Cadherin switch from E- to N-cadherin in melanoma progression is regulated by the PI3K/PTEN pathway through Twist and Snail.

BACKGROUND: Transition of normal melanocytic cells to malignant melanoma has characteristic features of epithelial to mesenchymal transition. This includes the disruption of the adherens junctions caused by the downregulation of E-cadherin and the upregulation of N-cadherin. The cadherins have functional importance in normal skin homeostasis and melanoma development; however, the exact mechanism(s) that regulate the 'cadherin switch' are unclear. OBJECTIVES: To determine the mechanistic role of the PI3K/PTEN pathway in regulating the change in cadherin phenotype during melanoma progression. METHODS: Using a panel of cell lines representative of the phases of melanoma progression, we determined cellular expressions of the components of the PI3K/PTEN pathway, E- and N-cadherin, and the transcriptional regulators Twist, Snail and Slug with Western blot and immunofluorescence analysis. Transcriptional regulation of E-cadherin, N-cadherin, Twist and Snail by the PI3K/PTEN pathway was confirmed using quantitative reverse transcription-polymerase chain reaction. RESULTS: Loss or inactivity of PTEN correlated with the switch in cadherin phenotype during melanoma progression. PTEN-null or inactive cells exhibited high levels of phosphorylated protein kinase B (PKB)/AKT (Serine 473) (PKB-Ser473-P), undetectable levels of E-cadherin and high levels of N-cadherin. Re-introduction of PTEN or treatment with the PI3K inhibitor Wortmannin resulted in the re-expression of E-cadherin and downregulation of N-cadherin. This cadherin switch was regulated at the transcriptional level by Twist and Snail which were, in turn, transcriptionally regulated by the PI3K pathway. Although E-cadherin was re-expressed, it failed to localize to the plasma membrane. CONCLUSIONS: The PI3K/PTEN pathway transcriptionally regulates the 'cadherin switch' via transcriptional regulation of Twist and Snail but does not regulate the localization of E-cadherin to the plasma membrane.

Drug utilization patterns in pregnant women: a case study at the Mount Hope Women's Hospital in Trinidad, West Indies.

OBJECTIVE: To explore drug (prescription, over-the-counter and herbal) utilization in pregnant women attending a public sector tertiary healthcare institution. METHODS: This was a cross-sectional case study in women attending antenatal clinics at the Mount Hope Women's Hospital. Women (506) who consecutively presented for routine care at the antenatal clinic were interviewed on the medication they took. Descriptive statistics and logistic regression for predictors of drug use were done using SPSS 16. RESULTS: There were 200 (39.5%) primigravidae, 306 (60.5%) multigravidae and 299 (59%) women were in the third trimester of pregnancy. Most women (69.8%) were between 20-35 years of age. Women took an average of 1.32, 1.22 and 0.94 prescribed drugs in each trimester respectively. Multivitamins (59.8%) and iron/folic acid (54.2%) were the most frequently prescribed drugs. Regardless of trimester only 20% of women took supplemental calcium. Very few women (2.4%) took herbal medications. Paracetamol was the most common over-the-counter (OTC) medication in all trimesters. Women with secondary level education were most likely to use OTC iron/folic acid (p = 0.02), paracetamol and histamine2 receptor antagonists [H2RAs] (p = 0.001). More primigravidae took non-steroidal anti-inflammatory drugs (p = 0.02) and more women in the first trimester used antiemetics (p = 0.001). Age group (p = 0.048), marital status (p = 0.001) and the trimester of pregnancy (p = 0.001) were predictors of drug utilization. CONCLUSION: Overall, women in tertiary healthcare institutions took medication as prescribed particularly multivitamins and iron/folic acid. More women with higher education took OTC paracetamol, iron/folic acid and vitamin supplements. Herbal supplements were rarely used. Research on drug utilization in primary care facilities is recommended.

Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Effects of ultraviolet-visible irradiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells.

