Participation of cAMP in tacrine-induced gastric smooth muscle relaxation

Tacrine, a well-known acetylcholinesterase inhibitor, applied in concentrations higher than 2×10⁻⁵ mol/l promoted Ca²⁺-independent relaxation of rat gastric smooth muscles in experiments in vitro. The relaxation was not cholinergic and was a result of influence of tacrine over intracellular signaling pathways regulating smooth muscle contraction/relaxation. The nature of this untypical muscle relaxation was studied by using smooth muscle strips isolated from rat stomach. Their bioelectrical and mechanical responses were recorded after treatment with tacrine and different activators or blockers of intracellular pathways involved in muscle contractility. Following the activation of adenylate cyclase with 1×10⁻⁶ mol/l forskolin and increase in the concentration of cyclic adenosine monophosphate (cAMP) after application of 4×10⁻⁵ mol/l SQ22536, a significant decrease in the muscle relaxation was observed. Theophylline (2×10⁻⁴ mol/l), a phosphodiesterase inhibitor, had no effect on the amplitude of tacrine-induced relaxation. The latter was also reduced by inhibition of protein kinase A (PKA) with 5×10⁻⁶ mol/l KT5720. These findings support the assumption that tacrine promoted smooth muscle relaxation through PKA-induced phosphorylation and inhibition of myosin light chain kinase activity. The reduction of spike-linked Ca²⁺ influx provoked by tacrine was probably a secondary contributing process, associated with an influence of increased cAMP level on Ca²⁺ channels.     

Resistin up-regulates fractalkine expression in human endothelial cells: Lack of additive effect with TNF-α

Resistin is a cytokine and fractalkine (Fk) a cell adhesion molecule and chemokine that contribute to human vascular inflammation by mechanisms not clearly defined. We questioned whether resistin induces Fk expression in human endothelial cells (HEC), compared the effect with that of the pro-inflammatory cytokine, TNF-α, and evaluated the consequences of co-stimulating HEC with both activators on Fk induction and on the signalling molecules involved. We found that resistin up-regulated Fk expression at comparable level to that of TNF-α by a mechanism involving P38 and JNK MAPK and NF-κB. Co-stimulation of cells with resistin and TNF-α did not increase Fk expression induced by every single inducer. Moreover resistin reduced the expression induced by TNF-α in HEC. The new data uncover Fk as a novel molecular link between resistin and inflammation and show that resistin and TNF-α have no additive effect in Fk up-regulation or on the signalling molecules implicated.     

Separation and quantitative recovery of mouse serum arylesterase and carboxylesterase activity

Paraoxonase-1 (PON1) is known to be associated with high density lipoproteins. We optimized buffer conditions to obtain quantitative recovery of PON1 (arylesterase) activity and analyzed the distribution of PON1 in mice using a combination of size-exclusion chromatography and ultracentrifugation. Size-exclusion chromatography of mouse serum separated the esterase activity into two peaks, one overlapping the high density lipoproteins and a second peak of lower molecular weight, consistent with serum carboxylesterase, which accounted for approximately 20% of the total esterase activity of normal mouse serum. Using conditions for the quantitative recovery of arylesterase activity, we fractionated serum by ultracentrifugation into d < 1.21 g/ml, d < 1.25 g/ml, d > 1.21 g/ml, and d > 1.25 g/ml fractions. We observed that PON1 arylesterase activity and mass were isolated in the d < 1.21 g/ml fraction and that serum carboxylesterase was recovered in the d > 1.25 g/ml fraction. The significance of the confounding of PON1 arylesterase activity by serum carboxylesterase was demonstrated by studying mice challenged with a high-fat, high-cholate diet for 14 days. It was shown that all of the decrease in arylesterase activity in response to this diet is attributable to the HDL-associated arylesterase activity (PON1). We conclude that mouse PON1 is quantitatively associated with high density lipoproteins. The contribution of serum carboxylesterase to the total esterase activity significantly confounds the interpretation of total arylesterase activity in mouse serum.     

Neoductular progenitor cells regenerate hepatocytes in severely damaged liver: a comparative ultrastructural study

In severely injured liver, stem cells give rise to progeny that tend to replace lost hepatocytes. Neoductular reaction appears as an inherent stage of liver reconstruction following severe damage caused by different pathological mechanisms. Few ultrastructural types of progenitor cells have been described, and some molecular phenotypes of progenitor stages have been characterized, but the details of the differentiation process are largely unknown. We prepared for light and electron microscopy examination human liver from biopsies of patients with chronic active hepatitis, and rat liver with allyl alcohol-induced periportal necrosis. We found that progenitor neoductular cells acquire the hepatocytic polarity pattern during a multi-step process apparently involving cell migration and dissolution of neoductular basement membrane. An intermediate stage with "mixed" ductular and hepatocytic polarity was described.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH

Purified serum paraoxonase (PON1) had been shown to attenuate the oxidation of LDL in vitro. We critically reevaluated the antioxidant properties of serum PON1 in the in vitro assays initiated with copper or the free radical generator 2,2'-azobis-2-amidinopropane hydrochloride (AAPH). The antioxidant activity of different purified PON1 preparations did not correlate with their arylesterase (AE), lactonase, or phospholipase A2 activities or with the amounts of detergent or protein. Dialysis of three of these preparations resulted in a 30-40% loss of their AE activities but in a complete loss of their antioxidant activities. We also followed the distribution of the antioxidant activity during human serum PON1 purification by two purification methods. The antioxidant activity of the anion-exchange chromatography fractions did not copurify with PON1 using either method and could largely be accounted for by the "antioxidant" activity of the detergent present. In conclusion, using the copper or AAPH in vitro assays, no PON1-mediated antioxidant activity was detected, suggesting that the removal of PON1 from its natural environment may impair its antioxidative activity and that this assay with highly purified PON1 may be an inappropriate method with which to study the antioxidative properties of the enzyme.     

A new hybrid theory of muscle contraction

This work represents an attempt to find a more complete and adequate interpretation of the phenomenon of muscle contraction than is presently available. Arguments are presented in favour of the idea that the principal groups of existing theories on contraction contain both elements that should be excluded from consideration and elements that are of particular interest to retain. In the present theory, it is accepted that the essential process in muscle contraction is a relative increase in long-range repulsive forces, exerted directly perpendicular to the myofilaments. It is then assumed that these forces of repulsion are converted into forces which shorten the fibre, by way of a passive mechanical action of obliquely arranged cross bridges between the thick and thin filaments. Analysis of important experimental data serves to emphasize the explicative potential of the new theory.     

Further evidence for BRCA1 communication with the inactive X chromosome

BRCA1, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (Ganesan et al., 2002). Xiao et al. (2007) (this issue of Cell) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.