Biomedical subjects

T Suda

Publications and source records attributed to T Suda.

At least 271 records · Page 15Linked to original sources

PubMed1997

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.

Amino Acid Sequence↗

PubMed1997

Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis.

Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis was shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. Moreover, IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity.

3T3 Cells↗

PubMed1997

Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1997

The effect of ashing on cells: spectromicroscopy of physiological elements.

We analyzed the effects of cold oxygen plasma ashing of neurobiological specimens on different elements with synchrotron spectromicroscopy. Our results demonstrate that while carbon is almost completely removed, phosphorus, calcium, potassium, sulfur, and, to some extent, nitrogen are retained and their relative concentration is enhanced.

Animals↗

PubMed1997

Smad1 and smad5 act downstream of intracellular signalings of BMP-2 that inhibits myogenic differentiation and induces osteoblast differentiation in C2C12 myoblasts.

Bone morphogenetic protein-2 (BMP-2) inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda T. (1994) J. Cell Biol. 127, 1755-1766). In the present study, we examined the possible involvement of Smad proteins, vertebrate homologues of Drosophila Mothers against decapentaplegic, in the BMP effects on the differentiation of C2C12 myoblasts. C2C12 cells expressed Smad1, Smad2, Smad4, and Smad5 mRNAs, and expression levels were not altered by treatment with BMP-2 or TGF-beta1. When Smads were transiently transfected into C2C12 cells, both Smad1 and Smad5 induced alkaline phosphatase (ALP) activity and decreased the activity of myogenin promoter/chloramphenicol acetyltransferase (myogenin-CAT) without BMP-2. When C-terminal-truncated Smad1 and Smad5 were transfected into constitutively active BMP receptor type IB (BMPR-IB)-expressing C2C12 cells, BMP signals were blocked, resulting in an increase in myogenin-CAT activity. On the other hand, Smad1 and Smad5 decreased myogenin-CAT activity but did not induce ALP activity in MyoD-transfected NIH3T3 fibroblasts. These results suggest that both Smad1 and Smad5 are involved in the intracellular BMP signals which inhibit myogenic differentiation and induce osteoblast differentiation in C2C12 cells, and that the conversion of the two differentiation pathways is regulated independently at a transcriptional level.

Animals↗

PubMed1997

Constitutively active BMP type I receptors transduce BMP-2 signals without the ligand in C2C12 myoblasts.

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, inhibits the terminal differentiation of C2C12 myoblasts and changes their differentiation pathway into cells expressing osteoblast phenotypes such as alkaline phosphatase (ALP) activity and osteocalcin production (Katagiri et al., 1994, J. Cell Biol. 127, 1755-1766). Two type I receptors for BMP-2 (BMPR-IA and BMPR-IB) have been cloned, but the role of the respective receptors in signal transduction is not clear. In the present study, we examined the signal transduction of BMP-2 in C2C12 cells using constitutively activated mutant BMPR-IA and BMPR-IB. C2C12 cells expressed BMPR-IA and BMPR-II mRNAs, but not BMPR-IB mRNA at detectable levels in Northern blotting. When mutated BMPR-IA and BMPR-IB were transiently transfected into C2C12 cells, both BMPR-IA and BMPR-IB similarly induced ALP activity in the absence of BMP-2. We also established subclonal cell lines of C2C12 cells by stably transfecting mutated BMPR-IB. When the mutated BMPR-IB-transfected cells were cultured in medium with low serum (differentiation medium) without BMP-2, the cells differentiated into ALP-positive mononuclear cells and not into myosin heavy chain-positive myotubes. These mutated BMPR-IB-transfected cells expressed ALP activity and osteocalcin mRNA in a time-dependent manner, but neither muscle creatine kinase nor myogenin mRNAs. These results indicate that the mutated BMP-2 type I receptors can constitutively transduce BMP-2 signals in the absence of the ligand in C2C12 cells.

Alkaline Phosphatase↗

PubMed1997

Administration of IL-12 induces a CD3+ CD4- CD8- B220+ lymphoid population capable of eliciting cytolysis against Fas-positive tumor cells.