The present study is an attempt to investigate the possibility that ultraviolet irradiation in the presence of pheomelanin may be more harmful to cells than the irradiation in the presence of eumelanin. The effects of UV-visible irradiation upon Ehrlich ascites carcinoma cells in the presence of the melanin isolated from human black hair (eumelanin) or from red hair (pheomelanin) were investigated. Irradiation of these cells was found to produce cell lysis, as observed by leakage of 51Cr from labeled cells and intracellular lactic dehydrogenase from the cells and decrease in cell viability demonstrated by the trypan blue exclusion test. The three parameters were quantitatively parallel to one another under various experimental conditions, namely different periods of irradiation and irradiation in the presence of different concentrations of melanin. The above effects were more pronounced when the irradiation was carried out in the presence of melanin from red hair than in the presence of black-hair melanin. In the absence of either melanin, the irradiation did not produce any significant effect in cell viability or cell lysis. Irradiation of the cells in the presence of red-hair melanin also decreased the transplantability of these cells. These observations clearly show that irradiation of cells in the presence of pheomelanin could produce cytotoxic effects. The present experimental design may have application in the development of in vitro models for the study of UV radiation-induced cutaneous carcinogenesis. The reactions of pheomelanin may be related to the susceptibility of "Celtic" skin to UV radiation-induced skin damage and carcinogenesis.

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

Effects of melanin-induced free radicals on the isolated rat peritoneal mast cells.

Pheomelanin from human red hair (RHM) produces considerably more cellular damage in Ehrlich ascites carcinoma cells when subjected to radiations of wavelength 320-700 nm than eumelanin from black hair (BHM). Irradiation of RHM generated large amounts of superoxide while BHM did not produce detectable amounts of superoxide. The present investigations describe the effects of irradiation of mast cells in the presence of various natural and synthetic melanins. Irradiation of mast cells in the presence of RHM and red hair melanoprotein released large amounts of histamine while BHM and synthetic melanins prepared from dopa, cysteinyldopa, or a mixture of dopa and cysteinyldopa did not release histamine. The release of histamine at lower concentrations of RHM was not accompanied by the release of 51Cr from chromium-loaded cells, suggesting that this release was of noncytotoxic nature. On the other hand, the release of histamine at higher concentrations of RHM was due to cell lysis since both histamine and cytoplasmic marker 51Cr were released to the same extent. The release evoked by large concentration RHM was not inhibited by superoxide dismutase or catalase. This suggests that the cell lysis under these conditions was not due to H2O2 or O-2. The finding that mast cells release histamine when irradiated in the presence of RHM suggests that the immediate and late-phase reactions seen in sunburn may in part be due to the release of mediators from these cells.

A paramagnetic agent causing ochronotic arthropathy.

Magnetic resonance imaging (MRI) identified a paramagnetic substance in the hyaline cartilage of the hips and knees in a patient with ochronosis. Chemical studies characterized the paramagnetic agent as melanin. The free radicals contained in melanin were shown to initiate cytotoxicity. The loss of cartilage in ochronotic arthropathy now can be explained at the electron level using the superoxide theory of oxygen toxicity. Inappropriate metabolism of oxygen also may explain early cartilage degeneration in hemochromatosis, hemosiderosis, and Wilson's disease.

A telemedicine consultative service for the evaluation of patients with urolithiasis.

OBJECTIVES: A 6-month pilot teleconsultative project linking Georgetown University Medical Center (GUMC) in Washington, DC, and City Hospital in Martinsburg, West Virginia, 90 miles away, was designed to assess the effectiveness of telemedicine on the clinical decision-making process for patients with urolithiasis. METHODS: The telemedicine system designed and tested for this project was based on a PC-based platform. Videoconferencing and review of the patient's imaging studies were performed over an Integrated Service Digital Network (ISDN) with 3 Basic Rate (BRI) ISDN lines providing a 336-kilobytes/s bandwidth through an Inverse Multiplexor (IMUX). Treatment options were recorded for the clinical trial group and a simulated study group by the consulting urologist after the initial telephone consultation, after the telemedicine consultation, and after examination of those patients transferred to GUMC. RESULTS: A total of 32 telemedicine consultations were performed: 14 in the clinical trial group and 18 in the simulated study group. The recommendation of the consulting urologist at the tertiary center was altered in 12 patients (37.5%) after the telemedicine consultation compared with the recommended treatment after the initial telephone consultation. CONCLUSIONS: In the evaluation of patients with urolithiasis, this telemedicine application enhanced the clinical decision-making process by allowing for improved quality of care through immediate access and effective transfer of information between the referring urologist, the patient, and the stone center specialist.

A comparison of the phototoxicity of protoporphyrin, coproporphyrin and uroporphyrin using a cellular system in vitro.

