In vitro spermicidal activity of peptides from amphibian skin: dermaseptin S4 and derivatives

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.     

Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.     

Role of nitric oxide in D-galactosamine-induced cell death and its protection by PGE1 in cultured hepatocytes.

Prostaglandin E(1) (PGE(1)) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE(1) protection against D-galactosamine (D-GalN) citotoxicity in cultured hepatocytes. PGE(1) was preadministered to D-GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite+nitrate (NO(x)) release, iNOS expression, and NF-kappaB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by D-GalN in hepatocytes. PGE(1) protection against D-GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by D-GalN. Nevertheless, iNOS inhibition showed that protection by PGE(1) was also mediated by NO. Low concentrations of a NO donor reduced D-GalN injury with a decrease in the extracellular NO(x) concentration. High concentrations of the NO donor enhanced NO(x) concentration and increased cell death by D-GalN. The present study suggests that low NO production induced by PGE(1) preadministration reduces D-GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.     

Photo oxidation of rice starch II. Using a water soluble photo initiator

A new photo oxidation system was established when rice starch was oxidized using UV irradiation and 4-(trimethyl ammoniummethyl) benzophenone chloride (BP2) as a photo initiator. BP2 is a water soluble photo initiator. The slurry prepared for photo oxidation contained rice starch, water and BP2 only. No oxidizing agents were added. Parameters affecting the photo oxidation process, i.e. temperature, concentration of BP2, material:liquor ratio and irradiation time were determined. The produced oxidized starch was evaluated by measuring the carboxyl content, carbonyl content and apparent viscosity. The produced photo oxidized rice starch showed sound increase in the carboxyl and carbonyl contents and sharp decrease in the apparent viscosity. The produced photo oxidized starch was tested for its suitability as a sizing agent for cotton yarns. Native starch and oxidized starch, used as a sizing agent by Misr Company of Spinning and Weaving in El-Mahalla El-Kubra (Egypt), were used for comparison. Sized cotton yarns were evaluated by measuring the tensile strength, elongation at break and percent of size removal. Cotton yarns sized using the prepared photo oxidized rice starch showed higher tensile strength, elongation at break and percent of size removal compared with native starch and oxidized starch used by Misr Company of Spinning and Weaving.     

Synthesis of potent antitumor and antiviral benzofuran derivatives

A new series of potent antitumor and antiviral benzofuran derivatives was synthesized by the reaction of the furochromone-6-carboxaldehydes 1 and 2 with different heterocyclic amines to yield the benzofuran-5-carbonyl derivatives 4–11. The synthesized compounds 1, 3–11 were tested against twelve different human cancer cell lines and all of the compounds were more potent than the comparative standards. The HIV inhibitory activity of the tested compounds 1, 3–11 showed that they have higher potency than Atevirdine. Moreover, compound 6 was significantly potent with wider therapeutic index. The HIV-1 RT inhibitory activity showed that compounds 10, 11, 3 and 4 were notably potent but with lower therapeutic index than Atevirdine. The HCV NS3-4A protease inhibitor activity of the tested compounds revealed that they have weaker potency and less therapeutic index than VX-950, although compounds 1, 4, 9 and 6, respectively exhibited significant activity.     

Inhibition of proinflammatory macrophage responses and lymphocyte proliferation in vitro by ethyl acetate leaf extract from Daphne gnidium

Medicinal plants are considered immunomodulatory as they display various biological activities. There is no report addressing the anti-inflammatory effects of Daphne gnidium. In this study, we investigated the effects of D. gnidium ethyl acetate (EA) leaf extract on mice immune cell function in vitro. Production of pro-inflammatory cytokines (IL-1β and TNF-α), cyclooxygenase-2-derived prostaglandinE2 (PGE2) and iNOS-II-synthesised nitric oxide (NO) were examined. EA extract effect on mitogen-induced lymphocyte proliferation was also investigated. We reported for the first time that D. gnidium EA leaf extract dose-dependently inhibits macrophage proinflammatory function by reducing LPS-induced production of IL-1β, TNF-α, COX-2-derived PGE2 and iNOS-II-synthesised NO. Mitogen-induced lymphocyte proliferation was also dose-dependently inhibited by the extract. Lectin-induced response appears to be more sensitive to the suppressive effects of the extract than LPS-stimulated response. Collectively, these results demonstrate that D. gnidium EA leaf extract acts as an in vitro anti-inflammatory factor by inhibiting mice macrophage and lymphocyte activities.     

PKCε regulates contraction‐stimulated GLUT4 traffic in skeletal muscle cells

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc‐epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol‐induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF‐109203X, Gö6983, and Ro‐31‐8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated‐EAVSLKPT), but not one of PKCδ (myristoylated‐SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol‐induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA‐mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction‐stimulated GLUT4 traffic in muscle cells. J. Cell. Physiol. 226: 173–180, 2010. © 2010 Wiley‐Liss, Inc.     

Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells

Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase–dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)–MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP–MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP–MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA–mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.