Soluble protein encoded by CD44 variant exons 8-10 in the serum of patients with colorectal cancer.
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Bacterial Proteins↗
Biomedical subjects
A Yamaguchi
Publications and source records attributed to A Yamaguchi.
Determination of a transmembrane segment using cysteine-scanning mutants of transposon Tn10-encoded metal-tetracycline/H+ antiporter.
The metal-tetracycline/H+ antiporter is a bacterial plasma membrane protein belonging to the 12-transmembrane transporter family. The boundaries of membrane-embedded domains can be clearly determined by means of a method involving cysteine-scanning mutants and their reactivity to N-ethylmaleimide. The results for 37 cysteine-scanning mutants around putative transmembrane helix 9 indicate that the residues from 266 to 275 and from 296 to 298 form water extruding loops, while residues from 276 to 295 and from 299 to 302 are embedded in the membrane. This method is generally useful for determination of the transmembrane regions of a polytopic membrane protein.
Amino Acid Sequence↗
Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5.
Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). Here we investigate the binding and signaling properties of GDF-5 through type I and type II receptors. GDF-5 induced alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 bound to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs revealed that GDF-5 bound to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells were transfected with type II receptor cDNAs, GDF-5 bound to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 bound to different sets of type I receptors, but the binding was most efficient to BMPR-IB compared with the other type I receptors. Moreover, a transcriptional activation signal was efficiently transduced by BMPR-IB in the presence of BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II.
Alkaline Phosphatase↗
Transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter of Staphylococcus aureus.
Three transmembrane aspartyl residues play essential roles in the transposon Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 7490-7498]. The tetK gene-encoding tetracycline resistance protein (Tet(K)) of Staphylococcus aureus mediates metal-tetracycline/H+ antiport similarly to Tet(B); however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu-30, Glu-152 and Glu-397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gln, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu-397, even the charge-conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu-30 and Glu-152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmembrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmembrane aspartyl residues in Tet(B).
Amino Acid Sequence↗
Mercaptide formed between the residue Cys70 and Hg2+ or Co2+ behaves as a functional positively charged side chain operative in the Arg70-->Cys mutant of the metal-tetracycline/H+ antiporter of Escherichia coli.
The bacterial tetracycline/H+ antiporter (TetA) mediates active efflux of a chelation complex of tetracycline with a divalent cation such as Mg2+, Co2+, or Mn2+ [Yamaguchi, A., Udagawa, T., & Sawai, T. (1990a) J. Biol. Chem. 265, 4809-4813]. The positive charge of Arg70 in the antiporter is important for the transport function [Yamaguchi, A., Someya, Y., & Sawai, T. (1992c) J. Biol. Chem. 267, 19155-19162]. Out of six site-directed mutants of Arg70, only the Lys70 mutant retained moderate transport activity, whereas the Ser70, Ala70, Trp70, Leu70, and Asp70 mutants had no or extremely low transport activity. In this study, we constructed the Cys70 mutant and found that the Cys70 mutant showed, unexpectedly, a significant activity comparable to that of the Lys70 mutant in the presence of Co2+ ions, whereas it showed very low activity as well as the Ala70 mutant in the presence of Mg2+ or Mn2+ ions. Hg2+, which is known to be a cysteine specific modifier but has no ability to form a complex with tetracycline, caused a dramatic increase in the Vmax value of Co(2+)-dependent tetracycline transport mediated by the Cys70 mutant without affecting the k(m) value, whereas activities of the wild-type and the Lys70 and Ala70 mutants were not affected by Hg2+, Hg2+ alone without Co2+ could not support the transport activity at all, because Hg2+ does not form a chelation complex with tetracycline. These observations suggest that a mercaptide formed between the SH group of Cys70 and Hg2+ or Co2+ works as a positively charged side chain like that of Arg or Lys. When the SH group of the Cys70 mutant was masked with modification by sulfhydryl reagents, the residual activity was no longer affected by Hg2+. Inversely, when the Cys70 mutant was preincubated with Hg2+, it was protected from the inactivation by sulfhydryl reagents. These observations also confirm the mercaptide formation between the Cys70 and a divalent cation as a functional side chain.
Antiporters↗
Asp-285 of the metal-tetracycline/H+ antiporter of Escherichia coli is essential for substrate binding.
The transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)) was preferentially photolabeled when [3H]tetracycline was irradiated in the presence of energized inverted membrane vesicles containing the TetA protein. The degree of labeling depended on the duration of irradiation and the energization of the membrane. Photolabeling was not observed in vesicles containing the Asp-285 --> Asn mutant TetA protein, indicating that Asp-285 participates in the substrate binding or the step(s) prior to substrate binding.
Antiporters↗
Bone morphogenetic protein-12 and -13 inhibit terminal differentiation of myoblasts, but do not induce their differentiation into osteoblasts.
Effects of bone morphogenetic protein (BMP)-12 and BMP-13, new members of the BMP family which belong to the transforming growth factor (TGF)-beta superfamily, on terminal differentiation of myoblasts were examined in C2C12 and L-6 myoblasts. When the myoblasts were cultured with BMP-12 or BMP-13, the expression of the myosin heavy chain and the formation of multinucleated myotubes mRNA in L-6 cells. The inhibitory effects of BMP-12 and BMP-13 on myogenic differentiation were similar to the effects of BMP-2, though their potencies were lower than BMP-2. Unlike BMP-2, neither BMP-12 nor BMP-13 induced alkaline phosphatase activity in C2C12 myoblasts. The differences in the biological activities of these new BMPs suggest that the intracellular signalling pathway used by BMP-12 and BMP-13 differs from that of BMP-2.
Alkaline Phosphatase↗
Stimulation by C-type natriuretic peptide of the differentiation of clonal osteoblastic MC3T3-E1 cells.
We examined the effects of C-type natriuretic peptide (CNP) and B-type natriuretic peptide receptor (NPR-B) system, which stimulates the intracellular production of cGMP, on osteoblastic differentiation using clonal murine calvarial MC3T3-E1 cells. CNP-like immunoreactivity was detected in the conditioned medium and in lysates of MC3T3-E1 cells. Exposure of cells to CNP caused an increase in the intracellular production of cGMP and the increase was dose-dependent, while ANP had no effect. These results imply that CNP regulates osteoblastic metabolism via NPR-B in an autocrine manner. Northern blot analysis revealed that treatment of MC3T3-E1 cells with CNP increased the steady-state levels of mRNAs for type-I collagen, cellular alkaline phosphatase (ALPase), and osteocalcin, which are well known as markers of osteoblastic differentiation. Our observations suggest the possibility that CNP functions as a local regulator of osteoblastic differentiation, acting via a cGMP-mediated pathway.
3T3 Cells↗
His257 is a uniquely important histidine residue for tetracycline/H+ antiport function but not mandatory for full activity of the transposon Tn10-encoded metal-tetracycline/H+ antiporter.
His257 is the only histidine residue located in the putative transmembrane region of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA) and contributes to the substrate/H+ coupling [Yamaguchi, A.. Adachi, K., Akasaka. T., Ono. N., & Sawai, T. (1991) J. Biol. Chem. 266, 6045-6051]. Tn10-TetA contains five histidine residues, including His257. When these histidine residues were replaced by alanine one by one, only the His257Ala mutant showed almost complete loss of the tetracycline transport activity, whereas the other four His --> Ala mutants, H42A, H158A, H329A, and H359A, retained transport activity comparable to that of the wild type. The mutant which contains only one histidine, His257, retained about 80% of the wild-type activity, whereas the histidine-less mutant, in which all five histidine residues were replaced by Ala, exhibited little activity. These results clearly indicated that His257 is a unique histidine residue in TetA responsible for the transport activity. The His257Tyr mutant, irrespective of the presence or absence of the other four histidine residues, retained about 30% of the wild-type tetracycline transport activity and showed corresponding tetracycline-coupled H+ translocation, indicating that an imidazole group is not necessary at position 257 for the substrate/H+ coupling. A histidine-specific reagent, diethyl pyrocarbonate (DEPC), equally inactivated the wild-type and one-histidine mutant TetA, whereas the H257Y mutant was hardly inactivated by DEPC irrespective of the presence or absence of the other four histidine residues, indicating that the inactivation by DEPC is due to the modification of His257.
Amino Acid Sequence↗
Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6.
