Screening of rifamycin producing marine sponge bacteria by LC-MS-MS

HPLC-MS-MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.     

Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.     

A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.     

Calcium entry mediated by SOCs and TRP channels: variations and enigma

Ca(2+) signals in response to receptors mediate and control countless cellular functions ranging from short-term responses such as secretion and contraction to longer-term regulation of growth, cell division and apoptosis. The spatial and temporal details of Ca(2+) signals have been resolved with great precision in many cells. Ca(2+) signals activated by phospholipase C-coupled receptors have two components: Ca(2+) release from endoplasmic reticulum (ER) stores mediated by inositol 1,4,5-trisphosphate (InsP(3)) receptors, and Ca(2+) entry from outside the cell. The latter remains largely a molecular and mechanistic mystery. The activation of "store-operated" Ca(2+) channels is believed to account for the entry of Ca(2+). However, debate now focuses on how much of a contribution emptying of stores plays to the activation of Ca(2+) entry in response to physiological activation of receptors. Here we discuss recent information and ideas on the exchange of signals between the plasma membrane (PM) and ER that results in activation of Ca(2+) entry channels following receptor stimulation and/or store emptying.     

Shortcomings of protein removal prior to high performance liquid chromatographic analysis-A case study using method development for BAY 11-7082

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS-MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082.     

Orai1 and STIM reconstitute store-operated calcium channel function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.     

Non-canonical signaling and localizations of heterotrimeric G proteins

Heterotrimeric G proteins typically transduce signals from G protein-coupled receptors (GPCRs) to effector proteins. In the conventional G protein signaling paradigm, the G protein is located at the cytoplasmic surface of the plasma membrane, where, after activation by an agonist-bound GPCR, the GTP-bound Gα and free Gβγ bind to and regulate a number of well-studied effectors, including adenylyl cyclase, phospholipase Cβ, RhoGEFs and ion channels. However, research over the past decade or more has established that G proteins serve non-canonical roles in the cell, whereby they regulate novel effectors, undergo activation independently of a GPCR, and/or function at subcellular locations other than the plasma membrane. This review will highlight some of these non-canonical aspects of G protein signaling, focusing on direct interactions of G protein subunits with cytoskeletal and cell adhesion proteins, the role of G proteins in cell division, and G protein signaling at diverse organelles.     

L<Subscript>2</Subscript>-norm multiple kernel learning and its application to biomedical data fusion

Background  

This paper introduces the notion of optimizing different norms in the dual problem of support vector machines with multiple kernels. The selection of norms yields different extensions of multiple kernel learning (MKL) such as L _∞, L ₁, and L ₂ MKL. In particular, L ₂ MKL is a novel method that leads to non-sparse optimal kernel coefficients, which is different from the sparse kernel coefficients optimized by the existing L _∞ MKL method. In real biomedical applications, L ₂ MKL may have more advantages over sparse integration method for thoroughly combining complementary information in heterogeneous data sources.