[Genetic and molecular principles for the selection of Pseudomonas and Staphylococcus therapeutic bacteriophages].

The content of empirically selected bacteriophage mixtures, produced by Microgen for the prevention and treatment of staphylococcal and pseudomonade infections, was investigated by negative stain electron microscopy. The main population of phages was shown to belong to the groups suitable for therapeutic purposes based on bioinformatics analysis of known genomes of Pseudomonas and Staphylococcus phages. However, the phage morphology studies did not always reveal the exact correspondence of the phage to the exact group. Therefore, we suggest group genotyping of the therapeutic bacteriophages on thebasis of genetic conservative locus.

Neuroprotective activities of carvedilol and a hydroxylated derivative: role of membrane biophysical interactions.

Carvedilol is a vasodilating beta-blocker and antioxidant approved for treatment of mild to moderate hypertension, angina, and congestive heart failure. SB 211475 (4-[2-hydroxyl-3-[[2-(2-methoxyphenoxy)ethyl]amino]propoxyl]-9H-++ +carbazol-3-ol), a hydroxylated carvedilol analogue, is an even more potent antioxidant in several assay systems. Carvedilol also has neuroprotective capacity with modulatory actions at N-methyl-D-aspartate (NMDA) receptors and Na+ channels. In the present study, we demonstrated that in cultured rat cerebellar neurons, SB 211475 has 28-fold greater antioxidant activity than carvedilol, but is 2- to 6-fold less potent, respectively, at inhibiting neurotoxic activities at Na+ channels and at NMDA receptor channels. To determine a biophysical rationale for these differential activities, small angle x-ray scattering data were obtained from model lipid and brain membrane bilayers containing either carvedilol, SB 211475, or dihydropyridine calcium channel blockers. Electron density profiles revealed that the location of SB 211475 was restricted to the glycerol backbone/hydrocarbon interface and significantly reduced membrane width by 5%, whereas the time-averaged location for carvedilol and flunarizine also extended to the hydrated surface of the bilayer. Comparison of carvedilol with several dihydropyridines showed a correlation between high ClogP values (lipophilicity), Na+ channel inhibitory potency, and bilayer localization. The antioxidant activity of SB 211475 could be explained by restricted intercalation into the glycerol phosphate/hydrocarbon interface, creating an increase in volume associated with the phospholipid acyl chains, which would then become resistant to lipid peroxidation. Differential channel modulation may also be explained by these membrane structural results, which indicate that carvedilol and the less spatially restricted dihydropyridine molecules are more likely to inhibit transmembrane receptor channels.

SB 211475, a metabolite of carvedilol, a novel antihypertensive agent, is a potent antioxidant.

The antioxidant effects of SB 211475, a metabolite of carvedilol, a novel antihypertensive agent, were studied and compared with carvedilol and other antioxidants such as U78517F, U74500A and probucol. SB 211475 inhibited Fe(2+)-vitamin C-initiated lipid peroxidation, assessed as thiobarbituric acid reactive substance, in brain-homogenate with an IC50 of 0.28 microM. Under the same conditions, the IC50s of probucol, carvedilol, U74500A and U78517F were 50, 8.1, 0.71 and 0.16 microM, respectively. SB 211475 inhibited oxidation of human low density lipoprotein by mouse macrophages with an IC50 of 0.043 microM. In the same model, the IC50s of carvedilol, U78517F and probucol were 3.8, 0.15, and 0.80 microM, respectively. SB 211475 protected cultured bovine pulmonary artery endothelial cells against hydroxyl radical-initiated lipid peroxidation (IC50 = 0.15 microM) and cell damage (lactate dehydrogenase release, IC50 = 0.16 microM), and promoted cell survival with an EC50 of 0.13 microM. SB 211475 also protected endothelial cells against xanthine/xanthine oxidase-initiated cytotoxicity and protected rat cerebellar neurons from hydroxyl radical-mediated cell death (EC50 = 0.19 microM). Moreover, SB 211475 inhibited superoxide (O2-) release from human neutrophils stimulated by phorbol myristate acetate. These observations indicate that SB 211475 is a potent antioxidant and may potentially contribute to the therapeutic effects of carvedilol in vivo.

