The mechanism of the interaction of α-crystallin and UV-damaged ßL-crystallin.

α-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native α-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged ßL-crystallin as the target protein. α-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged ßL-crystallin. DSC investigation has shown that refolding of denatured UV-damaged ßL-crystallin was not observed under incubation with α-crystallin. α-Crystallin and UV-damaged ßL-crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged ßL-crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged ßL-crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75 kDa complex is indicative of α-crystallin dissociation. We suppose that α-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly ß and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of α-crystallin, and thus preventing aggregation.

Discrimination between chewing of coca leaves or drinking of coca tea and smoking of "paco" (coca paste) by hair analysis. A preliminary study of possibilities and limitations.

BACKGROUND: Hair analysis is a suitable way to discriminate between coca chewers and consumers of manufactured cocaine using the coca alkaloids hygrine (HYG) and cuscohygrine (CUS) as markers. In the present preliminary study it was examined whether CUS and HYG can be detected in hair of occasional and moderate coca chewers or coca tea drinkers, whether CUS and HYG appear in hair of PACO consumers (smoking coca paste waste), and whether anhydroecgonine methyl ester (AEME) is a useful cocaine smoking marker in this context. METHOD: Three groups were included: 10 volunteers from Buenos Aires with occasional or moderate chewing of coca leaves or drinking coca tea, 20 Argentinean PACO smokers and 8 German cocaine users. The hair samples (1-4 segments) were analyzed by a validated LC-MS/MS method for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), AEME, CUS and HYG. For comparison, eight samples of coca leaves or coca tea were analyzed. RESULTS: Only low concentrations of COC were found in hair of seven occasional users of coca leaves or coca tea (0.010-0.051 ng/mg). For three moderate chewers of coca leaves all compounds were detected including AEME but except TRO. The hair samples of PACO smokers contained much higher concentrations of COC (0.027-341 ng/mg, mean 37.4 ng/mg) and its metabolites. CUS was not found in these samples but traces of HYG were seen in 8 of 37 hair segments. AEME as a marker for coca smoking was detected in hair of 15 smokers. In comparison to COC, the concentrations of EME and CIN were higher for PACO smokers than for German cocaine consumers. AEME (56 ± 20 µg/g) was detected in all coca leave and coca tea samples which explains the detection of this substance in hair of coca chewers. Therefore, its use for differentiation between coca chewers and PACO smokers is limited. CONCLUSION: CUS remains to be the most suitable marker in hair for chewing coca leaves or drinking coca tea more frequently than two times per month since it does not appear in hair of Argentinean PACO smokers and German cocaine users. Contrary to a previous proposal, the ratios CIN/COC and EME/COC appeared not to be applicable as criteria for this purpose because of the higher concentration of these alkaloids in hair of PACO smokers. More research is needed to assess the value of AEME in hair of South American coca leave or cocaine users.

Study of α-crystallin structure by small angle neutron scattering with contrast variation.

The structure of the oligomeric protein α-crystallin from bovine eye lens was investigated by small-angle neutron scattering (SANS) with contrast variation. Based on the SANS curves, the match point for α-crystallin (43% D2O) and its average scattering length density at this point (2.4·10(10) cm(-2)) were evaluated. The radius of gyration and the distance distribution functions for α-crystallin were calculated. On the basis of these calculations, it was concluded that α-crystallin is characterized by homogeneous distribution of scattering density in the domains inaccessible for water penetration, and all polypeptide subunits in α-crystallin oligomers undergo equal deuteration. The latter indicates that all α-crystallin subunits are equally accessible for water and presumably for some other low molecular weight substances. These conclusions on the α-crystallin structure (homogeneous distribution of scattering density and equal accessibility of all subunits for low molecular weight substances) should be taken into account when elaborating α-crystallin quaternary structure models.

Resistance of alpha-crystallin quaternary structure to UV irradiation.

The damaging effect of UV radiation (lambda > 260 nm) on bovine alpha-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an alpha-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, alpha-crystallin is able to prevent aggregation of destabilized proteins (especially of gamma- and beta-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of alpha-crystallin).