[Postoperative right ventricular function after repair of tetralogy of Fallot with respect to the method of right ventricular outflow tract reconstruction].

We evaluated postoperative right ventricular function in the sixty-four consecutive patients with tetralogy of Fallot underwent total correction. The patients were divided to three groups according to the method of right ventricular outflow tract reconstruction: transannular patching (TA group; n = 31); right ventricular outflow patching with preservation of pulmonary valve ring (RV group; n = 12) and transatrial-transpulmonary approach without right ventriculotomy (no-RV group; n = 21). The early results of postoperative cardiac catheterization and echocardiography were compared among the three groups. Degree of pulmonary regurgitation was significantly low in the RV group and no-RV group compared with TA group (p < 0.005). Right ventricular ejection fraction was the highest in the no-RV group (p < 0.002). The repair without right ventriculotomy for tetralogy of Fallot can provide the best results with respect to postoperative right ventricular function.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) is the major etiological agent of hepatocellular carcinoma, and HCV RNA-dependent RNA polymerase (RdRp) is one of the main potential targets for anti-HCV agents. HCV RdRp performs run-off copying replication in an RNA-selective manner for the template-primer duplex and the substrate, but the structural basis of this reaction mechanism has still to be elucidated. RESULTS: The three-dimensional structure of HCV RdRp was determined by X-ray crystallography at 2.5 A resolution. The compact HCV RdRp structure resembles a right hand, but has more complicated fingers and thumb domains than those of the other known polymerases, with a novel alpha-helix-rich subdomain (alpha fingers) as an addition to the fingers domain. The other fingers subdomain (beta fingers) is folded in the same manner as the fingers domain of human immunodeficiency virus (HIV) reverse transcriptase (RT), another RNA-dependent polymerase. The ribose-recognition site of HCV RdRp is constructed of hydrophilic residues, unlike those of DNA polymerases. The C-terminal region of HCV RdRp occupies the putative RNA-duplex-binding cleft. CONCLUSIONS: The structural basis of the RNA selectivity of HCV RdRp was elucidated from its crystal structure. The putative substrate-binding site with a shallow hydrophilic cavity should have ribonucleoside triphosphate (rNTP) as the preferred substrate. We propose that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps. The C-terminal region might exert a regulatory function on the initiation and activity of HCV RdRp.

CD34high+ CD38(low/-) cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation.

CD34+ cells isolated from human umbilical cord blood (HUCB) are thought to have potential in clinical applications such as transplantation and gene therapy. Recently, we developed a xenogenic coculture system involving HUCB-CD34+ cells and murine bone marrow stromal cells, HESS-5 cells, in combination with human interleukin-3 and stem cell factor. Under these xenogenic coculture conditions, the numbers of CD34high+ cells and primitive progenitor cells, such as CD34high+ CD38(low/-) cells and high proliferative potential colony-forming cells (HPP-CFCs), increased dramatically by a factor of 102.1, 66.5 and 104.9, respectively. In the present study, we used a secondary culture of B progenitor cells and long-term culture (LTC)-initiating cells to characterize and compare the progenitor capability of re-isolated CD34high+ CD38(low/-) cells, which have been identified as one of the most primitive progenitor cells, with that of freshly isolated CD34high+ CD38(low/-) cells. Compared with freshly isolated CD34high+ CD38(low/-) cells, the re-isolated CD34high+ CD38(low/-) cells were equally as capable of proliferating and differentiating into myeloid and B progenitor cells. No significant differences were observed in the frequency of LTC-initiating cells in the re-isolated CD34high+ CD38(low/-) cells compared with that in freshly isolated CD34high+ CD38(low/-) cells. Furthermore, the re-isolated CD34high+ CD38(low/-) cells were capable of long-term reconstitution and multiple differentiation in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). The results demonstrate that this xenogenic coculture system can be used for successful in vitro expansion of HUCB-progenitor cells that possess the capability for both long-term hematopoiesis as well as multipotent differentiation into myeloid and lymphoid cells both in vivo and in vitro.

