PET findings in a brain abscess associated with a silent atrial septal defect

Brain abscesses are classical complications of congenital heart disease (CHD) in children and adolescents. This association is rarely observed in adults. We report a 46-year-old man presenting a fronto-parietal abscess associated with an asymptomatic atrial septal defect. Positron emission tomography (PET) study revealed high uptake of L-[methyl-11C]methionine ([11C]methionine) and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) around the brain abscess. We suggest (1) to exclude a silent cardiac malformation in the presence of a cerebral abscess of unknown source occurring in adults; (2) to consider the diagnosis of brain abscess in cases of high uptake of [11C]methionine and FDG in relation to a brain lesion.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Purification and metal ion requirements of a candidate matrix metalloproteinase: a 41 kDa gelatinase activity in the sea urchin embryo

[11C]L-159,884 ([11C] N-[[4'[(2-ethyl-5,7-dimethyl-3H- imidazo[4,5-b]pyridin-3-yl) methyl] [1,1'-biphenyl]-2-yl] sulfonyl]-4-methoxybenzamide) and [11C]L-162,574 ([11C] N-[[4'[2-ethyl-5,7- dimethyl-3H-imidazo[4,5-b] pyridin-3-yl)methyl] [1,1'-biphenyl]-2-yl]sulfonyl]-3- methoxybenzamide), both potent and selective ligands for the AT1 receptor, were prepared by C-11 methylation of the corresponding desmethyl phenolic precursors. The radiotracers were purified by semi-preparative reverse-phase HPLC. Non-decay corrected radiochemical yields were 5 and 3% for L-159,884 and L-162,574 respectively, and the average specific activity was 2979 mCi/mumol at end-of-synthesis (EOS). The average time of synthesis was 18 min.     

硼替佐米作用不同时间对K562耐药细胞株核因子-κ B途径的影响

目的 观察硼替佐米(商品名:万珂,PS-341)对柔红霉素(DNR)诱导的K562耐药细胞株(K562/DNR)核因子-κ B(NF-κ B)、抑制蛋白κ B(I κ B)及P-糖蛋白(P-gp)表达的影响,探讨PS-341逆转耐药的分子机制.方法 以100μg/ml DNR单用或联合应用4μg/L PS-341分别作用于K562/DNR 12、24及36 h,检测不同时间点各组NF-κ B、Iκ B及P-gp表达情况,同时测定NF-κ B p65活性,检测各组细胞凋亡率.结果 Western blot结果显示:与阴性对照组相比,DNR可诱导NF-κ B表达上调及活性增强、I κ B表达下调、P-gp表达上凋;加用PS-341可显著抑制DNR诱导的NF-κ B及P-gp表达,使I κ B表达增加.加用PS-341后,NF-κ B活性12 h为(15.3±1.87)%[DNR组为(23.8±2.27)%],24 h为(10.2±1.69)%[DNR组为(25.4±1.98)%],36 h为(6.08±2.53)%[DNR组为(26.9±2.58)%],与相应单用DNR组相比均有明显下降,差异有统计学意义(P值均＜0.05).DNR与PS-341联用后,细胞凋亡率12 h为(35.23±5.15)%[DNR组为(15.56±4.12)%],24 h为(40.26±6.89)%[DNR组为(17.25±2.89)%],36 h为(43.58±7.69)%[DNR组为(22.47±4.58)%],与DNR组相比,细胞凋亡率均明显增加,差异具有统计学意义(P值均＜0.05).上述作用呈时间依赖性.结论 PS-341可减少K562/DNR细胞NF-κ B的活化,降低P-gp表达,逆转细胞耐药,促进细胞凋亡.     

