κ-卡拉胶琥珀酰基化衍生物的抗氧化活性研究

对κ-卡拉胶进行酸降解得到三种卡拉胶低聚糖,并进一步琥珀酰基化得到分子量分别为2720、4000和5960的κ-卡拉胶琥珀酰衍生物(A、B和C)。对产物进行FT-IR表征,并测得其琥珀酰基取代度(DS)分别为0.61、0.29和0.83。检测了三种κ-卡拉胶琥珀酰衍生物对超氧阴离子自由基O2.-、DPPH自由基、羟基自由基.OH以及过氧化氢的清除活性。结果表明:随着取代度的增加,其清除超氧阴离子自由基O2.-和DPPH自由基的能力增强;随着分子量的增加,其清除羟基自由基.OH和过氧化氢的能力增强。这可能与衍生物的羟基含量、取代基团的性质以及取代度等因素有关。     

Evaluation of functional state of crayfish <Emphasis Type="Italic">Pontastacus leptodactylus</Emphasis> in normal and toxic environment by characteristics of their cardiac activity and hemolymph biochemical parameters

The work deals with study of basic characteristics of cardiac activity (heart rate—HR and stress-index—SI) and hemolymph protein parameters in the crayfish Pontastacus leptodactylus. The main criteria of the crayfish selection are developed for formation of the animal reference groups available for subsequent toxicological experiments. The action of a model toxicant, hydroquinone (often present in waste water), at a concentration of 1 g/l on parameters of the crayfish cardiac activity at various exposition time, its effect on circadian cardioactivity and on the hemolymph total protein were studied. At its short-term action, hydroquinone produced a temporary increase of HR and stress-index, but had no marked effect on total protein parameters in hemolymph. At long action (for one day), hydroquinone led to a considerable tachycardia, disturbance of the circadian cardioactivity and to a decrease by 40% of the hemolymph total protein. In 50% of cases the toxicant caused death of the animals either in the course of its short-term action or at period of washout from the toxicant. Mechanisms of the toxic action of hydroquinone at various levels of organization are discussed.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Genetic polymorphism of glutathione-S-transferases and inhibition of DNA repair

A complex investigation of different cell defence systems, such as: DNA repair, antioxidant system (SOD), xenobiotic detoxification system (glutathione-S-transferases M1 and T1), radioadaptive response (RAR) in lymphocytes of patients with hereditary disease of connective tissue (Elers-Danlose syndrome) was carried out. The frequency of genotype GSTM1 (0/0) in children with Elers-Danlose syndrome (23%) is lower as compared to the control group (44%). The lymphocytes of children with Elers-Danlose syndrome were characterized by reduced ability to repair gamma-induced damage of DNA. At given size of the samples of examined children no correlative relationships between GST-status of organism and the condition of other cell defence systems were revealed. The data obtained demonstrate the individual peculiarities of the defence systems in repair-deficient cells of the examined children.     

Role of V1- and V2-receptors in mechanism of physiological paradox--an increase of reabsorption of the solute free water and simultaneous rise of diuresis

In experiments on non-anesthetized rats with administration into stomach of water (5 ml/100 g body mass) direct correlation has been found between an increase of diuresis and excretion of solute free water (r = 0.98, p < 0.01), while after injection to these animals of 5 x 10(-11) M arginine-vasotocin - between an increase of diuresis and simultaneous rise reabsorption of solute free water (r = 0.8, p < 0.01). The rise of diuresis after the vasotocin injection is due to inhibition of sodium re- absorption, with the solute excretion fraction increasing from 2.6 +/- 0.2 % to 11.9 +/- 1.2, p < 0.001. A similar physiological paradox - an increase of diuresis with the simultaneous increase of reabsorption of solute free water - has been revealed at night hours in children with tendency for nocturnal enuresis (r = 0.64, p < 0.01). Mechanism responsible for this phenomenon consists in a rise of diuresis due to a decrease of sodium ion reabsorption in the ascending Henle loop limb. A problem is discussed of the homeostatic significance of a decrease of sodium reabsorption combined with an increase of solute-free water reabsorption; it is suggested that this phenomenon is based on a redistribution of reabsorption inside the nephron - a decrease of ion and water reabsorption in the initial parts of the nephron distal segment and an increase of solute free water reabsorption with the antidiuretic hormone-stimulated high osmotic permeability of terminal parts of renal tubules. An intraperitoneal injection of V1-anatagonist (OPC-21268) decreased the natriuretic component of response to arginine-vasotocin, while injection of V2-antagonist (OPC-31260) eliminated the antidiuretic component.     

