Large-scale purification, crystallization and some physicochemical properties of extracellular guanyl-RNases C2 and Pch1]

The purification of RNase C2 from 76.5 1 of Asp. clavatus cultural fluid and RNase Pch1 from 160 1 of Pen. chrysogenum 152 A cultural fluid was described. 1150-fold purification of RNase C2 was attained by precipitation with ammonium sulfate, ion-exchange chromatography and rechromatography on DEAE-cellulose, gel chromatography on Sephadex G-75, and crystallization from diluted acidic buffer. During the preparation of RNase Pch1 additional chromatography on CM-cellulose was used before crystallization, the purification being 2220-fold. It was obtained 600 mg RNase C2 and 900 mg RNase Pch1. Some physico-chemical properties of crystalline RNases were studied.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Effect of magnetic nanoparticles Fe3O4 on viability, attachment and spreading of cells isolated from fetuses and new-born rats

This study shows that toxic effect of non-modified nanoparticles of Fe3O4 in vitro depends on metabolic and morphological condition of cells, derived from fetuses and newborn rats. During the cultivation of cells with magnetic nanoparticles, the new-born rats. During the cultivation of cells with magnetic nanoparticles, the latter bind to the cell surface and penetrate into the intracellular space. In this case, sorption of nanoparticles on the cell surface makes it difficult for cells to adhere to substrate, and the absorption by spread cells may prevent their proliferation. Magnetic nanoparticles are well absorbed by the upper layer of cellular aggregates. In this case, the cells of the inner layer remain intact. Consequently, the cell aggregates are able to respond to the constant magnetic field. These aggregates could potentially by used in cell transplantation for directed cell delivery.     

Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.     

The component analysis of tryptophan fluorescence spectra of melittin during its oligomerization

The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.     

Morphofunctional and cytogenetic characteristics of tripronuclear human zygotes in extracorporal fertilization programme

Morphofunctional and cytogenetic characteristics of 34 tripronuclear human zygotes obtained during in vitro fertilization have been studied. It has been shown that 50% of them were triploid. Possible mechanisms of formation of this pathology are discussed.     

Compensation for behavioral disorders caused by 6-hydroxydopamine administration via the transplantation of embryonic tissue from the locus coeruleus in rats

Neonatal injection of 6-OHDA produces defects of sensory attention and emotional reactions in the rats. Possibility of compensation of these behavioural defects by the grafts of embryonal locus coeruleus (LC) tissue into the neocortex was investigated in male rats of the Wistar strain. Histological analysis revealed spindle-shaped and oval cells typical of LC in the neocortex of all animals with LC grafts. Characteristic green fluorescence of these noradrenergic cells was demonstrated by Falck's method. Normalization of the orienting reaction to sensory (tactile and visual) stimuli, as well as of frustration effects was observed in the animals with LC grafts, but not in the control groups with saline injections or hippocampal grafts. The data show the possibility of stable compensation of the behavioural defects resulting from neonatal injection of 6-OHDA by the grafts of embryonal tissue containing noradrenergic neurones.