An automated liquid chromatographic plasma analysis of amino acids used in combination with positron emission tomography (PET) for determination of in vivo plasma kinetics

Quantification of physiological processes measured with positron emission tomography (PET) requires an "input" function which can be the concentration of administered radio-tracer in plasma. Radioactive nuclides used in PET have short half lives (2-20 min) and a limited time is available for the PET investigation including analysis of the composition of the radioactive signal in plasma. Therefore, an automated method for the analysis and separation of beta-[11C]-L-5-hydroxy-tryptophan ([11C]-L-5-HTP), beta-[11C]-L-3,4-dihydroxy-phenylalanine ([11C]-L-DOPA) and L-[methyl-11C]-methionine and their respective metabolites in plasma was developed. A size exclusion exclusion column was used for isolation of the low molecular weight fraction. In the case of [11C]-L-5-HTP and [11C]-L-DOPA, the low molecular weight fraction was injected onto a liquid chromatographic system for separation of radioactive tracer from in vivo formed radio-labelled metabolites. The elution volume from the size exclusion column was 7.0, 5.0 and 3.5 ml for [11C]-L-5-HTP, [11C]-L-DOPA and L-[methyl-11C]-methionine, respectively. An interaction with the column matrix and the solutes was observed for both [11C]-L-5-HTP and [11C]-L-DOPA. The yield in the isolation step was > 98%. Separation of [11C]-L-5-HTP and [11C]-L-DOPA from their respective metabolites was performed with high-performance liquid chromatography with automated collection of fractions of the eluate corresponding to those of administered tracer and metabolites. The fractions were measured for radioactivity in a well counter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of carbon-11-methionine is enhanced by insulin

The anabolic effects of insulin are not restricted to carbohydrate and lipid metabolism but also include protein metabolism. However, the effects of insulin on protein metabolism have been difficult to demonstrate in vivo. Amino acid transport is partly regulated by insulin according to the experimental data. PET provides a way to measure fractional uptake rates of amino acids. The purpose of this study was to measure the effect of insulin on amino acid transport from the plasma to the human parotid glands. METHODS: We compared the uptake of L-[methyl-11C]methionine ([11C]methionine) into the parotid glands and cerebellum in seven healthy volunteers during the fasting state and euglycemic insulin clamp technique (1 mU/kg per minute). RESULTS: The fractional uptake rate of [11C]methionine was increased by 31% for the right parotid gland (p = 0.003) and by 29% for the left parotid gland (p = 0.009) during insulin clamp, while the increase was 19% for the cerebellum (p = 0.01). The concentration of amino acids typical for the hormone-sensitive transport system A was 11% lower during insulin infusion than in the fasting state. CONCLUSION: The uptake of methionine into brain tissue does not seem to be under major control by insulin, while the transport of methionine in the parotid glands is stimulated by insulin. PET provides a sophisticated method to study the transport system of amino acids in vivo.     

Methylation status of DNA cytosine during the course of induction of liver cancer in hamsters by hydrazine sulfate

Hydrazine, which is toxic and carcinogenic to rodent liver, has been shown to react with endogenous formaldehyde in the liver to form formaldehyde hydrazone (CH2 = N-NH2), an alkylating intermediate that methylates DNA guanine at the N7- and O6-positions. Studies were conducted to investigate the role of chronic hydrazine-induced hepatotoxicity on DNA maintenance methylation (formation of 5-methyldeoxycytosine) and the development of liver cancer. Male Syrian golden hamsters were given hydrazine sulfate (0, 170, 340 and 510 mg/l) in drinking water for 21 months (average dose 0, 4.2, 6.7 and 9.8 mg/kg body wt hydrazine as the free base). Hepatotoxicity was evaluated histologically, and regenerative DNA synthesis and maintenance methylation were measured as the incorporation of [methyl-14C]thymidine into DNA and the methyl moiety of [methyl-3H]methionine into 5-methyldeoxycytosine in DNA, respectively. Methylguanines were detected in liver DNA at the first observation time of 6 months of treatment; levels of these aberrant bases decreased or became undetectable at 14 months, and increased in a dose-related manner for the remainder of the study. DNA adducts persisted in the highest dose group throughout the study, repeating the results of a similar study previously reported by this laboratory (Bosan et al., Carcinogenesis, 8, 439-444, 1987). Linear regression analysis of thymidine and methionine methyl moiety incorporation into liver DNA suggested impairment of maintenance methylation of DNA (5-methyldeoxycytosine) in the middle and high exposure animals. Hepatic adenomas and hepatocellular carcinomas developed in a dose-related manner and were highly correlated to decreased uptake of radiolabel from methionine into DNA 5-methylcytosine. These results are part of a continuing study on alteration of maintenance methylation during hydrazine induction of liver cancer.     

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).     

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.     

