Pax7 shows higher satellite cell frequencies and concentrations within intrafusal fibers of muscle spindles

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.     

Enhancement of the 5-HT neurotransmission by antidepressant treatments

The hypothesis of an etiopathogenic role of 5-HT and that of a mediation by the 5-HT system in the effect of antidepressant treatments have often been confused. Little unequivocal evidence exists for a 5-HT deficit in depression. However, several recent animal and clinical data suggest that the 5-HT system might contribute to the therapeutic effect of various antidepressant treatments. Long-term administration of tricyclic antidepressant (TCA) drugs induces a sensitization of rat forebrain neurons to iontophoretically-applied 5-HT. Repeated electroconvulsive shocks result also in an increased sensitivity of forebrain 5-HT receptors. However, chronic administration of a new antidepressant drug, zimelidine, a potent and long-lasting 5-HT uptake blocker, fails to modify 5-HT receptor sensitivity. These results suggest that enhancement of 5-HT neurotransmission obtained via either pre- or postsynaptic mechanisms might determine the antidepressant effect of these treatments. In a recent clinical study, we observed that lithium administration to TCA-resistant depressive patients induced a rapid relief of depression. It is possible that the presynaptic enhancing effect of lithium on the 5-HT system might unveil the TCA-induced sensitization of the postsynaptic 5-HT receptors. Most depressed patients exhibit marked diurnal variations of mood. Preliminary experiments in rats revealed that the responsiveness of hippocampal neurons to iontophoretically-applied 5-HT is enhanced in the evening. Similar diurnal variations of 5-HT receptor sensitivity might occur in human brain and be related to diurnal variation of mood in depression. Since normal individuals do not show these fluctuations of mood, it is proposed that the "mood regulating system" might become 5-HT dependent in depressed patients.     

Functional properties of sarcoplasmic reticulum Ca(2+)-ATPase after proteolytic cleavage at Leu119-Lys120, close to the A-domain

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu(119) and Lys(120), in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of "backdoor" formation of E2P from E2 and P(i). E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for P(i) or Mg(2+), while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca(2+) and exhibited a reduced rate for its Ca(2+)-dependent transition from E2 to Ca(2)E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca(2+)-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca(2+)-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.     

The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles

The FUR1 gene of Saccharomyces cerevisiae encodes uracil phosphoribosyltransferase (UPRTase) which catalyses the conversion of uracil into uridine 5'-monophosphate (UMP) in the pyrimidine salvage pathway. The FUR1 gene is included in a 2.1 kb genomic segment of DNA and is transcribed into a 1 kb poly(A)+mRNA. Sequencing has determined a 753 bp open reading frame capable of encoding a protein of 251 amino acids. The FUR1 genes for three recessive fur1 alleles, having different sensibilities to 5-fluorouridine (5-FUR) but identical levels of resistance to 5-fluorouracil (5-FU), were cloned and sequenced. Single bp changes located in different regions of the gene were found in each mutant. Two in vitro-constructed deletions of the FUR1 gene have been integrated at the chromosomal locus, giving strains with 5-FURR and 5-FURR mutant phenotype. Assays of UPRTase, uridine kinase, uridine ribohydrolase and uridine 5'-monophosphate nucleotidase enzymatic activities, in extracts of strains where the FUR1 gene is overexpressed or deleted, indicate that the FUR1 encoded protein possesses only UPRTase activity.     

Inhibition of RUNX2 Transcriptional Activity Blocks the Proliferation,Migration and Invasion of Epithelial Ovarian Carcinoma Cells

Previously, we have identified the RUNX2 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from serous epithelial ovarian cancer (EOC) patients, when compared to primary cultures derived from matched primary (prior to CT) tumors. However, we found no differences in the RUNX2 methylation in primary EOC tumors and EOC omental metastases, suggesting that DNA methylation-based epigenetic mechanisms have no impact on RUNX2 expression in advanced (metastatic) stage of the disease. Moreover, RUNX2 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. Knockdown of the RUNX2 expression in EOC cells led to a sharp decrease of cell proliferation and significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX2 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced.Taken together, our data are indicative for a strong oncogenic potential of the RUNX2 gene in serous EOC progression and suggest that RUNX2 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX2 and other members of the RUNX gene family in ovarian tumorigenesis.     

Iron induces Bcl-2 expression in human dermal microvascular endothelial cells

Iron is suspected to be involved in the induction and/or progression of various human tumors. The present study was designed to investigate the effects of iron on endothelial cells, keeping in mind that the homeostasis of microvessels plays a critical role in neo-angiogenesis. Applying a model of human dermal microvascular endothelial cell terminal differentiation and death induced by serum deprivation, we found that iron salts (iron chloride and ferric nitrilotriacetate) provided a survival advantage to endothelial cells. Using immunohistochemistry and Western Blot analysis, we found that the extended cellular life span induced by iron was paralleled by an increase of Bcl-2 protein expression. Taken together, these observations suggest that iron may give a survival advantage to endothelial cells and represent a novel mechanism through which iron may contribute to tumorigenesis.     

A High-Yield Co-Expression System for the Purification of an Intact Drs2p-Cdc50p Lipid Flippase Complex,Critically Dependent on and Stabilized by Phosphatidylinositol-4-Phosphate

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C₁₂E₈-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.