Cell sheet injection as a technique of osteogenic supply.

We previously reported a new cell transplantation method utilizing injections of mesenchymal stem cell (MSC) sheets that have osteogenic potential. After subcutaneous transplantation without any scaffold, the sheet demonstrated in vivo bone formation. In the present study, we transplanted such sheets by injection into implanted ceramics and assessed whether the injectable MSC sheets could stimulate osteogenic integration of the ceramics. To fabricate MSC sheets, bone marrow cells cultured from femur shafts of 7-week-old rats were subcultured in regular 10-cm dishes containing dexamethasone and ascorbic acid phosphate until confluent. Each cell sheet was then lifted using a scraper. Porous ß-tricalcium phosphate (ß-TCP) disks (5 mm Φ×2 mm) were transplanted subcutaneously into the backs of the rats. Immediately following implantation, the sheets were injected around the disks via a 16G needle (immediate group). Cell sheets were also injected into the remaining implanted disks 1 week after disk implantation (1-wk group). Four weeks following sheet injection, radiography and histology revealed calcification and bone tissue around the harvested disks of the immediate group (eight disks exhibited bone formation/eight implanted disks), whereas calcification and bone tissue were observed in 50% of the samples in the 1-wk group (four disks exhibited bone formation/eight implanted disks). The present study indicates that injected cell sheets can supply osteogenic potential to implanted ceramics. Owing to the usage of a needle for cell sheet transplantation, such an injection method can be applied as a minimally invasive technique of osteogenic supply to implanted ceramics.

Reactive oxygen species generation and photo-cytotoxicity of eugenol in solutions of various pH.

In order to clarify the mechanism of photo-damage caused by eugenol (4-allyl-2-methoxyphenol), we measured cell survival in the presence of eugenol at concentrations of 10(-3) - 10(-7) M, with and without VL (visible light) irradiation by a VL dental lamp and at various pHs (7.2, 7.8 and 8.2) using two different cells (HSG, a human submandibular gland tumor cell line; HGF, a human gingival fibroblast in primary culture). Also, ROS (reactive oxygen species) generation in the above adherent single cells was measured by ACAS laser cytometry combined with CDFH-DA, a peroxide probe. The survival of both HSG and HGF cells treated with eugenol was significantly decreased as the VL irradiation time and/or the pH of the medium was increased. The amount of ROS generated from eugenol was also enhanced by increasing the VL irradiation time and elevating the pH of the medium. Cytotoxicity and ROS generation of HGF cells were significantly lower than that of HSG cells. Glutathione (1 mM) or cysteine (1 mM) protected the photo damages. We conclude that the cytotoxicity of VL-irradiated eugenol possibly was caused by the generation of eugenol radicals and additionally by ROS, both of which were produced dependent on the dose of eugenol, length of irradiation time, and pH of the medium.

The production of reactive oxygen species by irradiated camphorquinone-related photosensitizers and their effect on cytotoxicity.

Camphorquinone (CQ) is widely used as an initiator in modern light-cured resin systems but there are few reports about its effects on living cells. To clarify the mechanism of photosensitizer-induced cytotoxicity, the production of initiator radicals and subsequent reactive oxygen species (ROS) by CQ, benzil (BZ), benzophenone (BP), 9-fluorenone (9-F) in the presence of the reducing agent (2-dimethylaminoethyl methacrylate or N,N-dimethyl-p-toluidine, DMT) with visible-light irradiation was examined in a cell or cell-free system. Initiator radical production was estimated by the reduction rate of 1,1-diphenyl-2-picrylhydrazyl and by the conversion of poly-triethyleneglycol dimethacrylate; the results indicated that CQ/DMT had the highest activity among them. The cytotoxic effects of the photosensitizers on both human submandibular gland (HSG) adenocarcinoma cell line and primary human gingival fibroblast (HGF) showed that the 50% toxic concentration (TC(50)) declined in the order: CQ>BP>9-F>BZ. ROS produced in HSG or HGF cells by elicited, irradiated photosensitizers were evaluated in two different assays, one using adherent cell analysis and sorting cytometry against adherent cells and the other, flow cytometry against floating cells, with fluorescent probes. ROS production was dose- and time- dependent, and declined in the order: BZ>9-F>BP>CQ. Cytotoxic activity was correlated with the amount of ROS. Cytotoxicity and ROS generation in HGF cells was significantly lower than in HSG cells. ROS induced by aliphatic ketones (CQ) were efficiently scavenged by hydroquinone and vitamin E, whereas those by aromatic ketones (9-F) were diminished by mannitol and catalase, suggesting that OH radicals were involved in ROS derived from 9-F. A possible link between the cytotoxic activity and ROS is suggested.