Neuroprotective effects of carvedilol, a new antihypertensive, at the N-methyl-D-aspartate receptor.

Carvedilol's potent antioxidant activity could explain its protective action in brain ischemia, but may not apply to glutamate-induced excitotoxicity in cultured cerebellar granule cells, since glutamate neurotoxicity was not associated with the formation of lipid peroxidative products. Rather, carvedilol diminished the N-methyl-D-aspartate (NMDA)/glycine-induced increase in intracellular calcium ([Ca2+]i), lowering [Ca2+]i by a maximum of 66 +/- 5% (n = 8) with a 50% inhibitory concentration of 0.8 microM. Prior addition of 5 microM dihydropyridines did not shift the dose-response of carvedilol, but did significantly lower the NMDA/glycine-stimulated response to 64% of untreated (n = 8, P = 0.014). Inclusion of 5 microM carvedilol before the additions of NMDA/glycine prevented 85% of the increase in [Ca2+]i. Furthermore, carvedilol displaced 3[H]MK-801 binding to rat brain cortical membranes with a Kd of 29.4 +/- 2.2 microM (n = 6) and no selectively for the glutamate or glycine binding sites. These data therefore suggest that, in addition to its antihypertensive and anti-lipid peroxidative functions, carvedilol has neuroprotective activity as a calcium channel blocker and as a non-competitive inhibitor at the NMDA receptor.

Neuroprotective effects of carvedilol, a new antihypertensive agent, in cultured rat cerebellar neurons and in gerbil global brain ischemia.

BACKGROUND AND PURPOSE: Free radical generation mediates part of the ischemic neuronal damage caused by the excitatory amino acid glutamate. Carvedilol, a novel multiple-action antihypertensive agent, has been shown to scavenge free radicals and inhibit lipid peroxidation in swine heart and rat brain homogenates. Therefore, we studied the neuroprotective effect of carvedilol on cultured cerebellar neurons and on CA1 hippocampal neurons of gerbils exposed to brain ischemia. METHODS: Neuroprotective mechanisms were studied using an in vitro ischemia model of cultured rat cerebellar granule cell neurons exposed to either glutamate or oxygen free radical-generating systems. Prevention of lipid peroxidation by carvedilol was studied by measuring the formation of thiobarbituric acid-reactive substance. Gerbil CA1 neuron survival was examined by direct neuronal count 7 days after 6 minutes of global ischemia with reperfusion. RESULTS: Carvedilol protected cultured neurons in a dose-dependent manner against glutamate-mediated excitotoxicity (inhibitory concentration [IC50] = 1.1 microM) as well as against a 20-minute oxidative challenge (IC50 = 5 microM). The IC50 against the oxidative challenge was lowered to 1.3 microM by growing neurons for 24 hours in the presence of carvedilol. At 10 microM carvedilol inhibited lipid peroxidation 50% and 73% (n = 4, p < 0.001) in neurons exposed to two different free radical-generating systems. Neuroprotection of 52% (n = 22, p = 0.009 versus vehicle) of gerbil CA1 hippocampal neurons was achieved by pretreatment and posttreatment with subcutaneous injection of 3 mg/kg carvedilol twice a day for 4 and 3 days, respectively. CONCLUSIONS: Carvedilol provided neuroprotection in both in vitro and in vivo models of neuroinjury, where oxygen radicals are likely to play an important role. Therefore, carvedilol may reduce the risk of cerebral ischemia and stroke by virtue of both its antihypertensive action and its antioxidative properties.

Carvedilol, a new vasodilator and beta adrenoceptor antagonist, is an antioxidant and free radical scavenger.