The photobiological effects of protoporphyrin (PP), coproporphyrin (CP) and uroporphyrin (UP) were investigated using an in vitro model. Suspensions of Ehrlich ascites carcinoma cells were labelled with 51Cr and irradiated in the presence of a wide range of concentrations of PP, CP and UP. It was found that PP was the most potent photosensitizer in this system; CP was less effective than PP and UP was the least potent. The cell lysis by CP was enhanced by superoxide dismutase (SOD) and inhibited by catalase; the lysis by UP was also inhibited by catalase; on the other hand, the lysis by PP was not affected by SOD or catalase. These indicate that the cell lysis by CP and UP was largely due to hydrogen peroxide produced from superoxide formed during the irradiation. The lysis produced by PP was not mediated by hydrogen peroxide. These differences in the mechanisms of the phototoxicity of the various porphyrins may have relevance in the etiology and treatment of the porphyrias.

Differences between depressed and nondepressed individuals in the recognition of and response to facial emotional cues.

The present study investigated the recognition of, and responses to, facial expressions of emotion. Participants were all women and consisted of the following groups: (a) Sixteen depressed college students; (b) 16 nondepressed college students; (c) 16 depressed psychiatric patients; and (d) 11 nondepressed psychiatric patients. Results suggest that both depressed groups, relative to the nondepressed college group, made more errors in recognizing the facial expressions and reported more freezing or tensing; higher fear and depression reactions; and less comfort with their own emotional reactions to these expressions and a stronger desire to change these reactions. Few differences were found between the depressed psychiatric patients and the psychiatric control subjects. It is concluded that inappropriate reactions to others' emotions may maintain or increase depression.

Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin.

Free porphyrins are strong photosensitizers. Previously reported findings indicate that the in vitro cell lysis induced by irradiation in the presence of coproporphyrin (CP) and uroporphyrin (UP) is mediated by H2O2 and that induced by irradiation with protoporphyrin (PP) is not mediated by H2O2. In the present study the possible role of H2O2 in the porphyrin photosensitization was investigated by direct measurement of the H2O2 formed during the irradiation of PP, CP and UP. Our results show that the amount of H2O2 formed decreased in the following order: UP, CP, PP. The amounts of H2O2 formed during irradiation of CP and PP were approximately 86% and 38% respectively in comparison to the H2O2 formed during the irradiation of UP. The formation of H2O2 was inhibited by sodium azide, a strong quencher of singlet oxygen. These observations are in good agreement with the previous report that the in vitro photolysis of Ehrlich ascites carcinoma cells by UP and CP, but not that by PP, was inhibited by catalase and clinical findings with patients with erythropoietic protoporphyria (EPP) and porphyria cutanea tarda (PCT). The patients with EPP, where the photosensitivity is due to PP, respond well to beta-carotene while beta-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin.

Is there any difference in the photobiological properties of melanins isolated from human blue and brown eyes?

Investigations were carried out to determine whether the melanin present in the blue and brown eyes were eumelanin, the melanin present in black hair and dark skin, or pheomelanin, the melanin present in red hair and the skin of people with red hair. Our results showed that UV-visible irradiation of blue or brown eye melanin did not produce any superoxide. Irradiation of 51Cr-labelled Ehrlich ascites carcinoma cells in the presence of blue or brown eye melanin did not produce significant cell lysis. The electron spin resonance (ESR) signals of blue and brown eye melanins were very similar to those of eumelanin. Comparison of these findings with our previous results indicated that the blue and brown eye melanins are essentially eumelanin. The ESR signals further suggested that in the case of both blue and brown eye melanins the iris, ciliary body, choroid, and retinal pigment epithelium did not differ.

Quantitation of cutaneous inflammation induced by reactive species generated by UV-visible irradiation of rose bengal.

The present studies were undertaken to quantitate the initial inflammatory response produced by the photo-generated reactive species in rabbit skin. Rose bengal (RB), a photosensitizer dye, was injected into the skin sites at various concentrations and exposed to UV-visible light for 30-120 min. The increase in vascular permeability and the accumulation of PMNs were investigated using 125I-labeled albumin and 51Cr-labeled PMNs. RB at a concentration of 1 nmol with 120-min exposure to light enhanced vascular permeability by 3.7 times and accumulation of PMNs by 3.3 times. As low as 0.01 nmol of RB produced discernible effects. beta-Carotene (0.1 nmole) inhibited the inflammatory response by 75-100%, suggesting that the reactive species involved in this response was predominantly singlet oxygen. The increase in vascular permeability was inhibited by 48-70% by 25 micrograms of chlorpheniramine maleate. It is therefore suggested that histamine plays a major role in the initial vascular response. The studies demonstrate that this rabbit model is suitable for the quantitation of photoinduced inflammatory response which is not observable by gross anatomic procedures.

