Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.     

Influence of DNA synthesis inhibition on the coordinate expression of core human histone genes during S phase

Core histone mRNA metabolism has been examined in S phase HeLa cells recovering from DNA synthesis inhibition by 1 mM hydroxyurea. Using cloned human histone genes as probes for histone mRNA quantitation, the response to and recovery from DNA synthesis inhibition is shown to depend on the position of the cell with respect to the initiation of DNA replication. The incorporation of 3H-uridine into multiple histone mRNAs in recovering cells does not exceed preinhibition levels, and as this incorporation is maximal in early S phase, the synthesis of core histone mRNA is apparently related to the ordered replication of the genome. The total histone mRNA present in interrupted S phase cells after recovery is not significantly different from that present in control cells, and a temporal and functional coupling between histone mRNA levels and the relative rate of DNA synthesis is maintained in perturbed cells.     

Method of extracting DNA from fine needle aspirates of human solid tumors for Southern blot analysis

A rapid, simple, convenient method for extracting DNA from fine needle aspiration (FNA) samples of human solid tumors for Southern blot hybridization studies is described. After the preparation of an air-dried cytologic smear, the remaining sample in the needle was rinsed directly into a test tube for DNA extraction. The extraction procedure, in which manipulation of the sample is minimized, produced sufficient DNA for Southern blot analysis within 24 hours of the FNA biopsy in the ten consecutive cases studied. The DNA bound to the nylon membranes can be washed and reexamined with a variety of probes, allowing studies of lymphoid cell lineage, oncogene amplification or tumor progression. The assessment of cellularity on the cytologic specimen at the time of FNA provided a reliable guide to the need for further passes to obtain sufficient cells for DNA hybridization; the cytologic diagnosis could also be made on the smears.     

DNA replication patterns of the early S phase from amniotic fluid cells as revealed by a Giemsa staining technique

Summary We report on DNA replication patterns of the early S phase demonstrated by a BUdR/Hoechst (33258)-fluorescence/Giemsa technique. Replication starts in R bands and spreads within 1 hr over the whole chromosome complement with exceptions concerning heterochromatin. The beginning and the duration of replication are discussed.Supported by the Deutsche Forschungsgemeinschaft.     

Influence of mature Douglas fir roots on the solid soil phase of the rhizosphere and its solution chemistry

Plants can induce significant changes in the rhizosphere through the uptake of water and ions, the exudation of organic compounds and the activities of micro-organisms. The aim of the present study was to assess the influence of tree roots on the chemistry (pH, exchangeable cations, total organic carbon) of both the solid phase of the soil and the soil solutions, extracted by centrifugation, under a mature Douglas fir stand over two distinct seasons (March and September, 1999). The chemical characteristics of either the solid soil phase or the soil solutions of the rhizosphere were found to be different from those of the bulk soil. The cation exchange capacity, base saturation and organic C were all greater in the rhizosphere than in the bulk soil, as a possible result of rhizodeposition, incorporation of decaying root material and micro-organism activity. The concentration of all elements increased in the rhizosphere solutions as compared to the bulk soil solutions, except for P. The pH was lower in the rhizosphere than in the bulk soil for both the solid soil phase and the soil solutions. Despite the greater overall Al concentration of the rhizosphere solutions, as compared to the bulk soil solutions, we suggest that in both, Al toxicity was efficiently restricted by both high Ca + Mg contents and Al complexation with various ligands.     

Evaluation of a compost obtained from forestry wastes and solid phase of pig slurry as a substrate for seedlings production

A composted material obtained from forestry wastes and solid phase of pig slurry was evaluated as a substrate component for the production of tomato and lettuce seedlings. Four different substrates were tested: compost (100C), a mixture of 75% compost and 25% peat substrate (75C), a mixture of 50% compost and 50% peat substrate (50C), and peat-based substrate (control). Compost increased the pH of the substrate from 6.3 (control) to 6.9 (100C) but did not affect the electrical conductivity (0.26 and 0.27 mScm(-1), respectively, for control and 100C). Germination and growth of lettuce seedlings were not affected by the substrate type, contrasting with tomato seedlings where the highest growth occurred at 100C substrate. Increasing compost percentage on substrate increased nitrogen, calcium and magnesium availability and, consequently, the concentration of these elements in plant tissues increased. On the contrary, potassium and manganese concentration decreased. Results from the study suggest that the compost studied is a good alternative to peat-based substrates for the production of vegetable seedlings.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims: To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples. Methods and Results: Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real‐time PCR assay that included an internal positive control. The FastDNA^® SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.^® Soil DNA and PowerSoil^® DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. Conclusions: The results of this study suggest that commercially available extraction kits can be used to extract PCR‐quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Significance and Impact of the Study: The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real‐time PCR detection of a pathogen in soil.     