The effects of bone morphogenetic protein-2 (BMP-2) on osteoblastic differentiation of bone marrow stromal cells were investigated using two bone marrow stromal cell lines, ST2 and MC3T3-G2/PA6 (PA6). BMP-2 stimulated ALP activity and induced parathyroid hormone (PTH)-dependent production of cAMP in both ST2 and PA6 cells, but these effects were more apparent in ST2 cells than in PA6 cells. BMP-2 induced the production of osteocalcin in ST2 cells, but not in PA6 cells. BMP-4 and BMP-6 stimulated ALP activity in ST2 cells, but the effect of BMP-6 was less marked than that of BMP-2 and BMP-4. BMP-4 induced PTH-dependent cAMP production of cAMP in ST2 cells, but BMP-6 did not. When ST2 cells were transplanted into the peritoneal cavities of athymic mice with BMP-2 in diffusion chambers, these cells generated mineralized bone in the chambers. These results indicate that BMPs induce the differentiation of bone marrow stromal cells into osteoblasts. However, the effects differ among the BMPs and among the types of cell exposed to these proteins.
Alkaline Phosphatase↗
Yearly variation of spontaneous somatic mutation frequency in the stamen hairs of Tradescantia clone KU 9 grown outdoors, which showed a significant increase after the Chernobyl accident.
Scoring of spontaneous somatic pink mutation frequency in the stamen hairs of Tradescantia clone KU 9, a heterozygote for flower color (blue/pink; the blue color being dominant), was carried out for 11 years on plants grown outdoors, during the period of May 11-31 (for 3 weeks) in every year from 1982 to 1992. Weekly and yearly variations of the spontaneous mutation frequency were observed, and such variations could mostly be correlated to the difference in temperature. That is, the mutation frequency was generally higher in the weeks and years when the temperature was relatively low, showing the strongest negative correlation with the average minimum temperature. The variations were also correlated to the diurnal temperature difference, the mutation frequency being higher with larger diurnal temperature difference in general. However, the mutation frequency observed in 1986 was exceptionally higher than that expected from the temperature for this year, and was very significantly higher than for other years. The scoring of mutation frequency was thus continued in 1986 for an additional 4 weeks (June 1-28), and it was confirmed that such higher mutation frequencies lasted for 6 weeks in total. The exceptionally high mutation frequency seemed to be related to the radioactive fallout which occurred in early to mid May of 1986, even in Japan, after the serious nuclear reactor accident at Chernobyl, and also to the biological concentrations of radioactive nuclides which subsequently occurred, although it was difficult to conclude this definitely. The mutation frequency in 1987 was second highest, and was also significantly higher than the lowest mutation frequency observed in 1990.
Climate↗
Prospective adjuvant therapy with mitomycin C and carmofur (HCFU) for colorectal cancer, 10-year follow-up: Tokai HCFU Study Group, the first study for colorectal cancer.
A joint study was performed by the Tokai HCFU study group, which included 41 institutions to study the usefulness of the concomitant therapy with Mitomycin C (MMC) and Carmofur (HCFU) as a postoperative adjuvant chemotherapy in patients with colorectal cancer who had curative resection. Patients were divided into two groups, Group MMC and Group MMC+HCFU, using the "envelope" method. Among the 172 patients who had the envelope opened, 149 evaluable cases were analyzed for evaluation of the drug. The cumulative 10-year survival rates of Group MMC+HCFU had a statistically significant increase in survival rate compared with Group MMC. In particular, the rate was statistically significant in patients with colorectal cancer who had lymph node invasion. There were no severe side effects due to the adjuvant chemotherapy with MMC+HCFU. Thus the adjuvant chemotherapy with MMC+HCFU is suggested to be a useful and safe postoperative adjuvant chemotherapy.
Adult↗
Oral adjuvant chemotherapy with carmofur (HCFU) for colorectal cancer: five-year follow-up. Tokai HCFU Study Group--third study on colorectal cancer.
BACKGROUND: A joint study was performed by the Tokai HCFU study group, which included seven institutions, to examine the value of oral administration of Carmofur (HCFU), a 5-fluorouracil (5-FU) derivative, for postoperative adjuvant chemotherapy in patients with colorectal cancer undergoing curative resection. METHODS: The patients were divided into two groups, a control group receiving no HCFU and a group administered HCFU for 1 year, using a centralized registration system by telephone. Among 173 patients entered into this study, 159 evaluable cases were analyzed for evaluation of the drug. RESULTS: The cumulative 5-year disease-free rate of patients who received HCFU was significantly increased compared with the control group. In particular, the rate was much higher in patients with colon cancer. No severe side effects arose from adjuvant chemotherapy with HCFU. CONCLUSION: Adjuvant chemotherapy with oral HCFU appears to provide a useful and safe postoperative treatment.
