[Analysis of serum antibodies to nerve tissue antigens in patients with lateral amyotrophic sclerosis]. / Analiz syvorotochnykh antitel k antigenam nervnoi tkani u bol'nykh bokovym amiotroficheskim sklerozom.

We used non-direct immunofluorescence microscopy, immunoblotting and affinity chromatography on A-protein Superose to study antibodies to neural tissue antigens in sera from 11 patients with ALS and from 10 healthy donors. In all sera the majoric antigens had molecular masses of 150-200kD, 70kD and 50kD. No consistent differences were found between ALS patients and controls. Antibodies to 50kD and 70kD proteins from patients with ALS were found to be mostly IgM, whereas antibodies from control sera were mostly IgG. Antibodies to high molecular weight proteins (150-200kD) in ALS and controls belonged to both classes of immunoglobulins. Immunoblotting studies of neural tissue proteins after treatment blots with alkaline phosphatase showed considerable decrease of antibodies binding to neural tissue antigens in sera of ALS patients. The same results were obtained by immunofluorescence assay. The alkaline phosphatase experiments suggest that in ALS patients the sera antibodies are directed mainly against phosphoepitopes in protein antigenic determinants of the neural tissue. This results can lead to conclusion of a role for the altered phosphorylation of the neural proteins in the ALS pathogenesis.

[45 kDa fragment of the kinesin molecule possesses high ATPase activity and binds to microtubules]. / 45 kDa-fragment molekuly kinezina obladaet vysokoi ATP-aznoi aktivnost'iu i sviazyvaetsia s mikrotrubochkami.

Kinesin is a mechano-chemical ATPase capable to move particles along microtubules and microtubules along the solid substrate. Molecule of bovine brain kinesin is a heterotetrameric unit consisting of two heavy (120 kDa) and two light (62 kDa) chains. We used limited proteolysis to study the location of the functional sites on the kinesin molecule. Chymotrypsin cleavage produced a stable 45 kDa fragment of the heavy chain which was purified from the digest using FPLC chromatography on a Superose 12 column. 45 kDa fragment contained both a microtubule-binding site and a ATPase site of the kinesin molecule. Cleavage of the 45 kDa fragment from the rest of the heavy chain significantly activated its ATPase activity. However, this activity remained fully dependent on microtubules. We suggest that the chymotrypsin cleavage uncouple ATPase activity of kinesin (found in the 45 kDa fragment) from its translocator activity (which, probably, required the presence of other parts of the molecule).

[Organization of stress fibrils in cultured fibroblasts studied by using an actin filament-fragmenting protein]. / Issledovanie organizatsii stress-fibrill kul'tiviruemykh fibroblastov s pomoshch'iu belka, fragmentiruiushchego aktinovye filamenty.

Earlier we isolated a 1:1 complex of 90 kD-protein and actin from bovine brain. This complex was able to fragment actin filaments. Effects of this complex on the cytoskeleton of mouse and quail embryo fibroblasts are described. The cells were extracted with Triton X-100, and the resulting cytoskeletons were treated with the complex. Tetramethylrhodaminylphalloin and actin antibody staining failed to detect actin in preparations treated with the 90 kD protein-actin complex. Electrophoretic data confirmed actin solubilization upon this treatment. Immunofluorescent microscopy showed that actin solubilization was accompanied by extraction of vinculin and alpha-actinin from focal contacts and stress-fibers. In contrast, myosin distribution in treated cytoskeletons did not differ significantly from that in control extracted cells: in both the cases myosin was found mainly in the stress-fibers. Thus, myosin localization in stress-fibers does not depend on actin and is probably controlled by some other cytoskeletal component(s).

[Study of a protein complex from the brain which reduces actin viscosity. I. Composition and various properties of the complex]. / Issledovanie belkovogo kompleksa iz mozga, ponizhaiushchego viazkost' aktina. I. Sostav i nekotorye svoistva kompleksa.

The method of isolation from bovine brain of a preparation containing 90 kDa- and 42 kDa-proteins is described. This preparation shortens actin filaments and therefore decreases viscosity of F-actin. The 42 kDa-component was identified as actin by one-dimensional peptide mapping. Quantitative densitometry has demonstrated that 90 kDa-protein and actin are present in the preparation in equimolar ratio. Fractionation of the preparation by gel-filtration, analytical centrifugation or electrophoresis under non-denaturing conditions showed that 90 kDa-protein and actin are in a light complex. This complex consists of one actin molecule and one molecule of 90 kDa-protein and has a sedimentation coefficient of 3.5S. Both beta- and gamma-isoelectric forms of actin are present in the complex.

[Study of a protein complex from the brain which reduces actin viscosity. II. The complex inhibits elongation of actin filaments at the end preferable for polymerization and fragments the filaments]. / Issledovanie belkovogo kompleksa iz mozga, ponizhaiushchego viazkost' aktina. II. Kompleks ingibiruet élongatsiiu aktinovykh filamentov na predpochtitel'nom dlia polimerizatsii kontse i fragmentiruet filamenty.