A murine stromal cell line promotes the expansion of CD34high+-primitive progenitor cells isolated from human umbilical cord blood in combination with human cytokines.

The in vitro expansion of CD34+ cells is important for clinical applications such as transplantation and gene therapy with CD34+ cells isolated from human umbilical cord blood. In the present study, we developed a xenogenic coculture system involving HUCB-CD34+ cells and a murine stromal cell line, HESS-5 cells, in the presence of recombinant human (rh) cytokines. We examined the effects of combinations of cytokines, such as rh-IL-3, rh-SCF, rh-granulocyte colony-stimulating factor (G-CSF), rh-granulocyte-macrophage-CSF and h-erythropoietin (EPO), on the expansion of CD34high+ cells and colony-forming progenitor cells (CFCs). The proliferation of CD34high+ cells and CFCs was dramatically promoted on coculture with HESS-5 cells, and the expansion ratio of the CD34high+ cells showed good correlation with that of high-proliferative potential colony-forming cells (HPP-CFCs). The most potent combination of cytokines in this xenogenic coculture system for the expansion of CD34high+ cells and HPP-CFCs was rh-IL-3 and rh-SCF. The proliferation of CD34high+ cells was supported in the presence of HESS-5 cells with direct cell contact, but not observed in the indirect coculture involving a microporous membrane. Furthermore, we developed a unique coculture method, designated as the bilayer coculture method, involving CD34+ cells and HESS-5 cells using a microporous membrane. This expansion system will be applicable to the expansion of the primitive progenitor cells of HUCB-CD34+ cells and is worthy of consideration for the clinical application of HUCB-CD34+ cells.

Cloning of the murine interferon-inducible protein 10 (IP-10) receptor and its specific expression in lymphoid organs.

To isolate the interferon-inducible protein 10 (IP-10) receptor gene, we searched for cells that respond to IP-10. Among several human and murine T cell lines, only CTLL2 cells ( a murine cytotoxic T cell line) responded to IP-10 with transient elevation of intracellular Ca2+. The murine IP-10 receptor gene has been cloned from cDNA derived from CTLL2 cells using the reverse transcriptase-polymerase chain reaction protocol with two degenerate primers corresponding to conserved regions of chemokine receptors. The cDNA encoding the murine IP-10 receptor has an open reading frame of 1101 bp corresponding to a protein of 367 amino acids that exhibits 86 % identity with the human IP-10 receptor. It mediates Ca2+ mobilization in response to IP-10, but does not recognize other rodent chemokines, including GRO, RANTES, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). Northern blot analysis revealed that murine IP-10 and its receptor mRNA were constitutively expressed in the spleen and thymus from normal mouse, while IP-10 and its receptor mRNA were derived from stromal cells and lymphocytes in both tissues, respectively. In vivo treatment with concanavalin A (Con A) for 12 hrs revealed that splenocytes significantly induce IP-10 receptor mRNA expression and show a good chemotactic response to IP-10. Therefore, it is supposed that IP-10 and its receptor are important for lymphocyte trafficking to lymphoid organs and that the IP-10 receptor on lymphocytes is rapidly inducible on inflammation or in immunological events.

Integrin beta2 (CD18)-mediated cell proliferation of HEL cells on a hematopoietic-supportive bone marrow stromal cell line, HESS-5 cells.