Administration, Oral↗
Cloning of rat type I receptor cDNA for bone morphogenetic protein-2 and bone morphogenetic protein-4, and the localization compared with that of the ligands.
A rat homologue cDNA of mouse (Koenig et al. [1994] Mol. Cell Biol. 14:5961-5974; Suzuki et al. [1994] Proc. Natl. Acad. Sci. USA 91: 10255-10259) and human (ten Dijke et al. [1994] J. Biol. Chem. 269:16985-16988) type I receptors for BMP-2 and BMP-4 was cloned. Tissue distribution of the receptor mRNA was studied by in situ hybridization using rats at embryonic days 9, 13, 15, and 18 as well as 1- and 5-day-old postnatal rats. In the rats at embryonic days 9, 13, and 15, the receptor mRNA was diffusely expressed over the embryonic bodies. At embryonic day 18, the receptor mRNA expression was high in the hair and whisker follicles, tooth bud, cartilage, bone, digestive organs, lung, kidney, heart, and meninges. The receptor mRNA was expressed over a much wider area than those of the ligands in many organs. In the lung and digestive organs, the receptor mRNA was diffusely expressed and most highly expressed in the bronchial epithelium and muscle layer, respectively, in both of which mRNA expression of the ligands was undetectable. The receptor mRNA was highly expressed in the meninges, although neither of the ligands was expressed in or near this region. These results suggest that this receptor participates in both mesoderm formation in early embryogenesis and differentiation of mesodermal cells during maturation of organs, and further suggest the presence of another factor(s) that binds the type I receptor.
Amino Acid Sequence↗
Elevated serum level of soluble HLA class I antigens in patients with systemic lupus erythematosus.
OBJECTIVE: To examine the clinical significance of serum soluble HLA class I antigens (sHLA class I) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). METHODS: Serum levels of sHLA class I were measured by enzyme-linked immunosorbent assay, using a monoclonal antibody against monomorphic determinant of HLA class I (W6/32) and an enzyme-labeled polyclonal antibody to human beta 2-microglobulin. RESULTS: The serum sHLA class I concentration was 1.85 +/- 1.15 micrograms/ml (mean +/- SD) in 27 patients with SLE (P < 0.0001 versus normal controls, P = 0.0001 versus RA), 0.61 +/- 0.34 micrograms/ml in 16 patients with RA (P = 0.02 versus normal controls), and 0.41 +/= 0.20 micrograms/ml in normal controls. The HLA class I levels were significantly correlated with the SLE Disease Activity Index (r = 0.62, P = 0.0004) and with a reduction of CH50 levels (r = -0.60, P = 0.0007). A longitudinal analysis of patients with SLE indicated that serum sHLA class I levels fluctuated in conjunction with other disease activity markers. CONCLUSION: Serum sHLA class I may be useful as a disease activity marker of SLE. The mechanism of secretion and the physiologic role of sHLA class I require further study.
Adolescent↗
Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation.
