[Bacteriophage enzymes for the prevention and treatment of bacterial infections: stability and stabilization of the Streptococcus pyogenes cell lysing enzyme].

The effect of various compounds on the activity and stability of a phage-associated enzyme lysing cells of streptococci of groups A and C (PlyC) was investigated. Substantial inhibition of the enzyme activity was revealed at an increased ionic strength (in the presence of NaCl) and upon the addition of carbohydrates (mono-, di-, and polysaccharides), i.e., agents stabilizing many enzymes. It was established that the enzyme activity was substantially reduced in the presence of positively charged polyelectrolytes and surfactants, whereas incubation with micelle-forming substances and negatively charged polyelectrolytes led to PlyC activation and stabilization. It was shown that, in the mycelial polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions (pH 6.3, room temperature), it practically completely lost its activity in 2 days. Characteristics of the enzyme thermal inactivation were found, in particular, its semiinactivation time at various temperatures; these allowed us to estimate its behavior at any temperature and to recommend conditions for its storage and use.

Basic fibroblast growth factor positively regulates hematopoietic development.

Recently identified BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, we show that BL-CFC expresses the receptor tyrosine kinase, Flk1, and thus we took advantage of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1(+) cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, we have analyzed ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, for their potential to generate BL-CFCs and Flk1(+) cells, to further define events leading to hemangioblast development. Our data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast.

Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151.

Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology.

Altered expression of ubiquitous kinesin heavy chain results in resistance to etoposide and hypersensitivity to colchicine: mapping of the domain associated with drug response.

The motor protein kinesin is a tetramer consisting of two heavy and two light chains. Expression of an antisense RNA fragment derived from the mouse ubiquitous kinesin heavy chain (uKHC) cDNA is associated with a unique type of multidrug resistance. We analyzed the effects of retroviral transduction of the human uKHC and its derivatives on drug sensitivity of the human fibrosarcoma cell line HT1080. Surprisingly, overexpression of full-length uKHC and its variants that were deficient in the NH2-terminal motor domain produced a phenotype similar to that of antisense RNA, characterized by resistance to etoposide and collateral sensitivity to colchicine. This altered drug response, therefore, appears to be a general consequence of kinesin deregulation. The genetic suppressor element approach was applied to map the determinants of drug response in the kinesin heavy chain. A sense-oriented genetic suppressor element conferring resistance to etoposide was isolated from a retroviral library of randomly fragmented uKHC cDNA. This element encodes the last 55 amino acids of uKHC, suggesting that the COOH-terminal tail domain of uKHC is involved in the cellular drug response.

A common precursor for hematopoietic and endothelial cells.

Embryonic stem cell-derived embryoid bodies contain a unique precursor population which, in response to vascular endothelial growth factor, gives rise to blast colonies in semi-solid medium. Upon transfer to liquid culture with appropriate cytokines, these blast colonies generate both hematopoietic and adherent, stromal-type cells. Cells within the adherent population display characteristics of endothelial lineage including the expression of CD31, flk-1, flt-1, tie-2, the capacity to take up acetylated LDL and the presence of cytoplasmic Weibel-Palade bodies. Mixing studies demonstrated that the hematopoietic and endothelial precursors within the blast colonies develop from the same cell, the blast colony-forming cell. Kinetic analysis showed that the blast colony-forming cell represents a transient cell population that develops early and is lost quickly during embryoid body development. These findings provide strong evidence that the blast colony-forming cell represents the long-hypothesized hemangioblast, the common precursor of the hematopoietic and endothelial lineages.

Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation.

Using a new strategy for tumour suppressor gene isolation based on subtractive hybridization and the subsequent selection of transforming 'genetic suppressor elements', we have cloned a novel gene called ING1 encoding a 33-kD protein (p33ING1) that displays characteristics of a tumour suppressor. Acute expression of transfected constructs encoding this gene inhibited cell growth while chronic expression of ING1 antisense constructs promoted cell transformation. Limited analyses of tumour cell lines show that mutation of the ING1 gene occurs in neuroblastoma cells and reduced expression was seen in some breast cancer cell lines. These results demonstrate that ING1 can act as a potent growth regulator in normal and in established cells and provide evidence for a role as a candidate tumour suppressor gene whose inactivation may contribute to the development of cancers.

Neu differentiation factor (Heregulin) activates a p53-dependent pathway in cancer cells.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by up-regulation of the p53 inducible gene, p21CIP1/WAF1, in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.

Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P. Rhoads Memorial Award Lecture.

Genetic suppressor elements (GSEs) are short biologically active gene fragments that encode dominantly acting peptides or inhibitory antisense RNAs. GSEs can be isolated from a single gene or from a multigene complex by constructing a library of short random fragments of the target gene(s) in an expression vector, followed by expression selection for the desired phenotype in a suitable cellular system. GSE selection from a single gene allows one to develop efficient and specific inhibitors of the gene function and to identify functional protein domains. GSE selection from a multigene complex, such as a normalized (uniform abundance) cDNA population from mammalian cells, makes it possible to identify genes that are involved in selectable cellular phenotypes. The potential of GSE selection for uncovering novel gene functions was first demonstrated using bacteriophage lambda as a model system. GSE selection in retroviral expression vectors has been applied in mammalian cells to identify genes responsible for sensitivity to etoposide and other chemotherapeutic drugs. GSE selection is also useful for cloning and analysis of tumor suppressor genes and can be applied to identifying tumor-specific targets for future anticancer drugs. Investigators should find this experimental strategy applicable to many different areas of medical and biological research.

Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization.

We describe a general strategy for cloning mammalian genes whose downregulation results in a selectable phenotype. This strategy is based on expression selection of genetic suppressor elements (GSEs), cDNA fragments encoding either specific peptides that act as dominant inhibitors of protein function or antisense RNA segments that efficiently inhibit gene expression. Since GSEs counteract the gene from which they are derived, they can be used as dominant selectable markers for the phenotype associated with downregulation of the corresponding gene. A retroviral library containing random fragments of normalized (uniform abundance) cDNA expressed in mouse NIH 3T3 cells was used to select for GSEs inducing resistance to the anticancer drug etoposide. Three GSEs were isolated, two of which are derived from unknown genes and the third encodes antisense RNA for the heavy chain of a motor protein kinesin. The kinesin-derived GSE induces resistance to several DNA-damaging drugs and immortalizes senescent mouse embryo fibroblasts, indicating that kinesin is involved in the mechanisms of drug sensitivity and in vitro senescence. Expression of the human kinesin heavy-chain gene was decreased in four of four etoposide-resistant HeLa cell lines, derived by conventional drug selection, indicating that downregulation of kinesin represents a natural mechanism of drug resistance in mammalian cells.

Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo II alpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo II alpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.

[The outpatient management of patients with a transplanted kidney]. / Ambulatornoe vedenie patsientov s transplantirovannoi pochkoi.

Organization of the management of outpatients with a transplanted kidney is described. The basis of such organization is formed by documentation of the patients' status during the entire observation period in a special card containing the data on the donor; distant monitoring of cyclosporin A for patients living far from the clinic; training of patients. The main complications that occur in patients with a transplanted kidney in the long-term postoperative period are related. They are pyelonephritis of the transplant, essential hypertension and symptomatic hypererythrocytosis. The 2-year survival of the patients is 70.8%, that of the transplant 73.1%.

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection.

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.

[A method for determining the cyclosporin A concentration in capillary blood]. / Metod opredeleniia kontsentratsii tsiklosporina A v kapilliarnoi krovi.

A new method is described, which enables one to follow the blood concentration of CyA in patients living in remote regions, being far away from the transplantation hospitals. The method is based on the use of chromatographic paper FN-17 intended for application of the patients' blood spots. The pieces of paper with dried blood spots are sent by post to the laboratory where CyA is extracted from the spots, followed by the CyA concentration measurement with the aid of RIA. A procedure has been elaborated, permitting the measurement of the CyA concentration in patients with a poor access to the veins.

[Relation between the characteristic activity of the beta-adrenergic agonist isoproterenol and the percolation properties of cell membrane]. / Zavisimost' kharakteristicheskoi aktivnosti beta-adrenergicheskogo agonista izoproterenola ot perkoliatsionnykh svoistv plazmaticheskoi membrany kletki.

Variations in the intrinsic activity of 1-isoproterenol and in the percentage of high affinity beta-receptor complexes have been studied under a changes in the macrostructure of reticulocyte plasma membranes. The fluid lipid fraction have been reduced in the membranes for this by phospholipase A2/BSA treatment. It was accompanied by a progressive decrease in the fraction of beta-receptors that are able to form the high affinity complexes with 1-isoproterenol (receptor--regulatory N-protein complexes). Evaluated with the ability to activate the adenylate cyclase, the intrinsic activity of 1-isoproterenol was decreased from 1 to 0 under with conditions. This variations proved to correlate strongly with each other. Thus, changes in the membrane macrostructure may directly determine an efficiency of hormone actions.

A simple metabolic system for selection of hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies.

A metabolic selective system has been proposed for the selection of hybrid hybridomas (tetradomas) based on the introduction in one of the parental cell lines of two traits simultaneously--a recessive one (resistance to 8-azaguanine) and a dominant one (multidrug resistance). Tetradomas were selected in the presence of two selective agents: aminopterin and actinomycin D. Using this approach we produced tetradomas secreting bispecific MAbs to horseradish peroxidase and human alpha-fetoprotein.

Application of percolation theory principles to the analysis of interaction of adenylate cyclase complex proteins in cell membranes.

