on cell growth.

Everted membrane vesicles were prepared from cells of Pseudomonas aeruginosa, and cation/H+ antiport was measured. We observed activities of Na+/H+ antiport, Li+/H+ antiport and K+/H+ antiport. Judging from the competition pattern, it seems that there are at least two types of antiporter, a Na+(Li+)/H+ antiporter and a K+/H+ antiporter. Na+ was a good substrate for the Na+(Li+)/H+ system, whereas Li+ was a poor substrate. Although the K(m) value for Na+ (or Li+) was similar to those in Escherichia coli Na+/H+ antiporters, the Vmax value for Na+ (or Li+) was much smaller in the P. aeruginosa antiporter than in the E. coli antiporters. Growth of P. aeruginosa was strongly inhibited by 0.4 M LiCl, but not by NaCl or KCl.

Amiloride↗
PubMed1997

Hepatic damage influences the decay of nitroxide radicals in mice--an in vivo ESR study.

To determine the role of the liver in the elimination of free radicals from the body, the clearance rate (K) of nitroxide radicals (Tempol) at the hepatic domain was compared with that at the pelvic domain of live mice, using L-band ESR spectroscopy. The reduction of Tempol in biopsy specimens (liver tissue and femoral muscle) and blood obtained from Tempol-treated mice was also monitored using X-band ESR spectroscopy. Results indicated that the reduction of nitroxide radicals was delayed in both the liver and peripheral tissues when the liver was damaged. The decrease in both blood supply and reductants in the damaged liver might be involved in delaying the reduction in the whole body, because the liver can reduce the radicals supplied via the blood from the peripheral tissues, and the reductants such as reduced, glutathione in the peripheral tissues are supplied from the liver.

Animals↗
PubMed1996

Evidence of a direct action of taurine and calcium on biological membranes. A combined study of 31P-nuclear magnetic resonance and electron spin resonance.

To determine the actions of taurine and calcium on biological membranes, the effects of these compounds on the mobility of phospholipids of resealed and sonicated ghosts of human erythrocytes were investigated, using 31P-NMR spectroscopy. In addition, the effects of taurine and calcium on lipid fluidity were investigated by ESR spectroscopy, using a spin-labeling method with 5-doxyl stearic acid. The mobility of the membranes decreased following treatment with 10 mM taurine, but coadministration of 2.5 mM calcium blocked this effect. The fluidity of the membranes was not changed following treatment with 10 mM taurine, but decreased following coadministration of 2.5 mM calcium. These actions of taurine and calcium on the dynamics of biological membranes might explain, in part, the observation that most of the pharmacological effects of taurine on mammalian organs occur in the presence of calcium ions.

Calcium↗
PubMed1996

An improved isolation method for murine migratory cutaneous dendritic cells.

Dendritic cells are highly specialized accessory cells for the initiation of primary immune responses. They occur as trace populations in non-lymphoid and lymphoid organs. Therefore, the isolation and enrichment of primary dendritic cells is difficult and time-consuming. This applies also to dendritic cells from skin, i.e. epidermal Langerhans cells and dermal dendritic cells. Recently introduced skin organ cultures serve as a convenient source for primary cutaneous dendritic cells. We report here a refinement of such cultures in which cutaneous dendritic cells emigrate spontaneously into the culture medium. Murine ear skin is cultured for a total of 3 days in three sequential 24 h steps. This simple modification doubles or triples the yields of dendritic cells that can be obtained and up to 30,000 dendritic cells can be recovered from one ear half. This represents 50-70% (range 30-80%) of all viable cells. The cells are mature dendritic cells that possess potent T cell stimulatory function. Compared to the classical methods of preparing epidermal Langerhans cells by trypsinization this technique is easier and quicker; it does not require enzymes such as trypsin, and it yields similar numbers of mature dendritic cells. It should prove useful for further studies of dendritic cells of the skin.

Animals↗
PubMed1996

Cloning and sequencing of the gene for Na+/H+ antiporter of Vibrio parahaemolyticus.

A gene encoding an Na+/H+ antiporter was cloned from vibrio parahaemolyticus into the plasmid pBR322 and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells to grow in the presence of 0.2 M NaCl (or 10 mM LiCl). These cells were originally unable to grow under such conditions because of the lack of major Na+(Li+)/H+ antiporters. We detected Na+/H+ antiporter activity due to the gene in membrane vesicles. The gene was sequenced and the deduced amino acid sequence was found to be 72% identical to the NhaB Na+/H+ antiporter of E. coli.

Amino Acid Sequence↗