Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases.

v-Abl is an oncogenic form of the c-Abl nonreceptor tyrosine kinase. v-Abl induces transcription of c-myc, and c-Myc function is a necessary but not sufficient component of the v-Abl transformation program. Previously we showed that the E2F site in the c-myc promoter is a v-Abl response element and that v-Abl appears to induce c-myc by initiating a phosphorylation cascade that ultimately activates E2F-binding proteins. In this work we have investigated the signaling pathway between the v-Abl tyrosine kinase and activated E2F proteins. We show that the Ras GTPase and Raf1 serine/threonine kinase are required in this pathway. However, in contrast to other aspects of v-Abl signaling, induction of c-myc transcription is independent of the Rac GTPase. Our results also establish a requirement for activated cyclin-dependent kinases (cdks), as v-Abl-dependent induction of c-myc transcription is blocked by cdk inhibitor p21 and induction of c-myc is accompanied by activation of cdk2 and cdk4. Finally, we show that v-Abl-dependent induction of c-myc is accompanied by hyperphosphorylation of pRb, p107, and p130. On the basis of these data, we propose a model for the signaling path from v-Abl to c-myc.

Comparative structural and functional features of the human fibrinogen alpha C domain and the isolated alpha C fragment. Characterization using monoclonal antibodies to defined COOH-terminal A alpha chain regions.

The alpha C domain of fibrinogen (A alpha-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the A alpha chain were employed as structural probes to: 1) isolate the human alpha C domain, 2) compare the topography of the eight epitopes within the alpha C domain of intact fibrinogen and in purified alpha C fragments, and 3) explore the degree to which the alpha C domain's role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated alpha C fragments. Five antibodies were raised against small, synthetic peptide immunogens (A alpha-(220-230), A alpha-(425-442), A alpha-(487-498), and A alpha-(603-610)), and three were generated against larger cyanogen bromide (A) alpha chain derivatives with each epitope subsequently localized to discrete A alpha chain sequences (A alpha-(259-276), A alpha-(529-539), and A alpha-(563-578)). Human alpha C preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-A alpha-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of alpha C fragments was either A alpha-220 or A alpha-231 and that, although the extreme COOH-terminal region, A alpha-(603-610), was absent, all molecules were intact at least through A alpha-(563-578). Solution phase competitive assays indicated that the release of the alpha C domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of A alpha-(220-230), A alpha-(259-276), A alpha-(487-498), and A alpha-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. A alpha-(425-442) and A alpha-(563-578). Immunoblotting analysis of alpha C cross-linking in vitro revealed that isolated alpha C fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the A alpha chain proteolysis that occurs during thrombolytic therapy indicated that alpha C fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen alpha C domain and its functional implications and also draw attention to the as yet unexplored role of alpha C fragments in the pathophysiology of thrombosis and hemostasis.

Alpha-Chain cross-linking in fibrin(ogen) Marburg.

The fibrinogen structural variant, Marburg (A alpha 1-460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461-610, can affect the process of fibrin stabilization, ie, the factor XIIIa-mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross-linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross-linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross-linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1-12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.

Flow sorting of hybrid hybridomas using the DNA stain Hoechst 33342.

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described for the selection of hybrid hybridomas producing bispecific monoclonal antibodies. Parental hybridoma cells were first labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC); heterofluorescent cells were recovered after fusion. After a period of growth in culture, the cells were then stained with the DNA-specific dye bis-benzimidazole Hoechst 33342 and sorted on the basis of their DNA content. The staining conditions (10 micrograms/ml of Hoechst 33342, 90 min incubation time at 37 degrees C) were found to be optimal for obtaining a well resolved DNA histogram with minimal effect on the growth properties of cells from different mouse hybridoma lines. Employing this method we have isolated hybrid hybridomas synthesizing bispecific monoclonal antibodies reacting with human low density lipoprotein and alkaline phosphatase from calf intestine.

Interleukin-2- and phytohemagglutinin-activated proliferation of human T-lymphocytes is accompanied by stimulation of phosphoinositide turnover.

Interleukin-2 (IL-2) was more effective than phytohemagglutinin (PHA) in increasing the proliferative activity of human T-lymphocytes. Unlike PHA, IL-2 stimulated phosphoinositide turnover (PI turnover) only in those T-lymphocytes which had been preactivated by PHA with IL-2 and expressed the IL-2 receptors. The effect of PHA on PI turnover was, in general, stronger than that of IL-2. These results indicate that IL-2 and PHA-activated proliferation of human T-lymphocytes is accompanied by stimulation of PI turnover. However, IL-2-induced proliferative response may not be a direct consequence of PI turnover stimulation in these cells.

Noradrenaline induces the polyploidization of smooth muscle cells: the synergism of second messengers.

The effect of noradrenaline (NA) on DNA replication of cultured smooth muscle cells (SMC) isolated from rat aorta was examined. It was found that 10 microM NA significantly increased (approximately by twofold) the frequency of tetraploid cells. Cultivation of 4C cells isolated by flow cytometric cell sorting revealed that they were true polyploid cells. This receptor-mediated effect of NA was blocked only by simultaneous action of alpha- and beta-adrenoreceptor antagonists. SMC polyploidization was also stimulated by simultaneous application of direct activators of "second messenger" systems forskolin and phorbolmyristate-acetate. Thus, NA may be one of mediators of the "hypertensive" response of vessel wall SMC, which probably occurs due to synergism of two second messenger systems.

