The Stanford Microarray Database

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77–80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73–76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10–14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332–333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45–48] and can be accessed at http://genome-www.stanford.edu/microarray.

MethDB—a public database for DNA methylation data

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

The KMDB/MutationView: a mutation database for human disease genes

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med.keio.ac.jp.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Calmodulin kinase is a molecular switch for cardiac excitation –contraction coupling

Signaling between cell membrane-bound L-type Ca2+ channels (LTCC) and ryanodine receptor Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation–contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for an ECC coordinating molecule: it is activated by Ca2+/calmodulin, it regulates LTCC and RyR, and it is enriched in the vicinity of LTCC and RyR. Intact cardiomyocytes were studied under conditions where CaMK activity could be controlled independently of intracellular Ca2+ by using an engineered Ca2+-independent form of CaMK and a highly specific CaMK inhibitory peptide. CaMK reciprocally enhanced L-type Ca2+ current and reduced release of Ca2+ from the SR while increasing SR Ca2+ content. These findings support the hypothesis that CaMK is required to functionally couple LTCC and RyR during cardiac ECC.

Integrated control of appetite and fat metabolism by the leptin-proopiomelanocortin pathway

Leptin deficiency results in a complex obesity phenotype comprising both hyperphagia and lowered metabolism. The hyperphagia results, at least in part, from the absence of induction by leptin of melanocyte stimulating hormone (MSH) secretion in the hypothalamus; the MSH normally then binds to melanocortin-4 receptor expressing neurons and inhibits food intake. The basis for the reduced metabolic rate has been unknown. Here we show that leptin administered to leptin-deficient (ob/ob) mice results in a large increase in peripheral MSH levels; further, peripheral administration of an MSH analogue results in a reversal of their abnormally low metabolic rate, in an acceleration of weight loss during a fast, in partial restoration of thermoregulation in a cold challenge, and in inducing serum free fatty acid levels. These results support an important peripheral role for MSH in the integration of metabolism with appetite in response to perceived fat stores indicated by leptin levels.

An important function of Nrf2 in combating oxidative stress: Detoxification of acetaminophen

Nrf2, a member of the “cap ‘n collar” group of transcription factors, is important for protecting cells against oxidative damage. We investigated its role in the detoxification of acetaminophen [N-acetyl-p-aminophenol (APAP)]-induced hepatotoxicity. When Nrf2 knockout (Nrf2−/−) and wild-type mice were given APAP by i.p. injection, the Nrf2−/− mice were highly susceptible to APAP treatment. With doses of APAP that were tolerated by wild-type mice, the Nrf2−/− mice died of liver failure. When hepatic glutathione was depleted after a dose of 400 mg/kg of APAP, the wild-type mice were able to compensate and regain the normal glutathione level. In contrast, the glutathione level in the Nrf2−/− mice was not compensated and remained low. This was because of the decrease in the gene expression of gcsH and gcsL as well as gss in the livers of the Nrf2−/− mice. In addition, the expression of ugt1a6 and gstpi that detoxify APAP by conjugation was also decreased. This increased susceptibility of the Nrf2−/− mice to APAP, because of an impaired capacity to replenish their glutathione stores, compounded with a decreased detoxification capability, highlights the importance of Nrf2 in the regulation of glutathione synthesis and cellular detoxification processes.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells1

Anoxia induces a rapid elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca2+ release is important for gene activation and survival in O2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca2+ to the anoxic [Ca2+]cyt elevation in maize cells. Imaging of intramitochondrial Ca2+ levels showed that a majority of mitochondria released their Ca2+ in response to anoxia and took up Ca2+ upon reoxygenation. We also investigated whether the mitochondrial Ca2+ release contributed to the increase in [Ca2+]cyt under anoxia. Analysis of the spatial association between anoxic [Ca2+]cyt changes and the distribution of mitochondrial and other intracellular Ca2+ stores revealed that the largest [Ca2+]cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca2+]cyt under aerobic conditions but prevented a [Ca2+]cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca2+]cyt and participate in the signaling of O2 deprivation.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.

Formation of junctions involved in excitation-contraction coupling in skeletal and cardiac muscle.

During excitation-contraction (e-c) coupling of striated muscle, depolarization of the surface membrane is converted into Ca2+ release from internal stores. This process occurs at intracellular junctions characterized by a specialized composition and structural organization of membrane proteins. The coordinated arrangement of the two key junctional components--the dihydropyridine receptor (DHPR) in the surface membrane and the ryanodine receptor (RyR) in the sarcoplasmic reticulum--is essential for their normal, tissue-specific function in e-c coupling. The mechanisms involved in the formation of the junctions and a potential participation of DHPRs and RyRs in this process have been subject of intensive studies over the past 5 years. In this review we discuss recent advances in understanding the organization of these molecules in skeletal and cardiac muscle, as well as their concurrent and independent assembly during development of normal and mutant muscle. From this information we derive a model for the assembly of the junctions and the establishment of the precise structural relationship between DHPRs and RyRs that underlies their interaction in e-c coupling.

Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD.

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.