Primary tumour characteristics predict the invasiveness of lymph node metastases in papillary thyroid carcinoma patients.

OBJECTIVE: This study evaluated whether primary tumour characteristics are associated with specific features of metastatic lymph nodes in papillary thyroid carcinoma patients. METHOD: A retrospective review of 411 patients with pathologically diagnosed cervical lymph node metastasis was conducted. RESULTS: A metastatic lymph node focus size of at least 2 mm was independently associated with a primary tumour size of at least 1 cm (hazard ratio 1.962) and with male sex (hazard ratio 1.947). A number of at least five lymph node metastases was independently associated with a primary tumour size of at least 1 cm (hazard ratio 2.863), extrathyroidal extension (hazard ratio 1.737) and male sex (hazard ratio 1.689). Extranodal extension was independently associated with a primary tumour size of at least 1 cm (hazard ratio 2.288), extrathyroidal extension (2.201) and male sex (hazard ratio 1.733). CONCLUSION: Primary papillary thyroid carcinoma characteristics are related to the pathological features of lymph node metastases.

Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis, towards different stress stimulations.

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green-lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non-metal oxidant (hydrogen peroxide; H2 O2 ) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10-I and 72 for MT10-II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two-dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10-I and MT10-II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10-I responded promptly to Cd but had a lagged induction to H2 O2 treatments, while MT10-II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H2 O2 , compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.

Essential roles of Crk and CrkL in fibroblast structure and motility.

Cytosolic proteins containing SH2 and SH3 domains, such as Crk and Crk-like (CrkL), are broadly expressed adapters that interact with a variety of proteins to fulfill key roles in signal transduction pathways triggered by activation of receptor and non-receptor tyrosine kinases. Crk and CrkL are similar to each other in structure and biochemical function, although they provide both distinct, as well as overlapping, biological roles during development. We developed a systematic approach to investigate Crk family functions at the cellular level by generating a conditional knock-out system for ablation of Crk and CrkL in cultured fibroblasts. The loss of both Crk and CrkL from fibroblasts resulted in reduced cell surface area and adoption of a rounded, refractile cellular phenotype. These morphological alterations were accompanied by a decrease in focal adhesion sites, reduced actin stress fibers and a collapse of microtubule structures. In addition, cells exhibited decreases in spontaneous motility and wound-healing behavior. Reduced p130Cas phosphorylation and actin levels closely followed the loss of Crk and CrkL, and stabilization of polymerized actin by jasplakinolide suppressed the morphological conversion. Ablation of Crk or CrkL alone conferred a much more modest phenotype suggesting that Crk and CrkL have overlapping functions that are critical for maintaining cell structure. The morphological alterations could be partially rescued by reintroduction of CrkII, and, to a lesser extent, CrkL. Taken together, our results suggest that Crk and CrkL have critical roles in cell structure and motility by maintaining cytoskeletal integrity.

Associations of CD6, TNFRSF1A and IRF8 polymorphisms with risk of inflammatory demyelinating diseases.

AIMS: Multiple sclerosis (MS) and neuromyelitis optica (NMO) are inflammatory autoimmune diseases that affect the central nervous system. Several genome-wide and candidate gene studies have identified genetic polymorphisms associated with the risk of MS or NMO. In particular, two recently published studies of meta-analysis in European-origin populations have suggested associations of single-nucleotide polymorphisms (SNPs) in CD6, TNFRSF1A and IRF8 with MS. The aim of our study was to assess the associations between SNPs in these three genes and the risk of inflammatory demyelinating disease (IDD) including MS and NMO. To the best of our knowledge, this is the first time such a study has been performed in an Asian population. METHODS: A total of 21 SNPs of CD6, TNFRSF1A and IRF8 were genotyped in 178 IDD cases (79 MS and 99 NMO patients) and 237 normal controls in a Korean population. RESULTS: Logistic analyses revealed that one SNP in CD6 (rs12288280, P = 0.04) and three SNPs in TNFRSF1A (rs767455, rs4149577 and rs1800693, P = 0.01-0.03) were associated with NMO. However, there was no association of IRF8 polymorphisms with IDD, including MS and NMO. Using further information from the SNP Function Prediction website, two exonic splicing enhancers (ESEs), including the polymorphic site of rs767455, were predicted to be binding sites for splicing factors (SRp55, SF2/ASF2 and SF2/ASF1). CONCLUSION: Although additional studies are needed, our findings could provide information regarding the genetic aetiology of IDD in the Korean population.

