Phospholipase D development during differentiation of human promyelocytic leukemic HL60 cells.
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Cell Differentiation↗
Biomedical subjects
S Nakashima
Publications and source records attributed to S Nakashima.
Molecular cloning of a cDNA encoding 14-3-3 protein from the protozoan Tetrahymena pyriformis and its mRNA expression during synchronous division.
The 14-3-3 proteins have been identified in a wide variety of eukaryotic cells and diverse biochemical properties have been ascribed to them. Here we have cloned a cDNA encoding 14-3-3 protein from a cDNA library of Tetrahymena pyriformis. This cDNA (Tp14-3-3) encoded an open reading frame consisting of 244 amino acids with predicated molecular mass as 28.1kDa. The predicted protein shares 59.2%, 56.5% and 59.2% identity to Entamoeba histolytica 14-3-3-1, Schizosaccharomyces pombe Rad 24 and human 14-3-3 zeta/delta respectively. On the basis of comparison with other 14-3-3 proteins, two of the putative functional domains (dimerization domain and annexin-similarity domain) were found in Tp14-3-3. In order to know the role of Tp14-3-3 in Tetrahymena, its mRNA levels during synchronous division were examined by Northern blot analysis. There was a marked increase in Tp14-3-3 mRNA level at 45min after the end of heat treatment, followed by a gradual decrease. These results suggest that the Tp14-3-3 mRNA level might vary during the cell cycle. The accumulation of Tp14-3-3 mRNA before cell division was assumed to be a prerequisite for the initiation of synchronous cell division.
14-3-3 Proteins↗
Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.
The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.
Adaptor Proteins, Signal Transducing↗
Increased expression of phospholipase D1 mRNA during cAMP- or NGF-induced differentiation in PC12 cells.
The rat pheochromocytoma PC12 cells have been employed as a useful model to study neuronal differentiation. To gain insight into the molecular events involved in extension of neurites, the differential expression of phospholipase D (PLD) isozymes were examined in cyclic AMP- or nerve growth factor (NGF)-treated PC12 cells. When the cells were incubated with dibutyryl cyclic AMP (dbcAMP), almost all the cells displayed prominent neurite outgrowth at 24 h. The neurites developed in NGF-treated cells at 24 h were shorter than those in the cells treated with dbcAMP. However, most of the NGF-treated cells extended long neurites at day 5. The rPLD1b mRNA increased within 6 h following dbcAMP treatment and maintained a high level up to 24 h. In contrast, the levels of rPLD1a and rPLD2 mRNAs were rather consistent throughout the time course examined. However, when the cells were treated with NGF, rPLD1a and rPLD1b mRNAs, but not rPLD2 mRNA, increased within 2 days and remained elevated up to 5 days. These results suggest the possible implication of PLD1 in PC12 cell differentiation.
Animals↗
Molecular cloning and expression of a stress-responsive mitogen-activated protein kinase-related kinase from Tetrahymena cells.
To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stress-activated protein kinase which are known to be involved in various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15 degrees C (0.8 degrees C/min). Interestingly, osmotic shock either by sorbitol (100-200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells.
Amino Acid Sequence↗
Axin prevents Wnt-3a-induced accumulation of beta-catenin.
When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel filtration column chromatography. In this fraction, GSK-3beta, beta-catenin, and APC were co-precipitated with Axin. Although beta-catenin was detected in the high molecular weight fraction in L cells on gel filtration column chromatography, addition of conditioned medium expressing Wnt-3a to the cells increased beta-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of beta-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to beta-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3beta, beta-catenin, and APC, resulting in the stimulation of the degradation of beta-catenin and that Wnt-3a induces the dissociation of beta-catenin from the Axin complex and accumulates beta-catenin.
Adenomatous Polyposis Coli Protein↗
Possible role of phospholipase D in cellular differentiation and apoptosis.