Phototoxicity of chlorpromazine on retinal pigment epithelial cells.

As it is known that chlorpromazine (CPZ) can bind to melanins as well as cause ocular phototoxicity, we investigated the cytotoxic effects of UV-visible irradiation of melanotic and amelanotic retinal pigment epithelial (RPE) cells in the presence of CPZ. At low concentrations (5 micrograms/ml) of CPZ a photosensitization reaction took place which lysed the cells as measured by the release of 51Cr from cells labelled with chromium. At concentrations of CPZ less than 5 micrograms/ml, no significant cell lysis occurred when the cells were incubated at 37 degrees C in the dark. As the concentration of CPZ was increased to 25 micrograms/ml or more, high percentages of cells were lysed. When the melanotic RPE cells were exposed to different concentrations of CPZ and grown in culture, the cell growth (multiplication) diminished drastically with low concentrations (less than 2 micrograms/ml CPZ). Vitamin E decreased the cell lysis both in the dark and upon irradiation. Oxygen radical scavengers such as glutathione, B-carotene, mannitol, D-penicillamine as well as superoxide dismutase and catalase did not decrease cell lysis. The phototoxic effects of CPZ was found not to be due to stable photoproducts formed during irradiation of CPZ.

Mutagenic activity in groundwater in relation to mobilization of organic mutagens in soil.

In this study, the presence of mutagenic activity (Ames Salmonella-microsome assay) in different types of uncontaminated Dutch soils is demonstrated. The mutagenic activity can be mobilized by eluting the soils with organic solvents. The highest mutagenic activity was obtained using dimethylsulfoxide. It is also shown that the organic mutagens can be mobilized by percolating the soils with rain water, although this phenomenon is not always observed. Finally, the results of this study suggest that the organic mutagens found in groundwater may, at least in part, arise from mobilization of organic mutagens in soil by rain water.

Ocular timolol levels after drug withdrawal: an experimental model.

Carbon 14-labelled timolol maleate was instilled into both eyes of 12 pigmented rabbits daily for 42 days. Drug levels in the aqueous humour and ocular tissues were measured up to 42 days after drug withdrawal. The results indicate that timolol concentrates mainly in melanotic tissues, with slow release. Even 42 days after withdrawal the drug was still present in pigmented ocular tissues. Timolol was detected in the aqueous up to 5 days after withdrawal. These findings explain the long-term depressant effect of topically administered timolol on aqueous production. We conclude that lower or less frequent doses of timolol should be considered in patients with glaucoma.

Drug utilization patterns in pregnant women: a case study at the Mount Hope Women's Hospital in Trinidad, West Indies / Patrones de utilización de medicamentos en las mujeres embarazadas: un caso de estudio en el Hospital de Mujeres Mount Hope en Trinidad, West Indies

OBJECTIVE: To explore drug (prescription, over-the-counter and herbal) utilization in pregnant women attending a public sector tertiary healthcare institution. METHODS: This was a cross-sectional case study in women attending antenatal clinics at the Mount Hope Women's Hospital. Women (506) who consecutively presented for routine care at the antenatal clinic were interviewed on the medication they took. Descriptive statistics and logistic regression for predictors of drug use were done using SPSS 16. RESULTS: There were 200 (39.5%) primigravidae, 306 (60.5%) multigravidae and 299 (59%) women were in the third trimester of pregnancy. Most women (69.8%) were between 20-35 years of age. Women took an average of 1.32, 1.22 and 0.94 prescribed drugs in each trimester, respectively. Multivitamins (59.8%) and iron/folic acid (54.2%) were the most frequently prescribed drugs. Regardless of trimester, only 20% of women took supplemental calcium. Very few women (2.4%) took herbal medications. Paracetamol was the most common over-the-counter (OTC) medication in all trimesters. Women with secondary level education were most likely to use OTC iron/folic acid (p = 0.02), paracetamol and histamine2 receptor antagonists [H2RAs] (p = 0.001). More primigravidae took non-steroidal anti-inflammatory drugs (p = 0.02) and more women in the first trimester used antiemetics (p = 0.001). Age group (p = 0.048), marital status (p = 0.001) and the trimester of pregnancy (p = 0.001) were predictors of drug utilization. CONCLUSION: Overall, women in tertiary healthcare institutions took medication as prescribed particularly multivitamins and iron/folic acid. More women with higher education took OTC paracetamol, iron/folic acid and vitamin supplements. Herbal supplements were rarely used. Research on drug utilization in primary care facilities is recommended.