Time-dependent exposure of nicotine and smoke modulate ultrasubcellular organelle localization of dopamine D1 and D2 receptors in the rat caudate-putamen.

The correlation of the subcellular localization of dopamine D(1) and D(2) receptors (DA D(1) R, DA D(2) R) with nicotine addiction has not been studied. We demonstrated the ultrasubcellular organelle localization of DA D(1) and D(2) Rs in the caudate-putamen (CPu) area of rat brain in vivo exposed to nicotine (3 mg/day; oral) and passive cigarette smoking (500 ml each; 3 times/day) for 1, 4, and 12 weeks, respectively. Our results revealed DA D(1) R localization in the presynaptic and postsynaptic dendrites, endocytic vesicles, and secretory granules, and DA D(2) R localization in the presynaptic dendrites and vesicles. DA D(1) R immunogold particles were highly decreased in the secretory granules of CPu, and increased in the postsynaptic area and vesicles after prolonged nicotine and smoking exposures, suggesting the strong influence of long time smoking and nicotine exposures on DA D(1) R subcellular organelle localization. DA D(2) R immunoreactivity was comparatively less changed than that of the DA D(1) R. Western blot analysis also showed the differential expression of DA D(1) and D(2) R proteins upon nicotine and smoking exposures as compared to the untreated controls. Taken together, the results for the first time suggests the execution of addictive behavior of nicotine through modulation of mesolimbic dopaminergic system targeting subcellular organelle of DA D(1) and D(2) Rs in the CPu of adult rat brain that may lead to novel therapeutic approaches related to nicotine's neuropsychological disorders including drug addiction.

In vivo and in vitro ethanol exposure in prenatal rat brain: GABA(B) receptor modulation on dopamine D(1) receptor and protein kinase A.

We have investigated the effects of prenatal ethanol exposure on GABA(B) receptors (GABA(B)Rs), protein kinase A (PKA), and DA D(1) receptor (DAD(1)R) expressions. GABA(B1)R and GABA(B2)R showed different age-dependent expressions in in vivo fetal rat forebrain from gestational days (GD) 15.5 to 21.5 upon 10% ethanol treatment to mother, with and without baclofen at a dose of 10 mg/kg body weight/day. The protein level changes could not be attributed to changes in the level of transcription since GABA(B)R mRNA presented different expression patterns upon in vivo ethanol treatment. Using in vitro cultivated cortical neurons from GD 17.5 fetuses, we also explored the modulatory effects of ethanol on PKA and DAD(1)R through GABA(B)Rs, under 50 microM baclofen and 100 microM phaclofen administrations, with or without 100 mM of ethanol treatment in the culture media. The results showed that 20 min ethanol treatment without baclofen or phaclofen had increasing effects on both the GABA(B)Rs. Further, baclofen and phaclofen administration significantly affected PKA and GABA(B)R levels upon 20 min and 1 h ethanol treatment. In contrast, DAD(1)R showed increasing effects upon ethanol treatment, which was modulated by GABA(B)R's agonist baclofen and antagonist phaclofen. Therefore the present study suggested that the GABA(B)R activity could modulate ethanol's cellular effects, which possibly including PKA and DAD(1)R activities, and may be an underlying cause of ethanol's effects.

Activity of sulphate reducing bacteria according to COD/SO4(2-) ratio of acrylonitrile wastewater containing high sulphate.

This study was performed to evaluate the biodegradability of acrylonitrile wastewater, microbial inhibition effect of acrylonitrile wastewater on removal efficiency and the activity of sulphate reducing bacteria (SRB) according to COD/sulphate ratio. Acrylonitrile wastewater was hardly biodegradable in a biodegradability test, however, SRB activity was 57% for overall consumption of electron donor and it was relatively high value compared to 17% of reference test with glucose. COD removal of acrylonitrile wastewater was improved to 57% and 61% from 20% as the COD/sulphate ratio were 0.5 and 0.3 by sulphate addition to acrylonitrile wastewater. First order reaction rate constants k on organic removal of acrylonitrile wastewater were 0.001, 0.004 and 0.004 at each COD/sulphate ratio of 0.9, 0.5 and 0.3. Thus it was suggested that the activity of SRB was a significant factor for removing organics and sulphate simultaneously in acrylonitrile wastewater.

