Phospholipase D development during differentiation of human promyelocytic leukemic HL60 cells.
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Cell Differentiation↗
Biomedical subjects
Y Nozawa
Publications and source records attributed to Y Nozawa.
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↗
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↗
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↗
Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.
We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.
1-Sarcosine-8-Isoleucine Angiotensin II↗
Chronic treatment with an ACE inhibitor, temocapril, lowers the threshold for the infarct size-limiting effect of ischemic preconditioning.
This study examined whether chronic inhibition of the angiotensin-converting enzyme (ACE) lowers the threshold of preconditioning (PC) and potentiates cardio-protection of subthreshold PC. Rabbits were orally administered either 0.5 mg/kg/day temocapril or placebo for 14 days and were randomly subjected to subthreshold PC (i.e., PC with 2 minutes ischemia/5 minutes reperfusion) or no PC before a 30-minute coronary artery occlusion and a 3-hour reperfusion. The size of the infarct was determined by tetrazolium staining and was expressed as the percent of the area at risk (%IS/AR). At the end of the experiment, the lungs were sampled for a tissue ACE activity assay. There was no significant difference in %IS/AR among the rabbits given placebo alone, placebo plus 2 minutes PC, and temocapril alone (%IS/AR = 59.5 +/- 5.8%, 43.6 +/- 3.7%, and 56.7 +/- 7.1%, respectively). However, %IS/AR was significantly reduced in the group that received temocapril and 2 minutes PC before infarction (%IS/AR = 35.4 +/- 4.8%, P < 0.05 vs. placebo control and temocapril control). The lung tissue ACE activity did not differ in the placebo-treated rabbits with and without PC (12.6 +/- 2.8 vs. 13.7 +/- 2.0 nmol/g protein/min) but was suppressed in temocapril-treated rabbits despite 2 minutes PC (0.9 +/- 0.1 vs. 1.5 +/- 0.2 nmol/g protein/min, both P < 0.05 vs. placebo-treated groups). The present results suggest that chronic inhibition of ACE is beneficial for potentiating the anti-infarct effect of subthreshold PC.
Administration, Oral↗
Distribution of sialic acid-dependent carbohydrate epitope in thyroid tumors: immunoreactivity of FB21 in paraffin-embedded tissue sections.
The reactivity of monoclonal antibody FB21, which recognizes a sialic acid-dependent carbohydrate epitope, was tested with 94 non-neoplastic and neoplastic thyroid tissue specimens using immunohistochemical methods on formalin-fixed, paraffin-embedded tissue sections. These specimens included 11 cases of adenomatous goiter, three cases of Basedow's disease, 30 cases of follicular adenoma, 20 cases of papillary carcinoma, 15 cases of follicular carcinoma, six cases of medullary carcinoma, and nine cases of anaplastic carcinoma. FB21 reacted with 14 of 15 cases of follicular carcinoma that showed a microfollicular or trabecular pattern, and with nine of 20 cases of papillary carcinoma. A positive reaction was found on the cell surface membranes or apical parts of neoplastic follicles. FB21 also reacted with five of 30 cases of follicular adenoma. These cases showed a follicular pattern and positive staining pattern similar to that in follicular carcinoma. Adenomatous goiters, Basedow's disease, medullary carcinomas, and anaplastic carcinomas were negative for FB21 reactivity. Although the different reactivities of FB21 with papillary carcinoma and follicular carcinoma remain to be investigated, the high frequency of reactivity with FB21 suggests that it may be useful as a complement to morphological diagnosis in follicular carcinoma.
Adenoma↗
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↗
Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.
Exposure of human keratinocyte HaCaT cells to ultraviolet B-irradiation induced apoptotic morphologic changes. In this study, we found that the ultraviolet B irradiation (0.25 J per cm2) induced phosphorylation of p38 mitogen-activated protein kinase and c-jun N-terminal protein kinase, and also significant activation of caspase-3 (CPP32-like protease) and a small increase of caspase-1 (ICE-like protease) activity in the early stages of ultraviolet B-induced apoptosis. Pretreatments of the cells with a p38 mitogen-activated protein kinase inhibitor, SB203580, and a caspase-3 inhibitor, Ac-Asp-Met-Gln-Asp-1-aldehyde, suppressed the ultraviolet B irradiation-induced apoptosis by approximately 60% as estimated by nuclear staining and DNA laddering. Pretreatment with caspase-1 inhibitor, Ac-Tyr-Val-Lys-Asp-aldehyde was without effect. Ultraviolet B-induced caspase-3 activation resulted in cleavage of poly(ADP) ribose polymerase, which was abolished by the caspase-3 inhibitor. SB203580 pretreatment prevented activation of caspase-3 and caspase-1, and also suppressed the cleavage of poly(ADP) ribose polymerase. Neither ceramide generation nor sphingomyelinase activation (neutral and acid) was observed in the ultraviolet B-irradiated HaCaT cells. Also various antioxidants did not affect the caspase activation induced by ultraviolet B irradiation. These results indicated that activation of p38 mitogen-activated protein kinase upstream of caspases may play an important part in the apoptotic process of keratinocytes exposed to ultraviolet B irradiation.
Apoptosis↗
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↗
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↗
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↗
[Membrane lipid signaling system participating in intracellular signal transduction--General concept].
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Animals↗
[Roles of phospholipase D in signal transduction].
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Aging↗
Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells.
Apoptosis is an active form of cell death that can be induced by a wide variety of agents and conditions. In response to actinomycin D, hydrogen peroxide (H2O2), or TNF-alpha, Jurkat T cells underwent typical apoptosis. Phospholipase D (PLD) activity in intact cells determined by phosphatidylbutanol generation was up-regulated by these agents. The PLD activation was in a time-dependent manner during apoptosis. It was also shown that the PLD activity measured by using exogenous substrate in the lysate from apoptotic cells was higher than that in the lysate from control untreated cells. The PLD activity in lysate from control untreated cells was stimulated by unsaturated fatty acids (UFA), but not by guanosine 5'-O-(3-thiotriphosphate). However, the PLD activity in the apoptotic cell lysate was no longer enhanced by the addition of oleate, suggesting that the increased PLD activity during apoptosis was attributed to the PLD of UFA-dependent type, but not the small G protein-dependent one. In fact, the release of free UFA was increased during apoptosis. The caspase inhibitors, z-DEVD and z-VAD, effectively suppressed PLD activation and apoptosis, but UFA release was unaffected. These results suggest the possibility that UFA-dependent type PLD may be implicated in apoptotic process in Jurkat T cells. This is the first demonstration that the PLD of UFA-dependent type would be involved in cellular responses.
Apoptosis↗