A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

A case-control study of tea/coffee consumption and lung cancer risk

Background. Despite the increasing attention to the effects of dietary factors on lung cancer risk, epidemiological research on the role of black/green tea and coffee intake and lung cancer risk is scarce. The purpose of this study was to explore the following three hypotheses: (1) the preventive (protective) effect from lung cancer is higher in green tea than in black tea and coffee consumption. (2) brewed tea (either black or green) daily drinkers have lower odds of lung cancer than non-drinkers of brewed tea (3) regular black and green tea have more preventive effect against lung cancer than decaffeinated teas due to the synergistic effect of caffeine and other tea components. ^ Methods. Data on 1,088 lung cancer cases and 1,127 controls from an ongoing epidemiological study of lung cancer by the Department of Epidemiology of the University of Texas M.D. Anderson Cancer were analyzed. Multiple logistic regressions were performed for testing associations between frequency of specific types of tea/coffee consumption and the risk of lung cancer. ^ Results. We observed that more than a cup a week of green tea and decaffeinated black tea were significantly associated with reduced odds of lung cancer by 64% for green tea (adjusted OR = 0.44; 95% CI = 0.31–0.64), 36% for decaffeinated black tea (OR = 0.64; 95% CI = 0.45–0.90), when compared with non-drinkers and those who drank less than a cup a week. On the other hand, increasing intake of regular coffee (more than 3 cups a day) was associated with a 30% higher odds ratio of lung cancer (OR = 1.30; 95% CI = 1.01–1.09). No association was found between regular black tea, decaffeinated coffee consumption and the odds ratio of lung cancer. However, when drinkers of other tea/coffee beverages were excluded from each model in order to explore the independent effect of each type of tea/coffee, green tea and decaffeinated black tea-lung cancer associations remained but no association was observed for drinkers of regular coffee. ^ Conclusion. We report the chemopreventive effects of more than a cup a week of green tea and decaffeinated black tea on lung cancer. ^

AN INVESTIGATION OF THE POSSIBLE RELATIONSHIP BETWEEN CERTAIN PHYSIOLOGICAL AND PSYCHOLOGICAL MEASURES AND SMOKING CESSATION IN A SELF - SELECTED POPULATION

This study focused on the possible relationship between certain physiological and psychological variables and the cessation of smoking. The population studied was employees enrolled in a multimodality smoking cessation program at the local offices of a major American corporation. In order to be eligible to participate, each individual must have become a non-smoker by the end of the smoking cessation program.^ Three physiological measures were taken on each individual while performing a relaxation exercise; (1) Electromyogram (EMG), (2) Galvanic Skin Response (GSR), and (3) Skin Temperature. The psychological measure consisted of the variable "anxiety" in the Cattell 16-PF personality inventory. Individual's self report of their smoking status was verified through a test for expired carbon monoxide levels.^ For the total population (N-31) no significant relationships were found between the physiological and psychological variable measured and cessation; however, with the removal of two cases discovered during the post-test interview to be influenced by external factors of high caffeine level and a severe family crisis, the measure of EMG, attained significance in discriminating between the successful and unsuccessful in Smoking Cessation. ^

Análisis de la estructura del juego y parámetros morfológicos y fisiológicos en bádminton

