An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of N-9F at 10-12-10-9M in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative, single positive and double positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF neutralized the increasing effect of NPF on immature thymocytes. All effects of NPF was inhibited by mAb QR6.6 to recognize a 100kDa surface molecule of N-9F. The amino-terminal 20 amino acid sequence of NPF was identified and identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A Hitrap-mouse recombinant His-tag-saposin A antibody column bound NPF, pulled down the NPF activity in TCGF, and the antibody recognized a 16kDa molecule in western-blotting of TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes. The physiological significance of NPF in the growth and differentiation of immature thymocytes was discussed in view of the characteristic distributions of NPF and the molecule recognized by its mAb QR6.6 in fetal thymi.

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.

Fetal liver (FL) hematopoiesis is thought to be important for expanding the cell number during ontogeny. In order to investigate the cellular interaction molecules among FL stromal and hematopoietic cells, we established a monoclonal antibody, Ndk-10, that reacts with FL stromal cells but not with dish non-adherent cells. When Ndk-10 was added to an FL stromal and hematopoietic cell-coculture, it inhibited the survival of c-kit+ cells. The inhibitory activity of Ndk-10 was also observed in the fetal liver organ culture. The Ndk-10 recognized a 150 kD molecule in the adherent cells of FL and kidney, and the N-terminal amino acid sequence was identical to that of mouse aminopeptidase N/CD13. The peptidase activity of CD13 was inhibited by Ndk-10, and addition of its specific inhibitor resulted in the same inhibitory activity as Ndk-10. We propose that aminopeptidase N/CD13 is a critical molecule that regulates the survival of c-kit+ cells in the FL microenvironment.

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of NPF at 10(-11) -10(-8) g/ml in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative thymocytes and CD8 single positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF decreased double negative thymocytes. The amino-terminal amino acid sequence of NPF was identified to be Ser-Leu-Pro-Cys-Asp-Ile-Cys-Lys-Thr-Val-Val-Thr-Glu-Ala-Cys-Asn-Leu-Leu-Lys-Asp- and was identical to that of rat saposin A. The apparent molecular weight of NPF, 16 000, was comparable to that of saposin A. A rabbit anti-mouse recombinant His-tag (mrH)-saposin A antibody recognized a 16 000 MW molecule in TCGF. A Hitrap-saposin A antibody column bound NPF and pulled down the NPF activity in TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes.

We have cloned a human counterpart to a guinea pig STE20-like kinase cDNA, designated human SLK (hSLK), from a human lung carcinomatous cell line A549 cDNA library. hSLK cDNA encodes a novel 1204 amino acid serine/threonine kinase for which the kinase domain located at the N-terminus shares considerable homology to that of the STE20-like kinase family. The C-terminal domain of hSLK includes both the coiled-coil structure and four Pro/Glu/Ser/Thr-rich (PEST) sequences, but not the GTPase-binding domain (GBD) that is characteristic of the p21-activated kinase (PAK) family, polyproline consensus binding sites, or the Leu-rich domain seen in the group I germinal center kinases (GCKs). Northern blot analysis indicated that hSLK was ubiquitously expressed. hSLK overexpressed in COS-7 cells phosphorylates itself as well as myelin basic protein used as a substrate. On the other hand, hSLK cannot activate any of the three well-characterized mitogen-activated protein kinase MAPK (ERK, JNK/SAPK and p38) pathways. Moreover, hSLK kinase activity is not upregulated by constitutive active forms of GTPases (RasV12, RacV12 and Cdc42V12). These structural and functional properties indicate that hSLK should be considered to be a new member of group II GCKs.

Green fluorescent protein (GFP) transgenic (GFP+) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP+ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP+ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3ε stimulation between GFP+ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP+ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP+ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP+ mice to the analysis of lymphocyte development. When bone marrow cells from GFP+ mice were transplanted, T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP+ and CD4+8+ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP+ mice is a powerful tool for visualizing lymphocyte development.

Background: Immunotherapy for cedar pollinosis makes use of multiple injections of allergens, but its effectiveness remains controversial. Recent studies indicate that immunization with certain protein antigens and immunostimulatory DNA sequence (ISS) oligodeoxynucleotides (ODNs) represent a potential approach to allergen-specific immunotherapy, Objective: We determined whether the coadministration of 2 major protein allergens, Cry j 1 and Cry j 2, of Japanese cedar pollen and ISS-ODN (5' TGACTCTGAACGTTCGAGATGA-3) improves the immune responses induced by protein allergens in BALB/c mice.Methods: Mice were primed intradermally with allergens or ISS-ODN in saline solution and boosted with allergens in alum, and other mice were primed with allergens in alum and boosted with allergens/ISS-ODN. Allergen-specific IgG2a and IgG1 antibody responses were measured by means of ELISA in sera after ODN injection, and allergen-specific IgE antibody production was measured by the passive cutaneous anaphylaxis reaction. IFN-gamma and IL-4 releases were also measured by ELISA in the supernatants of allergen-stimulated spleen cells,Results: The coadministration of allergens/ISS-ODN increased IgG2a titers and IFN-gamma release in both groups of mice, whereas it decreased IgG1 titers and IL-4 release in comparison with control mice injected with allergens/mutant ODN, The coadministration additionally inhibited IgE antibody production.Conclusion: The data demonstrate that the coadministration of cedar pollen allergens and ISS-ODNs before secondary T-H2 and IgE responses or during ongoing primary T-H2 and IgE responses brings about a T-H1-shifted immune response and inhibition of IgE antibody production, suggesting that this coadministration strategy may provide a novel type of immunotherapy for cedar pollinosis.