The present study investigates the effect of IL-12 administration on the generation of lymphoid cells that exhibit cytotoxicity against tumor cells expressing Fas Ag. Systemic injection of rIL-12 into BALB/c or (B6C3)F1 mice bearing syngeneic CSA1M or OV-HM tumor induced complete tumor regression. CSA1M tumor cells expressed Fas Ag, and exposure of these cells to IFN-gamma enhanced Fas expression. In contrast, Fas Ag was hardly detected on OV-HM cells even after IFN-gamma exposure. Only CSA1M cells were lysed by anti-Fas mAb or cells expressing Fas ligand (FasL), indicating that Fas on CSA1M cells is functional in mediating cell death. An increase in the frequency of lymphoid cells characterized as CD3+ CD4- CD8- B220+ was observed in spleens from both CSA1M and OV-HM tumor-bearing mice after IL-12 treatment. A splenic population enriched in cells with these unique phenotypes exhibited considerable degrees of cytotoxicity against Fas+ CSA1M, but not against Fas- OV-HM tumor cells. The lysis of CSA1M cells was almost completely blocked by addition of Fas-Fc, a fusion protein between the extracellular domain of mouse Fas and the Cgamma1 domain of human Ig. Regressing CSA1M and OV-HM tumor masses after IL-12 treatment exhibited a massive lymphoid cell infiltration and expressed significant levels of FasL mRNA, suggesting the infiltration of FasL-expressing cells to tumor sites. These results indicate that IL-12 induces the expansion of lymphoid cells that exhibit FasL-mediated cytolytic activity and accumulate into regressing tumor masses.

Animals↗

PubMed1997

A kinase domain-truncated type I receptor blocks bone morphogenetic protein-2-induced signal transduction in C2C12 myoblasts.

Members of the transforming growth factor (TGF)-beta superfamily bind the transmembrane serine/threonine kinase complex consisting of type I and type II receptors. Their intracellular signals are propagated via respective type I receptors. Bone morphogenetic protein (BMP)-2, a member of the TGF-beta superfamily, induces ectopic bone formation when implanted into muscular tissues. Two type I receptors (BMPR-IA and BMPR-IB) have been identified for BMP-2. We have reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts their differentiation pathway into that of osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994) J. Cell Biol. 127, 1755-1766). In the present study, we examined the involvement of functional BMP-2 type I receptors in signal transduction in C2C12 cells, which expressed mRNA for BMPR-IA, but not for BMPR-IB in Northern blotting. TGF-beta type I receptor (TbetaR-I) mRNA was also expressed in C2C12 cells. Subclonal cell lines of C2C12 that stably expressed a kinase domain-truncated BMPR-IA (DeltaBMPR-IA) differentiated into myosin heavy chain-expressing myotubes but not into alkaline phosphatase (ALP)-positive cells, even in the presence of BMP-2. In contrast, the differentiation of the DeltaBMPR-IA-transfected C2C12 cells into myotubes was suppressed by TGF-beta1, as in the parental C2C12 cells. BMP-2 did not efficiently suppress the mRNA expression of muscle-specific genes such as muscle creatine kinase, MyoD, and myogenin, nor did it induce the expression of ALP mRNA in the DeltaBMPR-IA-transfected C2C12 cells. In contrast, TGF-beta1 inhibited mRNA expression of the muscle-specific genes in those cells. When wild-type BMPR-IA was transiently transfected into the DeltaBMPR-IA-transfected C2C12 cells, a number of ALP-positive cells appeared in the presence of BMP-2. Transfection of wild-type BMPR-IB or TbetaR-I failed to increase the number of ALP-positive cells. These results suggest that the BMP-2-induced signals, which inhibit myogenic differentiation and induce osteoblast differentiation, are transduced via BMPR-IA in C2C12 myoblasts.

Affinity Labels↗

PubMed1997

Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: similarity to estrogen deficiency.