En este trabajo se ha realizado un análisis de la estructura del juego y de los parámetros morfológicos y fisiológicos en jugadores de bádminton. Para ello se han realizado 4 estudios aplicados. Objetivo: Los objetivos del trabajo han sido: (1) comprobar si existen diferencias entre el lado dominante y no dominante de las medidas antropométricas en jugadores de bádminton de máximo nivel nacional, así como verificar si el lado del cuerpo donde se realiza la medición puede influir en el cálculo de la composición corporal y del somatotipo. (2) Comparar la estuctura temporal y notacional en partidos de individual masculino entre los Juegos Olímpicos de Pekín y de Londres para observar como ha evolucionado el bádminton de 2008 a 2012. (3) Medir la ocurrencia de daño muscular después de un partido simulado de bádminton y su influencia en parámetros físicos y hematológicos. (4) Investigar la efectividad de una bebida energética que contiene cafeína para mejorar el rendimiento físico y el rendimiento en un partido en jugadores de élite de bádminton. Metodología: Para caracterizar el bádminton participaron en esta tesis un total de 78 jugadores de bádminton de élite (63 hombres y 15 mujeres), distribuidos en tres estudios y se analizaron 40 sets de bádminton de individual masculino usando los videos oficiales de los Juegos Olímpicos de Pekín 2008 y Londres 2012. En el primer estudio se tomaron medidas de pliegues cutáneos, diámetros, longitudes y perímetros del lado dominante y no dominante de los jugadores. Se calculó la composición corporal y el somatotipo. En el segundo estudio se analizaron los factores temporales y los factores notacionales de los partidos. En el tercer estudio se midieron la fuerza máxima isométrica, la velocidad en test específicos de bádminton y se tomaron muestras de sangre antes y después de jugar un partido de bádminton de 45 minutos. En el cuarto estudio se realizó un experimento a doble ciego, aleatorizado y controlado con placebo, los jugadores ingirieron 3 mg de cafeína por kilógramo de masa corporal en forma de bebida energética, o la misma bebida sin cafeína (placebo). En este estudio se registraron diferente tests específicos de bádminton (tests de salto, fuerza máxima y test de agilidad) y se jugó un partido simulado de 45 minutos. Resultados y discusión: (1) El porcentaje óseo fue mayor calculado a partir de las mediciones del lado dominante (dominante = 16.37 ± 1.14 %, no dominante = 15.66 ± 1.12 %; P < 0.001), mientras que el porcentaje muscular fue mayor calculado a partir de las mediciones del lado no dominante (dominante = 49.39 ± 2.60 %, no dominante = 50.18 ± 2.69%; P < 0.001). (2) La duración del set (Pekín: 1124.6 ± 229.9 s vs Londres: 1260.3 ± 267.1 s.; P < 0.05), el tiempo real de juego (Pekín: 306.9 ± 45.7 s vs Londres: 354.7 ± 86.5 s; P < 0.05), tiempo de rally, golpeos por rally, tiempo de descanso en el punto 11, tiempo de descanso entre sets y golpeos por rally fueron significativamente mayores en Londres que en Pekín. (3) El partido simulado de bádminton no afectó a la fuerza isométrica máxima (Pre: 1263.6 ± 245.5, Post: 1290.8 ± 240.4 N) o a la velocidad específica de bádminton (Pre: 21.0 ± 1.7, Post: 20.9 ± 1.8 s), sin embargo las concentraciones de mioglobina y de creatina quinasa en sangre aumentaron de 26.5 ± 11.6 a 197.3 ± 70.2 μg • L-1 y de 258.6 ± 192.2 a 466.0 ± 296.5 U • L-1, respectivamente después del partido de bádminton. (4) En comparación con la bebida placebo, la ingesta de la bebida energética con cafeína incrementó la altura del SJ (34.5±4.7 vs. 36.4±4.3 cm; P < 0.05) y del CMJ (37.7 ± 4.5 vs. 39.5 ± 5.1 cm; P < 0.05) y aumentó el número de aceleraciones totales durante el partido (7395 ± 1594 vs. 7707 ± 2033 aceleraciones; P < 0.05). Conclusiones: (1) Existen asimetrías corporales en los jugadores de bádminton de alto nivel, al encontrarse diferencias en los diámetros óseos y en los perímetros entre el lado dominante y no dominante. Al calcular la composición corporal con el lado dominante de los jugadores de bádminton se está sobreestimando el porcentaje óseo e infraestimando el porcentaje muscular. (2) El bádminton está evolucionando hacía rallies más largos con intervalos de descanso mayores, lo que resulta en partidos más largos. (3) El partido de bádminton generó daño muscular, sin embargo, el nivel de daño muscular alcanzado después de un partido de bádminton no produjo una disminución del rendimiento muscular. (4) El uso de una bebida energética