Phospholipase D (PLD) is widely distributed in mammalian cells and is implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, two related PLD isozymes, PLD1 and PLD2, were cloned. The former activity is regulated in vitro by protein kinase C and small molecular weight GTP-binding proteins (Arf and Rho family). By contrast, the basal activity of the latter is high and it is unresponsive in vitro to these activators. The cellular PLD activity and mRNA levels of these PLD isozymes drastically changed during differentiation and apoptosis in several types of cells. The general trend was that the mRNA level of PLD1 increased during differentiation, as did the observed GTP gamma S-dependent PLD activity which presumably derived from PLD1-specific catalysis. In contrast, the PLD activity and mRNA level of PLD1 were down-regulated during apoptosis. In addition to these PLD isozymes, there exists another PLD isozyme which is activated by unsaturated fatty acids such as oleic acid, although its molecular nature and physiological roles are not well defined. We have observed that this type of PLD activity is drastically increased during apoptosis of Jurkat T cells, which mainly possess this kind of PLD activity. These results suggest the possibility that PLD activity is controlled at the transcriptional level in certain circumstances, and that PLD plays roles in differentiation, survival and apoptosis in mammalian cells.
Animals↗
Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice.
BACKGROUND/AIMS: Tumor necrosis factor a (TNF-alpha) and Fas ligand are apoptotic cell-death mediators that act by binding to their responsive receptors. The aims of this study were to assess the differences between liver cell deaths induced by TNF-alpha and anti-Fas antibody, and to investigate the mechanism by which GalN sensitizes the hepatocyte to injury by TNF-alpha. METHODS: TNF-alpha or anti-Fas antibody was injected into BALB/c mice sensitized or unsensitized by D-galactosamine (GalN). Liver injury was assessed biochemically and histologically. The expressions of TNF receptor (TNFR)1 and TNFR2 mRNA in the liver were determined by Northern blot analysis. Nuclear factor-kappaB (NF-kappaB) DNA binding activity was determined by gel shift assay. RESULTS: In GalN-sensitized mice, hepatocyte apoptosis and liver failure were observed after TNF-alpha injection, but neither occurred in unsensitized mice. Microscopically, GalN preceding TNF-alpha caused massive hemorrhagic liver damage with fragmented hepatocyte nuclei resembling effects of anti-Fas antibody, but GalN largely failed to sensitize to injury by this antibody. TNFR1 mRNA expression in the liver was upregulated within 3 h after GalN administration, and anti-TNFR1 antibody protected GalN-sensitized mice from hepatotoxic effects of TNF-alpha. GalN treatment failed to affect TNF-alpha-induced NF-kappaB activation. CONCLUSIONS: Unlike Fas-related apoptosis, TNFR-mediated apoptosis requires hepatocyte sensitization involving TNFR1 upregulation.
Animals↗
Inhibition of neutral sphingomyelinase activation and ceramide formation by glutathione in hypoxic PC12 cell death.
Reduced glutathione (GSH) and N-acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor-differentiated PC12 cells. The level of intracellular total GSH decreased time-dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose-dependently by GSH and NAC. Activation of caspase-3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase-3 activation induced by C2-ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.
Acetylcysteine↗
Analysis of phospholipase C gene in patients with subarachnoid hemorrhage due to ruptured intracranial saccular aneurysm.
This study is designed to determine whether patients with aneurysmal subarachnoid hemorrhage have mutations in the phospholipase C-delta 1 (PLC-delta 1) gene, which was identified as a gene responsible for hypertension in spontaneously hypertensive rats. Seventy-two cases (31 male and 41 female) with intracranial saccular aneurysms were analyzed. The mean age was 60.1 +/- 11.5 years (mean +/- SD) (range 24-85 years). There were 35 patients (48.6%) with hypertension, 5 (6.9%) with diabetes mellitus, 12 (16.7%) with hyperlipidemia, 8 (11.1%) with ischemic heart disease, and 25 (34.7%) who were active smokers. The location of aneurysm was distributed as follows: 33 (33%) were at anterior cerebral artery, 23 (23%) were at middle cerebral artery, 28 (28%) were at internal carotid artery, and 16 (16%) were at vertebro-basilar artery. Six patients (8.3%) had a family history of intracranial aneurysms. There were 20 patients (27.8%) with multiple aneurysms, and 8 patients (11.1%) with a large or giant aneurysm. The four regions of PLC-delta 1 gene (bases 1099-1271, 1254-1401, 1343-1481, and 1882-2023) where genetic mutations were found in spontaneously hypertensive rats, were screened by PCR-SSCP analysis and their nucleotide sequences of all patients were determined. However, no mutations were detected in all patients. These results suggest that mutations of PLC-delta 1 gene previously implicated in hypertensive factor in rats may not be the case with human patients and therefore may be poorly related with aneurysmal subarachnoid hemorrhage.
