Lability of RNA from the large cytoplasmic ribosomal subunit of the protozoon Crithidia oncopelti

Cytoplasmic ribosomalRNA extracted from Crithidia oncopelti and analysed by gel electrophoresis at 4 degrees C consisted of two components, with molecular weights (relative to E. coli rRNA) of 1-30 X 10(6) and 0-83 X 10(6) daltons, present in equimolar amounts. On heating briefly at 51 degrees C followed by rapid cooling, the 1-30 X 10(6) RNA completely dissociated into two components of molecular weights 0-70 X 10(6) and 0-56 X 10(6) (present in equimolar amounts). Fifty per cent dissociation of the molecule occurred at 28 degrees C. That the integrity of the RNA molecule at low temperatures is maintained by its secondary structure was confirmed by electrophoresis under denaturing conditions (98%, v/v, formamide). To account for these phenomena, latent cleavage of the molecule in vivo is proposed.     

Tetracycline-regulated suppression of amber codons in mammalian cells

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitivity growth-suppressed accumulated the mRNA of IgG1 which is reported stable, and IgG1 production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM lambda 1 chain and cultured at nonpermissive temperature to enhance production of lambda 1. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.     

Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells

Between 1985 and 1987, 1837 primary total knee arthroplasty (TKA) prostheses were implanted in 1503 patients. Group I included 843 knee with metal-backed patellar components (MBPC), and group II included 994 knees with all polyethylene patellar components (APPC). Follow-up averaged 5.7 years (range, 2 to 11 years). Twenty-four MBPC (2.9%) and 16 APPC TKA cases (1.6%) developed deep infection. In the time interval between arthroplasty and 2-year follow-up, eight MBPC and 11 APPC knees developed deep periprosthetic infection. The difference in the cumulative probability of infection between the two groups during this time interval was not significant (relative risk, 0.9; 95% confidence interval [CI], 0.3-2.1; P = 0.73). However, after the 2-year follow-up, 16 MBPC and 5 APPC knees developed late infection, and the difference in the cumulative probability of infection between the MBPC and APPC knees during this time interval was significant (relative risk, 3.1; 95% CI, 1.1-8.5; P = 0.02). Although the mechanism for this increased risk of these late infections is not well understood, the attendant synovitis, effusion, and relative hyperemia of these knees in the presence of the particulate metal and polyethylene debris may increase the potential of bacterial seeding to these prostheses. Particulate metal debris has been previously shown to suppress bacterial phagocytosis and may play a role in the pathogenesis of these infections. We propose that the presence of metal-backed patellar failure represents a "prosthesis at risk" for the development of late prosthetic infection.     

The possibility of tinnitus suppression by electrical stimulation]

Coupling of an inert polymer to the surface of red cells was examined as a potential means of covering blood group antigens and producing cells that could serve as universal donor cells for transfusion. Effective blockade of red cell antigens was achieved with N-hydroxysuccinimide-activated esters of polyethylene glycol. It was possible to block all antigens tested, but lower concentrations of reactants were required to block peptide-defined antigens than carbohydrate-defined antigens. Red cells remained intact after modification but were significantly damaged. Our results demonstrate the feasibility of antigenic blockade of red cells, but there is a need to reduce damage during coupling reactions to produce viable red cells.     

Inhibition of chemiluminescence response of human mononuclear cells and suppression of mitogen-induced proliferation of spleen lymphocytes of mice by hispolon and hispidin

The effects of two phenolic compounds, hispolon and hispidin isolated from fruit bodies of the basidiomycete Inonotus hispidus (Bull. ex Fr.) Karst, were investigated on the chemiluminescence response by LPS- or zymosan-activated human mononuclear cells (MNC) and on the concanavalin A-induced proliferation of spleen lymphocytes of mouse in vitro. Both compounds showed inhibitory activity in the chemiluminescence-test with an IC50 (the concentration of test compound causing 50% effect) ranging from 4.4 to 4.6 micrograms/ml (20.3 to 21.2 microM) for hispolon and < 0.1 to 1.5 micrograms/ml (from < 0.4 microM to 6.0 microM) for hispidin. Antiproliferative effects have been achieved in the lymphocyte transformation test (LTT) by hispolon with an IC50 of 3.4 micrograms/ml (15.5 microM).     

