Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Regulation of Visceral and Subcutaneous Adipocyte Lipolysis by Acute AICAR‐induced AMPK Activation

This study investigated the role of adenosine monophosphate–activated protein kinase (AMPK) in the regulation of lipolysis in visceral (VC) and subcutaneous (SC) rat adipocytes and the molecular mechanisms involved in this process. VC (epididymal and retroperitoneal) and SC (inguinal) adipocytes were isolated from male Wistar rats (160–180 g). Adipocytes were incubated either in the absence or in the presence of the AMPK agonist 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR, 0–500 µmol/l). AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, basal and epinephrine‐stimulated (100 nmol/l) glycerol release, and hormone‐sensitive lipase (HSL) phosphorylation and activity were determined. AICAR‐induced (500 µmol/l) AMPK activation inhibited basal glycerol release by ～42, 41, and 44% in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Epinephrine‐stimulated glycerol release was almost completely prevented by AICAR treatment in adipocytes from all fat depots. The AMPK inhibitor compound C (20 µmol/l) prevented AICAR‐induced phosphorylation of AMPK and significantly increased basal (～1.3‐, 1.4‐, and 1.7‐fold) and epinephrine‐stimulated (～1.3‐, 1.2‐, 1.4‐fold) glycerol release in epididymal, retroperitoneal, and inguinal adipocytes, respectively. AICAR increased phosphorylation of HSL_Ser565 and inhibited epinephrine‐induced phosphorylation of HSL_Ser563 and HSL_Ser660. This was also accompanied by a 73% reduction in epinephrine‐stimulated HSL activity. Compound C prevented the phosphorylation of HSL_Ser565 induced by AICAR and partially prevented the inhibitory effect of this drug on basal and epinephrine‐stimulated lipolysis in adipocytes in VC and SC fat depots. In summary, despite different fat depots eliciting distinct rates of lipolysis, acute AICAR‐induced AMPK activation suppressed HSL phosphorylation/activation and exerted similar antilipolytic effects on both VC and SC adipocytes.     

The Legionella pneumophila F‐box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication

The environmental pathogen Legionella pneumophila encodes three proteins containing F‐box domains and additional protein–protein interaction domains, reminiscent of eukaryotic SCF ubiquitin–protein ligases. Here we show that the F‐box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella‐containing vacuole. Single, double and triple mutants of the F‐box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP‐1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo , and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two‐hybrid screen and co‐immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein–protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.     

Calcium occlusion in plasma membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.     

Dynamic lipid-protein stoichiometry on E1 and E2 conformations of the Na+/K+ -ATPase

Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.     

Experimental and theoretical study of a Diels-Alder reaction between a sugar-derived nitroalkene and cyclopentadiene

The Diels–Alder reaction of a pyranose-derived nitroalkene 1 with cyclopentadiene is described, and the unexpected facial selectivity of the cycloaddition is analyzed by a computational study.     

3D Stationary electric current density in a spherical tumor treated with low direct current: an analytical solution

Electrotherapy with direct current delivered through implanted electrodes is used for local control of solid tumors in both preclinical and clinical studies. The aim of this research is to develop a solution method for obtaining a three-dimensional analytical expression for potential and electric current density as functions of direct electric current intensity, differences in conductivities between the tumor and the surrounding healthy tissue, and length, number and polarity of electrodes. The influence of these parameters on electric current density in both media is analyzed. The results show that the electric current density in the tumor is higher than that in the surrounding healthy tissue for any value of these parameters. The conclusion is that the solution method presented in this study is of practical interest because it provides, in a few minutes, a convenient way to visualize in 3D the electric current densities generated by a radial electrode array by means of the adequate selection of direct current intensity, length, number, and polarity of electrodes, and the difference in conductivity between the solid tumor and its surrounding healthy tissue.