Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects. (9/4528)

Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested.  (+info)

Stability of gentamicin in serum. (10/4528)

Patients' sera were divided into three portions when the initial gentamicin level was determined and were stored at -20, 4, and 25 degrees C in plastic or glass tubes. Gentamicin levels were repeated after 1 and 2 days of storage at the respective temperatures. There was no significant difference in gentamicin levels among portions, except those from a patient in renal failure with high serum concentrations of carbenicillin.  (+info)

Control of lipid membrane stability by cholesterol content. (11/4528)

Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content.  (+info)

Dynamical properties of phospholipid bilayers from computer simulation. (12/4528)

We present the results of a 10-ns molecular dynamics simulation of a dipalmitoylphosphatidylcholine/water system. The main emphasis of the present study is on the investigation of the stability over a long time and the dynamic properties of the water/membrane system. The motion of the lipid molecules is characterized by the center of mass movement and the displacement of individual atom groups. Because of the slow movement of the headgroup atoms, their contributions to the dipole potential vary slowly and with a large amplitude. Nevertheless, the water molecules compensate the strong fluctuations and maintain an almost constant total dipole potential. From the lateral displacement of the center of masses, we calculate the lateral diffusion coefficient to be Dlat = (3 +/- 0.6) x 10(-7) cm2/s, in agreement with neutron scattering results. The rotational motion is also investigated in our simulations. The calculated value for the rotational diffusion coefficient parallel to the molecular long axis, D = (1.6 +/- 0.1) x 10(8) s-1, is in good agreement with the experiment.  (+info)

Western blot analysis of bile or intestinal fluid from patients with septic shock or systemic inflammatory response syndrome, using antibodies to TNF-alpha, IL-1 alpha and IL-1 beta. (13/4528)

Septic shock or systemic inflammatory response syndrome (SIRS) often develops in patients following burns, traumatic injury, surgery or biliary obstruction. Although the inflammatory cytokines TNF-alpha and IL-1 have been strongly implicated in the development of these syndromes, treatment of patients by the systemic administration of inhibitors of TNF-alpha or IL-1 has shown limited effectiveness. Recent reports suggest that septic shock may be perpetuated by inflammatory cytokines secreted by the liver in response to bacterial translocation resulting from cytokine-induced gastrointestinal damage. The present study sought to demonstrate the presence of high levels of inflammatory cytokines in the bile or small intestine of patients suffering from septic shock or SIRS, with a view to the development of strategies for the reduction of gastrointestinal damage through intraduodenal administration of cytokine inhibitors. Western blot analysis of human bile or intestinal fluid using anti-TNF-alpha antibodies resulted in the detection of a number of bands in samples from patients with septic shock or SIRS. However, these proteins differed antigenically from human recombinant TNF-alpha (rTNF-alpha) and showed no activity in a biological assay for TNF-alpha. Antibodies to IL-1 alpha and IL-1 beta detected several strong bands, some of which appeared to be identical to recombinant IL-1 alpha and IL-1 beta. It is concluded that proteins resembling several known inflammatory cytokines are present in the bile and intestine of septic shock patients, but it is suggested that further work is required to determine the nature and function of these molecules.  (+info)

Solubilization of diglyceride acyltransferase from the membrane of Mycobacterium smegmatis. (14/4528)

Diglyceride acyltransferase [acyl-CoA : 1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20] was found to be localized in the membrane of Mycobacterium smegmatis, and this enzyme could be solubilized from the membrane by treatment with aqueous acetone. The solubilized enzyme required either 1,2-diolein or 1, 3-diolein as an acceptor for palmitoyl-CoA. The apparent Km value for 1,2- or 1,3-diolein and that for palmitoyl-CoA were about 1.4 X 10(-5) M and 6 X 10(-6) M, respectively. Several sulfhydryl reagents were inhibitory to the enzyme activity, suggesting the existence of a thiol group(s) in its active site. The solubilized enzyme, which was more labile than that membrane-bound one, could be stabilized to some extent with antichaotropic salts such as phosphate, pyrophosphate, and sulfate.  (+info)

Ribonucleases from porcine brain. Partial purification and properties. (15/4528)

1. An acid ribonuclease was partially purified from an acetone powder of porcine brain. This enzyme was an acidic protein with a molecular weight of aroung 70,000. It acted on yeast RNA optimally at about pH 5.9, yielding only a mixture of 3'-mononucleotides, and therefore appears to be an exonuclease. It did not hydrolyze heat-denatured calf thymus DNA or bis(rho-nitrophenyl) phosphate. It was fairly unstable to heat and acid. 2. An alkaline ribonuclease was partially purified from the same source simultaneously. This enzyme was a basic protein with a molecular weight of 25,000-26,000. It was a pyrimidine-specific endoribonuclease, and acted on yeast RNA optimmally at around pH 7.4. It did not hydrolyze heat-denatured calf thymus DNA or bis(rho-nitrophenyl) phosphate. It was fairly stable to heat and acid.  (+info)

Proteolytic activation and inactivation of chitin synthetase from Mucor rouxii. (16/4528)

Crude chitin synthetase preparations from the mycelial and yeast forms of Mucor rouxii behaved differently. The mycelial preparations, incubated at 28 degrees C, lost virtually all chitin synthetase activity in a few hours; by contrast, the activity of enzyme preparations from yeast cells increased several fold during similar incubations. These spontaneous changes were probably caused by endogenous protease(s). Seemingly, the chitin synthetase in yeast preparations was present mainly in a latent, 'zymogenic', form that was activated by proteases. In the mycelial preparations, chitin synthetase was present mainly in an active state and was rapidly degraded by endogenous proteolysis. Exogenous proteases accelerated activation and destruction of chitin synthetase; an acid protease from Rhizopus chinensis was the most effective activator. The activation of chitin synthetase was inhibited by a soluble protein in the cell-free extract. Treatment with the detergent Brij 36T stabilized the chitin synthetase of crude preparations against spontaneous changes. Stabilized preparations were rapidly activated by exogenous proteases. The different behaviour of chitin synthetases in crude extracts of mycelium and yeast cells is consistent with, and perhaps partially responsible for, the differences in wall construction between mycelial and yeast forms of M. rouxii.  (+info)