Comparison of the interaction of methionine and norleucine-containing peptides with phospholipid bilayers

Norleucine is a structural analog of methionine with a methylene group replacing the thio ether. Despite the close structural similarity of these two amino acids, norleucine-containing peptides have markedly different behaviour with phospholipids compared with methionine-containing peptides. For example, HCO-L-Ahx-L-Leu-L-Phe-OMe behaves as a hydrophobic peptide when mixed with dimyristoylphosphatidylcholine. This peptide lowers the enthalpy of the lipid phase transition. The effect is independent of the rate of heating. With the homologous peptide, HCO-L-Met-L-Leu-L-Phe-OMe, the results are markedly dependent on scan rate with a higher enthalpy observed at faster scan rates. Only at a scan rate of 0.2 K min-1 do the two peptides approach similar behaviour. The higher enthalpy observed for samples with the methionine peptide at higher scan rates can be explained assuming that the peptide aggregates at low temperature. As the phase transition temperature is approached, the more hydrophilic methionine peptide partitions more slowly into the membrane than the norleucine peptide. Partitioning of the peptides between aqueous and lipid phases was measured at 37 degrees by centrifuging down the lipid-bound fraction. At a peptide concentration of 15 microM and a lipid concentration of 1.4 mM, 89% of the HCO-L-Ahx-L-Leu-L-PheOMe and 97% of the HCO-L-Met-L-Leu-L-PheOMe remained in the supernate; indicating a greater tendency of the norleucine-containing peptide to partition into the lipid phase. The peptides Ac-L-Phe-L-Met-L-Arg-L-Phe-NH2 and Ac-L-Phe-L-Ahx-L-Arg-L-Phe-NH2 are readily soluble in water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nalpha-trinitrophenyl glucagon: an inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis.

Nalpha-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AM-independent process.     

Structural properties of acidic phospholipids in complexes with calcitonin: a Fourier transform infrared spectroscopic investigation

The interaction of the polypeptide hormone calcitonin with two acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA), was investigated by Fourier-transform infrared spectroscopy. The association of calcitonin with DMPG results in a broadening of the lipid phase transition, accompanied by a marked decrease in the conformational order of the acyl chains at temperatures below the phase transition region. Infrared bands due to carbonyl ester and phosphate group vibrations of DMPG molecules are not significantly affected by the presence of calcitonin. The effect of calcitonin on the conformation of acyl chains in DMPA is much smaller compared with DMPG. The different susceptibility of DMPG and DMPA to perturbation by calcitonin is suggested to be related to different degrees of intermolecular interactions between the headgroups of these two phospholipids.     

Histopathological effects of cisplatin,doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats

Cisplatin, doxorubicin and fluorouracil (5-FU), drugs belonging to different chemical classes, have been extensively used for chemotherapy of various cancers. Despite extensive investigations into their hepatotoxicity, there is very limited information on their effects on the structure and ultra-structure of liver cells in vivo. Here, we demonstrate for the first time, the effects of these three anticancer drugs on rat liver toxicity using both light and electron microscopy. Light microscopic observations revealed that higher doses of cisplatin and doxorubicin caused massive hepatotoxicity compared to 5-FU treatment, including dissolution of hepatic cords, focal inflammation and necrotic tissues. Interestingly, low doses also exhibited abnormal changes, including periportal fibrosis, degeneration of hepatic cords and increased apoptosis. These changes were confirmed at ultrastructural level, including vesiculated rough endoplasmic reticulum and atrophied mitochondria with ill-differentiated cisternae, dense collection of macrophages and lymphocytes as well as fibrocytes with collagenous fibrils manifesting early sign of fibrosis, especially in response to cisplatin and doxorubicin -treatment. Our results provide in vivo evidence, at ultrastructural level, of direct hepatotoxicity caused by cisplatin, doxorubicin and 5-FU at both light and electron microscopi. These results can guide the design of appropriate treatment regimen to reduce the hepatotoxic effects of these anticancer drugs.     

The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue

Introduction  

Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.     

Interaction of NDPK-D with cardiolipin-containing membranes: Structural basis and implications for mitochondrial physiology

Nucleoside diphosphate kinases (NDPKs/Nm23), responsible for intracellular di- and tri-phosphonucleoside homeostasis, play multi-faceted roles in cellular energetic, signaling, proliferation, differentiation and tumor invasion. The mitochondrial NDPK-D, the NME4 gene product, is a peripheral protein of the inner membrane. Several new aspects of the interaction of NDPK-D with the inner mitochondrial membrane have been recently characterized. Surface plasmon resonance analysis using recombinant NDPK-D and different phospholipid liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin, a phospholipid located mostly in the mitochondrial inner membrane. Mutation of the central arginine (R90) in a surface exposed cationic RRK motif unique to NDPK-D strongly reduced phospholipid interaction in vitro and in vivo. Stable expression of NDPK-D proteins in HeLa cells naturally almost devoid of this isoform revealed a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the enzyme. Owing to its symmetrical hexameric structure exposing membrane binding motifs on two opposite sides, NDPK-D could bridge liposomes containing anionic phospholipids and promote lipid transfer between them. In vivo, NDPK-D could induce intermembrane contacts and facilitate lipid movements between mitochondrial membranes. Most of these properties are reminiscent to those of the mitochondrial creatine kinase. We review here the common properties of both kinases and we discuss their potential roles in mitochondrial functions such as energy production, apoptosis and mitochondrial dynamics.     

Mammalian diacylglycerol kinases: Molecular interactions and biological functions of selected isoforms

The mammalian diacylglycerol kinases (DGK) are a group of enzymes having important roles in regulating many biological processes. Both the product and the substrate of these enzymes, i.e. diacylglycerol and phosphatidic acid, are important lipid signalling molecules. Each DGK isoform appears to have a distinct biological function as a consequence of its location in the cell and/or the proteins with which it associates. This review discusses three of the more extensively studied forms of this enzyme, DGKα, DGK?, and DGKζ. DGKα has an important role in immune function and its activity is modulated by several mechanisms. DGK? has several unique features among which is its specificity for arachionoyl-containing substrates, suggesting its importance in phosphatidylinositol cycling. DGKζ is expressed in many tissues and also has several mechanisms to regulate its functions. It is localized in several subcellular organelles, including the nucleus. The current state of our understanding of the properties and functions of these proteins is reviewed.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Cell-Wall Interactions and the Selective Bacteriostatic Activity of a Miniature Oligo-Acyl-Lysyl

The oligo-acyl-lysyl, C_12(ω7)K-β₁₂, is comprised of only three Lys residues. Despite its small size, it exhibits potent bacteriostatic activity against Gram-positive bacteria, but it is ∼10-fold less potent against Gram-negative bacteria. We followed the interactions of C_12(ω7)K-β₁₂ from its initial contact with the bacterial surface across the cell wall down to the cytoplasmic membrane. Binding to anionic lipids, as well as to negatively charged LPS and LTA, occurs with very high affinity. The C_12(ω7)K-β₁₂ does not cross the outer membrane of Gram-negative bacteria; rather, it achieves its action by depositing on the LPS layer, promoting surface adhesion and blocking passage of solutes. In Gram-positive bacteria, the thick peptidoglycan layer containing LTA allows passage of C_12(ω7)K-β₁₂ and promotes its accumulation in the small periplasm. From that location it is then driven to the membrane by strong electrostatic interactions. Despite its high potency against Gram-positive bacteria, this agent is not capable of efficiently breaking down the permeability barrier of the cytoplasmic membrane or of reaching an intracellular target, as suggested by the fact that it does not interact with DNA.