Energetic localization of saxitoxin in its channel binding site

Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional -OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na(+) channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC(50)s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains.     

New class of steroidal alkaloids from Fritillaria imperialis

Two members of a new class of C-nor-D-homo steroidal alkaloids, impranine (1). and dihydroimpranine (2). along with a new pyridyl-pregnane-type steroidal alkaloid, fetisinine (3). and a known base, korsevine (4). were isolated from the bulbs of Fritillaria imperialis. The structures of the compounds were established on the basis of spectroscopic techniques and some chemical transformations. Compounds 1 and 2 form a new class of steroidal alkaloids, named as "impranane."     

Tamoxifen,protein kinase C and rat sperm mitochondria

Tamoxifen at a dose of 400 microg/kg/day has been reported to reduce the fertility of adult male rats and alter the pattern of cauda sperm motility from forward progressive to circular yawing type. Since sperm motility is powered by mitochondria, the effect of tamoxifen on mitochondrial function was studied. Tamoxifen treatment significantly increased rhodamine 123 fluorescent dye uptake by sperm mitochondria, reflecting an altered mitochondrial membrane potential. ATP and DAG levels, activities of glycolytic enzymes, creatine kinase and PKC all remained unaffected by tamoxifen. This is also the first report describing the presence of PKC alpha and beta in rat sperm. Morphological and biochemical integrity of sperm membranes was determined by electron microscopy and malondialdehyde levels, which were unaltered after tamoxifen treatment. This study indicates that the altered sperm motility induced by tamoxifen is accompanied by changes in mitochondrial membrane potential, but in the absence of any detectable change in membrane integrity, lipid peroxidation, ATP levels and activities of glycolytic enzymes, creatine kinase and PKC.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).     

Calcium regulates the expression of a<Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> asparaginyl tRNA synthetase gene

In a screen for calcium-regulated gene expression during growth and development ofDictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. TheddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the firstD. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of theAsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution ofGlnRS andAsnRS in eukaryotes.     

In vivo pharmaco-proteomic analysis of hydroxyurea induced changes in the sickle red blood cell membrane proteome

Hydroxyurea (HU) is an effective drug for the treatment of sickle cell disease (SCD). The main clinical benefit of HU is thought to derive from its capacity to increase fetal hemoglobin (HbF) production. However, other effects leading to clinical benefit, such as improved blood rheology, have been suggested. In order to understand HU-induced changes at the proteomic level, we profiled sickle RBC membranes from of HU-treated and untreated patients. Our previous in vitro profiling studies on sickle RBC membranes identified a significant increase in predominantly anti-oxidant enzymes, protein repair and degradation components and a few RBC cytoskeletal proteins. In the present study, using 2D-DIGE (Two-Dimensional Difference In-Gel Electrophoresis) and tandem mass spectrometry, we detected 32 different proteins that significantly changed in abundance in the HU treatment group. The proteins that significantly increased in abundance were mostly membrane skeletal components involved in the regulation of RBC shape and flexibility, and those showing a significant decrease were components of the protein repair and degradation machinery. RBC palmitoylated membrane protein 55 (p55) is significantly increased in abundance at low (in vitro) and high (in vivo) concentrations of HU. Palmitoylated p55 may be an important target of HU-dependent regulation of the sickle RBC membrane, consistent with our earlier in vitro studies.     

Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.