con cafeína puede ser una ayuda nutricional eficaz para aumentar el rendimiento en el salto y patrones de actividad durante el juego en jugadores de élite de bádminton. ABSTRACT: This study analyzes the structure of the game and the morphological and physiological parameters in badminton players, investigated in four applied studies. Purpose: The purposes of the study were: (1) To check if there are differences between the dominant and non-dominant side in the anthropometric measures of badminton players at the highest national level and verify if the side of the body where the measurements are performed can influence the calculation of the body composition and the somatotype. (2) To compare the temporal and notational structure in men’s singles matches between the Olympic Games in Beijing and London to observe the evolution of badminton between 2008 and 2012. (3) To asses the occurrence of muscle damage after a simulated badminton match and its influence on physical and haematological parameters. (4) To determine the effectiveness of a commercially available energy drink that contains caffeine to improve match performance in elite badminton players. Methods: A total of 78 elite badminton players (63 men and 15 women) participated in this thesis to characterize the sport of badminton distributed in three studies and 40 sets of men’s singles badminton analyzed using the official videos of the Olympic Games of Beijing 2008 and London 2012. In the first study skinfolds, diameters, lengths and perimeters of the dominant and non-dominant side of the players were measured and body composition and somatotype were calculated. In the second study the temporal and notational factors were analyzed. In the third study maximal isometric force and speed in badminton specific tests were measured and blood samples were taken before and after a badminton match of 45 minutes. In the fourth study, a double-blind, randomized placebo-controlled experiment, players ingested 3 mg of caffeine per kilogram of body mass in the form of an energy drink or an identical drink with no caffeine content (placebo). In this study different badminton specific tests (jump tests, handgrip force test and an agility test) were recorded and a simulated badminton match of 45 minutes was played. Results and discussion: (1) The percentage of bone was higher when calculated from measurements of the dominant body side (dominant = 16.37 ± 1.14 %, nondominant = 15.66 ± 1.12 %; P < 0.001), while the muscle percentage was higher when calculated from measurements of the non-dominant side (dominant = 49.39 ± 2.60 %, non-dominant = 50.18 ± 2.69%; P < 0.001). (2) Set duration (Beijing: 1124.6 ± 229.9 s vs. London: 1260.3 ± 267.1 s.; P < 0.05), real time played (Beijing: 306.9 ± 45.7 s vs. London: 354.7 ± 86.5 s; P < 0.05), rally time, shots per rally, rest time at point 11, rest time between sets and shots per rally were significantly higher in London than in Beijing. (3) A simulated badminton match did not affect maximal isometric force (Pre: 1263.6 ± 245.5, Post: 1290.8 ± 240.4 N) or specific badminton speed (Pre: 21.0 ± 1.7, Post: 20.9 ± 1.8 s), however, concentrations of myoglobin and creatine kinase in blood increased from 26.5 ± 11.6 to 197.3 ± 70.2 μg • L-1 and from 258.6 ± 192.2 to 466.0 ± 296.5 U • L-1, respectively after the badminton match. (4) In comparison to the placebo drink, the caffeinated beverage increased height in the SJ (34.5±4.7 vs. 36.4±4.3 cm; P < 0.05) and in the CMJ (37.7 ± 4.5 vs. 39.5 ± 5.1 cm; P < 0.05) and increased the number of total accelerations during the match (7395 ± 1594 vs. 7707 ± 2033 accelerations; P < 0.05). Conclusions: (1) Body asymmetries were found in high level badminton players, due to the differences found in bone diameters and perimeters between the dominant and non-dominant body side. When calculating body composition with the dominant side of the badminton players we are overestimating bone percentage and underestimating muscle percentage. (2) Badminton is evolving towards longer rallies with greater rest intervals, resulting in longer matches. (3) The badminton match generated muscle damage, however, the level of muscle damage reached after a badminton match did not produce a decrease in muscle performance. (4) The ingestion of an energy drink containing caffeine might be an effective ergogenic nutritional supplement to increase jump performance and activity patterns during the game in elite badminton players.