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D-3 (ST-630) is a newly developed agent to maintain the levels of calcium and phosphorus in blood. Herein, we investigated the effect of this compound on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice. ST-630 was more effective than 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] with respect to the induction of Cyp24 and calbindin-D-9k mRNAs in the kidney and in the small intestine. Moreover, the increase in mRNA levels of vitamin-D-responsive genes induced by ST-630 lasted longer than that induced by 1,25(OH)(2)D-3. These results indicate that ST-630 was more effective in inducing Cyp24 and calbindin-D-9k gene expression than 1,25(OH)(2)D-3 when both compounds were injected into vitamin-D-deficient mice.

cDNA fragments encoding mouse ferredoxin and ferredoxin reductase were simultaneously introduced into COS7 cells by using an expression vector, pUC-SR alpha plasmid. When using the mitochondrial fraction prepared from the transfected cells, cytochrome-c reductase activity was detected. This activity was highest when 7.5 micrograms of the ferredoxin expression plasmid (pSR alpha F) and 2.5 micrograms of the ferredoxin reductase expression plasmid (pSR alpha FR) were transfected into COS7 cells. In this system, NADPH could be replaced by NADH as a cofactor for the reduction of cytochrome-c although the cytochrome-c reductase was more dependent on NADPH than NADH at a low concentration. When CYP24 expression plasmid was transfected into COS7 cells along with both pSR alpha F and pSR alpha FR, the transfected cells revealed a 3-fold higher 25-hydroxyvitamin D3-24-hydroxylase activity than COS7 cells transfected with CYP24 expression plasmid.

Protein kinases play a key role in cell growth and differentiation. We have isolated the cDNA of a novel protein serine/threonine kinase (referred to as STE20-like kinase (SLK)) from a guinea pig liver cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction. The encoded polypeptide (1231 amino acids,Mr141,079) contains all conserved subdomains characteristic of the protein serine threonine kinase family. A hemagglutinin-tagged SLK expressed artificially in COS7 cells was hyperphosphorylated by anisomycin. By Northern blot analysis, SLK mRNA was detected in all organs examined: brain, lung heart, liver, kidney, spleen, testis, and eosinophils. Sequence comparisons of its catalytic domain related SLK to p21-activated kinase family of protein serine/threonine kinases. Its noncatalytic domain comprises several intriguing structural features, including the acidic region and the nuclear targeting sequence. This noncatalytic domain exhibited no extended similarity with other proteins. Thus, SLK is a protein serine/threonine kinase which contains an unknown regulatory domain(s).

Tuna muscle-derived angiotensin-converting enzyme inhibitory peptide, PTHIKWGD, inhibited the injury of bovine aorta endothelial cells (BAECs) induced by N-formyl-methionyl-leucyl-phenylalanine-activated-polymorpho-nuclear leukocytes and the adhesion of a leukemia cell line, THP-1 cells, to BAECs stimulated by lipopolysaccharides. It is possible that this peptide directly affects BAECs, protecting them from activated leukocytes.

The effect of a cytotoxic substance (bis(2-hydroxyethyl) trisulfide; BS-1), isolated from Bacillus stearothermophilus UK563, on messenger ribonucleic acid (mRNA) level in mouse macrophage-like J774A.1 cells was investigated by the method of differential display. The treatment of J774A.1 cells with BS-1 led us to detect the inducible gene. The sequence analysis revealed that the gene was identical to mouse mitochondrial cytochrome b. In fact, Northern blot analysis showed that cytochrome b mRNA in J774A.1 cells was increased by BS-1.

Two full-length cDNAs (F1-1 and F41-1) complementary to mouse kidney mRNA coding for cytochrome P-450 (P450) linked ferredoxin were isolated and completely sequenced. The coding sequences between F1-1 and F41-1 were identical. However, the 3' untranslated regions of F1-1 and F41-1 were 228 and 27 bases long due to the presence of alternative polyadenylation sites, respectively. The deduced amino acid sequence of mouse cytochrome P-450 linked ferredoxin showed 92.5, 75.0, 71.2 and 71.0% identities with those of rat, human, pig and bovine cytochrome P-450 linked ferredoxin, respectively. The cytochrome P-450 linked ferredoxin mRNA was detected in adrenal, kidney and ovary among the organs examined. The treatment of Y-1 cells with dibutyryladenosine 3',5'-cyclic monophosphate or forskolin induced the transcript of cytochrome P-450 linked ferredoxin mRNA.