Usefulness of aspiration surgery for elderly patients with hypertensive cerebellar hemorrhage

Synthesis of 1-[11C]methylpiperidin-4-yl propionate ([11C]PMP), an in vivo substrate for acetylcholinesterase, is reported. An improved preparation of 4-piperidinyl propionate (PHP), the immediate precursor for radiolabeling, was accomplished in three steps from 4-hydroxypiperidine by (a) protection of the amine as the benzyl carbamate, (b) acylation with propionyl chloride, and (c) deprotection of the carbamate by catalytic hydrogenation. The final product was obtained in an overall 82% yield. Reaction of the free base form of PHP with [11C]methyl trifluoromethanesulfonate at room temperature in N,N-dimethylformamide, followed by high performance liquid chromatography (HPLC) purification, provided [11C]PMP in 57% radiochemical yield, > 99% radiochemical purity, and > 1500 Ci/mmol at the end of synthesis. The total synthesis time from end-of-bombardment was 35 min. [11C]PMP can thus be reliably prepared for routine clinical studies of acetylcholinesterase in human brain using positron emission tomography.     

Formation of methylated and phosphorylated metabolites during the fermentation process of verdamicin

In an attempt to understand the biosynthetic processes leading to the formation of verdamicin (end product), we have examined the patterns of the formation of methylated and phosphorylated metabolites, which resulted from either the addition of l-[methyl-(14)C]methionine or [(32)P]KH(2)PO(4) to the fermentation. Incorporation of label from l-[methyl-(14)C]methionine into the bioactive sisomicin, verdamicin, and the chromatographically polar components increased with the progression of time. Two methylated bioinactive metabolites were found in the culture broth after removal of the methylated bioactive metabolites. In contrast to the bioactive metabolites, incorporation of the methyl-(14)C label into the two methylated bioinactive metabolites decreased with the progression of time. A phosphorylated bioinactive metabolite (nonmethylated) was also found in the culture broth, fermented in the presence of [(32)P]KH(2)PO(4). The role of the phosphorylated metabolite in the biosynthesis of the bioactive metabolites cannot yet be explained.     

Utilization of phosphocholine from extracellular complex polysaccharide as a source of cytoplasmic choline derivatives in Penicillium fellutanum

Penicillium fellutanum produces a phosphorylated, choline-containing extracellular polysaccharide, peptidophosphogalactomannan (pP(x)GM) [where x is the number of phosphodiester residues]). The 13C-methyl-labeled pP(x)GM ([methyl-13C]pP(x)GM) was prepared from the cultures supplemented with L-[methyl-13C]methionine and was used as a probe to monitor the fate of phosphocholine in this polymer. The addition of [methyl-13C]pP(x)GM to growing cultures in low-phosphate medium resulted in the disappearance within 5 days of [methyl-13C]phosphocholine and N,N'-dimethylphosphoethanolamine from the added [methyl-13C]pP(x)GM. Two 13C-methyl-enriched cytoplasmic solutes, choline-O-sulfate and glycine betaine, were found in mycelial extracts, suggesting that phosphocholine-containing extracellular pP(x)GM of P. fellutanum is a precursor of intracellular choline-O-sulfate and glycine betaine. The mycelia cultured in low-phosphate (2 mM) medium contained glycine betaine and 1.5-fold more choline-O-sulfate than those grown in high-phosphate (20 mM) medium. The high levels of extracellular nonspecific phosphocholine:phosphocholine hydrolase and acid phosphomonoesterase observed in the low-phosphate culture medium are likely related to the release of phosphocholine from pP(x)GM and hydrolysis of phosphocholine, respectively. These results suggest that extracellular pP(x)GM of P. fellutanum provides phosphate needed as the environment becomes depleted of this nutrient. Choline, in excess of that needed immediately, is stored in the cytoplasm in forms that can be reutilized.     