Regulation of Na(+)-K(+)-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein.

Na(+)-K(+)- ATPase alpha-subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were (32)P-labeled within 5 s by incubation with [gamma-(32)P]ATP at 37 degrees C in the presence of cAMP, but no labeling occurred without cAMP. Phosphorylation of Na(+)-K(+)-ATPase was associated with a decrease in its activity. This alpha-subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [gamma-(32)P]ATP, indicating that catalytic subunit of protein kinase A (PKA) associated to BLMVs via binding with its RII regulatory subunit anchored on the membrane. In the absence of cAMP, a PKA catalytic subunit readily reassociated with the membrane-bound RII subunit. HT-31 peptide inhibited the Na(+)-K(+)-ATPase phosphorylation by membrane-bound endogenous PKA, indicating an involvement of A-kinase anchoring protein (AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an RII overlay assay and was coimmunoprecipitated by anti-RII antibody. These results show that Na(+)-K(+)-ATPase of rat parotid gland acinar cells is regulated in vivo by membrane-anchored PKA via AKAP rather than by free cytosolic PKA.

Cytotoxicity and radical intensity of eugenol, isoeugenol or related dimers.

To investigate the possible link between radicals and cytotoxicity of eugenol-related compounds, dimer compounds were synthesized from eugenol (4-allyl-2-methoxyphenol) or isoeugenol (4-propenyl-2-methoxyphenol): bis-eugenol (3,3'-dimethoxy-5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol); dehydrodiisoeugenol (2-(3-methoxy-4-hydroxyphenyl)-3-methyl-5-(1-propenyl)-7-methoxy-2,3- dihydrobenzofuran) and alpha-di-isoeugenol (r-l-ethyl-5-hydroxy-t-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-c-2- methylindane). Both the cytotoxic activity and the DNA synthesis inhibitory activity of these compounds against a salivary gland tumor cell line (HSG) and normal human gingival fibroblast (HGF) were decreased in the order of: dehydrodiisoeugenol, alpha-di-isoeugenol > isoeugenol > eugenol > bis-eugenol. Electron spin resonance (ESR) spectroscopy showed that dehydrodiisoeugenol, alpha-di-isoeugenol and eugenol, but not isoeugenol and bis-eugenol, produced phenoxyl radicals under alkaline condition (pH > 9.5). However, benzyl radicals were produced during the dimerization of isoeugenol to dehydrodiisoeugenol. The radical intensity of alpha-di- and dehydrodiisoeugenol appeared at relatively later incubation time than eugenol, suggesting that their phenoxyl radical was more stable than that of eugenol. Such a phenoxyl radical is produced by scavenging free radicals, during the inhibition of lipid peroxidation. Higher cytotoxic activity of isoeugenol dimers was thought to be induced by interaction with cell membranes via the lipophilic radical. The present study supports the notion that relative cytotoxicity of chemicals can be evaluated by measuring the radical intensity using ESR.

Cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines.