Multiple parthenoforms of Empoasca leafhoppers from Madeira Island: where are these unisexual forms coming from?

There are controversial opinions on whether asexual reproduction is more common on islands than on the mainland. Although some authors consider that the evidences of geographical parthenogenesis support the view that asexual reproduction is more common on islands, comparative data on the modes of reproduction of insular and continental taxa confirming this statement are very limited. In this work, we report the presence of three unisexual forms and three bisexual species of the genus Empoasca (Cicadelloidea, Hemiptera, Insecta) from Madeira Island. Experimentally, the unisexual forms reproduced in the absence of males for several generations. The chromosome analysis has shown that the bisexual species differ from one another in chromosome number, and unisexual forms are apomictic and also each have different chromosome numbers. Of parthenoforms, one is triploid and two are of obscure level of ploidy, 2n or 3n. The results obtained show that for this genus unisexual forms are more common on Madeira Island than in the nearby continental areas. It is suggested that unisexual forms may be more plentiful on islands than on the mainland because if an asexual reproduction event occurs, the relaxing competition in these underexploited and enemy-free habitats may favor the establishment of new parthenogenetic lineages.     

High-resolution organization of mouse centromeric and pericentromeric DNA

We studied the organization of mouse satellite 3 and 4 (MS3 and MS4) in comparison with major (MaSat) and minor (MiSat) DNA sequences, located in the centromeric and pericentromeric regions of mouse telocentric chromosomes by fiber-FISH. The centromeric region consists of a small block of MiSat and MS3 followed by a pericentromeric block of MaSat with MS4. Inside the block of the long-range cluster, MaSat repeats intermingle mostly with MS4, while MiSat intermingle with MS3. The distribution of GC-rich satellite DNA fragments is less strict than that of AT-rich fragments; it is possible to find MS3 fragments in the MaSat array and MS4 fragments in the MiSat array. The methylation pattern does not fully correspond to one of the four families of satellite DNA (satDNA). In each satDNA fragment only part of the DNA is methylated. MS3 and MS4 are heavily methylated being GC-rich. Pericentomeric satellite DNA fragments are more methylated than centromeric ones. Among the four families of satDNA MS4 is the most methylated while MiSat is methylated only to a minimal extent. Estimation of the average fragment length and average distance between fragments shows that the range of the probes used does not cover the whole centromeric region. The existence of unknown sequences in the mouse centromere is likely.     

A new format of electrodes for the electrochemical reduction of cytochromes P450

New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropriate substrates for P450s ("reverse" electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2-1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20-60 microl of electrolyte volume. The enzyme-substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates.     

Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system

The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.     

Proteins associated with mitochondrial DNA protect it against X-rays and hydrogen peroxide

The high susceptibility of mitochondrial DNA to reactive oxygen species and other damaging agents has been supposed to result from the absence of histones. Here we show that DNA-binding proteins of mitochondrial nucleoids can shield mtDNA from X-ray radiation and hydrogen peroxide just as nuclear histones do. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver and assessed for mtDNA protection by the yield of PCR products. In vitro, mtDNA in complex either with nucleoid proteins or with nuclear histones proved to be much less damaged than naked mtDNA, with little difference in protective efficacy. Most probably, in mitochondria the nucleoid proteins also protect mtDNA against reactive oxygen species and thus attenuate the oxidative damage.