Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca2+/recoverin-dependent inhibition of phosphorylation of rhodopsin.

Antibodies AB(60-72) and AB(80-92) against two immune-dominant epitopes of photoreceptor Ca(2+)-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca(2+)-dependent manner, were obtained. The presence of AB(60-72) or AB(80-92) results in a slight increase in Ca(2+)-affinity of recoverin and does not affect significantly a Ca(2+)-myristoyl switch mechanism of the protein. However in the presence of AB(60-72) or AB(80-92) recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca(2+)-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

Preparation and characteristics of Ca(2+)-dependent monoclonal antibodies to recoverin.

Thirty-four primary hybridoma clones were prepared which expressed monoclonal antibodies to the Ca(2+)-binding protein recoverin. Among the resulting monoclonal antibodies, two Ca(2+)-dependent clones (mAb3 and mAb19) recognizing recoverin were detected by solid-phase immunoenzyme assay. In the presence of Ca(2+), antibodies of the mAb3 and mAb19 clones bound to recoverin several times better than in the absence of Ca(2+). The mAb3 and mAb19 antibodies recognized epitopes located inside the sequences Pro61-Met91 and Pro57-Tyr64 of the recoverin molecule, respectively. The possible mechanism of the Ca(2+)-dependent recognition of recoverin by the prepared monoclonal antibodies is discussed.

Detection of annexin IV in bovine retinal rods.

The fraction of proteins capable of binding to photoreceptor membranes in a Ca2+-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.

[An immunohistochemical study of localization of the calcium-binding protein recoverin in retina of the newt Pleurodeles waltl]. / Immunokhimicheskoe izuchenie lokalizatsii kal'tsii-sviazyvaiushchego belka rekoverina v setchatke tritona Pleurodeles waltl.

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studied of retina regeneration in newts.

Retinal degeneration under the effect of antibodies to recoverin.

Serious changes in the retina, diagnosed as retinal degeneration and uveitis, are observed only in the presence of high titers of antibodies to recoverin (Ca2+-binding protein, a paraneoplastic antigen) in the blood of rabbits. Negligible changes in the retina of rabbits with low antibody titers are detected only by cytohistochemical analysis of the retina. No changes in the retina develop in rabbits intravenously injected with antibodies to recoverin.

Low titre autoantibodies against recoverin in sera of patients with small cell lung cancer but without a loss of vision.

To date, many authors have described the presence of autoantibodies against various neuronal proteins, paraneoplastic antigens (PNA), in a serum of patients with different kinds of malignant tumors located outside the nervous system. These autoantibodies may cross-react with the corresponding PNA or their epitopes present in neurons and thus initiate the development of a variety of neurological disorders, paraneoplastic syndromes (PNS), even though the primary tumor and its metastases have not invaded the nervous system. Cancer-associated retinopathy (CAR) is a rare ocular PNS induced by autoantibodies against several retinal antigens, one of which is a photoreceptor calcium-binding protein, recoverin. Only several CAR patients with a few kinds of cancer (endothelial carcinoma, breast cancer, epithelial ovarian carcinoma) have so far been found to contain autoantibodies against recoverin in their sera. As for lung cancer, the majority of CAR cases mediated by anti-recoverin autoantibodies have been revealed in patients with the most malignant lung cancer, small cell lung carcinoma (SCLC), and only one similar case has been described for a patient with non-small lung carcinoma. The common feature of all these anti-recoverin-positive patients, irrespective of the type of cancer, is the presence of both the CAR syndrome and high titres (as a rule, more than 1:1000) of the underlying autoantibodies in their serum. In this study, we have used recombinant myristoylated recoverin to screen serum samples of 50 patients with SCLC by Western blot and revealed 5 individuals with low titres of anti-recoverin antibodies, who have no manifestation of a loss of vision. To our knowledge, this is the first report on the presence of low titre autoantibodies against recoverin in a serum of patients with cancer, but without visual dysfunction.

[Organization of antituberculous work with homeless in Saint Petersburg]. / Organizatsiia protivotuberkuleznoi raboty s litsami bez opredelennogo mesta zhitel'stva v Sankt-Peterburge.

The paper deals with organizational measures for detection and treatment of homeless persons with tuberculosis in Saint Petersburg. A total of 1653 homeless tuberculosis patients were followed up in 1991-1997. There was high incidence of tuberculosis in homeless persons (30 detected per 1000 examinees. All agencies, facilities, and services should participate in detecting and treating tuberculosis among homeless subjects. Large-scale solution of this problems requires a package of measures by providing additional funds stipulated by a regional goal-oriented tuberculosis control programme.

[A new method of isolation and a new form of transketolase from baker's yeast]. / Novyi metod vydeleniia i novaia forma transketolazy iz pekarskikh drozhzhei.