A total of 150 chemically-defined natural and synthetic polyphenols (flavonoids, dibenzoylmethanes, dihydrostilbenes, dihydrophenanthrenes and 3-phenylchromen-4-ones), with molecular weights ranging from 224 to 824, were investigated for cytotoxic activity against normal, tumor and human immunodeficiency virus (HIV)-infected cells. They showed higher cytotoxic activity against human oral squamous cell carcinoma HSC-2 and salivary gland tumor HSG cell lines than against normal human gingival fibroblasts HGF. Many of the active compounds had a hydrophilic group (hydroxyl group) in the vicinity of a hydrophobic group (prenyl, phenyl, methylcyclohexene or methylbenzene moiety), similar to isoprenoid-substituted flavones. Substitution of hydrophobic group (prenyl or geranyl group) did not significantly change the cytotoxic activity of flavanones, isoflavans, chalcones or 5-hydroxy-3-phenoxychromen-4-ones. However, the prenylation(s) of an isoflavone and a 2-arylbenzofuran significantly enhanced the cytotoxic activity. Agarose gel electrophoresis showed that active components induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in HSC-2 cells. Most of the polyphenols failed to reduce the cytophathic effect of HIV infection in MT-4 cells.

Cytotoxic activity of hydrolyzable tannins against human oral tumor cell lines--a possible mechanism.

Hydrolyzable tannins showed higher cytotoxic activity against human oral squamous cell carcinoma and salivary gland tumor cell lines than against normal human gingival fibroblasts, whereas gallic acid, a component unit of tannins, showed much weaker selective cytotoxicity. The cytotoxic activity of dimeric compounds was generally higher than that of monomeric compounds. Macrocyclic ellagitannin oligomers, such as oenothein B, woodfordin C and woodfordin D showed the greatest cytotoxic activity, and their activity (per given number of molecules) was one order higher than those of gallic acid and epigallocatechin gallate, a major component of green tea. These compounds induced apoptotic cell death characterized by DNA fragmentation (as demonstrated by the TUNEL method) and cleavage of cytokeratin 18 by activated caspase(s) (as demonstrated by M30 monoclonal antibody). ESR spectroscopy revealed that these macrocyclic compounds at higher concentrations produced their own radicals and significantly enhanced the radical intensity of sodium ascorbate, possibly by their prooxidant actions. Catalase failed to eliminate their apoptosis-inducing activity, reducing the possibility of the involvement of hydrogen peroxide production in the extracellular fraction. These observations suggested that the antitumor activity of macrocyclic ellagitannin oligomers reported previously might be explained by their apoptosis-inducing activity.

Induction of apoptosis by flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavonoids in human oral tumor cell lines.

Various flavones, flavonols (3-hydroxyflavones) and isoprenoid-substituted flavones (flavonols) were investigated for their cytotoxic activity. Most of these compounds were more cytotoxic against human oral squamous cell carcinoma and salivary gland tumor cell lines than human gingival fibroblasts. The cytotoxic activity of flavonoids was generally higher than that of tannin-related compounds. Flavonoids induced apoptotic cell death characterized by DNA fragmentation (as identified by TUNEL method) and activation of caspase(s) (as identified by degradation products of cytokeratin 18 with M30 monoclonal antibody). ESR spectroscopy revealed that higher concentrations of flavonoids produced radicals under alkaline conditions. However, not all of them enhanced the radical intensity of sodium ascorbate, suggesting that the redox potential of flavonoids differs considerably from samples to samples. Catalase failed to eliminate the cytotoxic activity of flavonoids, reducing the possibility of the involvement of hydrogen peroxide for the cytotoxicity induction by them.

Thyroid hormone (3,5,3'-triido-L-thyronine) masking/inversion of stimulatory effect of androgen on expression of mk1, a true tissue kallikrein, in the mouse submandibular gland.