OBJETIVO: Explorar el uso de los medicamentos (con prescripción, sin receta médica, herbarios) en mujeres embarazadas que asisten a una institución terciaria de atención a la salud pública dentro del sector público. MÉTODOS: Se trató de un estudio transversal de mujeres que asisten a las clínicas prenatales en el Hospital de Mujeres Mount Hope. Las mujeres (506) que consecutivamente se presentaron para cuidados de rutina en la clínica prenatal, fueron entrevistadas acerca de la medicación que tomaban. Se hicieron estadísticas descriptivas y se hizo una regresión logística para los predictores del uso del medicamento usando SPSS 16. RESULTADOS: Había 200 (39.5%) primerizas, 306 (60.5%) multíparas, y 299 (59%) embarazadas en su tercer trimestre. La mayoría de las mujeres (69.8%) tenían entre 20-35 años de edad. Las mujeres tomaban un promedio de 1.32, 1.22 y 0.94 medicamentos prescritos en cada trimestre, respectivamente. Las multivitaminas (59.8%) y el hierro/ácido fólico (54.2%) fueron los medicamentos más frecuentemente prescritos. Con independencia del trimestre, sólo 20% de las mujeres tomaron suplemento de calcio. Muy pocas mujeres (2.4%) tomaban medicaciones herbarias. El paracetamol fue el medicamento sin receta más común en todos los trimestres. Las mujeres con nivel de educación secundaria presentaban una mayor probabilidad de usar hierro/ácido fólico (p = 0.02), el paracetamol y los antagonistas de los receptores de la histamina-2- [H2RAs] (p = 0.001). Un mayor número de primerizas tomaron medicamentos anti-inflamatorios no esteroideos (p = 0.02) y más mujeres en el primer trimestre usaron anti-eméticos (p = 0.001). El grupo etario (p = 0.048), el estado matrimonial (p = 0.001) y el trimestre de embarazo (p = 0.001) fueron predictores de la utilización de medicamentos. CONCLUSIÓN: En general, las mujeres en las instituciones terciarias de atención a la salud tomaron la medicación como fue prescrita, en particular las multivitaminas y el hierro/ácido fólico. Más mujeres con mayor escolaridad tomaron medicamentos sin recetas: paracetamol, hierro/ácido fólico y suplementos de vitamina. Raramente se usaron suplementos herbarios. Se recomienda la investigación del uso de medicamentos en centros de atención primaria.

Abnormal synthesis of N-methylated phospholipids during calcium paradox of the heart.

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activity that synthesizes phosphatidylcholine (PtdCho) via formation of methylated intermediates (phosphatidyl-N-monomethylethanolamine, PtdEtnMe and phosphatidyl-N,N-dimethylethanolamine, PtdEtnMe2) was comparatively studied in rat heart sarcolemmal (SL), sarcoplasmic reticular (SR) and mitochondrial fractions during Ca2+ paradox. Perfusion (5 min) with Ca(2+)-free medium followed by reperfusion (5 min) with Ca(2+)-containing medium produced a marked rise in resting tension without any recovery of contractile force. Methyltransferase catalytic sites I, II and III which synthesize PtdEtnMe, PtdEtnMe2 and PtdCho, respectively, were assayed by measuring the [3H] methyl group incorporation from 0.055, 10 and 150 microM S-adenosyl-L-[3H-methyl] methionine into membrane PtdEtn molecules. Five minutes of perfusion with Ca(2+)-free medium did not affect either SL or SR N-methyltransferase systems. Ca(2+)-readmission for 1 to 5 min induced a selective, time-dependent depression of SL site II and SR site I methyltransferase activities. Individual N-methylated phospholipids specifically formed at the two sites reflected these changes. The above abnormalities were differently influenced by the duration (1-5 min) of Ca(2+)-free perfusion and were characterized by different kinetic alterations. The mitochondrial methylation system was not affected under Ca2+ paradox. The results suggest that reduced synthesis of SL N-methylated phospholipids may contribute to the contractile dysfunction observed in Ca2+ paradox.