Methylation of O(6)-methylguanine-DNA methyltransferase gene is associated significantly with K-ras mutation, lymph node invasion, tumor staging, and disease free survival in patients with gastric carcinoma.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase (MGMT) can remove O(6)alkylG DNA adducts. If they are not removed, then the adducts mispair with T during DNA replication, resulting in G-to-A mutation. Interrelations between MGMT gene inactivation by promoter methylation, K-ras mutation, and clinicopathologic features in patients with gastric carcinoma were studied. METHODS: Surgically removed tumor tissues from 79 patients were analyzed with MGMT methylation by genomic DNA modification and methylation specific polymerase chain reaction analysis, K-ras mutation by mutant allele specific amplification, TNM classification according to the International Union Against Cancer system, and MGMT protein expression by immunohistochemistry. RESULTS: MGMT-promoter methylation was found in 18 of 79 tumors. Among those 18 tumors, K-ras mutations were found in 33% and 11% of tumors at codons 12 and 13, respectively, corresponding to 20 times and 7 times greater rates of mutation compared with unmethylated tumors. MGMT methylation was associated significantly with lymph node invasion (P < 0.01), tumor stage (P < 0.03) and 5-year disease free survival (P < 0.02). MGMT protein expression was detected in intestinal metaplasia and adenocarcinoma samples, whereas no expression was detected in normal foveolar cells. CONCLUSIONS: MGMT-promoter methylation in patients with gastric carcinoma was associated significantly with point mutations of K-ras at codons 12 and 13, lymph node invasion, tumor stage, and disease free survival. These associations indicate a significant role of MGMT methylation during gastric carcinogenesis.

Coupling of L-type voltage-sensitive calcium channels to P2X(2) purinoceptors in PC-12 cells.

Extracellular ATP elevates cytosolic Ca(2+) by activating P2X and P2Y purinoceptors and voltage-sensitive Ca(2+) channels (VCCCs) in PC-12 cells, thereby facilitating catecholamine secretion. We investigated the mechanism by which ATP activates VSCCs. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP) and UTP were used as preferential activators of P2X and P2Y, respectively. Nifedipine inhibited the ATP- and 2-MeS-ATP-evoked cytosolic Ca(2+) concentration increase and [(3)H]norepinephrine secretion, but not the UTP-evoked responses. Studies with Ca(2+) channel blockers indicated that L-type VSCCs were activated after the P2X activation. Mn(2+) entry profiles and studies with thapsigargin revealed that Ca(2+) entry, rather than Ca(2+) release, was sensitive to nifedipine. Although P2X(2) and P2X(4) receptor mRNAs were detected, studies with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid revealed that P2X(2) was mainly coupled to the L-type VSCCs. The inhibitory effect of nifedipine did not occur in the absence of extracellular Na(+), suggesting that Na(+) influx, which induces depolarization, was essential for the P2X(2)-mediated activation of VSCCs. We report that depolarization induced by Na(+) entry through the P2X(2) purinoceptors effectively activates L-type VSCCs in PC-12 cells.

Sequential changes in hepatocarcinogenesis induced by diethylnitrosamine plus thioacetamide in Fischer 344 rats: induction of gankyrin expression in liver fibrosis, pRB degradation in cirrhosis, and methylation of p16(INK4A) exon 1 in hepatocellular carcinoma.

To clarify the sequential changes in pRB and p16 during different stages of hepatocarcinogenesis such as fibrosis, cirrhosis, hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC), male Fischer 344 rats were singly injected with diethylnitrosamine (DEN), immediately followed with phenobarbital for 1 wk and then thioacetamide (TAA) for 39 wk in drinking water. Rats were killed at 9, 20, 30, and 40 wk after DEN initiation and changes of pRB level, p16 gene hypermethylation, and in vivo gankyrin expression were examined. Histologic examination showed stepwise appearances of fibrosis, cirrhosis, HCA, and HCC at weeks 9, 20, 30, and 40, respectively. Hypermethylation of p16 exon 1 was not found until HCA but appeared in 50% of the rats with HCC accompanied by complete loss of its mRNA expression. The amount of glutathione S-transferase--gankyrin bound to pRB and pRB degradation in the liver depended on the concentration of gankyrin and incubation time. Gankyrin expression preceded pRB degradation in liver cirrhosis. In conclusion, gankyrin expression induced in liver fibrosis accelerated the degradation of pRB during liver cirrhosis, and inactivation of p16 exon 1 by DNA hypermethylation occurred during the progression of tumor cells to poorly differentiated HCC. Inactivation of pRB and/or p16 resulted in complete loss of regulation in the cell-division cycle during early and late stages, respectively, of hepatocarcinogenesis. Mol. Carcinog. 30:138--150, 2001.

