Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

Uneven modulation of the annexin 1 system in osteoblast-like cells by dexamethasone

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.     

Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica

The production of benzophenanthridine alkaloids (sanguinarine, chelerythrine and macarpine) in cells of Eschscholtzia californica is enhanced by sodium alginate and by entrapment in Ca2+-alginate. Tyrosine decarboxylase, a key enzyme of alkaloid biosynthesis, is induced by the treatments. Alginate- entrapped cells are elicited over an extended period of time which leads to increased alkaloid biosynthesis (up to 800-fold enhancement). A major portion of alkaloids produced are released into the growth medium.     

Endoglin is expressed in the chicken vasculature and is involved in angiogenesis.

Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex, highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant vascular disorder caused by a haploinsufficiency mechanism. Vascular lesions (telangiectasia and arteriovenous malformations) in HHT1 are associated with loss of the capillary network, suggesting the involvement of endoglin in vascular repair processes. Using the chick chorioallantoic membrane (CAM) as an angiogenic model, we have analyzed the expression and function of chicken endoglin. A pan-specific polyclonal antibody (pAb) recognized chicken endoglin as demonstrated by immunostaining and Western blot analysis. In ovo treatment of chicken embryos with this pAb resulted in a significantly increased area of CAM. This effect was likely mediated by modulation of the ligand binding to endoglin as this pAb was able to inhibit TGF-beta1 binding. These results support the involvement of endoglin in the angiogenic process.     

Comparative Study of Biological Properties and Electrophoretic Characteristics of Lipopolysaccharide from Eel-Virulent and Eel-A virulent Vibrio vulnificus Strains

In Vibrio vulnificus, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.     

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.     

Paranoia,self-deception and overconfidence

Self-deception, paranoia, and overconfidence involve misbeliefs about the self, others, and world. They are often considered mistaken. Here we explore whether they might be adaptive, and further, whether they might be explicable in Bayesian terms. We administered a difficult perceptual judgment task with and without social influence (suggestions from a cooperating or competing partner). Crucially, the social influence was uninformative. We found that participants heeded the suggestions most under the most uncertain conditions and that they did so with high confidence, particularly if they were more paranoid. Model fitting to participant behavior revealed that their prior beliefs changed depending on whether the partner was a collaborator or competitor, however, those beliefs did not differ as a function of paranoia. Instead, paranoia, self-deception, and overconfidence were associated with participants’ perceived instability of their own performance. These data are consistent with the idea that self-deception, paranoia, and overconfidence flourish under uncertainty, and have their roots in low self-esteem, rather than excessive social concern. The model suggests that spurious beliefs can have value–self-deception is irrational yet can facilitate optimal behavior. This occurs even at the expense of monetary rewards, perhaps explaining why self-deception and paranoia contribute to costly decisions which can spark financial crashes and devastating wars.     

Sodium transport systems in human chondrocytes. II. Expression of ENaC, Na+/K+/2Cl- cotransporter and Na+/H+ exchangers in healthy and arthritic chondrocytes.

In this article, the second of two, we continue our studies of sodium-dependent transport systems in human cartilage from healthy individuals and with osteoarthritis (OA) and rheumatoid arthritis (RA). We demonstrate the presence of the epithelial sodium channel (ENaC), previously undescribed in chondrocytes. This system is composed of three subunits, alpha, beta and gamma. We have shown that the human chondrocytes express at least the alpha and the beta subunit of ENaC. The expression of these subunits is altered in arthritic chondrocytes. In RA samples the quantity of alpha and beta is significantly higher than in control samples. On the other hand, ENaC alpha and beta subunits are absent in the chondrocytes of OA cartilage. Human chondrocytes also possess three isoforms of the Na+/H+ exchanger (NHE), NHE1, NHE2 and NHE3. The NHE system is composed of a single protein and is believed to participate in intracellular pH regulation. Furthermore, our studies indicate that at least one isoform of the electroneutral Na+/K+/2Cl- cotransporter (NKCC) is present in human chondrocytes. There are no obvious variations in the relative expression of NHE isoforms or NKCC between healthy and arthritic cartilage. Our data suggests that chondrocytes from arthritic cartilage may adapt to changes in their environmental sodium concentration through variations in ENaC protein levels. ENaC is also likely to serve as a major sodium entry mechanism, a process that, along with cytoskeletal proteins, may be part of mechanotransduction in cartilage.