Carboxamide-methylated light chain (G1L) from human serum IgG inhibited the secretion of tumor necrosis factor (TNF-alpha), one of the inflammatory cytokines, from adherent splenocytes and thioglycolate-induced peritoneal macrophages. The inhibition of TNF-alpha secretion by G1L was associated with disappearance of tyrosine phosphorylation on about 40 kDa protein when thioglycolate-induced peritoneal macrophages were stimulated with lipopolysaccharide (LPS). It is possible that this G1L anti-inflammatory activity occurs through the blockage of the phosphorylation of about 40 kDa protein.

A cDNA encoding ferredoxin reductase has been isolated from a mouse kidney cDNA library using human ferredoxin reductase cDNA as a probe. Mouse ferredoxin reductase coded for 494 amino acid residues. The mouse mature enzyme which comprises 460 amino acid residues shared 87.8-89.1% amino acid identities with the bovine and human enzyme. Northern blot analysis showed that ferredoxin reductase mRNA was expressed in the adrenal, testis and ovary and to a lesser extent in the liver and kidney. However, this mRNA in the adrenal cell line, Y-1 cell, was not induced by adenosine 3',5'-cyclic monophosphate (cAMP) in contrast with ferredoxin mRNA.

T lymphocyte unresponsiveness, induced in mice by a single gastric intubation of 0.2 ml cedar pollen extract (CPE, containing 4 micrograms protein/ml)/mouse daily for 3 to 28 consecutive days, was evaluated by the absence of a proliferative response of popliteal lymph node (PLN) T lymphocytes to CPE in vitro. T lymphocyte unresponsiveness increased with the period of gastric intubation of CPE and reached more than 80% of the control on day 28. The unresponsiveness to CPE was antigen-specific and T lymphocyte-mediated. In vitro CPE-specific T lymphocyte proliferation was significantly suppressed by intestinal intraepithelial lymphocytes (IELs) and hepatic mononuclear cells (MNCs), but not by spleen cells or PLN T lymphocytes from mice fed CPE for 28 days. The effector activity of IELs and MNCs was obviously antigen-specific. These results suggest that lymphocytes in the intestine and liver of mice fed CPE would be involved in the induction and maintenance of CPE-specific T lymphocyte unresponsiveness.

A cDNA encoding a 25-hydroxyvitamin D-3 24-hydroxylase, Cyp-24, has been isolated from mouse kidney cDNA library by hybridization screening. Mouse Cyp-24, coding for 514 amino acid residues, shared 82.1 and 94.7% amino acid identity with human and rat CYP24s, respectively. Among mouse organs examined, Cyp-24 mRNA could be detected in the kidney. When mice were treated with vitamin D-3, Cyp-24 mRNA was induced in the kidney.

We determined the copper (Cu) and metallothionein (MT) concentrations in the liver and kidney supernatants of Long-Evans rats with a cinnamon-like coat color (LEC rats), and also measured the Cu and MT levels in the serum of these rats. Seven-week-old rats had abnormally high levels of both substances in the liver. The levels in the liver supernatant were over 80- and 16-fold higher, respectively, in LEC rats than in normal 7-week-old Wistar rats. LEC rats suffering from acute hepatitis or hepatoma had a much higher level of hepatic MT, but the Cu level was higher only in the liver of those with hepatoma. The serum levels of Cu and MT in LEC rats with acute hepatitis were more than 10-fold higher than those in normal LEC rats. These levels were decreased in the rats with chronic hepatitis or hepatoma. In the liver of LEC rats with hepatoma, the area of hepatocellular carcinoma and of noncancerous liver showed over twice higher Cu and MT levels than the area of cholangiofibrosis. The Sephadex G-75 elution profile from the liver supernatant of a normal LEC rat showed that the peak of Cu closely corresponded to that of MT recognized with anti-MT antiserum. The levels of Cu and MT in the kidney supernatant of LEC rats with acute hepatitis were more than 25-fold higher than in that of normal LEC rats. However, there were no marked increases in the levels in the kidney supernatant of LEC rats with chronic hepatitis or hepatoma.

The effect of carboxamidemethylated Fc fragment (CM-Fc) from human immunoglobulin G (IgG) on type II collagen (C II)-induced arthritis (CIA) in DBA/1J mice was studied. CM-Fc suppressed the development of CIA while Fc fragment did not. CM-Fc inhibited the increase in serum anti-C II IgG. CM-Fc suppressed C II-induced delayed type hypersensitivity (DTH) in DBA/1J mice. Moreover, the C II-induced proliferation of splenocytes from mice immunized with C II was suppressed by the administration of CM-Fc. Following an analysis of the lymphocyte population, a decreased percentage of B220+ cells and ratio of L3T4+/Ly-2+ cells was observed in lymph nodes of CM-Fc administered mice as compared with those of control group. These results suggest that CM-Fc inhibits CIA by regulating lymphocyte function.