Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.Compared to the traditional TRAP assay that utilize ³²P labeled radioactive dCTP''s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.     

Endonuclease of high specific activity in a purified mouse interferon preparation

We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 10⁹ NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA₂^Gln species from $\text{E. coli}$, or tRNA^Val species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of $\text{in vitro}$-synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established.     

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

Purification and Characterization of Mouse L Cell Interferon

Interferon was produced in high yields in mouse L cell cultures infected with Newcastle disease virus, with a specific activity of the order of 10⁶ units per mg protein. It was partially purified by zinc acetate precipitation, carboxymethyl Sephadex chromatography, Sephadex gel filtration and pressure dialysis. On electrophoresis in polyacrylamide gel, it consisted of a fast-moving sharp component and a slow, broadly distributed component(s). The highest specific activity of the former component so far obtained was 8 × 10⁷ units per mg protein, numerically the highest value ever reported for interferon. It was considered likely, however, that the protein components in the purified samples, revealed by staining of the electrophoresis gel, still represented mostly impurities. Gel filtration experiment indicated some heterogeneity of interferon in molecular weight but the major component was estimated to be 30 000 in molecular weight. Interferon activity could be maintained without added preservatives for prolonged periods, provided that the protein concentration of the sample itself was high. One interferon unit as defined in this paper was found to correspond to 0.3 international unit.     

A nucleolus-specific phosphoprotein in mouse ascites tumor cells

Analysis of proteins in nucleoli and chromatin of mouse ascites tumor cells labeled with [³²P]orthophosphate by SDS-polyacrylamide gel electrophoresis showed that a highly radioactive protein was localized in the nucleoli. This protein was purified and the final preparation appeared as a single component on hydroxylapatite column chromatography with or without SDS. This protein was found to be a nucleolus-specific phosphoprotein with a molecular weight of 120 000. The phosphate moiety in this protein turned over very rapidly whereas the protein itself was stable. When the nucleoli were disrupted by EDTA treatment, this unique protein was found as a major protein constituent of the ultracentrifugal supernatant.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Synthesis of two non-phosphorylated proteins induced in mouse L-cells by homologous interferon

We have shown that two proteins P1 and P2 of M_r 43000 and 40800 are always detected by two-dimensional gel electrophoresis of interferon-treated mouse L-929 cell extract. These two proteins have an isoelectric point of pH 4.6 and pH 4.7 respectively. If Pl is detectable in small amount in the control gels, P2 is completely absent. Actinomycin D added at the same time as interferon, prevents both P1 and P2 synthesis, but enhances their production when added between 4 to 6 h after interferon. Using molecular weight and isoelectric point as criteria, we have tried to compare P1 and P2 to enzymes induced by interferon. With double-labelled two-dimensional gels by |³⁵S| methionine and |γ³²P| ATP, we have shown that neither P1 nor P2 is phosphorylated. This experimental procedure has allowed us to obtain new data on substrates phosphorylared by interferon induced protein kinase.     

Construction of a novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 10⁸ IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against¹²⁵I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in inhibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malignant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ -treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2^−/− mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2^−/− red cells and reduced Prx2^−/− red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2^−/− mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2^−/− mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2^−/− mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

The Synthesis In Vitro of Flagellin by a Cell-Free Extract from Bacillus Pumilus

Abstract

Bacterial flagella are constructed mainly, or perhaps exclusively, of protein subunits, the flagellins. Demonstration is given in this report for the in vitro incorporation of radioactive amino acids into flagellin, it also appears that part of such incorporation reflects de novo synthesis of flagellin moleculus. Cell-free extracts were prepared from flagellated cells of Bacillus pumilus, by digestion of the cell wall with lysozyme, lysis in the Standard buffer of NIRENBERG and MATTHAEI (1961), treatment with deoxyribonuclease and centrifugation at 15.000×g. The reaction mixtures contained the cell-free extract, one or more [¹⁴C]-amino acids and the usual components required for cell-free protein synthesis. After incubation at 37° carrier flagellin was added and the pH of the reaction mixture adjusted to 2. Flagellin, which is soluble at this pH, was purified by disc electrophoresis or by reconstitution of flagellar filament at pH 5.4 followed by electrophoresis on a column of ethanolized cellulose. When an amino acid absent from B. pumilus flagellin (such as tyrosine) was used, the amount of radioactivity incorporated into the flagellin fraction was negligible as compared to that incorporated when radioactive leucine, arginine and lysine were used. The identity of the purified radioactive protein was established more conclusively by tryptic digestion and chromatographic separation of the resulting peptides. The ninhydrin positive peaks were shown to be coincident with the radioactive peaks. The radioactive peaks disappeared when a cell-free extract from non-flagellated mutant cells was used. The incorporation of radioactive methionine in the N-terminal position of the molecule indicated that at least some of the molecules had been synthesized de novo.     

The Mode of Production of Endotoxin-Induced Interferon in Rabbit Tissue Cells

In vitro production of endotoxin-induced interferon in rabbit tissue cell cultures could be enhanced by pretreatment with interferon. The enhancible state developed from the first hr of incubation at 37 C and a maximal priming effect was attained at 6 hr of incubation. Yields of interferon from unprimed cultures were usually 20–200 units/ml. In contrast, the primed cultures constantly yielded 1,000–2,500 units/ml of interferon. The pretreatment with interferon seemed to cause an earlier appearance of detectable interferon and the primed cells became more sensitive to endotoxin. It turned out that 10–30 units/ml of rabbit interferon were enough to develop the maximal priming. Even when cells were pretreated with higher doses of rabbit interferon such as 1.0 × 10⁴–1.0 × 10⁵ units/ml, the same level of priming effect was always observed without diminution. Various types of homologous (rabbit) and heterologous (human and mouse) interferon preparations showed similar dose-dependent enhancement of interferon production in proportion to the antiviral titers of these preparations as tested with RK-13 cells of rabbit origin.     

