Cyclosporin A-sensitive permeability transition pore is involved in Cd(2+)-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd(2+)-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca(2+) and P(i), which is prevented by Sr(2+). Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P(i), and Ca(2+)) affect in a corresponding way Cd(2+)-promoted membrane permeabilization in NH(4)NO(3), KCl, and sucrose media. Maximal depression of Cd(2+)-induced swelling is achieved by simultaneous addition of ADP, Mg(2+), and CsA that produces either synergistic (NH(4)NO(3)) or additive (KCl and sucrose media) action. Sustained activation by low [Cd(2+)] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone+oligomycin) CsA inhibits completely Cd(2+) activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd(2+) effects are discussed in terms of comparison with those of Zn(2+) and PhAsO.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

The treatment of isolated genera

Problems presented by genera, or small groups of genera, which have been given family rank are reviewed, and the genera are divided into a number of categories according to the closeness of their affinity to other genera or families. Satellite genera that stand in close relation to families should be united with them. Binary families, that have been divided into two (or more) related families, should be re–united. Families connected by linking genera, should, logically, be united but practical considerations usually prevent this. Clusters of diverse but more or less distantly related genera present unusual problems, being treated either as several, often monogeneric families or as a loosely structured family. Truly isolated genera must be given family and often ordinal rank.     

Interrelation of conformational changes of crustacin and its capacity to bind specifically with carcino-embryonic antigen

Temperature-, ionic strength-, calcium ion- and pH-dependence of spatial structure of crustacin have been studied using CD and fluorescent spectroscopy. Secondary structure of crustacin was estimated by CD spectra. An irreversible conformational transition of crustacin's protein moiety connected with the loss of CEA-binding activity has been found at ca. 50 degrees C. Crustacin is shown to be calcium-binding protein, stability of the native crustacin conformation being markedly enhanced by calcium ions (1 mM Ca2+ shifted up the transition temperature by approximately 10 degrees C). Calcium binding and ionic strength increase led to alteration of both secondary and tertiary structures of crustacin. The highest CEA-binding activity was observed for the calcium-bond form of crustacin. A lack of specific interaction of crustacin with some saccharides was shown. Interrelation between conformation and immunochemical activity of crustacin is discussed.     

Conformational stability and immunochemical properties of yersinin--a basic protein of the outer membrane of the Pseudotuberculosis microbe

Using spectroscopic methods (circular dichroism and intrinsic protein fluorescence) and immunoenzyme assay, changes in the spatial and antigenic structure of yersinin, porin from outer membrane of Yersinia pseudotuberculosis, were studied in solutions of ionic and non-ionic detergents at various temperatures and low pH values. Yersinin was shown to retain its secondary structure under various denaturation conditions, the content of regular structural patterns depending on specific action of the denaturation agent. Process of yersinin denaturation similarly to other membrane proteins appears to occur via two structural transitions: dissociation of oligomers and denaturation of monomers. At the first stage changes of quaternary structure accompanied by the loss of so called conformational determinants were observed. Temperature-dependent changes of monomers' tertiary structure affect antigenic activity of yersinin in a smaller degree.     

Antitumor and anticoagulant activities of collagen protein from the holothurian <Emphasis Type="Italic">Apostichopus japonicas</Emphasis> modified by proteolytic enzymes

A collagen preparation with antitumor and anticoagulant activity was obtained from the body of the holothurian (sea cucumber) Apostichopus japonicus treated with a complex of proteolytic enzymes. Administration of the collagen preparation inhibited in vivo growth of Ehrlich solid adenocarcinoma 2-fold (P < 0.05) and contributed to the survival of ascitic tumor-bearing mice, increasing their life span by 29.4 ± 3.2% (P < 0.05). With its moderate anticoagulant activity, this preparation is a preferential inhibitor of the initial link of the blood coagulation system. The biomedical properties and the character of the chemical structure of the collagen protein obtained from A. japonicus suggest that the mechanism of its action is similar to that of the collagen-like protein endostatin and sulfated polysaccharides. The obtained protein may be applied as a nontoxic agent of supplementary therapy for oncological and cardiovascular diseases.     

Effects of Cd2+ and two cadmium organic complexes on isolated rat liver mitochondria

Effects of Cd2+ and two complexes of bivalent cadmium with 1,3-bis(4-chlorbenzylidenamino)-guanidine and anabasine on ion permeability of the inner membrane and respiration of isolated rat liver mitochondria were studied. Starting from 5 microM, Cd2+ decreased state 3 and DNP-stimulated respiration of mitochondria and increased their state 4 respiration. At 30 microM, Cd2+ decreased state 4 respiration. The complexes, particularly complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine, inhibited the mitochondrial respiration at lower concentration of Cd2+. Nonenergized mitochondria incubated in media containing 125 mM of NH4NO3 or KNO3 showed more pronounced swelling in experiments with 10 microM of the complexes than with Cd2+. The complexes produced swelling of the mitochondria energized by 5 mM of succinate and incubated in medium containing 25 mM K-acetate and 100 mM sucrose. Uptake of 137-Cs by succinate-energized mitochondria in the presence of 10(-8) M of valinomycin was substantially decreased in experiments with 10 microM of the complexes than with Cd2+. Ruthenium red (7.5 microM) prevented this effect with 10 microM of complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine and especially complex of Cd2+ with anabasine and Cd2+. These results indicate that the cadmium organic complexes affect respiration and perturb ion permeability significantly stronger than Cd2+.