[Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms]. / Razlichiia v kharaktere vzaimodeistviia normal'nykh i transformirovannykh keratinotsitov cheloveka s izoformami laminina.

Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated.

The p65/RelA subunit of NF-kappaB interacts with actin-containing structures.

Nuclear factor-kappa B (NF-kappaB) is a universal transcription factor that participates in induction of a wide variety of cellular genes. In nonstimulated cells, NF-kappaB is sequestered in the cytoplasm. However, little is known about where NF-kappaB is located. We have studied the effect of inducing a reorganization of the actin filament system on NF-kappaB distribution, using normal and E1A+cHa-ras-transformed rat fibroblasts. This paper demonstrates that the p65/RelA subunit of NF-kappaB interacts with actin-containing structures. Immunofluorescence reveals that p65 is concentrated in focal contacts and along stress fibers in normal fibroblasts. Restoration of actin stress fibers in transformants spread on fibronectin is followed by reallocation of p65 to focal contacts and stress fibers, as in normal cells. The p65 is accumulated at the edge of leading lamellae in transformants spread on laminin and is redistributed to cell-to-cell contacts after a prolonged cultivation. Treatment of cells with Cytochalasin D leads to redistribution of p65 into the actin-containing aggregates. Affinity chromatography on matrix-bound F-actin confirms that p65 can bind to filamentous actin. Taken together, these data indicate that distribution of p65 in the cytoplasm depends on the state of the actin cytoskeleton and suggest an additional, yet unknown, function of the NF-kappaB in the cytoplasm.

[The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts]. / Osobennosti strukturnoi organizatsii aktinovogo tsitoskeleta hormal'nykh, immortalizovannykh i transformirovannykh fibroblastov krysy i ee izmeneniia pod vliianiem belkov bnekletochnogo matriksa.

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.

[Detection and identification of Mycoplasma infections by DNA hybridization]. / Obnaruzhenie i identifikatsiia mikoplazmennykh zarazhenii metodom DNK-gibridizatsii.

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.