Validation of 19F-magnetic resonance determination of myocardial blood volume

Site-specific regeneration of the liver after 70% partial hepatectomy was investigated noninvasively in terms of protein synthesis using PET with L-[methyl-11C]methionine in a living animal. Protein synthesis in rat liver at 24 hr after partial hepatectomy did not occur uniformly in the whole liver but intensely in the middle part of the regenerating organ in comparison with the other parts. The activity was significantly low at the posterior aspect of the liver. On the other hand, site-specific protein synthesis was not observed in normal liver. These results were confirmed by bioimaging analyzer system (BAS) analysis, an invasive method that indicates radioactivities of precise intrahepatic sites. DNA synthesis in regenerating liver was also monitored with [2-14C]thymidine and analyzed by BAS 24 hr after 70% hepatectomy. Site-specific DNA synthesis in regenerating liver corresponding to the protein synthesis was also observed by BAS analysis. These results indicate that liver regeneration occurs intensely in the middle part of the liver and that PET enables noninvasive in vivo biochemical analysis.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Studies on substances with antiblastic activity. IX. Anthramycin and analogs. IX. Synthesis of 7-methoxy-8-hydroxy-10,11-dihydro-5H-pyrrolo[2,1-c] [1,4] benzodiazepine and other compounds related to oxotomaymycin

The synthesis of some pyrrolobenzodiazepine derivatives related to oxotomaymycin, an antibiotic recovered together with tomaymycin from fermentation broths of Streptomyces achromogenes var. tomaymycetics, is described. Reaction between 2-nitro-4-benzyloxy-5-methoxybenzylbromide and pyrrole-2-carboxyaldehyde afforded 1-(2-nitro-4-benzyloxy-5-methoxybenzyl)pyrrole-2-carboxyaldehyde. Catalytic reduction of this compound with hydrogen in the presence of Pd/C gave 10,11-dihydro-8-hydroxy-7-methoxy-5H-pyrrolo[2.1-c] [1,4]benzodiazepine. Amides obtained from condensation between 2-nitro-4-benzyloxy-5-methoxybenzoic acid chloride and proline or hydroxyproline were reduced catalytically to 2,3-dihydro-8-hydroxy-7-methoxy-1H-pyrrolo [2,1-c] [1,4]benzodiazepine-5,11 (10H, 11aH)-dione and its 2-hydroxyderivative respectively. The synthesis of 10,11-dihydro-8-hydroxy-9-methoxy-5-pyrrolo [2,1-c] [1,4]benzodiazepine is also reported.     

Effect of the multidrug inhibitor GG918 on drug sensitivity of human leukemic cells

The drug GG918 has been specifically developed for overcoming MDR phenotype and is now in use in clinical trials. In this study, the effects of GG918 on leukemic cell were investigated using a 3 day MTT assay. Results showed that, in a highly resistant P-gp(+) leukemic cell line, 0.1 microM of GG918 gives rise to a 40-fold sensitization to daunorubicin (DNR) (residual resistance: 2.1), a 57-fold sensitization to mitoxantrone (residual resistance: 1.5), and a 3.3-fold sensitization to idarubicin (residual resistance: 2.9). When human AB serum was added to the incubation medium, 1 microM of GG918 was needed to observe the full P-gp modulation potency described above. The effect of 1 microM of GG918 was tested on 27 samples of poor prognosis acute leukemia (25 AML, two ALL). DNR sensitization (using the MTT assay) and modulation of rhodamine 123 uptake were monitored and used as criteria for comparing the in vitro modulation potency of this new compound to the potency of 10 microM of verapamil, which was used as reference. A good correlation (r = 0.8, P = 0.001) was observed between the results of the two tests. Eleven out of the 26 cases tested were MDR1(+) (42%), and showed a higher IC50 for DNR than the negative cases (861 +/- 1284 nM vs 187 +/- 246 nM, P = 0.05). GG918 was able to modulate the in vitro resistance to DNR in eight cases (seven MDR1(+), no MDR1(-), one non-tested). Verapamil did not increase DNR toxicity in four of these eight cases, but was more efficient in one other MDR1(+) case. In conclusion, the DNR sensitivity of the majority of the fresh AML samples expressing P-gp could be modulated in vitro by 1 microM of GG918.     

Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation

The organoselenium compounds benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC), as well as sodium selenite, are effective chemopreventive agents for various chemically induced tumors in animal models at both the initiation and postinitiation stages. The mechanisms involved at the postinitiation stage are not clear. Because several lines of evidence indicate that inhibition of excess DNA (cytosine-5)-methyltransferase (Mtase) may be a sufficient factor for the suppression or reversion of carcinogenesis, we examined the effects of sodium selenite, BSC, p-XSC and benzyl thiocyanate (BTC), the sulfur analog of BSC, on Mtase activity in nuclear extracts of human colon carcinomas, and of p-XSC on the Mtase activity of HCT116 human colon carcinoma cells in culture. For this purpose, we developed an improved Mtase assay, in which the incorporation of the methyl-[3H] group from S-adenosyl[methyl-3H]methionine into deoxycytidine of poly(dI-dC)-poly(dI-dC), is specifically determined by HPLC with radioflow detection after enzymatic hydrolysis, enhancing specificity and reliability. In a variation, using SssI methyltransferase and labeled S-adenosylmethionine, the overall methylation status of DNA in various tissues can also be compared. Selenite, BSC and p-XSC inhibited Mtase extracted from a human colon carcinoma with IC50s of 3.8, 8.1 and 5.2 microM, respectively; BTC had no effect. p-XSC also inhibited the Mtase activity and growth of human colon carcinoma HCT116 cells, with an IC50 of approximately 20 microM. The improved Mtase assay should prove to be a reliable method for screening potential Mtase inhibitors, especially using cells in culture. We suggest that inhibition of Mtase may be a major mechanism of chemoprevention by selenium compounds at the postinitiation stage of carcinogenesis.     

Imaging epileptogenic tubers in children with tuberous sclerosis complex using alpha-[11C]methyl-L-tryptophan positron emission tomography

Several reports have indicated that cortical resection is effective in alleviating intractable epilepsy in children with tuberous sclerosis complex (TSC). Because of the multitude of cortical lesions, however, identifying the epileptogenic tuber(s) is difficult and often requires invasive intracranial electroencephalographic (EEG) monitoring. As increased concentrations of serotonin and serotonin-immunoreactive processes have been reported in resected human epileptic cortex, we used alpha-[11C]methyl-L-tryptophan ([11C]AMT) positron emission tomography (PET) to test the hypothesis that serotonin synthesis is increased interictally in epileptogenic tubers in patients with TSC. Nine children with TSC and epilepsy, aged 1 to 9 years (mean, 4 years 1 month), were studied. All children underwent scalp video-EEG monitoring, PET scans of glucose metabolism and serotonin synthesis, and EEG monitoring during both PET studies. [11C]AMT scans were coregistered with magnetic resonance imaging and with glucose metabolism scans. Whereas glucose metabolism PET showed multifocal cortical hypometabolism corresponding to the locations of tubers in all 9 children, [11C]AMT uptake was increased in one tuber (n=3), two tubers (n=3), three tubers (n=1), and four tubers (n=1) in 8 of the 9 children. All other tubers showed decreased [11C]AMT uptake. Ictal EEG data available in 8 children showed seizure onset corresponding to foci of increased [11C]AMT uptake in 4 children (including 2 with intracranial EEG recordings). In 2 children, ictal EEG was nonlocalizing, and in 1 child there was discordance between the region of increased [11C]AMT uptake and the region of ictal onset on EEG. The only child whose [11C]AMT scan showed no regions of increased uptake had a left frontal seizure focus on EEG; however, at the time of his [11C]AMT PET scan, his seizures had come under control. [11C]AMT PET may be a powerful tool in differentiating between epileptogenic and nonepileptogenic tubers in patients with TSC.     

Increasing incidence of childhood class V lupus nephritis

1. Homozygously mdr1a gene disrupted mice (mdr1a(-/-) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo. 2. [11C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P-gp function, cyclosporin A was administered. 3. Biodistribution studies revealed 9.5-fold (P < 0.001) and 3.4-fold (P < 0.001) higher [11C]verapamil in the brain and testes of mdr1a(-/-) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [11C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3-fold (P < 0.01 (brain); P < 0.001 (testes)). Fifty mg/kg cyclosporin A increased [11C]verapamil in the brain 10.6-fold (P < 0.01) and in the testes 4.1-fold (P < 0.001). No increases were found in the mdr1a(-/-) mice. This indicates complete inhibition of P-gp mediated [11C]verapamil efflux. 4. Positron camera data showed lower [11C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(-/-) mice. [11C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(-/-) mice, indicating that reversal of P-gp mediated efflux can be monitored by PET. 5. We conclude that cyclosporin A can fully block the P-gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P-gp function and reversal of its function non-invasively.