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss due to stimulated bone resorption by osteoclasts. During our investigations of the pathogenesis of bone loss in estrogen deficiency, we found that OVX selectively stimulates B-lymphopoiesis which results in marked accumulation of B220-positive pre-B cells in mouse bone marrow. To examine the possible correlation between stimulated B-lymphopoiesis and bone loss, 8-week-old female mice were treated with interleukin (IL) 7, which stimulates B-lymphopoiesis in bone marrow. We also examined bone mass in IL-7 receptor-knockout mice that exhibit marked suppression of B-lymphopoiesis in the bone marrow. The increased B-lymphopoiesis induced by IL-7 administration resulted in marked bone loss by stimulation of osteoclastic bone resorption in mice with intact ovarian function. The changes in both B-lymphopoiesis and bone mass in IL-7-treated female mice were similar to those in age-matched OVX mice. In contrast, the trabecular bone volume of the femur was greatly increased in both female and male IL-7 receptor-knockout mice when compared with the respective wild-type and heterozygous littermates. These results show that the perturbation of B-lymphopoiesis in the bone marrow is closely linked to the change in bone mass. We propose here that the increased B-lymphopoiesis due to estrogen deficiency is involved in the mechanism of stimulated bone resorption.

Animals↗

PubMed1997

Ca2+-ATPase inhibitors and Ca2+-ionophore induce osteoclast-like cell formation in the cocultures of mouse bone marrow cells and calvarial cells.

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+ levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+ levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+ levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.

Animals↗

PubMed1997

Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells.

It has been reported that PTH exerts bone-forming effects in vivo when administered intermittently. In the present study, the anabolic effects of PTH(1-34) on osteoblast differentiation were examined in vitro. Osteoblastic cells isolated from newborn rat calvaria were cyclically treated with PTH(1-34) for the first few hours of each 48-h incubation cycle. When osteoblastic cells were intermittently exposed to PTH only for the first hour of each 48-h incubation cycle and cultured for the remainder of the cycle without the hormone, osteoblast differentiation was inhibited by suppressing alkaline phosphatase activity, bone nodule formation, and mRNA expression of alkaline phosphatase, osteocalcin, and PTH/PTHrP receptor. Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and Ca2+/PKC demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, the intermittent exposure to PTH for the first 6 h of each 48-h cycle stimulated osteoblast differentiation. Both cAMP/ PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. Continuous exposure to PTH during the 48-h incubation cycle strongly inhibited osteoblast differentiation. Although both cAMP/PKA and Ca2+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. A neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin mRNA induced by the 6-h intermittent exposure. The inhibitory effect induced by the 1-h intermittent exposure was not affected by anti-IGF-I antibody. These results suggest that PTH has diverse effects on osteoblast differentiation depending on the exposure time in vitro mediated through different signal transduction systems. These in vitro findings explain at least in part the in vivo action of PTH that varies with the mode of administration.

Alkaline Phosphatase↗

PubMed1997

Expression and function of murine receptor tyrosine kinases, TIE and TEK, in hematopoietic stem cells.

Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin- c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin- c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

Animals↗

PubMed1997

1alpha,25-dihydroxyvitamin D3-24-hydroxylase (CYP24) hydroxylates the carbon at the end of the side chain (C-26) of the C-24-fluorinated analog of 1alpha,25-dihydroxyvitamin D3.

The sequential oxidation and cleavage of the side chain of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) initiated by the hydroxylation at C-24 is considered to be the major pathway of this hormone in the target cell metabolism. In this study, we examined renal metabolism of a synthetic analog of 1alpha,25(OH)2D3, 24, 24-difluoro-1alpha,25-dihydroxyvitamin D3 (F2-1alpha,25(OH)2D3), C-24 of which was designed to resist metabolic hydroxylation. When kidney homogenates prepared from 1alpha,25(OH)2D3-supplemented rats were incubated with F2-1alpha,25(OH)2D3, it was mainly converted to a more polar metabolite. We isolated and unequivocally identified the metabolite as 24,24-difluoro-1alpha,25,26-trihydroxyvitamin D3 (F2-1alpha,25,26(OH)3D3) by ultraviolet absorption spectrometry, frit-fast atom bombardment liquid chromatography/mass spectroscopy analysis, and direct comparison with chemically synthesized F2-1alpha,25,26(OH)3D3. Metabolism of F2-1alpha,25(OH)2D3 into F2-1alpha,25,26(OH)3D3 by kidney homogenates was induced by the prior administration of 1alpha,25(OH)2D3 into rats. The C-24 oxidation of 1alpha,25(OH)2D3 in renal homogenates was inhibited by F2-1alpha,25(OH)2D3 in a concentration-dependent manner. Moreover, F2-1alpha,25,26(OH)3D3 was formed in ROS17/2.8 cells transfected with a plasmid expressing 1alpha,25(OH)2D3-24-hydroxylase (CYP24) but not in the cells transfected with that expressing vitamin D3-25-hydroxylase (CYP27) or containing inverted CYP27 cDNA. These results show that CYP24 catalyzes not only hydroxylation at C-24 and C-23 of 1alpha,25(OH)2D3 but also at C-26 of F2-1alpha,25(OH)2D3, indicating that this enzyme has a broader substrate specificity of the hydroxylation sites than previously considered.