The number of unidentified amacrine cells in the mammalian retina

The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit's retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.     

Excision of 7-bromomethylbenz[a]anthracene--DNA adducts in replicating mammalian cells

The excision of 7-bromomethylbenz[a]anthracene--DNA adducts was studied in two cell lines (HeLa S-3 and Chinese hamster V-79379A). In both cell lines, carcinogen-modified adenine residues were excised more readily than the modified guanine residues and the percentage of the total products excised decreased after treatment with higher concentrations of carcinogen. At the highest concentrations used in the Chinese hamster cells, neither DNA synthesis nor excision was detected. The lowest concentration used for these cells permitted almost 100% survival and all the DNA was replicated in a 30-h interval even though 50% of the initial damage was still present. The two- to threefold lower sensitivity of the Chinese hamster cells (compared with the Hela cells) to the carcinogen is attributed to this capacity for replication of DNA on a damaged template since the two cell lines' capacities for excision of the chemical damage were found to be comparable.     

Molecular biology of mutagenesis of mammalian cells by ionizing radiation

Although the genotoxic potential of styrene is known, very limited information is available regarding its dose-dependent genotoxic response to human blood lymphocytes and how such response correlates with different metabolic events in whole blood lymphocytes. The present study was therefore carried out to study such a relationship using in vitro human blood lymphocytes from healthy volunteers. To study genotoxic response to styrene, sister chromatid exchanges (SCEs), cell cycle, and cell survival were analyzed. Lymphocytes were cultured for 72 hr in the presence of different concentrations of styrene (0-1,000 microM). Twenty-four hr before harvest, BrdU (5 micrograms/ml) was added to assess the increase in SCEs and cell cycle delay. Both the SCE frequency and the cell cycle length were increased linearly with increasing concentrations of styrene up to 200 microM, without addition of any exogenous metabolizing system. Above 200 microM, no further increase in genotoxic response occurred. The range of concentrations (10-200 microM) at which increase of cell cycle length due to styrene was observed did not impair the viability of the cells, suggesting that such cell cycle delay is a genotoxic-related event and not caused by cytotoxicity. In vitro metabolic transformation of styrene in whole-blood lymphocyte cultures without the presence of any exogenous metabolic activation system showed the formation of a reactive intermediate, styrene 7,8-oxide, to be capacity-limited, as verified from a nonlinear increase in the formation of styrene glycol. The value of such metabolic parameter reached a plateau above 200 microM styrene. The same phenomenon of saturation has also been observed with regard to other metabolic effects due to styrene in whole blood lymphocytes in culture, such as dose-dependent increase in lipid peroxidation and depletion of blood lymphocyte glutathione. Based on the relationship between the formation of different metabolic events and the genotoxicity of styrene, it may be possible that the genotoxic properties of styrene in human blood lymphocytes may be mediated initially not only by the formation of the presumably reactive styrene 7,8-oxide, but also by that of a reactive oxygen species as well. However, the present data are not sufficient enough to definitely identify the role of reactive oxygen species in such toxicity and therefore it warrants further study.     