Functional properties of the protein complex from bovine brain that shortens actin filaments are described. In the presence of Ca2+ complex shortens actin filaments and increases the initial rate of actin polymerization. In the absence of free calcium ions the complex loses its accelerating effect on actin polymerization, but still possesses actin filament shortening activity. Neither phalloidin nor tropomyosin prevent the shortening of actin filaments induced by the protein complex. Therefore the protein complex causes the fragmentation of actin filament. The data on actin polymerization in the presence of F-actin nuclei have indicated that the protein complex inhibits the elongation step of actin polymerization. The analysis of elongation in the presence of both the protein complex and cytochalasin D has demonstrated that the inhibition occurs on the fast-growing end of actin filaments.

[Glycerol models of the ciliated epithelium of bronchial mucosa and their use in the diagnosis of chronic nonspecific lung diseases]. / Glitserinovye modeli mertsatel'nogo épiteliia slizistoi obolochki bronkhov i ikh ispol'zovanie dlia diagnostiki khronicheskikh nespetsificheskikh zabolevanii legkikh.

The method of extraction of ciliated epithelium from biopsy samples of human bronchial mucosa with glycerol is suggested. Permeabilized cilia of glycerol-extracted cells can be easily reactivated by exogenous ATP. This method was used for the study of ciliary dyskinesia in patients with chronic lung diseases. It was shown that in patients with Kartagener's syndrome neither freshly-isolated, nor glycerol-extracted ATP-treated cilia were motile. On the other hand, in some patients with bronchial asthma ATP reactivated glycerol-extracted cilia, while cilia of freshly-isolated cells remained immotile. The study shows that glycerol permeabilization and reactivation by ATP can be used for the analysis of cilial contractile apparatus in patients with chronic lung disease.

[Regulation of microtubule assembly and disassembly by nucleotides]. / Reguliatsiia sborki i razborki mikrotrubochek nukleotidami.

The role of nucleotides in microtubular assembly and disassembly has been reviewed. Two possible functions of GTP hydrolysis during assembly are discussed: (1) hydrolysis renders sensitivity to factor(s) regulating microtubule depolymerization; (2) the energy of GTP hydrolysis is utilized for the subunit flow from one end of the microtubule to the other. In the second part of the review, experiments are considered showing that microtubular disassembly takes place in the cells only in the presence of ATP, and, therefore, this process is regulated via some ATP-dependent mechanism (most probably, phosphorylation of microtubule-associated proteins).

[Possible role of centrioles in stabilizing cytoplasmic microtubules]. / Vozmozhnaia rol' tsentriolei v stabilizatsii tsitoplazmaticheskikh mikrotrubochek.

Small fragments of the peripheral cytoplasm were obtained from cytochalasine B-treated mouse embryo fibroblasts and studied for distribution of microtubules by indirect immunofluorescence. Microtubules were demonstrated to progressively depolymerize in these fragments which did not contain any tubules after 6 hours of incubation in the growth medium. This effect was specific for microtubules, since the distribution of intermediate filaments remained unchanged during incubation. The fragments remained viable during incubation, inasmuch no changes were detectable in the membrane potential of the mitochondria stained with rhodamine 123. Progressive destruction of microtubules in the tiny cell fragments is likely to be related to the lack of centrioles in such fragments.

[Active region of the lamellar cytoplasm and focal contacts--the most cytochalasin-sensitive structures of fibroblasts]. / Aktivnaia oblast' lamelliarnoi tsitoplazmy i fokal'nye kontakty--naibolee chuvstvitel'nye k tsitokhalazinam struktury fibroblastov.

Effects of low doses of cytochalasin B (CB, 2 micrograms/ml) and cytochalasin D (CD, 0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated. CB desorganized the cortical layer of actin microfilaments to cause partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Low doses of CB did not inhibit the control, on the upper surface of these lamellas distal zones with convex outer edges ruffles were lacking. The disappearance of these ruffling active edges was accompanied with the loss of ability to clear the surface of lamellas from the concanavalin A receptors cross-linked by a corresponding ligand. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of cross-linked surface receptors.

[Structure of the cytoskeleton of embryonic muscle fibroblasts. Electron microscopic study of platinum replicas]. / Struktura tsitoskeleta myshinykh émbrional'nykh fibroblastov. Elektronno-mikroskopicheskoe issledovanie platinovykh replik.

Cultured mouse embryo fibroblasts were extracted with 1% Triton X-100 at pH 6.7 in buffer containing EGTA and stabilizing supplements. Exposed during this extraction cytoskeletons were fixed, dried with the critical point technique, and shadowed with platinum. The platinum replicas of cytoskeleton were used for electron microscopic characterization of the three-dimensional distribution of cytoskeletal fibrils in cytoplasm. Four cytoplasmic regions are revealed with different cytoskeletal structure; these being a "mesh-work zone", a "loose zone", which together formed an active edge of cell lamelloplasm, the "lamella proper" and endoplasm. The two former zones are occupied with dense three-dimensional or loose planar actin network, respectively. The "lamella proper" contains two-dimensional network of different fibrils: microfilaments, microtubules, intermediate filaments and thin connective filaments. The central perinuclear area has a thick microfilament sheath at dorsal cell surface. The most important cytoplasmic elements of fibroblasts are actin microfilament bundles. The fine structure of these bundles, their junctions with each other ("organizing centers") and their terminal parts corresponding to cell-substrate focal contacts are described.