Animals↗

PubMed1997

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.

Animals↗

PubMed1997

Focal adhesion kinase upregulated by granulocyte-macrophage colony-stimulating factor but not by interleukin-3 in differentiating myeloid cells.

The involvement of focal adhesion kinase (FAK) in myeloid differentiation was investigated in primary murine bone marrow (BM) cells. In unstimulated BM, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid precursors. When the BM cells were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), FAK expression showed a remarkable difference depending on the cytokine. Although FAK was upregulated in the cells stimulated by GM-CSF (GM-treated cells), the kinase was barely detectable in the cells cultured with IL-3 (IL-3-treated cells). Morphology and flow cytometry analysis showed GM-CSF promoted the growth and differentiation of monocyte/macrophage lineage stronger than IL-3. In addition, motility of the cytokine-differentiated cells showed an overt distinction between the cultures, which was closely correlated with FAK expression. After 7 days of stimulation, GM-treated cells showed active migration and chemoattractant-induced morphologic polarization. In contrast, IL-3-treated cells showed minimal migration and polarization. These results suggest an important role of GM-CSF in the terminal differentiation of monocytes/macrophages, and possible involvement of FAK in functional maturity of this lineage.

Animals↗

PubMed1997

Structural characterization of lipid A obtained from Pantoea agglomerans lipopolysaccharide.

Lipopolysaccharide isolated from Pantoea agglomerans showed higher priming and triggering activities for macrophages in terms of tumor necrosis factor production than other lipopolysaccharides. To identify the difference in biological activities of lipopolysaccharide of Pantoea agglomerans from other lipopolysaccharides on the basis of structure, we determined the structure of the lipid A part, which is the biological center of lipopolysaccharides, by quantitative analysis, nuclear magnetic resonance spectroscopy and mass spectrometry. Lipopolysaccharide of Pantoea agglomerans is constructed with at least two kinds of lipid A of different levels of acylation. One is of the same type as that of Escherichia coli with hexa-acyl lipid A and the other is the Salmonella minnesota type with hepta-acyl lipid A.

Bacterial Proteins↗

PubMed1997

Selective expression of the receptor tyrosine kinase, HTK, on human erythroid progenitor cells.

HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34(low), and glycophorin A(-/low). Assays of progenitors showed that HTK+ c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK- c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

Antibodies, Monoclonal↗

PubMed1997

Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes.

Fas ligand (FasL) is a type II membrane protein. Binding of FasL to its receptor, Fas, induces apoptosis. Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form. Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris. The purified rFasL was found to be glycosylated and to exist as a trimer. The rFasL was effective in inducing apoptosis in a Fas-expressing T cell or a fibroblast cell line. The ID50 of rFasL for mouse Fas-expressing T cells was about 0.5 ng/ml. The killing process with rFasL was quick. That is, >80% Fas-expressing mouse cells were killed within 1 h by a saturation concentration of human rFasL. Intravenous administration of 500 microg of human rFasL had a lethal effect in mice. When the mice were pretreated with Propionibacterium acnes, the subsequent injection of 30 microg of human rFasL induced hepatic failure and killed the mice within 24 h. These results indicated that the soluble human FasL is active in inducing apoptosis in vitro and in vivo, and its deleterious effect may be strengthened in patients who are suffering from bacterial infection.

Animals↗