[High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus].

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

[Virions and membrane proteins of the potato X virus interact with microtubules and enables tubulin polymerization in vitro]. / Viriony i belok obolochki X-virusa kartofelia vzaimodeistvuiut s mikrotrubochkami i sposobstvuiut polimeriaztsii tubulina in vitro.

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.

[Translation initiation factor eIF3 is able to bind with microtubules in mammalian cells]. / Faktor initsiatsii transliatsii eIF3 mozhet sviazyvat'sia s mikrotrubochkami v kletkakh mlekopitaiushchikh.

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170 kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170 kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170 kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along cellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.

A major 170 kDa protein associated with bovine adrenal medulla microtubules: a member of the centrosomin family?

Microtubules isolated from bovine adrenal medulla cells contain a major 170 kDa protein (p170). p170 is heat-labile and is associated with microtubules in an ATP-insensitive manner. This protein was purified to near homogeneity using FPLC. A preparation containing purified p170 caused bundling of microtubules. By microsequencing of p170, two polypeptides were identified which appeared to be identical to a recently sequenced p167 centrosomin-related protein. Polyclonal affinity-purified anti-p170 antibody was found to immunostain microtubules and to recognize the 170 kDa polypeptide in culture cells. We suggest that p170 is a new member of a centrosomin family and is a new structural protein associated with microtubules in some cell types.

[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.

MAP2-mediated binding of chromaffin granules to microtubules.

We have examined the interaction of chromaffin granules from bovine adrenal medulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose-purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12,000 x g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules; (i) was not sensitive to ATP; and (ii) was completely inhibited by the cleavage of C-terminal peptides of alpha- and beta-subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule-associated proteins rather than by microtubule-dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X-100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol-stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the microtubules.

[Effects of bronchial secretions of patients with bronchial asthma and chronic bronchitis on the motility of ciliated cells of the ciliated epithelium]. / Vliianie bronkhial'nogo sekreta bol'nykh bronkhial'noi astmoi i khronicheskim bronkhitom na podvizhnost' resnichek kletok mertsatel'nogo épiteliia.

It has been shown that bronchial mucus from patients with bronchial asthma and chronic bronchitis can produce a ciliostatic effect when incubated with strips of frog palate mucosa. This effect can more often be found during clinical exacerbation and is supposed to be reversible. The ciliostatically active samples of bronchial mucus taken from asthmatic patients markedly inhibit reactivation glycerol models of frog's ciliated epithelium. It can therefore be suggested that the found activity acts directly on tubulin-dynein complex of the cilia. In contrast to these findings, the ciliostatically active samples of bronchial of glycerol models and therefore possibly act through some other mechanism.

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.

[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.

[Study of dynein ATPase by oxygen isotope exchange H2(18)O in equilibrium with [16O]-Pi]. / Issledovanie dineinovoi ATPazy metodom izotopnogo obmena kisloroda H2 18O ekvilibr [16O]--Pi.

The oxygen isotope exchange reactions catalyzed by sea urchin Strongylocentrotus intermedius spermatozoa dynein I were studied with a view of comparing molecular mechanisms of ATP hydrolysis by dynein and myosin ATPases. It was demonstrated that the isotope exchange takes place during ATP hydrolysis and during enzyme incubation with ADP and Pi and is absent when the enzyme is incubated with Pi. It was assumed that the molecular mechanisms of ATP hydrolysis by dynein I and myosin are identical.