[Application of heat shock proteins as stress markers in aquatic organisms using endemic Baikal amphipods as an example].

When heat shock proteins (HSPs) are used as biomarkers in monitoring studies of aquatic ecosystems, it is necessary to take into account the specificity of synthesis of these proteins in various organisms. This especially applies to endemic species and species with narrow ranges of adaptation for specific conditions in certain water bodies. In this study, we assessed the possibility to use HSPs as molecular stress markers in species with a narrow niche breadth using endemic Baikal amphipods (Crustacea, Amphipoda) as an example. The effect of stress induced by toxicants and temperature has been assessed. Proteins of families HSP70 and low-molecular-weight HSP related to alpha-crystallins were used as biomarkers. Temperature- and toxicant-induced stresses induced low-molecular-weight HSP synthesis in the endemic amphipod species studied. However, induction of HSP70 synthesis in the same species after temperature stress has not been detected. The specificity of synthesis of HSP70 is discussed. The results obtained in this study suggest that low-molecular-weight HSPs can be used as stress markers in Baikal species and species with a narrow niche breadth.

Evaluation of biochemical responses in Palearctic and Lake Baikal endemic amphipod species exposed to CdCl2.

This study evaluated small heat shock proteins (sHSP) (related to alpha-crystallin) and antioxidant enzymes (POD, peroxidase and CAT, catalase) as possible biomarkers for use in toxicological studies. Biochemical responses to cadmium chloride in two Lake Baikal endemic amphipods (Eulimnogammarus verrucosus, Eulimnogammarus cyaneus) and Palearctic species (Gammarus lacustris) were compared. Our findings showed that cadmium chloride toxicity directly influenced POD activity and sHSP synthesis in all amphipod species. The Baikalean endemic and the Palearctic amphipod species responded by decreasing activity of POD and they exhibited a dose-dependent activation of sHSP synthesis. All measured parameters differed among species and depended on the species' ability to resist cadmium chloride toxicity. CAT activity in the Palearctic species responded significantly to cadmium chloride exposure; however, responses were negligible for both Baikalean species. We suggest that synthesis of sHSP, together with changes in POD activity, could be used as biomarkers for further studies of amphipod species including endemics from Lake Baikal.

Non-phosphorylating bypass of the plant mitochondrial respiratory chain by stress protein CSP 310.

Recently, it has been reported that the cold-stress protein CSP 310, discovered in the cytoplasm of cold-resistant winter cereals, causes uncoupling of oxidative phosphorylation during cold stress. To understand how the uncoupling mechanism of CSP differs from that of cyanide-insensitive alternative oxidase and plant mitochondrial uncoupling protein, we determined the effect of respiratory-chain inhibition on winter wheat (Triticum aestivum L. cv. Zalarinka) mitochondria. Our data show a possible involvement of stress protein CSP 310 in mitochondrial electron transport in winter wheat. CSP 310 shunts electrons around the main cytochrome pathway of the mitochondrial respiratory chain, i.e. electron flow bypasses ubiquinone and complex III via CSP 310 to complex IV.

Influence of stress protein CSP 310 and antiserum against this protein on oxygen uptake, lipid peroxidation, and temperature of winter wheat seedling shoots during cold stress.

It is determined that infiltration of winter wheat seedling shoots by anti-CSP 310 antiserum caused a significant decrease of oxygen uptake in winter wheat shoots during short-term cold stress. On the other hand, infiltration of winter wheat seedling shoots by stress protein CSP 310 caused an increase of oxygen consumption. The comparison of the influence of infiltration of winter wheat shoots by CSP 310 and anti-CSP 310 antiserum on the rate of lipid peroxidation showed that, if infiltration by CSP 310 caused a decrease of conjugated diene formation, infiltration by anti-CSP 310 antiserum did not cause any significant changes in the rate of lipid peroxidation. The study of the influence of infiltration of winter wheat shoots by CSP 310 and anti-CSP 310 antiserum on the temperature of winter wheat shoots during cold stress showed that CSP 310 caused the increase of their temperature while anti-CSP 310 caused a decrease of their temperature.

Stress-induced protein CSP 310: a third uncoupling system in plants.

Addition of the cold-stress-related protein CSP 310 to mitochondria isolated from winter wheat ( Triticum aestivum L. cv. Zalarinka), winter rye ( Secale cereale L. cv. Dymka), maize ( Zea mays L. cv. VIR 36) and pea ( Pisum sativum L. cv. Marat) caused an increase in non-phosphorylative respiration. This increase was inhibited by KCN, indicating that the protein is not a CN-resistant alternative oxidase. Unlike plant mitochondrial uncoupling proteins such as PUMP, the uncoupling action of CSP 310 did not depend on the presence of free fatty acids in the incubation medium. We propose that the mechanism of the uncoupling action of CSP 310 differs from that of other known plant uncoupling systems and that the CSP 310 uncoupling system is a third uncoupling system in cereals.

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo.

