Analysis of long-lived substates of the conductivity of single ion channels

Using the patch-voltage-clamp method kinetic parameters of single ionic channels were studied. It was found that the channels have long-lasting conductance substates along with the short-living ones. The conductance of a long-lasting substate fluctuates near an average sublevel in the boundaries of a restrict number (k) of elementary conductance steps. There is a direct relationship: the greater k, the longer is average duration (tau k) of the substate. For a given k the falling phase of tau k value distribution is approximately one-exponential. The substates of k-th order result in the multi-exponentiality of the ion current kinetics.     

Structural and physico-chemical properties of amino acids of functionally important regions of proteins

A correlation of the localization of functionally important regions with places having low and high values on twelve profiles built on a basis of amino acid sequences was analysed using a broad set of proteins. The profiles of hydrophilicity, resemblance to the sequences of human proteins, flexibility, mutability and others were considered. The resemblance profile was plotted by the program fixing short similar fragments in the testing protein and 92 human ones. The active centres were shown to be located in the primary structure regions having relatively low values on the resemblance profiles. Similar effect was observed in the mutability and alpha-helicity profiles. The potential functionally important sites of the human leukocyte interferon and interleukin-2 isolated on the basis of the analysis of this profiles were in accord with the available literary data.     

The isolation and characterization of 2-carboxyethyl adducts following in vitro reaction of acrylic acid with calf thymus DNA and bioassay of acrylic acid in female Hsd:(ICR)Br mice

Reaction of acrylic acid (AA) at pH 7.0 and 37 degrees C for 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo) and thymidine (dThd) resulted in the formation of 2-carboxyethyl (CE) adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (percent yield after 40 days) were 1-CE-dAdo (5%), N6-CE-dAdo (11%) (via Dimroth rearrangement of 1-CE-dAdo), 3-CE-dCyd (7.5%), 7-CE-Gua (4%), 7,9-bis-CE-Gua (0.9%) (formed by reaction of AA with depurinated 7-CE-Gua during the course of the reaction) and 3-CE-dThd (0.5%). The products isolated following in vitro reaction of AA with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-Ade (9.9), N6-CE-Ade (8.2), 7-CE-Gua (7.2) and 3-CE-Thy (1.9). Compound 3-CE-Cyt was not detected. Thus the adducts formed following in vitro reaction of AA with DNA are identical to those formed by in vitro reaction of the carcinogen beta-propiolactone (BPL) with DNA as reported in an earlier paper. Structures were assigned on the basis of identical UV spectra, Rf values on paper chromatograms and Rt values on HPLC as marker compounds prepared from reactions of BPL with 2'-deoxynucleosides and 2'-deoxynucleotides-5'-monophosphoric acids. AA was assayed for carcinogenic activity by s.c. injection (20 mumol, once a week for 52 weeks) in female Hsd: (ICR)Br mice. Two mice with sarcomas at the site of application were observed out of 30 mice. Malignancies were not observed in solvent and no-treatment controls. The bioassay results reported in this paper and elsewhere in the same strain of mice suggest that AA is a weak carcinogen in female Hsd:(ICR)Br mice.     

Labeling of phosphatidylcholines of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n-6)), docosapentaenoate (22:5(n-3)) and docosahexaenoate (22:6(n-3))

The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas incubated for 2 h with (1-14C)-labeled (n-6) eicosatetraenoate (n-3) docosapentaenoate and (n-3) docosahexaenoate (20:4, 22:5 and 22:6, respectively) and in four subcellular fractions isolated after such incubations. Of the total radioactivity incorporated in PC, the following percentages of the above fatty acids, respectively, are found in its dipolyunsaturated species: 58, 56 and 53% in rod outer segments; 29, 41 and 49% in mitochondria; 24, 28 and 39% in microsomes; 12, 14 and 16% in postmicrosomal supernatants; 28, 36 and 58% in entire retinas. The remainder percentages are in tetra-, penta- and hexaenoic species of PC, respectively. The levels of pentaenoic species in the PCs of all fractions are similar, while tetraenes are lowest and hexaenes highest in photoreceptor membranes. Dipolyunsaturated species are highly concentrated in photoreceptor membranes, but are minor components of mitochondrial, microsomal and cytosolic PC. The specific radioactivities of tetraenoic, pentaenoic and hexaenoic PCs are decreasingly lower in the following order: postmicrosomal supernatants, microsomes, mitochondria, photoreceptor membranes. In contrast, the specific radioactivities of dipolyunsaturated PCs are higher in mitochondria and microsomes than in the other fractions, especially with 22:5 and 22:6. It is suggested that mitochondria as well as the endoplasmic reticulum could play a role in the synthesis and further modifications of dipolyunsaturated PCs before being supplied to photoreceptor membranes.     

A novel method of protein analysis for prediction of biological function: application to tumor toxins

Informational spectra method was applied to the analysis of lymphotoxin and tumor necrosis factor. The correlation between the information contained in primary structure of these tumor toxins and some oncogen transforming proteins was established. This correlation implies the possibility of a competitive action between these two groups of proteins. "Hot spots" positions in the primary structure of lymphotoxin and tumor necrosis factor for the functional "up" and "down" mutations were predicted.     

An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

Orosomucoid (ORM) typing by isoelectric focusing: evidence for gene duplication of ORM1 and genetic polymorphism of ORM2

Summary It has been demonstrated that the genetic polymorphism of human serum orosomucoid (ORM) is controlled by polymorphic ORM1 and monomorphic ORM2 loci. In this study a Japanese family was encountered in which several members had puzzling electrophoretic patterns consisting of four bands. The ORM patterns were due to the products of a duplicated ORM1 locus haplotype (ORM1 ^* 2·1) or the products of new variant alleles at the ORM2 locus. The ORM1 ^* 2·1 haplotype is very common in the Japanese population, occurring at an allele frequency of 0.16. The increased occurrence of ORM1 2-1 and the heterogeneity in band intensity among ORM1 2-1 phenotypes could be explained in terms of a duplicated gene ORM1 ^* 2·1. The ORM2 locus proved to be polymorphic, with six alleles in the Japanese population. Dedicated to Professor Dr. K. Nishigami on the occasion of his 60th birthday     

Role of protein kinase C in regulating smooth muscle electrical and contractile activity: the effect of phorbol ester

The effects of protein kinase C activation by 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) on the functions of guinea-pig smooth muscle taenia coli have been studied, using double-sucrose-gap method. A 15-20-min treatment of the muscle with 2 X 10(-8) M TPA caused a progressing inhibition of spontaneous electrical activity and mechanical tension, suppression of post-hyperpolarizing electrical and contractile "off-responses", a decrease in the number of action potentials during superthreshold membrane depolarization, depression of electrical and mechanical responses induced by acetylcholine, histamine, bradykinin mediators. The treatment of pre-depolarized (140 mM kappa+) muscle with 2.10(-8) TPA has led to a considerable reduction in contractile responses induced by the above mediators. The results obtained indicate that protein kinase C is capable of regulating both voltage-sensitive and receptor-operated ionic channels in smooth muscle cells.