Enzymic iodination. A probe for accessible surface proteins of normal and neoplastic lymphocytes

1. Radioactive iodide was covalently bound to living cells from normal mouse spleen and a variety of lymphoid tumours by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. About 3x10(5)-6x10(5) molecules of [(125)I]iodide/cell could be incorporated without affecting cell viability. 3. Electron-micrographic radioautography showed that the radioactive label was associated with the outer surfaces of the cells. 4. Radioiodinated proteins were solubilized in 9m-urea-0.2m-mercaptoethanol and analysed by gel-filtration and disc electrophoresis. 5. Comparison of distinct tumour lines by disc electrophoresis showed qualitative and quantitative differences in protein distribution patterns.     

Purification of mouse cell interferon induced by isotopically labelled double-stranded f2 phage RNA

The dynamics of interferon formation by an established cell line of mouse fibroblast (L cells) and by mouse peritoneal leukocytes induced by double-stranded RNA extracted from E. coli f2 phage is described. The L cells produced interferon at a lower rate, the maximum values were obtained at 12 to 20 hours after induction, and the production was ultimately dependent on the established cell line used and on the presence of DEAE-dextran during induction. The mouse peritoneal leukocytes (MPL), on the other hand, did not require DEAE-dextran and the maximum of interferon production was reached between 6 and 12 hours after induction. Both the L cell- and the MPL-interferons were purified and concentrated so that the final specific biologic activity was 100-to 300-fold higher than that of the initial preparations (1 to 5 X 10(6) interferon units per mg protein). Polyacrylamide gel electrophoresis showed similar migration profiles for the preparations of both interferons. The smaller part of the activity was situated in a broader, slow-moving peak and the greater part formed a sharp, high and fast-moving peak. Using 3H uridine-labelled f2 ds-RNA for induction of interferon it was found that one of the radioactivity zones coincided with the fast-moving activity peak of the purified and concentrated interferon.     

Gene expression of foreign metaphase chromosomes introduced into cultured mammalian cells?

Transfer of genetic information from isolated hamster chromosomes to mouse cells is described. Metaphase chromosomes isolated from Chinese hamster diploid cells were incubated with mouse Cl. 1-d cells deficient in thymidine kinase activity. Two viable colonies appeared from the treated mouse cells after HAT selection with a frequency of about 10⁻⁸. The first colony isolated (Cl. 1) failed to grow, however. The second colony isolated (Cl. 2) grew well in HAT medium and was subcultured for more than 70 generations. Cl. 2 cells possessed an elevated tetrahydrofolate dehydrogenase activity of molecular species resembling that of Chinese hamster cells, as shown by disc electrophoresis. The cell line also expressed surface antigen(s) specific to hamster species, as shown by mixed hemadsorption test and immune cell electrophoresis. This latter phenotype disappeared after prolonged cultivation (59 generations) of the cells in non-selective medium. The karyotype of Cl. 2 cells corresponded to that of the mouse species and was quite different from that of hamster cells. Hamster chromosomes could not be identified in any of the cell clones by detailed analysis by the banding method (Q- and C-band). Not one revertant cell was obtained among 4.2×10⁸ Cl. 1-d cells in the control.     

Virus-mediated inhibition of the ppp(A2′p)n A system and its prevention by interferon

Encephalomyocarditis virus infection of mouse Ehrlich ascites tumour or L-cells or human HeLa cells inhibits the activation of the ppp(A2′p)_n A-dependent nuclease and prevents the binding of a radioactive analogue of ppp(A2′p)_n A to the ppp(A2′p)_n A binding protein. This loss or inactivation of the ppp(A2′p)_n A-dependent nuclease in response to virus infection is prevented by interferon pretreatment of the cells.     

In vitro binding of labeled 20-methylcholanthrene to a specific cytosol-protein

In vitro binding kinetics and specificity of [20-³H]methylcholanthrene ([³H]MC) interaction with mouse epidermal cytosol in the presence or absence of microsomal metabolizing systems were investigated. After an incubation period of 30 roin at 22°, the samples were dialyzed, subjected to gel filtration, ultrafiltration, and analyzed by 7 % basic polyacrylamide gel electrophoresis. A major portion of the binding appeared in a single peak on the gel which had the same mobility as bovine or mouse serum albumin. In vitro competition by other hydrocarbons or by promoters for binding of MC to this cytosol receptor protein showed an impressive correlation to carcinogenic and promoting activities. Essentially 100% of the MC bound to cytosol without a microsomal metabolizing system was non-covalent. However, binding Of MC to cytosol in the presence of microsomes plus reduced NADPH was party covalent which has previously been reported by Grover and Sims and Meunier and Chaveau. When bovine (BSA) or mouse serum albumin (MSA) was incubated with radioactive MC in the presence of competing non-radioactive carcinogens or promoters, little or no inhibition of binding was found. The finding that both a powerful tumor promoter and a strong carcinogen are competitors for the specific MC binding to the cytosol receptor protein indicates that it may represent a critical interaction for the promoting stage of chemical carcinogenesis in mouse skin.     

Stabilisation of human interferon β synthesis in Escherichia coli by zinc ions

Human interferon β synthesized in Escherichia coli is unstable and toxic for the bacterial cell. Zinc ions are able to stabilise interferon β in E. coli probably by inhibiting the action of cell internal proteinase(s) which affect the half-life of this foreign protein. As a result up to one order of magnitude more active IFN-β can be detected in Zn²⁺-treated E. coli cells.     

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.