Noncompetitive inhibition by camphor of nicotinic acetylcholine receptors.

The effect of camphor, a monoterpenoid, on catecholamine secretion was investigated in bovine adrenal chromaffin cells. Camphor inhibited [3H]norepinephrine ([3H]NE) secretion induced by a nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), with a half-maximal inhibitory concentration (IC50) of 70 +/- 12 microM. In addition, camphor inhibited the rise in cytosolic calcium ([Ca2+]i) and sodium ([Na+]i) induced by DMPP with IC50 values of 88 +/- 32 and 19 +/- 2 microM, respectively, suggesting that the activity of nAChRs is also inhibited by camphor. On the other hand, binding of [3H]nicotine to nAChRs was not affected by camphor. [Ca2+]i increases induced by high K+, veratridine, and bradykinin were not affected by camphor. The data suggest that camphor specifically inhibits catecholamine secretion by blocking nAChRs without affecting agonist binding.

Selective left-lobe atrophy by nodularin treatment accompanied by reduced protein phosphatase 1/2A and increased peroxisome proliferation in rat liver.

The effect of nodularin on selective atrophy of left lobes in the liver was investigated in F344 rats. Nodularin was injected for 10 weeks from the third week of initiation with saline or N-nitrosodiethylamine (DEN), grouped as S/N and D/N, respectively. Nodularin significantly decreased weights of left (LL) and caudate (CL) lobes but increased right (RL) and middle (ML) lobes in S/N rats. Activity of protein phosphatases [types 1 (PPI) and 2A (PP2A)] was more severely reduced in S/N than D/N rats; moreover, in LL compared with RL of S/N rats, activity was significantly inhibited by nodularin treatment from week 4, which corresponded to 2 weeks after nodularin injection. However, nodularin significantly induced peroxisomal palmitoyl-CoA oxidase and cytochrome P-450 4A1 expression in S/N compared with D/N rats. An effect of nodularin on apoptosis was evident since expression of Bcl-Xs was clearly induced in LL of S/N rats as opposed to various inductions of Bcl-XL. However, Bcl-XL in RL was persistently induced, with undetectable Bcl-Xs expression. These results demonstrate biochemical evidence of selective atrophy of LL by inhibition of PP1 and PP2A activity, increase of peroxisomal enzymes and induction of Bcl-Xs expression, in contrast to proliferation of RL in rats treated with nodularin alone. However, nodularin endowed DEN-altered hepatocytes with regenerating power and concomitant restoration of phosphatase activity as well as persistent expression of Bcl-XL in D/N rats.

Phosphorylation of methylated-DNA-protein-cysteine S-methyltransferase at serine-204 significantly increases its resistance to proteolytic digestion.

In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125, 493-499], we showed two major forms of active DNA-6-O-methylguanine:protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24 kDa species. Here we show that a 2 kDa C-terminal fragment was cleaved from the 26 kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser(204) of the 26 kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser(204) to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca(2+)-independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).

Inhibition of nicotinic receptor-mediated catecholamine secretion by Dryobalanops aromatica in bovine adrenal chromaffin cells.

Effect of the aqueous extract from a medicinal plant Dryobalanops aromatica(Dipterocarpaceae) on catecholamine secretion was investigated in bovine adrenal chromaffin cells. The aqueous extract inhibited [(3)H]norepinephrine ([(3)H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, with a half-maximal inhibitory concentration (IC(50)) of 8.4 +/- 1.7 microgml(-1). Increases in cytosolic calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) induced by DMPP were also inhibited by the extract. However, the binding of [(3)H]nicotine to nAChRs was not affected by the addition of the extract in receptor binding competition analysis, suggesting that active components in the extract and nicotine do not share the binding site in the nAChR. On the other hand, [Ca(2+)](i)increases induced by high K(+), ionomycin, bradykinin, angiotensin II, and thapsigargin were not inhibited by the extract. The data suggest that the extract from D. aromatica specifically inhibits catecholamine secretion by blocking nAChR in a noncompetitive manner.

Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells.

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.

