Radiolabelling of lipid-based nanocarriers with fluorine-18 for in vivo tracking by PET.

Organic nanoparticles made out of biodegradable and biocompatible materials have attracted increased attention in the therapeutic and diagnostic fields. In this study, we attempted to explore a new radiolabelling chelating free strategy for biodegradable sphingomyelin nanometric emulsions with fluorine-18 (18F), a radioisotope regularly used in clinic. [18F]fluoride was produced by the cyclotron and was incorporated into 4-[18F]fluorobenzamido-N-ethylmaleimide ([18F]FBEM), which was coupled next to the emulsions previously functionalized with a thiol group, via inclusion of either a thiol-PEG-lipid (SH-PEG12-C18), or a peptide-PEG-lipid (Cys-Pro-Ile-Glu-Asp-Arg-Pro-Met-Cys-PEG8-C18) derivative. Radiolabelled emulsions were obtained in a rapid and efficient fashion through facile-conjugated chemistry without the use of organic solvents, and characterized in terms of size, polydispersity, surface charge, pH, and osmolarity. PET imaging and biodistribution studies in BALB/c mice allowed obtaining the pharmacokinetics of the radiolabelled emulsions and determining the clearance pathways. Altogether, we confirmed the potential of this new technique for the radiolabelling of lipid-based drug nanosystems for application in PET imaging diagnosis.

Increase in molecular rigidity of the protein conformation of brain Na+-K+-ATPase by modification with 4-hydroxy-2-nonenal.

The effect of lipid peroxidation product 4-hydroxy-2-nonenal (HNE) on the protein conformation of porcine cerebral cortex Na(+)-K(+)-ATPase was examined in term of the intrinsic tryptophanyl fluorescence measurement. Treatment of ATPase with HNE resulted in a decrease in the fluorescence intensity and an increase in the fluorescence anisotropy in a concentration-dependent manner. The difference in the fluorescence intensity and fluorescence anisotropy observed between the control and HNE-modified ATPase completely disappeared after treatment of the protein with guanidine hydrochloride (1 M). These results suggest that HNE-modification of the Na(+)-K(+)-ATPase induces alterations in the conformation of the enzyme molecule. This interpretation was further supported by a decrease in fluorescence quenching efficiency with acrylamide and sulfhydryl (SH) content. The decrease in quenching efficiency suggests that the proximity of the quencher molecule to the fluorophores located in the enzyme is suppressed. Modification of the enzyme with N-ethylmaleimide (NEM) also resulted in a decrease in quenching efficiency with the loss of SH groups. Furthermore, a good relationship between the SH content and these fluorescence parameters (fluorescence anisotropy and quenching efficiency) were observed. On the other hand, treatment of the Na(+)-K(+)-ATPase with other aldehydes such as malondialdehyde (MDA), 1-hexanal and nonanal did not affect either the quenching efficiency or SH content. Based on these results, the possibility of alterations in the physical properties of the Na(+)-K(+)-ATPase associated with modification by HNE has been discussed.

Gastroprotective effects of phenylpropanoids from the rhizomes of Alpinia galanga in rats: structural requirements and mode of action.

The effects of 1'S-1'-acetoxychavicol acetate and related phenylpropanoids isolated from the rhizomes of Alpinia galanga on ethanol-induced gastric lesions in rats were examined. Among them, 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate markedly inhibited the ethanol-induced gastric mucosal lesions (ED(50)=0.61 and ca. 0.90 mg/kg). In addition, 1'S-1'-acetoxychavicol acetate inhibited the lesions induced by 0.6 M HCl (ED(50)=0.73 mg/kg) and aspirin (ED(50)=0.69 mg/kg) but it did not show a significant effect on indomethacin-induced gastric lesions and acid output in pylorus-ligated rats at doses of 0.5-5.0 mg/kg. From the gastroprotective effects of various related compounds, the 1'-acetoxyl group of 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate was found to be essential for their strong activity. With regard to the mode of action, the gastroprotective effects of 1'S-1'-acetoxychavicol acetate were attenuated by pretreatment with indomethacin and N-ethylmaleimide, and 1'S-1'-acetoxychavicol acetate significantly increased the glutathione levels of gastric mucosa in rats. These findings suggest that endogenous prostaglandins and sulfhydryl compounds are involved in the protective effect of 1'S-1'-acetoxychavicol acetate.

Aqueous access channels in subunit a of rotary ATP synthase.

The role of subunit a in proton translocation by the Escherichia coli F(1)F(o) ATP synthase is poorly understood. In the membrane-bound F(o) sector of the enzyme, H(+) binding and release occurs at Asp(61) in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp(61) via an aqueous access pathway formed at least in part by one or more of the five TMHs of subunit a. In this report, we have substituted Cys into a 19-residue span of the fourth TMH of subunit a and used chemical modification to obtain information about the aqueous accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side of the membrane and may lie on the H(+) transport route. Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are Ag(+)-accessible but N-ethylmaleimide-inaccessible and may form part of an aqueous pocket extending from Asp(61) of subunit c to the periplasmic surface.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.

Absorption and excretion in the experimental animal of a 14C-ethylmaleimide labelled peptide fraction of bovine factor VIII with antihaemorrhagic activity.

In this study the absorption and excretion of a peptide fraction from bovine Factor VIII (Vueffe) with antihaemorrhagic activity were investigated in rats and rabbits. The results obtained suggest that the peptide fraction may be absorbed from the gastrointestinal tract through an active transport or a process of nonselective pinocytosis. The radioactivity elimination was rapid and almost complete within 24 h. In addition no difference was observed between single or repeated dosing in the pharmacokinetic parameters. The evidence following routes of administration shows the good bioavailability of this peptide fraction of low molecular weight and confirms the efficacy after oral administration at very low doses.

Characterization of M1- and M2-muscarinic receptors in calf retina membranes.

Muscarinic receptors are identified in bovine retina membranes by the specific binding of 1-[benzilic-4,4'-3H]-quinuclidinyl benzilate [3H]-QNB. Binding occurs to one population of non-cooperative binding sites: KD = 0.11 +/- 0.02 nM and Bmax = 0.61 +/- 0.07 pmol/mg protein. Competition binding curves of the M1-selective antagonist pirenzepine are shallow. Computer-analysis reveals the presence of 45 +/- 1% M1-receptors (high affinity sites for pirenzepine, Ki = 31 +/- 10 nM). The remaining low affinity sites (Ki = 1.0 +/- 0.3 microM) are denoted as M2-receptors. Competition binding curves with the agonist carbachol are shallow as well. 1 mM GTP causes a rightward shift and a steepening of the carbachol curve, whereas 1 mM N-ethylmaleimide (NEM) provokes a leftward shift and also a steepening of the curve. The GTP effect is abolished by NEM. Binding of the antagonists [3H]-QNB, atropine or pirenzepine is not modulated by GTP nor by NEM.