We studied hormonal regulation of the expression of mkl, a true tissue kallikrein, in the submandibular gland (SMG) of ICR, C3H/ HeN, and F1 (mice from male C3H/HeN x female ICR and in the ones from male ICR x female C3H/HeN). In these mouse strains, mk1 was low in content in males, abundant in females, and increased remarkably by castration of males. In the case of ICR and both F1 mice, injection of 5alpha-dihydrotestosterone (DHT) reduced the mkl level of castrated and female mice. However, the mkl content in female C3H/ HeN mice (or castrated C3H/HeN) was further increased by DHT. To investigate the real action of DHT on mk1 expression, we examined the effects of adrenoectomy/glucocorticoid (dexamethasone, Dex) administration; DHT administration into castrated and adrenoectomized mice; ovariectomy/female hormone (17beta-estradiol, progesterone) administration; and hypophysectomy/combinatory administration of DHT, Dex, and thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the mk1 expression in the SMG of ICR mice. Adrenoectomy or ovariectomy did not change the characteristic pattern of mk1 expression in male and female ICR mice. In hypophysectomized (Hypox) ICR male mice, the mk1 content was increased to the same level as in normal ICR females, and DHT administration into the Hypox mice further increased the mk1 level. However, combinatory administration of DHT + T3 or of DHT + T3 + Dex into the Hypox mice lowered the mkl content to the level of normal ICR males, whereas T3 single administration had no effect. Dex single administration into the Hypox mice increased the mkl level to an even higher than that observed with DHT administration. The mk1 level in Hypox mice was not significantly changed by coadministration of Dex with T3. From these results, we conclude that 1) mk1 expression is fundamentally stimulated by androgen (DHT) as are other mk isozymes, such as mk9, mk13, mk22, and mk26 in the mouse SMG, 2) the effect (stimulatory) of DHT on mk1 expression becomes, however, inverted (inhibitory) in the presence of T3. Although the serum T3 level of C3H/HeN female (0.52 ng/ml) was not significantly different from that of C3H/HeN males or ICR mice, coadministration of T3 into C3H/HeN females with a fixed amount of DHT (20 mg/kg body weight) dose dependently repressed the DHT-induced increase in mkl expression, suggesting the lower sensitivity of C3H/HeN females to T3.

Application of bis-eugenol to a zinc oxide eugenol cement.

OBJECTIVES: To assess the usefulness of dimerized eugenol (bis-eugenol) in dentistry, the physical properties of zinc oxide eugenol cement (ZOE) with bis-eugenol and the cytotoxicity of bis-eugenol were studied. METHODS: Setting time, compressive strength, solubility and disintegration of ZOE cement with bis-eugenol according to the specifications of JDMAS315 were evaluated. The cytotoxicity of bis-eugenol and eugenol toward two different cell types, HGF (a primary culture of human gingival fibroblast) and HSG (a human epidermoid carcinoma cell line derived from a salivary gland) was evaluated by the MTT test and in terms of cell survival. RESULTS: Addition of bis-eugenol to ZOE did not decrease the physical properties when employed at the ratio of 9:1 or 6:1 (liquid ND:bis-eugenol, w/w). Bis-eugenol was less toxic than eugenol in the cell culture tests. CONCLUSIONS: The results of this assay demonstrated that bis-eugenol is useful in ZOE.

Radical intensity and cytotoxicity of butylated hydroxyanisole and its orthobisphenol dimer.

The radical intensity of BHA (4-Hydroxy-3-t-butylanisole) and its dimer (3,3'-Di-t-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol, Bis-BHA) were compared with their cytotoxic activity. ESR spectroscopy showed that BHA produced characteristic five peaks of radicals under alkaline conditions (pH > 9.5). At higher pH, BHA radical rapidly disappeared, and progressively transformed into new radical species, as detected by the splitting of the ESR signal. BHA showed higher cytotoxic activity against salivary gland tumor cell line than against normal human gingival fibroblast. On the other hand, Bis-BHA did not produce any detectable amounts of radicals at wide ranges of pH, corresponding with its weaker cytotoxic activity as compared with BHA. BHA scavenged DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical and superoxide anion, more efficiently than Bis-BHA. The present study demonstrated that BHA is more cytotoxic, produces higher amounts of radicals and more efficiently scavenges various radical species, as compared with Bis-BHA. This suggests the possible link between the cytotoxic activity and radical generation/scavenging activity in BHA-derived compounds.