Cellular interactions between hematopoietic cells and stromal cells play important roles in the proliferation and differentiation of hematopoietic cells. The proliferation of a human erythroleukemia cell line, HEL cells, which can differentiate into macrophage- and megakaryocyte-like cells, and erythroid precursors was dramatically induced on coculture with a hematopoietic-supportive stromal cell line, HESS-5 cells, which can support long-term hematopoiesis in vitro without fetal bovine serum. HEL cells proliferated when they were cocultured with but not without direct cell contact. Because the coculture supernatants with direct cell contact and cytokines such as interleukins and growth factors did not exhibit growth-stimulating activity toward HEL cells, it was suggested that some molecule that has growth-stimulating activity exists on the surface of the cells. Extracellular matrix components such as fibronectin, laminin, vitronectin, and collagen did not affect the proliferation of HEL cells. An anti-CD18 monoclonal antibody, which recognizes the common beta chain of the beta2 integrin subfamily, induced dramatic proliferation of HEL cells. Moreover, the proliferation of HEL cells was inhibited by an antisense oligonucleotide of CD18 mRNA. As judged from these observations, the proliferation of HEL cells was mediated by CD18 molecules expressed on HEL cells. On the contrary, the common counter-receptor of the beta2 integrin subfamily, intercellular adhesion molecule-1, which is expressed on CHO-K1 cells, did not stimulate the growth of HEL cells. It is known that other counter molecules of the beta2 integrin subfamily, such as complement C3bi and fibrinogen, are not produced by stromal cells. These findings suggest that the proliferation of HEL cells may be induced through an interaction between a novel molecule of the beta2 integrin subfamily on HEL cells and the counter-receptor on HESS-5 cells. The beta2 integrin subfamily may regulate the growth of hematopoietic cells in hematopoiesis in vivo and/or cause the abnormal growth of leukemia cells.

Small left coronary arteries after arterial switch operation for complete transposition.

BACKGROUND: Myocardial perfusion is not completely normal and ventricular function is depressed in some patients after the arterial switch operation. The basic mechanism has not yet been defined totally. METHODS: The diameters of the right, left main trunk, anterior descending, and circumflex coronary arteries were measured by computer-assisted densitometry at 8 to 86 months (mean, 47.5 months) after the arterial switch operation in 86 patients. RESULTS: The Z scores, compared with control, were +2.0 +/- 0.3, -1.8 +/- 0.3, and -1.5 +/- 0.3 for the right, left anterior descending, and circumflex coronary arteries, respectively. The Z score for the total cross-sectional area of the three vessels was -1.5 +/- 0.3. These parameters did not correlate with left ventricular ejection fraction. CONCLUSIONS: At the midterm follow-up after the arterial switch operation for complete transposition of the great arteries, the left coronary arteries are small. A careful follow-up study is mandatory to clarify the clinical significance of this finding.

Identification of multiple regulatory elements of human L-histidine decarboxylase gene.

Previously, we reported the structure of human L-histidine decarboxylase gene. To identify the regions that regulate the tissue-specific expression of HDC, we constructed a fusion DNA with the 5'-flanking region from -1003 to +99 of the HDC gene and chloramphenicol acetyltransferase (CAT) gene, which was then transfected into human basophilic leukemia KU-812-F cells or human epithelial carcinoma HeLa cells. The 1102 bp DNA fragment stimulated the CAT activity in KU-812-F cells, but not in HeLa cells. CAT analysis with a series of 5'-deletion constructs of the HDC-CAT gene revealed the existence of two positive and one negative regulatory elements at -855 to -841 and -532 to -497 and -829 to -821, respectively. Sequence analysis showed a nuclear factor c-Myb binding motif, TAACTG, at position -520. Gel mobility shift analysis showed that the nuclear extract of KU-812-F cells, but not that of HeLa cells, contains a factor which can bind to this motif. These results suggest that the 5'-flanking region of the HDC gene contains multiple regulatory elements for HDC gene expression and that at least one element, including a c-Myb binding motif, is responsible for the tissue-specific expression of HDC.

[A case report of one-stage repair for transposition of the great arteries with intact ventricular septum and coarctation of the aorta in neonate].