BACKGROUND: The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria. RESULTS: We studied the effects of CSP 310-like native cytoplasmic proteins from a number of cereal species (winter rye, winter wheat, Elymus and maize) on the energetic activity of winter wheat mitochondria. This showed that only CSP 310 (cold shock protein with molecular weight 310 kD) caused a significant increase of non-phosphorylative respiration. CSP 310-like proteins of other cereals studied did not have any significant influence on mitochondrial energetic activity. It was found that among CSP 310-like proteins only CSP 310 had prooxidant activity. At the same time, Elymus CSP 310-like proteins have antioxidant activity. The study of an influence of infiltration by different plant uncoupling system activators (pyruvate, which activates AOX, and linoleic acid which is a substrate and activator for PUMP and CSP 310) showed that all of these decreased lipid peroxidation during cold stress. CONCLUSIONS: Different influence of CSP 310-like proteins on mitochondrial energetic activity and lipid peroxidation presumably depend on the various subunit combinations in their composition. All the plant cell systems that caused an uncoupling of oxidation and phosphorylation in plant mitochondria can participate in plant defence from oxidative damage during cold stress.

Stress protein CSP 310 causes oxidation and phosphorylation uncoupling during low-temperature stress only in cereal but not in dycotyledon mitochondria.

It was found that an addition of antiserum obtained against stress protein 310 kD increased coupling of oxidation and phosphorylation in mitochondria isolated from cold-stress winter rye shoots and had no influence on dycotiledon mitochondria (pumpkins and sunflower). The data obtained showed a difference between molecular weights of dycotyledon polypeptides with immunochemical affinity to CSP 310 and CSP 310 subunits. It was shown that low-temperaturestress caused a transition to a low-energy state ("cold uncoupling") of free from endogenous free fatty acid cereal mitochondria. At the same time, this "cold uncoupling" in mitochondria of dycotyledon species investigated was not detected. We suppose that a special mechanism of low-temperature stress reaction in mitochondria dealing with uncoupling activity of stress protein CSP 310 exists in cereals.

An influence of stress protein CSP 310 and antiserum against this protein on lipid peroxidation in cereal mitochondria.

It is determined that an addition of an anti-CSP 310 antiserum to isolated winter wheat and maize mitochondria caused more significant increasing of spontaneous lipid peroxidation than the addition of stress protein CSP 310. It is shown that, at function of different mitochondrial respiratory chain complexes, the lipid peroxidation in winter wheat and maize mitochondria take place with different intensities. Under the functioning of mitochondrial respiratory chain complex IV, the maximum output of lipid peroxidation products, dienic conjugates is detected. The presence of antiserum against CSP 310 in incubation media induces lipid peroxidation more than the presence of CSP 310 in mitochondria isolated from stressed plants under these conditions. Based on data obtained, it is possible to conclude that in vivo endogenous CSP 310, during a cold stress, has an antioxidant activity the same as other known uncoupling proteins.

An influence of antiserum against winter wheat stress uncoupling protein, CSP 310, on energetic activity of some plant species mitochondria.

It is determined that addition of an anti-CSP 310 antiserum to isolated mitochondria of cereals (winter rye, winter wheat, and maize) caused an increasing of mitochondrial respiratory control. In a similar manner, addition of this antiserum to isolated pea mitochondria did not cause this effect. It is shown that coupling effect of antiserum is not dependent upon the presence of bovine serum albumin in mitochondria incubation media. Therefore, these results show that the mechanism of oxidation and phosphorylation uncoupling, and participation of immunochemically related CSP 310 proteins that can be precipitated by anti-CSP 310 antiserum is specific for cereals.

The search for proteins with immunochemical affinity to plant stress proteins at cold-adapted endemic Baikal fishes.

The search for proteins with immunochemical affinity to plant stress proteins in endemic Baikal fishes shows the presence of proteins, immunochemically related to plant heat-stabile proteins and plant uncoupling protein CSP 310. Western blotting showed that among the native cytoplasmic proteins of endemic Baikal fishes there are proteins immunochemically related to heat-stabile plant proteins with molecular weights about 480, 200-290, 150, 140 and about 90-100kD. SDS-electrophoresis showed the presence of polypeptides with molecular weights 23, 17 and 14kD in all species investigated and an additional 35kD polypeptide in Cottocomephorus grewingki. The search for polypeptides with immunochemical affinity to plant stress uncoupling protein CSP 310 in endemic Baikal fishes shows the presence of a 14kD polypeptide, immunochemically related to it.

The comparison of uncoupling activity of constituently synthesised and stress-induced forms of winter rye stress uncoupling protein CSP 310.

A difference between the uncoupling action of constituently synthesised and stress-induced forms of winter rye stress uncoupling protein CSP 310 on winter wheat mitochondria in vitro was found from the initiation of incubation. The uncoupling activity of CSP 310 depended on its concentration in the incubation media. The addition of anti-CSP 310 antiserum to isolated mitochondria from stressed winter rye shoots caused coupling of oxidation and phosphorylation. Western-blot analysis did not locate dehydrins K-segment in CSP 310 subunits.

[Dental caries in preschoolers living in a rural locality]. / Karies zubov u detei doshkol'nogo vozrasta, prozhivaiushchikh v sel'skoi mestnosti.

The examination has involved 211 children aged 3 to 6, whose mothers were exposed to pesticides when pregnant. Clinical examinations have shown a much higher incidence rate of caries and intensity of the teeth involvement in the children whose mothers were exposed to pesticides than in those whose mothers had no contacts with these chemicals. To prevent caries in children, one should persuade their mothers to breast-feed their babies during the first year. It is desirable that contacts with pesticides be ceased during pregnancy, i.e. that pregnant women be given other work; if that is impossible reliable protection means should be employed.