Bioactive recombinant neuregulin-1, -2, and -3 expressed in Escherichia coli

The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1 isoforms have been extensively studied, little is yet known about the other genes of the family. We report the expression of recombinant NRG1beta1, NRG2alpha, NRG2beta, and NRG3 as recombinant fusion proteins in Escherichia coli. The cDNA encoding for the EGF-like domain of each protein was cloned from the mouse olfactory bulb and inserted into the pET-19b vector allowing for bacterial expression of the protein fused to an N-terminal His tag. The recombinant NRGs expressed in the inclusion bodies were solubilized under denaturing conditions, purified by affinity chromatography, and refolded via dialysis in the presence of reducing agents. Purified recombinant NRGs were active as they bound to their receptors and induced their phosphorylation. In particular, and in agreement with data on the native proteins, all the molecules were able to bind and activate ErbB4 while only the rNRG1 and the two rNRG2 (but not rNRG3) bound ErbB3.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.     

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor- (TNF-) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg·kg^–1·day^–1 and 1.0 mg·kg^–1·day^–1) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF- and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF- and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity. apoptosis; cytokines; myosin heavy chains     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.