First synthesis of phosphonobile acids and preliminary studies on their aggregation properties

The synthesis of three novel phosphonobile acids from natural bile acids is reported. The CMC of phosphonodeoxycholic acid (PDCA) at pH 8.2 was found to be lower than that of the parent deoxycholic acid (DCA). PDCA micelles were also found to have higher microviscosity compared to DCA micelles, suggesting higher hydrophobicity and tighter packing in the interior of PDCA micelles. PDCA aggregated further to form an aqueous gel at pH 4.     

Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via naOH for two-dimensional electrophoresis

In preparing intracellular microbial samples for one- or two-dimensional electrophoresis, trichloroacetic acid (TCA) precipitation is frequently used to remove interfering compounds. Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is presented. Precipitated proteins are pretreated with 0.2 M NaOH for less than 5 min, followed by addition of standard sample solubilization buffer (SSSB). When compared to solubilization with SSSB alone, NaOH pretreatment of TCA-precipitated intracellular protein from Aspergillus oryzae and Escherichia coli shows an approximate 5-fold increase in soluble protein. In addition, two-dimensional gel electrophoresis on resolubilized proteins shows an equivalent number of proteins in samples with and without NaOH pretreatment.     

Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.     

Synthesis of 2'-spiro ribo- and arabinonucleosides

Four conformationally restricted bicyclic 2'-spiro nucleosides were synthesised and incorporated into oligonucleotides. These spiro nucleotides induced decreased duplex thermostabilities.     

Acid-labile subunit regulation during the early stages of liver regeneration: implications for glucoregulation

The initiation of liver regeneration is regulated by endogenously produced growth factors and cytokines and is accompanied by suppression of growth hormone (GH) binding to hepatocytes. We have demonstrated some of these factors, particularly GH, which modulate acid-labile subunit (ALS) expression in vitro. Consequently, we investigated ALS hepatic mRNA and serum levels in rats for 24 h after partial hepatectomy (PHx). There was a significant suppression of ALS gene expression (approximately 50%, P < 0.005) and serum levels (approximately 30%, P < 0.02) by 12 h in PHx rats relative to controls. Relative to intact animals, hepatic mRNA and serum levels of ALS were suppressed by approximately 60% at 24 h. Similarly, hepatic GH receptor mRNA levels were significantly reduced in PHx animals. Moreover, hepatocytes isolated from PHx animals were less responsive to GH than those from controls. Overall, our results demonstrate that suppression of ALS gene expression and serum levels during liver regeneration relates to lowered hepatic GH sensitivity. Suppressed circulating ALS may alter insulin-like growth factor bioavailability and constitute a mechanism to maintain relatively normal glucoregulation after loss of liver mass.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.     

Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor. Role in hormone signaling and cell proliferation

Follicle-stimulating hormone (FSH) regulated growth and function of the ovarian follicle was previously thought to be mediated solely through activation of G(s)-coupled receptors. In this study, we show for the first time that this function is predominantly mediated through the alternatively spliced and novel growth factor type 1 receptor (oFSH-R3) that is also present in the ovary. Immortalized granulosa cells lacking endogenous FSH receptors, when transfected with either oFSH-R3 cDNA (JC-R3) or the G(s)-coupled oFSH-R1 (JC-R1), expressed the corresponding glycosylated receptor. In JC-R3 or JC-R1 cells labeled with bromodeoxyuridine or [(3)H]thymidine, FSH stimulated the cells to progress through S-phase and divide. The growth promoting effect of recombinant FSH in JC-R3 cells was preceded by the rapid activation of ERK1 and ERK2. This effect was hormone-specific and transient. In JC-R3 cells inhibitors like calphostin C, PD98059, Ag 18, or calcium chelators EGTA or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM inhibited both mitogen-activated protein kinase activation and bromodeoxyuridine incorporation. FSH induced phosphorylation of the FSH-R3 receptor was blocked by pretreating cells with calphostin C. There was no cAMP induction by FSH in JC-R3 cells. The cAMP independent growth promoting effect of FSH is mediated by activation of Ca(2+) and mitogen-activated protein kinase-dependent pathways. Thus, alternative splicing of a G-protein coupled receptor creates the expression of a novel receptor motif that can mediate a widely recognized function of the glycoprotein hormone.     

Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.