Modulation of thyroid hormone-dependent Na+,K(+)-ATPase induction in cultured human submandibular gland cell lines, HSG cells.

The hormonal regulation of Na+,K(+)-ATPase enzyme activities and induction of the alpha subunit protein of the enzyme in the human submandibular gland (HSG) were studied by use of cultured HSG cells. We treated HSG cells with thyroid hormone, androgen, mineralocorticoid, and glucocorticoid, singly or in combination. 3,5,3'-Triiodothyronine (T3), 5 alpha-dihydrotestosterone (DHT), and aldosterone (Ald) induced neither Na+,K(+)-ATPase enzyme activity nor its protein. On the other hand, dexamethasone (Dex) induced both Na+,K(+)-ATPase enzyme activity and the alpha subunit protein level to 128% of the control. The effects of Dex in combination with either T3 or DHT were similar to the effect of Dex alone. Treatment in combination with Dex and Ald increased the enzyme activity and alpha subunit protein level to 160%, synergistically. These increased Na+,K(+)-ATPase enzyme activities were shown to be dependent on their protein levels induced by the hormones. Contrary to the previous evidence that Na+,K(+)-ATPase of ducts in the salivary gland are thyroid hormone inducible, HSG cells had an insignificant response to thyroid hormone in the present study. Also, Na+,K(+)-ATPase enzyme activity and its alpha subunit protein were not induced by any kind of combined treatments with T3. Furthermore, T3 did not cause intracellular calcium mobilization in HSG cells. In view of all data taken together, we suggest that HSG cells lack the thyroid hormone receptor, which is necessary for Na+,K(+)-ATPase induction in human salivary gland.

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+.

OBJECTIVES: The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. MATERIALS AND METHODS: Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). RESULTS: Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. CONCLUSIONS: The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.

A calcium channel in human submandibular duct cell line, HSG cells, not regulated by P2U purinergic receptor-mediated intracellular calcium mobilization.

Signal transduction via P2 purinergic receptors was investigated in HSG cells, a continuous cell line originally derived from an irradiated human salivary gland. Ligand specificity for nucleotide receptors in HSG cells was investigated with various nucleotides and their analogues. Inositol 1,4,5-trisphosphate (IP3) production was significantly increased by ATP, UTP and ATP gamma S. The ligand specificity of this effect agreed well with that of the P2U purinergic receptor. On the other hand, 45Ca2+ influx was stimulated by ATP, UTP > ATP gamma S, ADP, UDP > ADP beta S > AMPPNP, GTP, TTP > CTP, GDP, TDP, AMPPCP, AMPCPP. This ligand specificity of 45Ca2+ influx was much broader than IP3 production. Also pertussis and cholera toxin had no effect on both IP3 production and 45Ca2+ influx by ATP or UTP. 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) stimulates 45Ca2+ influx more effectively than IP3 formation. A 53-kDa membrane protein was photolabelled with [alpha-32P]Bz-ATP. This 53-kDa protein is a putative P2 purinergic receptor. In particular, the labelling was inhibited by a ligand profile that corresponded to that for 45Ca2+ influx. These findings suggest that nucleotides stimulate 45Ca2+ influx and IP3 formation by separate pathways via pertussis and cholera toxin-insensitive G proteins. Thus, in HSG cells, IP3 formation is coupled to the P2U subclass, while 45Ca2+ influx is coupled to another subclass, such as P2X, that regulates calcium channels.

Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzyme's activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.

Simultaneous measurement of Ca2+ and pH by laser cytometry using fluo-3 and SNARF-1.