Regulation of selection of liver nodules initiated with N-nitrosodiethylamine and promoted with nodularin injections in fischer 344 male rats by reciprocal expression of transforming growth factor-beta1 and its receptors.

To investigate how glutathione-S-transferase placental form (GST-P)+ hyperplastic nodules (HNs) are selected and to determine the driving force for progression or regression of HNs, changes in transforming growth factor-beta1 (TGF-beta) and its receptors were examined during hepatocarcinogenesis initiated by N-nitrosodiethylamine (DEN) and promoted by nodularin. The induction of TGF-beta1 expression in the GST-P+ HNs was dependent on nodularin injections for 10 wk, which started the third week after DEN initiation. The kinetics of TGF-beta1 induction during carcinogenesis were quite different from that of simple regeneration after partial hepatectomy (PH): hepatocytes initiated with DEN alone induced TGF-beta1 expression for 24 d, and subsequent stimulation by PH on the fourteenth day after DEN initiation super-induced TGF-beta1 mRNA (50 times that of the control level), as opposed to a transient expression for less than 5 d by PH alone. GST-P+ HNs did not express TGF-beta receptors I (RI) and II (RII) during the early stage of carcinogenesis, whereas the surrounding hepatocytes strongly expressed both of these receptors. On cessation of nodularin injection, however, the expression of RI and RII in the HNs changed significantly: RII+ nodules appeared, and the number and area of RII+/- nodules were significantly increased at 10 wk after the cessation. These findings indicate that induction of TGF-beta expression in GST-P+ HNs might be a strong selection pressure that allows outgrowth of RII- nodules during liver carcinogenesis.

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.

Phospholipase C-delta1 is activated by capacitative calcium entry that follows phospholipase C-beta activation upon bradykinin stimulation.

To characterize the regulatory mechanism of phospholipase C-delta1 (PLC-delta1) in the bradykinin (BK) receptor-mediated signaling pathway, we used a clone of PC12 cells, which stably overexpress PLC-delta1 (PC12-D1). Stimulation with BK induced a significantly higher Ca(2+) elevation and inositol 1,4,5-trisphosphate (IP(3)) production with a much lower half-maximal effective concentration (EC(50)) of BK in PC12-D1 cells than in wild type (PC12-W) or vector-transfected (PC12-V) cells. However, BK-induced intracellular Ca(2+) release and IP(3) generation was similar between PC12-V and PC12-D1 cells in the absence of extracellular Ca(2+), suggesting that the availability of extracellular Ca(2+) is essential to the activation of PLC-delta1. When PC12-D1 cells were treated with agents that induce Ca(2+) influx, more IP(3) was produced, suggesting that the Ca(2+) entry induces IP(3) production in PC12-D1 cells. Furthermore, the additional IP(3) production after BK-induced capacitative calcium entry was detected in PC12-D1 cells, suggesting that PLC-delta1 is mainly activated by capacitative calcium entry. When cells were stimulated with BK in the presence of extracellular Ca(2+), [(3)H]norepinephrine secretion was much greater from PC12-D1 cells than from PC12-V cells. Our results suggest that PLC-delta1 is activated by capacitative calcium entry following the activation of PLC-beta, additively inducing IP(3) production and Ca(2+) rise in BK-stimulated PC12 cells.

Differential expression of O6-methylguanine-DNA methyltransferase during diethylnitrosamine-induced carcinogenesis and liver regeneration in Sprague-Dawley male rats.

Differential expression of DNA-O6MeG: protein-L-cysteine S-methyltransferase (MGMT) activity and posttranslational modification of the protein during liver regeneration and carcinogenesis were compared in Sprague-Dawley male rats after partial hepatectomy and/or single i.p injection of diethylnitrosamine (DEN, 200 mg/kg). Regenerating hepatocytes after partial hepatectomy induced MGMT transiently within 3 days; however, the induction of MGMT was persistent for 2 weeks after DEN injection, and the combined treatment of DEN and partial hepatectomy maintained the elevated MGMT level for up to 4 weeks. The increased activity was transcriptionally regulated, when analyzed by Northern blot hybridization. The major active form of MGMT protein in the partially hepatectomized or DEN-treated rats was a 26-kDa or 24-kDa species respectively, which was confirmed by Western blot analysis and gel slice assay. The biological significance of the differential induction of MGMT during partial hepatectomy or DEN-induced carcinogenesis is not obvious; however, further studies on possible posttranslational modifications of MGMT protein might shed some light on the functional aspect of MGMT induction.