Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.     

Antimicrobial Resistance, Virulence Genes, and Genetic Lineages of Staphylococcus pseudintermedius in Healthy Dogs in Tunisia

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec _canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg _v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.     

Dual inhibition of DNA polymerase PolC and protein tyrosine phosphatase CpsB uncovers a novel antibiotic target

Increasing antibiotic resistance is making the identification of novel antimicrobial targets critical. Recently, we discovered an inhibitor of protein tyrosine phosphatase CpsB, fascioquinol E (FQE), which unexpectedly inhibited the growth of Gram-positive pathogens. CpsB is a member of the polymerase and histidinol phosphate phosphatase (PHP) domain family. Another member of this family found in a variety of Gram-positive pathogens is DNA polymerase PolC. We purified the PHP domain from PolC (PolC_PHP), and showed that this competes away FQE inhibition of CpsB phosphatase activity. Furthermore, we showed that this domain hydrolyses the 5′-p-nitrophenyl ester of thymidine-5′-monophosphate (pNP-TMP), which has been used as a measure of exonuclease activity. Finally, we showed that FQE not only inhibits the phosphatase activity of CpsB, but also ability of PolC_PHP to catalyse the hydrolysis of pNP-TMP. This suggests that PolC may be the essential target of FQE, and that the PHP domain may represent an as yet untapped target for the development of novel antibiotics.     

Facile synthesis and characterization of novel pyrazole-sulfonamides and their inhibition effects on human carbonic anhydrase isoenzymes

In the current study, a series of pyrazole-sulfonamide derivatives (2–14) were synthesized, characterized, and the inhibition effects of the derivatives on human carbonic anhydrases (hCA I and hCA II) were investigated as in vitro. Structures of these sulfonamides were confirmed by FT-IR, ¹H NMR, ¹³C NMR and LC–MS analysis. ¹H NMR and ¹³C NMR revealed the tautomeric structures. hCA I and hCA II isozymes were purified from human erythrocytes and inhibitory effects of newly synthesized sulfonamides on esterase activities of these isoenzymes have been studied. The K_i values of compounds were 0.062–1.278 μM for hCA I and 0.012–0.379 μM for hCA II. The inhibition effects of 7 for hCA I and 4 for hCA II isozymes were almost in nanomolar concentration range.     

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.