The intestinal epithelial cells of ground squirrel (Citellus undulatus) accumulate at G2 phase of the cell cycle throughout a bout of hibernation.

1. The mitotic index was found to be greatly reduced in the intestinal crypt cells of ground squirrel during bout of hibernation. The percentage of mitosis rose abruptly at least 2 hr after arousal. 2. An increase in the number of G2 cells was found in the intestinal tract of ground squirrel during bouts of hibernation. 3. The conclusion can be drawn that the cells are progressing steadily through the cell cycle. The cells accumulate at G2 in hibernation. 4. It was assumed that the block in G2 prevents the cells from possible damage in mitosis under hypothermia accompanying hibernation and, therefore, it represents an adaptive reaction.

Seasonal variations of DNA-synthesis in intestinal epithelial cells of hibernating animals--2. DNA-synthesis in intestinal epithelial cells of ground squirrel (Citellus undulatus) during autumn and late hibernation season.

1. DNA-synthesis was found to be gradually reduced in the intestinal crypt cells of ground squirrel during autumn at the time of preparation for hibernation and gradually rose during late hibernation season in February-March at the time of preparation for arousal. 2. A conclusion is made on the seasonal variations in cell replacement of true hibernators.

[Effect of extracorporeal sorption on the content of free intracellular calcium of the leukocytes in atopic bronchial asthma]. / Issledovanie vliianiia ékstrakorporal'noi sorbtsii na soderzhanie svobodnogo vnutrikletochnogo kal'tsiia v leikotsitakh pri atopicheskoi bronkhial'noi astme.

Antigen-IgE-antibody interaction on the cell surface in an allergic reaction increases methylation of phospholipids of a cell membrane making it permeable for Ca2+ ions. Therefore of interest is a study of free intracellular Ca2+ concentration from a view-point of development of atopic bronchial asthma and the use of extracorporal methods of its therapy permitting the elimination of IgE-antibodies from the blood flow. The level of intracellular Ca2+ in the leukocytes of patients with atopic bronchial asthma was studied. It has been shown that 1. the fraction of leukocytes with a higher level of intracellular Ca2+ in patients with atopic bronchial asthma was increased as compared to healthy donors; 2. extracorporal sorption results in a decrease in the number of leukocytes with high Ca concentration.

[Concentration of erythrocyte-based magnetic carriers in the vascular bed]. / Kontsentrirovanie magnitnykh nositelei na osnove éritrotsiov v sosudistom rusle.

The in vivo and in vitro experiments have shown that magnetic erythrocytes (loaded with ferromagnetic material) can be retained in certain sites of the vascular bed. Magnetic erithrocytes could be concentrated in the abdominal part of the dog aorta using a small permanent magnet fixed on the outer wall of the aorta.

Determination of total intracellular lipid content by flow cytofluorometry.

It was demonstrated that perylenoyl-labeled triglyceride, rac-1,2-dioleoyl-3-[9(3-perylenoyl)nonanoyl]glycerol (PLTG), specifically stains the intracellular lipids in formalin-fixed and non-fixed cells in situ and in suspension. The fluorescence of labeled cells was registered on a FACS-II cytofluorometer. It was found that the mean intensity of fluorescence in PLTG-labeled cells of various types, both fixed and non-fixed, correlates with their total lipid content. There was no correlation, however, between the mean intensity of fluorescence and intracellular levels of lipids belonging to different classes: phospholipids, triglycerides, cholesterol or cholesteryl esters.

Cellular composition of atherosclerotic and uninvolved human aortic subendothelial intima. Light-microscopic study of dissociated aortic cells.

Alcoholic-alkaline dissociation was used in the study of cellular composition of human aorta. Cells were isolated from an uninvolved intima and intima with different types of atherosclerotic lesions: fatty infiltration, fatty streak, and atherosclerotic plaque. In the isolated suspension we evaluated the ratio of four previously described morphologic forms of cells: stellate, elongated, elongated with side processes, and flat cells of irregular shape. It was demonstrated that the quota of stellate cells in an atherosclerotic lesion considerably exceeds that of the normal intima. For elongated cells the opposite is true. The other two cell forms are represented in the uninvolved and atherosclerotic intima in approximately equal proportions. Alteration of the ratio of different morphologic forms occurs because of the fact that the number of cells belonging to different morphologic forms increases disproportionately in the lesion zone. Specifically, the number of stellate cells is increased much more substantially, compared with elongated cells.

Heterogeneity of human aortic cells in regard to RNA content.

Cells of human aorta were isolated by dispersing the tissue with collagenase and elastase. The isolated cells were stained in suspension by the acridine orange fluorescent stain. The intensity of red fluorescence (greater than 580 nm) corresponding to the RNA content was measured in each individual cell and registered in a FACS II flow cytofluorometer. It was established that a cell population of human aorta is heterogenous with respect to RNA content. In a population of isolated cells, one can distinguish two subpopulations: (A) small cells with low RNA content, and (B) large cells with high RNA content. The ratio of both cell types varies in intima and media, and different types of atherosclerotic lesions. The share of cells belonging to subpopulation A is lower in media compared to intima. In intima, the number of these cells grows with the degree of atherosclerotic lesion. Possible reasons for the discovered metabolic heterogeneity of human aortic cells and prospects for the application of flow cytofluorometry to a research into cellular mechanisms of atherosclerosis is discussed.