Chronic immune responses and inflammatory reactions in rheumatoid arthritis (RA) often cause severe destruction of cartilage and bone, but its mechanism is still a matter of controversy. We reported that interleukin-6 (IL-6) alone does not induce osteoclast formation, but soluble interleukin-6 receptors (sIL-6R) triggered the formation in the presence of IL-6 in cocultures of murine osteoblastic cells and bone marrow cells. In this study, we examined the involvement of sIL-6R and IL-6 in joint destruction in patients with RA. Although the frequency of patients having osteoclast-like multinucleated cells in synovium derived from the knee joint was not significantly different between RA (65%) and osteoarthritis (OA) patients (43%), the number of osteoclast-like cells found in the synovium was greater in the former than in the latter. Multinucleated cells obtained from RA synovium expressed the osteoclast-specific phenotype such as tartrate-resistant acid phosphatase, carbonic anhydrase II, vacuolar proton-ATPase and vitronectin receptors at similar levels to those from a human giant cell tumor of bone. The concentration of both IL-6 and sIL-6R was significantly higher in the synovial fluids from patients with RA than with OA. The concentration of IL-6 and sIL-6R correlated well with the roentgenologic grades of joint destruction. Dose-response curves for human IL-6 and human sIL-6R in inducing osteoclast-like cell formation in cocultures indicated that the RA synovial fluids contained sufficient IL-6 and sIL-6R to induce osteoclastogenesis. When synovial fluids from RA and OA patients were added to the cocultures, some of the RA synovial fluids containing high levels of IL-6 and sIL-6R stimulated osteoclast-like cell formation, which was strikingly inhibited by adding anti-IL-6R antibody simultaneously. These results suggest that IL-6 in the RA synovial fluids is at least in part responsible for joint destruction in the presence of sIL-6R through osteoclastogenesis.
Acid Phosphatase↗
Changes in biological activity of bone cells in ovariectomized rats revealed by in situ hybridization.
Twelve-week-old female rats were ovariectomized (OVX) and compared with sham-operated control rats at 3, 5, 7, 10, 14, 30, and 60 days postoperation with respect to the expression of type I collagen and osteopontin mRNAs, as well as bone structure and the number of osteoclasts. The trabecular number and separation were significantly decreased and increased, respectively, in the metaphyseal trabecular bones of OVX rat femurs. The number of osteoclasts was significantly increased in the same region of OVX rats at 3 and 5 days postoperation. Type I collagen mRNA was expressed in osteoblasts, and osteopontin mRNA was expressed in some osteoclasts, in mononuclear cells on the bone resorption surface, and in osteocytes near the resorption surface. In the metaphyseal trabecular bone, type I collagen and osteopontin mRNA expression levels in individual cells was initially increased in OVX rats from 7 to 10 days postoperation, and this was sustained for 60 days. The number of osteopontin mRNA-expressing osteocytes was also significantly increased at 10 days postoperation, which lasted until 60 days. In the epiphysis, an increase in type I collagen mRNA expression was initially observed in OVX rats at 14 days postoperation, which lasted until 60 days. The number of osteopontin mRNA-expressing osteocytes was virtually identical until 30 days postoperation in the epiphysis. These findings indicated that the biological activities of osteoblasts and osteocytes are stimulated in bones of the OVX rat and that the response for OVX differs between the metaphysis and epiphysis. Furthermore, the number of osteopontin mRNA-expressing osteocytes was increased only in bones that tended to be resorbed after OVX. This indicates that some osteocytes were stimulated to express osteopontin mRNA by estrogen deficiency and suggests that these osteopontin mRNA-expressing osteocytes may be involved in regulation of bone metabolism.
Analysis of Variance↗
Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages.
The effects of parathyroid hormone (1-34) (PTH (1-34) on osteoblast differentiation were investigated using primary osteoblast-like cells isolated from newborn mouse calvaria. The osteoblast-like cells cultured at low cell densities, in which the cells remained in a subconfluent state at the end of culture, were exposed for 7 days to PTH. This stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner. In contrast, PTH dose-dependently inhibited both ALP activity and osteocalcin production in cells inoculated at high cell densities, in which they had reached a confluent state before the end of culture. The changes of ALP activity by PTH were accompanied with the expression of ALP messenger RNA. PTH induced no changes of the hydroxyproline content in the cell layer when the cells were exposed to the hormone at a subconfluent state, but reduced the content at a postconfluent state. The stimulation of ALP activity by PTH at a preconfluent state was retained even after the removal of PTH from the culture media. The opposite effect of PTH, observed between the preconfluent and the postconfluent state, was reproduced by adding dibutyryl cyclic adenosine monophosphate (cAMP) or forskolin, but not by adding phorbol myristate acetate. In a colony-forming unit fibroblastic (CFU-F) assay, using bone marrow cells isolated from tibiae of 10-week-old mice, PTH induced no changes in the total number of CFU-Fs, but increased the proportion of ALP-positive colonies. These results indicate that PTH exerts opposite effects on the phenotypic expression of osteoblasts, depending on their differentiation stages of osteoblasts. PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts but inhibit it in more mature cells.
Alkaline Phosphatase↗