Lateral protein movement in cell membranes takes place in a medium with 'obstacles'. These obstacles are: (a) aggregates of major integral proteins immobilized by submembranous structures and cytoskeleton, and (b) membrane lipids in the gel phase. Hormonal activation of the adenylate cyclase complex is associated with lateral mobility of the constiutent proteins. Modification of the interaction of these proteins due to variation of the 'fluid' lipid fraction in reticulocyte membranes has been studied. A decrease in the percentage of 'fluid' lipids in membranes resulted in the inhibition (up to the full cessation) of the interaction of beta-adrenoreceptors with regulatory NS-proteins. The interaction of NS-proteins with catalytic proteins stopped as well. On the other hand, an increase in the 'fluid' lipid fraction led to a more intensive interaction. These facts do not arise from the functional damage of interacting proteins. Consequently, hormonal activation of the adenylate cyclase complex depends on the fraction of 'fluid' lipids in the membrane. The data obtained are in conformity with the percolation theory which makes it possible to characterize long-distance protein movement in a medium ('fluid' lipids) containing obstacles. Thus, interacting proteins prove to diffuse within distances greatly exceeding protein sizes. As a consequence, the intrinsic activity of a beta-agonist, isoproterenol, varies from 1 to 0 depending on the 'fluid' lipid fraction. Our findings also suggest that in vitro there are no beta-receptors precoupled with NS-proteins in rat reticulocyte membranes in the absence of guanine nucleotides.

Cloning and characterization of DNA sequences amplified in multidrug-resistant Djungarian hamster and mouse cells.

Five recombinant phages containing different parts of genomic regions amplified in multidrug-resistant (MDR) cells have been isolated from genomic libraries of colchicine- and actinomycin D-resistant Djungarian hamster cells. Fragments of these clones together with a part of Chinese hamster mdr gene (plasmid pDR4.7 a gift of Dr. I. Roninson) were used as hybridization probes to study composition and variations of amplified DNA in a large number of MDR cell lines. Two of the six probes used (pC52 and pDR4.7) showed DNA amplification in a large number of MDR cell lines tested (commonly amplified clones) regardless of their origin (Djungarian hamster or mouse), type of selective agent used (colchicine, actinomycin D, or anthracyclines), and mode of selection (in vitro or in vivo). These clones hybridized with two different RNA transcripts (pDR4.7, 5kb; pC52, 3.5 kb) that were overproduced in MDR cells. Degrees of amplification of both commonly amplified sequences correlated with levels of resistance in all but one of the cell lines. Other cloned sequences (sporadically amplified clones) were amplified to different extents (but never greater than the commonly amplified sequences) in some of the Djungarian hamster MDR cell lines. Such differential amplification is not the result of heterogeneity of cell population since 20 cell clones tested showed identical ratios of amplification of different amplified sequences. Sporadically amplified sequences usually coamplified with the commonly amplified ones at first steps of selection, but then they would cease to amplify and, at the later stages of selection, they could even be completely deamplified. It seems that disappearance of unnecessary parts of amplicons is a regular process accompanying stepwise gene amplification.

[Endogenous MMTV in the normal mammary gland and in dimethylanthracene-induced mammary cancer in BALB/c mice]. / Endogennye MMTV v normal'noi molochnoi zheleze i indutsirovannom dimetilbenzantratsenom kantserogeneze molochnoi zhelezy myshei BALB/c.

Immunization of DMBA-treated mice by the glycoprotein fraction from mammary glands of mice BALB/c did not decrease the frequencies of induction of mammary tumors. This is in contrast to the results obtained during immunization by the formaldehyde treated preparation of Mouse Mammary Tumor Virus (MMTV). EcoRI and HindIII cleaved DNA from the DMBA-induced mammary tumors did not contain the additional virus specific fragments. In mammary tumors the expression of p27 MMTV was registered in contrast to normal mammary glands and mammary epithelium cultures in which the proteins of MMTV are not expressed even after induction.

[Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells]. / Zakonomernaia évoliutsiia amplikonov v khode razvitiia mnozhestvennoi lekarstvennoi ustoichivosti kletok dzhungarskogo khomiachka.

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.

[Dependence of hormonal stimulation of adenylate cyclase on the fraction of plasma membrane accessible to the lateral transport of adenylate cyclase complex proteins]. / Zavisimost' gormonal'noi stimuliatsii adenilattsiklazy ot doli plazmaticheskoi membrany, dostupnoi dlia lateral'nogo peremeshcheniia belkov adenilattsiklaznogo kompleksa.

Hormonal activation of the adenylate cyclase complex is associated with lateral mobility of proteins constituting this complex. Modification of interaction of the adenylate cyclase complex proteins due to variations in the "fluid" lipid fraction in cell membranes was studied. The decrease of percentage of "fluid" lipids in rat reticulocyte plasma membranes resulted in a decrease (up to a full stop) of interaction of beta-adrenoreceptors with regulatory N-proteins. The interaction of N-proteins with catalytic proteins was also blocked. On the other hand, an increase in the "fluid" lipid fraction led to a more intensive interaction. The observed phenomena do not result from functional damages of interacting proteins. Analysis of experimental results within the framework of the percolation theory suggests that hormonal activation of the adenylate cyclase complex depends on the "fluid" lipid fraction in the membrane and that the interacting proteins diffuse at distances comparable with the size of the cell membrane. The intrinsic activity of the beta-agonist isoproterenol changes from 1 to 0, depending on the "fluid" lipid fraction. The experimental data also suggest that there are no beta-receptors precoupled with N-proteins in rat reticulocyte membranes in vitro.