The role of proliferation in the origin of mutations in mammalian cells

Biliary obstruction, produced by common bile duct ligation or alpha-naphthylisothiocyanate (ANIT) treatment in rats, has been associated with the development of type I biliary epithelial cell (BEC) hyperplasia. However, the exact mechanism(s) by which bile duct obstruction lead(s) to this proliferative lesion are not clear. The present studies were designed to determine if cholestasis, in the absence of biliary obstruction, would result in type I BEC hyperplasia. Male Sprague-Dawley rats were given a single oral dose of 150 mg/kg ANIT or i.v. doses of estradiol glucuronide (E2-17G; 21 mumol/kg/h for 48 h) to produce obstructive and non-obstructive cholestasis, respectively. E2-17G treatment resulted in cholestasis that was comparable in extent and duration to that observed following ANIT treatment. E2-17G and ANIT treatments produced comparable increases in serum bile acids (55- to 60-fold) and activities of ALT (36- to 38-fold), ALP (4- to 5-fold), and 5'-nucleotidase (7- to 11-fold), respectively, compared to controls. Both ANIT and E2-17G also increased serum bilirubin concentrations. ANIT treatment resulted in significant increases in biliary glucose concentrations that were associated with BEC damage/necrosis and obstruction of the bile duct lumen. Conversely, no evidence of BEC damage was observed in E2-17G-treated rats. Nonetheless, BEC hyperplasia was observed in the majority of rats following treatment with either ANIT or E2-17G, assessed by light microscopy and by BrdU immunohistochemistry. These data indicate that E2-17G treatment produces nonobstructive cholestasis and type I BEC hyperplasia, suggesting that biliary obstruction is not a prerequisite for type I BEC hyperplasia in rats. Differences in the time of onset of hyperplasia were observed: hyperplasia was noted immediately following 48 h of E2-17G-induced cholestasis but occurred several days after ANIT-induced cholestasis had subsided. Since the magnitude/duration of cholestasis was similar in the two models but the temporal association between cholestasis and type I BEC hyperplasia were different, these data suggest that the proliferative stimulus may be different in the two models and that E2-17G-induced type I BEC hyperplasia may not be attributed solely to cholestasis.     

Signal transduction by protein kinase C in mammalian cells

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca(2+)-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC isoform often causes redistribution of the isoform from one subcellular compartment to another compartments where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

Malignant transformation of mammalian cells initiated by constitutive expression of the polo-like kinase

Polo-like kinase (Plk) is the mammalian homologue of the Drosophila polo and Saccharomyces cerevisiae CDC5 genes, which are thought to be involved in regulating chromosomal segregation. Previously, we showed that transient ectopic expression of Plk could induce DNA synthesis in quiescent NIH 3T3 cells, suggesting that Plk might also have a function during G1 or S phase. Here we report that microinjection of Plk mRNA is sufficient to drive quiescent cells into mitosis and that constitutive expression of Plk in NIH 3T3 cells causes oncogenic focus formation. These transformed cells grow in soft agar and form tumors in nude mice. Because Plk expression has been shown to be high in various human tumors, we suggest that Plk may contribute to the promotion and/or progression of human cancers.     

Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II

Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase (up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important for segregation of newly replicated DNA.     

Aminopeptidase activities on the surface of mammalian cells

Activities of hydrolytic enzymes on the surface of monkey kidney, canine kidney, L. FM3A and various tumor cells were determined and compared with those in the cell homogenate. Although aminopeptidase (EC 3.4.11.-) activities were always detected on the surface membrane in mammalian cells, trypsin, chymotrypsin and elastase activities were not detected while slight glycosidase activity was detected in a suspension of cultured cells. The activities of alanine-, leucine-, methionine- and phenylalanine-aminopeptidases were rather high but aminopeptidase A, proline-, valine-, glycyl propline dipeptidyl-and glycyl propyl leucine-tripeptidyl-aminopeptidases showed relatively low activities. Aminopeptidase activity was also demonstrated in the isolated membrane fractions. The specific activities of enzymes in these membrane fractions were not significantly greater than in cell homogenate so it was concluded that these enzyme activities were rather loosely bound to the cell membrane. Further evidence for the localization of the aminopeptidase activities on the cell surface was obtained by using glass-bead-bound substrate and detecting the release of the terminal residues. When bestatin, a specific inhibitor against aminopeptidase B and leucine aminopeptidase, was included in the assay system for the enzyme activities on the cell surface, the enzymes were commonly inhibited in all types of cells.     

Functional complementation by electroporation of human BACs into mammalian fibroblast cells

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.