Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

CACHONINA ILLDEFINA SP. NOV. (DINOPHYCEAE): CHLOROPLAST TUBULES AND DEGENERATION OF THE PYRENOID1

A new species of the dinoflagellate genus Cachonina, C. illdefina sp. nov., was isolated from a red tide off El Capitan State Park, Santa Barbara County, California, in October 1973. The organism is light yellowgreen in color with deeply incised girdle and sulcal grooves. Electron microscopy of the organism, revealed a typical dinokaryotic nucleus. The chloroplasts of the organism are connected, and often contain microtubule-like elements, 25 nm diam. The pyrenoids are characterized as excluding chloroplast thylakoids and ribosomes, although containing an amorphous matrix and numerous tubular invaginations from the cytoplasm. The pyrenoids become detached from the chloroplasts and degenerate into small vesicles. C. illdefina is not bioluminescent.     

EFFECTS OF LOW NITROGEN LEVELS AND VARIOUS NITROGEN SOURCES ON GROWTH AND WHORL DEVELOPMENT IN ACETABULARIA (CHLOROPHYTA)1

Nitrate, ammonia, urea, and glycine were compared as nitrogen sources for Acetabularia mediterranea. Cells grew normally in media containing nitrate or urea, while cells did not grow at all when the same amount of N was supplied as ammonium ion. The utilization of glycine remains questionable. Cells in medium without added N (NDM) increased in length and some formed reproductive caps. The whorls of vegetative cells showed considerable hypertrophy in NDM and in glycine. This hypertrophy was due to the elongation of only the first-(a₁) and second- (a₂) order articles. When cut, the basal portion of cells without added N regenerated new apices with whorls. The development of these whorls was inversely proportional to the NO₂ concentration. Analyses showed that the intracellular nitrogen pool in young cells and regenerating bases was very small, about 1/10 of that of fully grown cells. Therefore we suggest that trace amounts of N contaminants in the medium supported growth and development, the uptake of which was facilitated by the hypertrophied whorls, under N-limited conditions.     

Assessment of binding indices and physiological responsiveness of the 5-HT2 receptor on human platelets

This is the first report of parallel studies of binding indices and physiological responsiveness of the "Serotonin-two" (5-HT2) receptor on the human platelet membrane. Binding indices were measured by a microassay employing [125I]ILSD as radioligand and ketanserin to define specific binding. A single receptor population was found, characterized by a KD of 1.69 +/- 0.45 nM and Bmax of 14.5 +/- 6.0 pmol/g protein in healthy subjects. Functional responsiveness of the platelet 5-HT2 receptor complex was assessed by measurement of the extent to which serotonin (10uM) augmented platelet aggregation induced by threshold concentrations of adenosine diphosphate (ADP). A statistically significant positive correlation was found between the number of platelet 5-HT2 receptor sites (Bmax) and the magnitude of the serotonin-amplified aggregation response (r = .70, n = 38, p less than 0.001). Assessment of binding indices and physiological responsiveness of the platelet 5-HT2 receptor complex should facilitate study of age, hormonal, disease, and drug effects on 5-HT2 receptor function in human subjects.     

Regulation of fatty acid uptake and metabolism in L6 skeletal muscle cells by resistin

Resistin has been proposed as a potential link between obesity and insulin resistance. It is also well established that altered metabolism of fatty acids by skeletal muscle can lead to insulin resistance and lipotoxicity. However, little is known about the effect of resistin on long chain fatty acid uptake and metabolism in skeletal muscle. Here we show that treating rat skeletal muscle cells with recombinant resistin (50 nM, 24 h) decreased uptake of palmitate. This correlated with reduced cell surface CD36 content and lower expression of FATP1, but no change in FATP4 or CD36 expression. We also found that resistin decreased fatty acid oxidation by measuring 14CO2 production from [1-14C] oleate and an increase in intracellular lipid accumulation was detected in response to resistin. Decreased AMPK and ACC phosphorylation were observed in response to resistin while expression of ACC and AMPK isoforms was unaltered. Resistin mediated these effects without altering cell viability. In summary, our results demonstrate that chronic incubation of skeletal muscle cells with resistin decreased fatty acid uptake and metabolism via a mechanism involving decreased cell surface CD36 content, FATP1 expression and a decrease in phosphorylation of AMPK and ACC.     

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC₅₀ of 1.2?μM compared to an IC₅₀ of 1015?μM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.     

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.