We present a new convenient method for simultaneous measurement of intracellular calcium concentration ([Ca2+]i) and intracellular pH (pHi) using laser cytometry with a mixture of fluo-3 (for [Ca2+]i) and SNARF-1 (for pHi), with iso excitation (488 nm)-dual emission (530 nm for fluo-3 and > 630 nm for SNARF-1). By using this technique, we measured the changes in [Ca2+]i and pHi in A-431 human epidermoid carcinoma cells and HSG human salivary gland cells stimulated by ATP. We found that alkalization in A-431 cell occurred with the elevation of [Ca2+]i; in contrast, alkalization in HSG cells did not occur at all, even though the elevation of [Ca2+]i was observed. Using BAPTA (a chelating agent of Ca2+) and amiloride (an inhibitor of the Na+/H+ exchanger), we found that the elevation of pHi requires the elevation of [Ca2+]i but that the elevation of [Ca2+]i does not always require a rise in pHi. From our results we conclude that elevation of [Ca2+]i takes precedence over the elevation of pHi in ATP-stimulated signal transduction.

T-cell lymphoma presenting with pericardial and pleural effusion as the initial and primary lesion: cytogenetic and molecular evidence.

A 90-year-old woman was admitted with progressive dyspnea. Chest roentgenogram and computed tomography revealed a massive pericardial effusion and bilateral pleural effusion, but no lymphomatous lesion was seen. A diagnosis of malignant lymphoma was made by cytological and immunological studies of the cells obtained from the pericardial effusion. Chromosome analysis showed a clonal abnormality and T-lineage clonality was determined by the rearrangement of the T-cell receptor gamma gene. The patient achieved remission with chemotherapy, but she later relapsed, with right pleural effusion, and died. She exhibited no lymphomatous features throughout the clinical course, indicating the possibility of malignant lymphoma originating from the pericardium and/or pleura.

ATP- and EGF-stimulated phosphatidulinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells.

The [(3)H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [(3)H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca(2+)]i elevation, and inositol trisphosphate (IP(3)) production by ATP, suggesting that the phospholipase C(PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [(3)H]choline and [(32)P)phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP(3) production and [Ca(2+)]i elevation, this fact suggests that the lAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P(2)-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.

Effects of protein kinase inhibitors and protein phosphatase inhibitors on cyclic AMP-dependent down-regulation of vesicular monoamine transport in pheochromocytoma PC12 cells.

Cyclic AMP down-regulates vesicular monoamine transport in PC12 cells and thereby decreased catecholamine reuptake from the extracellular fluid. We examined the effects of protein kinase inhibitors and protein phosphatase inhibitors on this cAMP action. Treatment of cells with a protein kinase inhibitor, K252a, increased vesicular amine transport and cellular amine uptake, thereby antagonizing the regulatory action of cAMP. In contrast, a protein phosphatase inhibitor, okadaic acid, had the opposite effect on the amine transport, i.e. it enhanced the cAMP action. These results suggest the involvement of a protein phosphorylation process in the cAMP-dependent modulation of vesicular monoamine transport.

Effect of tri-n-butyltin on intracellular Ca2+ concentration of mouse thymocytes under Ca(2+)-free condition.

Effect of tri-n-butyltin at concentrations ranging from 100 nM to 1 microM on the intracellular Ca2+ concentration of mouse thymocytes was examined under Ca(2+)-free conditions in comparison with those of 50 nM A23187, 100 nM thapsigargin and 10 microM cyclopiazonic acid, using the fluorescent dye for intracellular Ca2+, fluo-3. Tri-n-butyltin persistently increased the intensity of fluo-3 fluorescence while A23187, thapsigargin and cyclopiazonic acid produced a transient augmentation of the fluorescence. Pretreatment with A23187 greatly decreased the fluorescence responses induced by 1 microM tri-n-butyltin. However, the effect of thapsigargin and cyclopiazonic acid on the tri-n-butyltin-induced response was much weaker than that of A23187. In the presence of tri-n-butyltin, the transient response produced by A23187 was greatly prolonged. Results may suggest that tri-n-butyltin increases the membrane Ca2+ permeability of the intracellular organelles (cellular calcium stores) and decreases the Ca2+ pump activity of thymocyte membrane, resulting in a sustained increase in the intracellular Ca2+ concentration under Ca(2+)-free concentration.