A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed. The enzyme yield is 90% of its total content in the original yeast extract. The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex.

A new form of baker's yeast transketolase. An enzyme-RNA complex.

Using an immunosorbent, a new form of transketolase, namely, an enzyme-RNA complex, was isolated from a baker's yeast extract. Spontaneous fission of RNA (or its enzymic hydrolysis by RNase) is accompanied by a sharp increase in the catalytic activity of transketolase, which may be directly related to the enzyme's regulation mechanism.

Purification of transketolase from baker's yeast by an immunoadsorbent.

Baker's yeast transketolase has been purified by immunoaffinity chromatography on specific TK antibodies covalently linked to CNBr-agarose. Affinity chromatography allows a 480-fold purification of TK from yeast extracts and about 80% recovery of the original activity. The isolated enzyme has a specific activity of 12-60 U/mg and during polyacrylamide gel electrophoresis performed at pH 8.9 migrates as two protein bands possessing a transketolase activity which corresponds to two isoforms of the enzyme.

[The nature of binding between the coenzyme and protein in transketolase from the swine liver]. / O kharaktere sviazi mezhdu kofermentom i belkom v transketolaze pecheni svin'i.

An analysis of a proteolytic hydrolysate of pig liver transketolase by thin-layer chromatography revealed the presence of a coenzyme-containing material which differed from free thiamine pyrophosphate in chromatographic behaviour. This coenzyme-containing material is distinct from the free coenzyme in terms of other properties as well, e.g., stability, pH dependence of thiochrome fluorescence, etc. It was demonstrated that incubation of enzyme preparations possessing a high specific activity (on the average, 2 E/mg) in acidic acetate buffer caused no or little detachment of the coenzyme, mainly in the composition of the heterogeneous material which, at least partly, was not represented by thiamine pyrophosphate.

Characterization and properties of pig liver transketolase.

Some properties of homogeneous transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) from pig liver were studied. It was shown that the pH optimum of the transketolase reaction lies within the range of 7.8-8.2; the isoelectric point is at pH 7.6-7.8. The molecular weight of transketolase is 138 000 +/- 3000 as determined by the sedimentation equilibrium method. The enzyme molecular is a tetramer of the alpha 2 beta 2 type. The molecular weights of the alpha- and beta-subunits determined by polyacrylamide gel in the presence of sodium dodecyl sulphate are 52 000-56 000 and 27 000-29 000, respectively. Transketolase contains about 2 mol of thiamine pyrophosphate per mol of protein and does not require metal ions for its catalytic activity.

[Pig liver transketolase: covalent binding between the coenzyme and protein]. / Transketolaza iz pecheni svin'i: kovalentnaia sviaz' mezhdu kofermentom i belkom.

Transketolase from pig liver contains a tightly bound coenzyme, which is not split off after severe treatment (e.g. boiling for 12 hrs in 1 n. HCl, 30 min boiling in 2 n HCl, boiling in 1% DS-Na, standing in 6 M solution of quanidine chloride, etc.). The proteolysis of the enzyme results in a coenzyme-containing material differing in some of its properties from free thiamine by pyrophosphate. The data obtained suggest that pig liver transketolase contains covalently bound thiamine pyrophosphate.

[Electromechanical model of an enzyme-substrate kompleksa]. / Elektromekhanicheskaia model' ferment-substratnogo kompleksa.

An electro-mechanical model is suggested for studying the hydrolytic reaction catalyzed by an enzyme having a domain structure. The fixation of the necessary set of reagents configurations by the enzyme molecule allows the reaction rate acceleration. The model suggests specificity of the enzyme action. The model makes more clear the phenomenon of complementarity between the unchanging part of the substrate molecule and the active site of the enzyme which require not only the spatial fit but also the agreement of some physical properties, such as atom and group partial charges. The model is selfconsistent, i. e. it is not contrary to the existing data on the structural and functional properties of enzyme proteins having the domain structure.

[Properties of pig liver transketolase]. / Svoistva transketolazy iz pecheni svinyi.

Some properties of homogeneous transketolase from pig liver were studied. It was shown that the pH optimum of the transketolase reaction lies within the range of 7.8--8.2. The isoelectric point is at pH 7.6--7.8. The molecular weight of transketolase is 138,000 +/- 3,000 as determined by the sedimentation equilibrium method and about 152,000 according to the data from gel filtration through Sephadex G-200. The enzyme molecule is a tetramer of the alpha 2 beta 2 type. The molecular weights of the alpha- and beta- subunits determined by polyacrylamide gel in the presence of sodium dodecyl sulfate are 52,000--56,000 and 27,000--29,000, respectively. Transketolase contains about two moles of TPP per mole of protein and does not require metal ions for its catalytic activity.