The antioxidant effect of carvedilol, a new vasodilating, beta adrenoceptor blocker was studied and compared with five other beta blockers. Carvedilol rapidly inhibited Fe(++)-initiated lipid peroxidation, measured as thiobarbituric acid reactive substance (TBARS), in rat brain homogenate with an IC50 of 8.1 microM. Under the same conditions, the IC50 values of atenolol, pindolol propranolol, celiprolol and labetalol were over 1.0 mM. Carvedilol protected against Fe(++)-induced alpha-tocopherol depletion in rat brain homogenate with an IC50 of 17.6 microM; propranolol, celiprolol and labetalol, up to 200 microM, did not show any effect. Using dihydroxyfumarate/Fe(++)-ADP as a OH.radical generating system and 5,5-dimethyl pyrroline-N-oxide (DMPO) as a trapping agent, the characteristic DMPO-OH signals were monitored by electron paramagnetic resonance. Carvedilol dose-dependently decreased the intensity of the DMPO-OH signal, with an IC50 of 25 microM, whereas propranolol, at 500 microM, and U74500A, a 21-aminosteroid, at 100 microM, had no effect. The antioxidant effect of carvedilol mainly resides in the carbazole moiety, and the substitution of a hydroxyl group at certain positions on the phenyl ring of either carbazole or the ortho-substituted phenoxylethylamine part of carvedilol resulted in an increase in antioxidant activity. Furthermore, the protective effect of carvedilol analogs against OH.-mediated neuronal death positively correlated to their antioxidant effect. We conclude that carvedilol is a far more potent antioxidant than other commonly used beta blockers. The apparent mechanism of carvedilol's inhibition of lipid peroxidation is mainly via scavenging free radicals. This novel property of carvedilol may contribute to the known cardioprotective activity of this compound.

Protein involvement in structural transition of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation.

In this study, it is shown that systematic temperature-induced protein aggregation occurs on the erythrocyte membrane by intermolecular disulfide bond formation. Specific protein bands disappear from acrylamide gel profiles over rather narrow temperature regions. The aggregation appears to be the result of irreversible structural transitions of the membrane, which can be seen in a sensitive scanning calorimeter. When this method of thermal gel analysis is used, the results suggest that spectrin is a participant in the A transition, that bands 2.1, 4.1, and 4.2 and the cytoplasma portion of 3 are involved in the B transition, and that the transmembrane portion of band 3 may undergo changes in the C transition, previously shown to occur in the anion transport domain of the membrane. The aggregation of specific proteins in the narrow temperature region of these transitions persists as the transitions are moved around on the temperature axis by varying solution conditions. The assignment of particular proteins to specific transitions is reinforced by selective extraction of membrane proteins. Large variations in both the calorimetry and the aggregation pattern occur as salt concentration is increased from 77 mosm to 310 mosm, which is manifested in the splitting of the B transition into two separate transitions, B1 and B2. It is speculated that this occurs as the result of a structural change which may involve components of the cytoskeletal network.

Calorimetric studies of the structural transitions of the human erythrocyte membrane. Studies of the B and C transitions.

Differential scanning calorimetry has been used to study several structural transitions of the human erythrocyte membrane. Earlier studies have shown that one of these transitions (the A transition) is due to the thermal unfolding of spectrin on the membrane. In this paper, it is shown that two of the other transitions (B and C) exhibit a high sensitivity to a local anesthetic, benzyl alcohol. Increasing the ionic strength of the suspending medium results in a splitting of the B transition into two indepent transitions (B1 and B2). It is found that one of these (B2) is associated with titrating groups, since the midpoint for the transitions shifts by about 20 degrees C, with an apparent pK near 7.5 Extensive bilateral proteolysis by papain causes a drastic decrease in the size of all transitions except the C transition, which remains unaltered. On the other hand, treatment with phospholipase by A2 largely affects the C transition, causing its disappearance. Because of the lack of sensitivity to proteolysis and the high sensitivity to phospholipase, it appears that the C transition has a large extent of 'lipid involvement'. It might result from the melting of a small fraction of phospholipid which exists in a crystalline state under physiological conditions. Alternatively, the C transition could arise from changes in protein-lipid interactions or from lipid-dependent changes in protein-protein interactions, providing one assumes that only protease-resistant portions of membrane proteins are participating.