Adult↗
Local anesthetics inhibit muscarinic receptor-mediated activation of extracellular signal-regulated kinases in rat pheochromocytoma PC12 cells.
BACKGROUND: Because protein phosphorylation is a key mechanism for controlling cellular functions and extracellular signal-regulated kinase (ERK) plays a role in cellular signal transduction, the authors wanted to determine whether local anesthetics interfere with biochemical signaling molecules. METHODS: Protein tyrosine phosphorylation and ERK activation induced by carbachol, an agonist for muscarinic acetylcholine receptors, were examined in rat pheochromocytoma PC12 cells, a model for investigating signal transduction. Carbachol-induced tyrosine-phosphorylated proteins of 44 and 42 kd were determined by Western blot analysis and identified as activated ERK1 and ERK2 using anti-ERK antibody. The ERK activation was blocked by preincubation with atropine or an M3 muscarinic acetylcholine receptor antagonist 4-diphenyacetooxy-1, 1-dimethylpiperidinium, indicating that is was mediated by M3 muscarinic acetylcholine receptor activation. Then, in the presence of local anesthetic, the carbachol-induced tyrosine phosphorylation and ERK activation were evaluated. The effects of three Na+ current-modifying reagents on carbachol-induced ERK activation were also evaluated. RESULTS: Procaine (10(-4) to 10(-3) M) inhibited carbachol-induced tyrosine phosphorylation and ERK activation in a concentration-dependent manner. Although tetracaine, lidocaine, and bupivacaine similarly suppressed carbachol-induced tyrosine phosphorylation and ERK activation, neither tetrodotoxin, veratridine, nor ouabain affected the carbachol-induced ERKs activation. Both ERKs were also activated by 4beta-phorbol 12-myristate 13-acetate, an activator of protein kinase C, and fluoroaluminate (AlF4-), respectively, but procaine did not affect ERK activation induced by these two substances. The inhibition of carbachol-induced ERK activation by procaine was not modified by a phosphatase inhibitor, calyculin A. CONCLUSIONS: The current results indicate that local anesthetics inhibit the activity of the signal-transducing molecule(s) leading to M3 muscarinic acetylcholine receptor-mediated ERK activation in PC12 cells. Such action is unlikely to be a result of the drug's action on Na+ channels or on the electrochemical gradients of the neuronal cell membrane.
Anesthetics, Local↗
Overexpression of MDM2 in MCF-7 promotes both growth advantage and p53 accumulation in response to estradiol.
The overexpression of the oncogene product MDM2 is often observed in human breast cancer cells, especially in estrogen receptor (ER)-positive ones. To study the role of MDM2 protein in ER-positive breast cancer, we have established cell lines derived from MCF-7 which stably express increased and decreased levels of MDM2 by transfection of a mammalian expression vector containing human mdm2 cDNA in sense and antisense orientations, respectively. Interestingly, MDM2 overexpression in MCF-7 cells afforded a remarkable growth advantage under estradiol (E2)-supplemented condition. Then, we analyzed the expression of p53, which is an important regulator of growth and the cell cycle. Unexpectedly, the p53 accumulation induced by E2 was remarkably higher in MCF-7 cells stably overexpressing MDM2 than in the parent MCF-7 cells. On the other hand, reduction of MDM2 suppressed the E2-induced increase in p53 protein. Moreover, mdm2 antisense oligonucleotides prevented E2-induced accumulation of p53. In the steady state, the cellular levels of p53 were also correlated with those of MDM2. These interactions are not consistent with the well-known role of MDM2, which acts as a negative regulator for p53 by inhibiting its function and promoting its rapid degradation. These results suggest that MDM2 may regulate the expression of p53 in the steady state and in response to E2 in breast cancer cells, and imply a novel and important role of MDM2 during breast carcinogenesis.
Breast Neoplasms↗
Polymorphism of the endoglin gene in patients with intracranial saccular aneurysms.