We report a neonatal case of one-stage repair for transposition of the great arteries with intact ventricular septum (TGA with IVS) and coarctation of the aorta (CoA). The surgery was performed at 27 days of age when the patient, a male, weighed 3408 g. Preoperative cardiac catheterization and angiography confirmed the diagnosis of TGA with IVS, CoA, atrial septal defect, patent ductus arteriosus, persistent left superior vena cava, left sided juxta-positioning of appendages and dextrocardia. The procedure was simultaneous subclavian flap aortoplasty through a left thoracotomy and an arterial switch operation through a median sternotomy. The patient's postoperative course was uneventful and he has grown well. Postoperative cardiac catheterization revealed mild pulmonary stenosis (pressure gradient of 19 mmHg between the right ventricle and the pulmonary artery) and mild aortic arch kinking from the arterial switch maneuver. One-stage repair, the combination of subclavian flap aortoplasty and arterial switch operation, provides a good clinical result in this complex malformation.

Comparative studies of human recombinant 74- and 54-kDa L-histidine decarboxylases.

We have expressed and characterized human recombinant 74-kDa (rHDC74) and 54-kDa (rHDC54) L-histidine decarboxylases (HDCs) in Sf9 cells. By immunoblot analysis, rHDC74 and rHDC54 were shown to be localized predominantly in the particulate and soluble fractions, respectively. rHDC74 exhibited histamine-synthesizing activity equivalent to that of rHDC54. The existence of 74- and 54-kDa HDCs was also confirmed in the particulate and supernatant fractions of the cell lysate, respectively, from the human basophilic leukemia cell line KU-812-F. The ratio of HDC activity to immunoreactivity was similar for the two forms of the enzyme. The specific activity of purified rHDC54 (1.12 mumol/mg/min) was comparable to those of HDCs from other mammalian tissues or cells. The purified rHDC54 was eluted as a monomer form from a Superdex-200 column; the molecular mass of the enzyme was approximately 54 kDa on SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol. The HDC activity of rHDC54 significantly decreased on dialysis against buffer without pyridoxal 5'-phosphate; addition of pyridoxal 5'-phosphate to the dialysate readily increased in the enzyme activity to the original activity. Taken together, these results suggest that human HDC functions as both 74- and 54-kDa forms having equivalent HDC activity, which are localized in the particulate and soluble fractions, respectively, and that the latter form exhibits its activity as a monomer form.

Expression and characterization of human recombinant parental and mature L-histidine decarboxylases.

Human recombinant 74 kD parental (rHDC74) and 54 kD mature (rHDC54) histidine decarboxylases (HDCs) have been expressed in Sf9 cells and characterized. By immunoblot analysis, rHDC74 and rHDC54 were shown to be localized predominantly in the particulate and soluble fractions, respectively. rHDC74 exhibited histamine-synthesizing activity equivalent to that of rHDC54. An active particulate HDC was also detected in the pellets obtained from 10,000 and 100,000 g centrifugation of a cell lysate from the human basophilic leukemia cell line, KU-812-F (14 and 18% of the total activity, respectively). By four purification steps, rHDC54 was purified to homogeneity, as judged by silver staining of the SDS-polyacrylamide gel. The purified rHDC54 was eluted as a monomer form from a Superdex-200 FPLC column. The molecular mass of the enzyme was found to be approximately 54 kD on SDS-poly-acrylamide electrophoresis in the absence of 2-mercaptoethanol. Taken together, these results suggest that human HDC functions as both 74 and 54 kD forms having equivalent HDC activity, which are localized in the particulate and soluble fractions, respectively, and that the latter form exhibits its activity as a monomer form.

[Molecular biology of L-histidine decarboxylase].

