Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 µM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Activation of RNase L by 2',5'-oligoadenylates. Kinetic characterization.

Ribonuclease L (RNase L), the 2',5'-oligoadenylate-dependent ribonuclease, is one of the cellular antiviral systems with enhanced activity in the presence of interferon. A reaction scheme has been developed to model the sequence of steps necessary for the activation of RNase L (Cole, J. L., Carroll, S. S., Blue, E. S., Viscount, T., and Kuo, L. C. (1997) J. Biol. Chem. 272, 19187-19192). The model comprises three sequential binding steps: the binding of activator to enzyme monomer, the subsequent dimerization of the activated monomer to form the active enzyme dimer, followed by the binding of substrate prior to catalysis. The model is used to evaluate the activation of RNase L by several synthetic analogs of the native activator. The 5'-phosphate of the activator has been determined to be an important structural determinant for the efficient activation of RNase L, and its loss caused a loss of activator affinity of 2-3 orders of magnitude. The length of activator is not an important determinant of activator potency for the activator analogs examined. The specific activity of the enzyme under conditions of saturation of activator binding and complete dimerization of the activated monomers varies only by about a factor of 3 for the activators examined, indicating that once dimerized in the presence of any of these activators, the enzyme exhibits a similar catalytic activity.

Cleavage of oligoribonucleotides by the 2',5'-oligoadenylate- dependent ribonuclease L.

RNase L, the 2',5' oligoadenylate-dependent ribonuclease, is one of the enzyme systems important in the cellular response to interferon. When activated in the presence of 2',5'-linked oligoadenylates, RNase L can catalyze the cleavage of synthetic oligoribonucleotides that contain dyad sequences of the forms UU, UA, AU, AA, and UG, but it cannot catalyze the cleavage of an oligoribonucleotide containing only cytosines. The primary site of the cleavage reaction with the substrate C11UUC7 has been defined to be 3' of the UU dyad by labeling either the 5' or the 3' end of the oligoribonucleotide and by examining the reaction products on polyacrylamide sequencing gels. Reaction time courses have been used to determine the kinetic parameters of the cleavage reactions. The effect of the overall length of the oligomeric substrate as well as the sequence of the bases around the position of the cleavage site on the kinetics of the cleavage reaction has been examined. The efficiency with which activated RNase L catalyzes the cleavage of the substrate C11UUC7 is 1.9 x 10(7) m-1 s-1. Because the cleavage of the synthetic oligoribonucleotide can be used to monitor the steady-state kinetics of catalysis by activated RNase L, this method offers an advantage over previous methods of assay for RNase L activity.

Solution oligomerization of the rev protein of HIV-1: implications for function.

rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts. Oligomerization of rev is thought to be associated with RNA binding and rev function. Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation. rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations. The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal [K = 1.08 x 10(6) M-1 at 4 degrees C]. The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9. Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy. Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay. rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function.

Endothelium-derived nitric oxide reduces baseline venous tone in awake instrumented rats.

To determine whether nitric oxide, which is likely endothelium-derived relaxing factor (EDRF), modulates baseline venous tone, the effects of intravenous NG-monomethyl-L-arginine (L-NMMA) (3-25 mg/kg), an EDRF inhibitor, on mean circulatory filling pressure (MCFP) were determined in 10 awake instrumented rats. MCFP, the equilibrated systemic pressure occurring when the circulation is arrested by transient inflation of a balloon in the right atrium, is a measure of total venous capacitance. L-NMMA caused a dose-dependent increase in mean arterial pressure and a dose-dependent decrease in heart rate. MCFP rose from 6.6 +/- 0.2 to 7.6 +/- 0.2 mmHg at the highest L-NMMA dose. The effects of L-NMMA on MCFP were reversed with L-arginine. In an additional four rats, in which hexamethonium was administered to induce ganglionic blockade, L-NMMA (25 mg/kg) caused a similar increase in MCFP (4.1 +/- 0.6 to 5.0 +/- 0.7 mmHg, P = 0.22) during the ganglionic blocked state as during the control unblocked state. These findings suggest that nitric oxide, which is likely EDRF, reduces baseline venous tone.

Aging increases venous compliance in awake instrumented rats.

To determine the effects of aging on total venous compliance, mean circulatory filling pressure (MCFP) was determined at several different blood volumes in 10 young (10-mo-old) and 10 older (30-mo-old) awake instrumented male Fischer 344/Brown Norway hybrid rats. Baseline weight and mean arterial pressure were similar in the two groups; heart rate was higher in the young (426 +/- 9 beats/min) than in the older rats (376 +/- 8 beats/min). Although MCFP was similar in the two groups at baseline blood volume, MCFP rose less with transient volume expansion and fell less with transient volume depletion in the older rats. The calculated venous compliance (reciprocal of the slope of the MCFP-to-volume relation) for the older rats was 25% greater than in the younger rats (3.28 +/- 0.21 vs. 2.63 +/- 0.12 ml.kg-1.mmHg-1; P = 0.014). In this conscious instrumented rat model, baseline total venous compliance is increased in older rats.

Adenosine increases total venous capacitance in awake instrumented rats.

To determine the effect of adenosine on the venous system, mean circulatory filling pressure (MCFP) was determined during infusion of intravenous (i.v.) adenosine (66.5 to a maximum of 532 microgram.kg-1.min-1) in 9 awake instrumented rats before and during ganglionic blockade with i.v. hexamethonium, 0.6 mg.kg-1.min-1. MCFP, the equilibrated pressure (mm Hg) occurring when the circulation is arrested by transient inflation of a balloon in the right atrium, is inversely related to total venous capacitance. Both adenosine and hexamethonium caused a reduction in mean arterial pressure (MAP); heart rate (HR) decreased during adenosine infusion in the blocked, but not the unblocked, state. In the unblocked state, baseline MCFP was 6.5 +/- 0.3 mmHg; hexamethonium caused baseline MCFP to decrease to 5.3 +/- 0.3 mm Hg. In both the unblocked and the blocked state, adenosine caused a dose-related decrease in MCFP [6.5 +/- 0.3 to 5.5 +/- 0.6 mm Hg (532 microgram.kg-1.min-1 adenosine dose) unblocked state; and 5.3 +/- 0.3 to 4.3 +/- 0.3 mm Hg (400 microgram.kg-1.min-1 adenosine dose) blocked state]. This decrease in MCFP induced by adenosine was highly significant. Intravenous adenosine, in an awake instrumented rat model, increases venous capacitance, with and without ganglionic blockade.

Intracoronary adenosine and papaverine do not increase myocardial systolic thickening.

STUDY OBJECTIVE: The aim was to determine if myocardial systolic thickening increases when coronary flow is augmented by infusing intracoronary vasodilators (adenosine and papaverine). DESIGN: Systolic thickening fraction was measured with pulsed Doppler crystals and sonomicrometer crystals before and during the intracoronary infusion of adenosine and papaverine. SUBJECTS: Sixteen anaesthetized mongrel dogs were studied. MEASUREMENTS AND MAIN RESULTS: Intracoronary adenosine did not alter systemic haemodynamics, but did induce a three- to fourfold increase in myocardial blood flow. Intracoronary papaverine caused a slight decrease in systemic arterial pressure and rise in heart rate. Neither intracoronary adenosine nor intracoronary papaverine increased systolic thickening: control thickening fraction (TF%) = 20 (SEM 1)%, adenosine TF% = 18(1)%; control TF% = 22(2)%, papaverine TF% = 20(2)%. CONCLUSIONS: These experiments do not support the hypothesis that an increase in myocardial blood flow induced by intracoronary vasodilators causes an increase in myocardial systolic function.