OBJECT: Endoglin, a transforming growth factor beta-binding protein, is a glycoprotein expressed on the surface of human vascular endothelial cells. Mutations of this gene are responsible for hereditary hemorrhagic telangiectasis and are associated with sporadic intracerebral hemorrhage as a risk factor. The purpose of this study was to examine the polymorphism of this gene in patients with intracranial aneurysms. METHODS: The authors identified the mutations and insertion polymorphism around exon 7 of the endoglin gene in 82 patients with intracranial saccular aneurysms (aneurysm group) and 114 control volunteers (control group). A 6-base insertion (GGGGGA) was found in intron 7 at 26 bases beyond the 3' end of exon 7. The homozygous insertion of intron 7 of the gene was present in 20.7% of the aneurysm group compared with 6.1% of the control group (chi2 = 9.837, p = 0.0073). The insertion allele frequency was significantly higher in the aneurysm group (67 [40.8%] of 164) than that in the control group (63 [27.6%] of 228) (chi2 = 7.48, p = 0.0062). The most notable clinical characteristic of the 17 patients with homozygous insertion in the aneurysm group was the relatively high percentage of patients with hypertension and of those with multiple aneurysms. CONCLUSIONS: The data provide evidence of an association between aneurysm development and a polymorphism at a genetic variant of endoglin in patients with these lesions.
Adult↗
[Mechanism of action of anti-fungal drugs].
Clinical application of recently developed anti-fungal drugs including fluconazole and itraconazole has provided great advantages in the treatment of deep mycoses. However, pathogenic fungi belong to eukaryotes including humans and are phylogenetically apart from prokaryotes, i.e. bacteria. In other words, the components and metabolic pathways of fungi and mammals are very similar. This sometimes makes it difficult to treat severe mycoses with the anti-fungal drugs available at present. Therefore, new drugs which are more selective for fungal components, such as new azoles, are desired by clinicians and some of them are now under clinical trial. Fungal factors involved in dimorphic change including transcription factors and members of MAP kinase cascades as well as virulence factors including proteases, phospholipases and catalase have recently been identified. These factors and enzymes responsible for cell wall construction could be selective targets to develop new anti-fungal drugs.
Animals↗
Molecular cloning of a second phospholipase B gene, caPLB2 from Candida albicans.
Accumulating evidence suggests that phospholipase B, secreted by pathogenic fungi such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, functions as one of the virulence factors. In the present study, we have attempted to clone phospholipase B gene from C. albicans. By RT-PCR analysis with degenerate primers based on conserved regions of phospholipase B from Saccharomyces cerevisiae, Penicillium notatum and Torulaspora delbrueckii two similar but different cDNA fragments were obtained. One corresponded to the partial sequence of caPLB1, recently cloned phospholipase B gene from C. albicans by a different approach (Leidich et al.: J Biol Chem 1998; 273: 26078-86). The other fragments contained sequences similar to the corresponding sequences of phospholipase B from other fungi. The presence of two related genes was confirmed by Southern and Northern blot analyses. The full length of the second C. albicans phospholipase B gene (caPLB2) encoded a putative protein with 608 amino acids and contained a potential signal peptide sequence and a putative catalytic region, which are found in phospholipase B from other fungi. Consistent with the findings of caPLB1, caPLB2 also lacks a cluster of hydrophobic amino acids at the COOH-terminal, which may function as a signal of glycosylphosphatidylinositol anchor.
Amino Acid Sequence↗
[Roles of phospholipase D in signal transduction].
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Aging↗
Renal angiomyolipoma associated with inferior vena caval and right atrial thrombus.
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Adult↗
Expression of p21WAF1/Cip1 in the p53-dependent pathway is related to prognosis in patients with advanced esophageal carcinoma.
The proteins p53 and p21 are important components that regulate G1-S transition through the cell cycle. We immunohistochemically investigated p53 and p21 expression in 111 patients with esophageal squamous cell carcinoma. We also evaluated whether the expression of either of these proteins is a prognostic factor according to the p53-dependent and -independent pathways. The positive rates of p53 and p21 expression were 42.8 and 43.2%, respectively. Clinicopathological findings according to p53 and p21 expression did not differ significantly. The 5-year-survival rates between p21 positive and negative expression did not differ significantly in the p53-positive group. In the p53-negative group, the 5-year-survival rate of patients with p21-positive expression was 22.9%, which was significantly better than that of patients with p21-negative expression (12.7%; P<0.05). Multivariate analysis revealed that p21 expression in the p53-dependent pathway was an independent prognostic factor. Accordingly, the prognostic values of p21 expression between the p53-dependent and -independent pathways differed. Examination of p21-positive expression in the p53-dependent pathway will help to estimate the favorable prognosis of patients with advanced esophageal carcinoma.
Adult↗