L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine. This biogenic amine is known to exert various effects in physiological and pathological reactions. In contrast to the well-known mechanism of histamine action through its interaction with specific receptors, the mechanisms regulating HDC gene expression are not elucidated. We have purified HDC from mouse mastocytoma cells, and isolated mouse HDC cDNA, and found that the primary translated product is posttranslationally processed to yield a mature active enzyme. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred on treatment with dexamethasone+TPA, and also cAMP+Ca2+. To clarify the mechanism of up-regulation by these stimuli of the transcription of the HDC gene, we have isolated a genomic DNA clone encoding 5'-flanking region sequence and the first two exons. The transcription start site and the nucleotide sequences of the promoter regions including TATA- and GC-boxes were determined. With mastocytoma cells transiently transfected with 5' deletion constructs of HDC-CAT fusion gene, it was found that the sequences from -132 to -53 and -267 to -53 are essential for the regulatory elements involved in the increased transcription of the HDC gene with dexamethasone+TPA and cAMP+Ca2+, respectively. Furthermore, we have isolated a genomic DNA from human basophilic cells, and analysed its structure to elucidate the mechanisms regulating the tissue specificity of HDC gene expression.

Structure of the L-histidine decarboxylase gene.

Two species of L-histidine decarboxylase (HDC) mRNA were found in the KU-812-F basophilic cell line, but only the 2.4-kilobase (kb) one encodes the functional HDC (Mamune-Sato, R., Yamauchi, K., Tanno, Y., Ohkawara, Y., Ohtsu, H., Katayose, D., Maeyama, K., Watanabe, T., Shibahara, S., and Takishima, T. (1992) Eur. J. Biochem. 209, 533-539). The 3.4-kb one encodes a truncated HDC protein and is also found in human leukemia-derived cell lines HEL and KCL-22. To clarify the mechanisms that regulate transcription of the HDC gene and generate the two species of mRNA, we have isolated genomic DNA clones coding for the HDC from human genomic libraries. Structural analysis of the isolated clones revealed that the human HDC gene is composed of 12 exons spanning approximately 24 kb. Genomic DNA blot analysis suggested that HDC is encoded by a single copy gene. The structural analysis also demonstrated that the heterogeneity of the HDC mRNA is caused by an insertion of the seventh intron sequence and alternative use of the splicing acceptor site at the 12th exon. The transcription start site of the HDC gene and the nucleotide sequences of the promoter and first exon regions were determined. We found a TATA-like sequence, a GC box, four CACC boxes, four GATA consensus sequences, and six leader-binding protein-1 binding motifs in the promoter region of the HDC gene.

Expression and characterization of recombinant mouse mastocytoma histidine decarboxylase.

The possibility of post-translational processing of mouse mastocytoma histidine decarboxylase (HDC; EC 4.1.1.22) was investigated. The molecular mass of the recombinant HDC expressed in Sf9 cells using HDC cDNA from mouse mastocytoma cells was determined to be 74 kDa by SDS-PAGE. In contrast to the native HDC from mastocytoma cells, the recombinant 74 kDa HDC was essentially inactive and precipitable in Sf9 cells. On the other hand, deletion mutants of the recombinant HDC lacking a C-terminal region equivalent to 10 (64 kDa) or 20 kDa (54 kDa) in size were present as active forms in the soluble fraction of Sf9 cells. To examine the C-terminal deletion of the 74 kDa species yielding the 53 kDa species by means of the immunoblotting analysis, two peptides (corresponding to residues 323-337 and 572-586 of the recombinant 74 kDa HDC peptide) were synthesized, and rabbit antiserum specific for each peptide was prepared. On immunoblotting analysis, anti-peptide 323-337 antiserum recognized both the recombinant 74 kDa and native enzyme subunit peptides, but anti-peptide 572-586 antiserum recognized only the recombinant 74 kDa peptide, i.e., not the native enzyme subunit peptide. Furthermore, HDC activity in the crude extract from Sf9 cells was not precipitable with antipeptide 572-585 antiserum. These results strongly suggest that the 53 kDa subunit peptide of native mastocytoma HDC is derived from the unidentified inactive 74 kDa HDC peptide, probably by post-translational processing of HDC in its C-terminal region.

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.

Synergistic effects of 12-O-tetradecanoylphorbol-13-acetate and dexamethasone on de novo synthesis of histidine decarboxylase in mouse mastocytoma P-815 cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.