Determination of selected pharmaceutical compounds in biosolids bysupported liquid extraction and gas chromatography?tandem massspectrometry

In this work, an analytical method was developed for the determination of pharmaceutical drugs inbiosolids. Samples were extracted with an acidic mixture of water and acetone (1:2, v/v) and supportedliquid extraction was used for the clean-up of extracts, eluting with ethyl acetate:methanol (90:10, v/v).The compounds were determined by gas chromatography?tandem mass spectrometry using matrix-match calibration after silylation to form their t-butyldimethylsilyl derivatives. This method presentsvarious advantages, such as a fairly simple operation for the analysis of complex matrices, the use ofinexpensive glassware and low solvent volumes. Satisfactory mean recoveries were obtained with thedeveloped method ranging from 70 to 120% with relative standard deviations (RSDs) ? 13%, and limitsof detection between 0.5 and 3.6 ng g?1. The method was then successfully applied to biosolids samplescollected in Madrid and Catalonia (Spain). Eleven of the sixteen target compounds were detected in thestudied samples, at levels up to 1.1 ?g g?1(salicylic acid). Ibuprofen, caffeine, paracetamol and fenofibratewere detected in all of the samples analyzed.

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. Comparison of the time courses of the rapid rise in cytosolic calcium with the slower increase in ERK1 and ERK2 activation suggests one or more intermediate stages in this pathway. Chelation of cytosolic calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid abolished the FCCP-stimulated rise in internal calcium, as well as the tyrosine phosphorylation and the activation of the ERKs. Surprisingly, caffeine, which releases calcium from different internal stores, did not increase the tyrosine phosphorylation and did not activate the ERKs. The FCCP effect on calcium storage may be related to mitochondrial dysfunction in Alzheimer disease, which might result in ineffective buffering of cytosolic calcium that leads to mitogen-activated protein kinase activation and subsequent protein phosphorylations.

Roles for Basal and Stimulated p21Cip-1/WAF1/MDA6 Expression and Mitogen-activated Protein Kinase Signaling in Radiation-induced Cell Cycle Checkpoint Control in Carcinoma Cells

We investigated the role of the cdk inhibitor protein p21Cip-1/WAF1/MDA6 (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0–10 min) and secondary (90–240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G1 that were p21 and MAPK dependent, whereas the elevation of cells present in G2/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G2/M phase 24–48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G2/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G2/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G2/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G1/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G2/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

The role of the D2 dopamine receptor (D2R) in A2A adenosine receptor (A2AR)-mediated behavioral and cellular responses as revealed by A2A and D2 receptor knockout mice

The A2AR is largely coexpressed with D2Rs and enkephalin mRNA in the striatum where it modulates dopaminergic activity. Activation of the A2AR antagonizes D2R-mediated behavioral and neurochemical effects in the basal ganglia through a mechanism that may involve direct A2AR–D2R interaction. However, whether the D2R is required for the A2AR to exert its neural function is an open question. In this study, we examined the role of D2Rs in A2AR-induced behavioral and cellular responses, by using genetic knockout (KO) models (mice deficient in A2ARs or D2Rs or both). Behavioral analysis shows that the A2AR agonist 2–4-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine reduced spontaneous as well as amphetamine-induced locomotion in both D2 KO and wild-type mice. Conversely, the nonselective adenosine antagonist caffeine and the A2AR antagonist 8-(3-chlorostyryl)caffeine produced motor stimulation in mice lacking the D2R, although the stimulation was significantly attentuated. At the cellular level, A2AR inactivation counteracted the increase in enkephalin expression in striatopallidal neurons caused by D2R deficiency. Consistent with the D2 KO phenotype, A2AR inactivation partially reversed both acute D2R antagonist (haloperidol)-induced catalepsy and chronic haloperidol-induced enkephalin mRNA expression. Together, these results demonstrate that A2ARs elicit behavioral and cellular responses despite either the genetic deficiency or pharmacological blockade of D2Rs. Thus, A2AR-mediated neural functions are partially independent of D2Rs. Moreover, endogenous adenosine acting at striatal A2ARs may be most accurately viewed as a facilitative modulator of striatal neuronal activity rather than simply as an inhibitory modulator of D2R neurotransmission.

Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.