Effect of hypoxic cell sensitizer metronidazole on the radiation reaction of clonogenic cells from solid tumors

The clonogenic capacity of cells from peripheral and central zones of solid NKLy tumors of mice treated with metronidazole, a sensitizer of hypoxic cells, and with a mixture of metronidazole and radiation was studied by cloning in diffusion chambers. The cytotoxic effect of metronidazole was only noted during the prolonged interaction with cells under acute hypoxia that was observed in central tumor zones. Metronidazole increased by more than two times the radiosensitivity of cells from the central zones of the tumor and did not influence the radiation response of cells from the peripheral zones. Metronidazole was shown to inhibit the repair of potentially lethal radiation damages.     

A novel Fmoc-based anchorage for the synthesis of protected peptides on solid phase

A novel bifunctional compound, 9-(hydroxymethyl)-2-fluoreneacetic acid, was synthesized, coupled to benzhydrylamine-resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor-bond cleavage was achieved with 15% piperidine/DMF. A protected heptapeptide, Boc-Val-Val-Ser(Bzl)-His(Tos)-Phe-Asn-Lys-(Z)-OH, corresponding to the sequence (1-7) of rat-transforming growth factor-alpha, was synthesized using this new support with an overall yield of 46%.     

Flow cytometric DNA analysis using cytokeratin labeling for identification of tumor cells in carcinomas of the breast and the female genital tract.

Flow cytometric assessment of DNA-ploidy and S-phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC-conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively) prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA-aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA-diploid tumors, a significantly improved detection of S-phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating "normal cells". S-phase fraction following tumor cell enrichment was increased by 10% (mean) following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA-analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.     

Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation

DNA damage causes cell cycle arrest in G(1), S, or G(2) to prevent replication on damaged DNA or to prevent aberrant mitosis. The G(1) arrest requires the p53 tumor suppressor, yet the topoisomerase I inhibitor SN38 induces p53 after the G(1) checkpoint such that the cells only arrest in S or G(2). Hence, SN38 facilitates comparison of p53 wild-type and mutant cells with regard to the efficacy of drugs such as 7-hydroxystaurosporine (UCN-01) that abrogate S and G(2) arrest. UCN-01 abrogated S and G(2) arrest in the p53 mutant breast tumor cell line MDA-MB-231 but not in the p53 wild-type breast line, MCF10a. This resistance to UCN-01 in the p53 wild-type cells correlated with suppression of cyclins A and B. In the p53 mutant cells, low concentrations of UCN-01 caused S phase cells to progress to G(2) before undergoing mitosis and death, whereas high concentrations caused rapid premature mitosis and death of S phase cells. UCN-01 inhibits Chk1/2, which should activate the mitosis-inducing phosphatase Cdc25C, yet this phosphatase remained inactive during S phase progression induced by low concentrations of UCN-01, probably because Cdc25C is also inhibited by the constitutive kinase, C-TAK1. High concentrations of UCN-01 caused rapid activation of Cdc25C, which is attributed to inhibition of C-TAK1, as well as Chk1/2. Hence, UCN-01 has multiple effects depending on concentration and cell phenotype that must be considered when investigating mechanisms of checkpoint regulation.     

A technique for determining the proportion of the clonogenic cells in S phase in ENT6 cell cultures and tumors.

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (3H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by 3H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of 3H-TdR. Under these conditions, either 3H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or 3H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by 3H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogeneic tumor cells in S phase. Incubation of tumor cell suspensions with 3H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of 3H-TdR during the incubation for colony formation.     

Purification of allergenic proteins from peanut for preparation of the reactive solid phase of a specific IgE radioimmunoassay

Peanut is one of the most allergenic foods. Detection of specific IgE in the serum of allergic patients requires the purification of allergenic proteins. In the present work, proteins were recovered from peanut kernel after successive treatment in acetone and diethy ether. The proteins were dissolved in 0.05% TFA and analysed by RP-HPLC with a 0–100% gradient of methanol containing 0.05% TFA. The protein peaks were recovered and tested in SDS-PAGE. Eleven proteins were identified with a M_r ranging from 13 to 81. Western blotting was performed with sera from allergic patients. Allergenic proteins had a M_r of 15, 18, 19, 33, 41 and 67. By comparison, a protein fraction from peanut shell contained seven proteins with M_r ranging from 15 to 81. Only two proteins with M_r of 18 and 41 were detected in a Western blot. The protein fractions were coupled to epoxy-Sepharose and the gels were used as a solid reactive phase for detection by IgE-RIA of specific IgE from the serum of allergic patients.     

Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei.

A one-step procedure for the preparation of nuclei for flow microfluorometric DNA analysis is described. The membranes of the cells were lysed by the non-ionic detergent Nonidet P40. Single-cell suspensions, and specimens of solid tissues obtained with fine-needle biopsy, could be prepared equally well as the nuclei of solid tissue cells were released separately. Lysis was performed in the staining solution containing either ethidium bromide or propidium iodide. Fluorescence due to fluorochrome binding to RNA, was abolished instantaneously by the presence of RNA-se, and fluorochrome binding to secondary binding sites in DNA was inhibited with NaCl. The preparation time was 10 min and the samples were stable for a minimum of 12 h. With the basic version of the method, usable, but not always optimal, results were obtained in all the cell types tested: four different mouse ascites tumors, leucocytes, bone-marrow, liver cells, human lymphomas, human carcinomas of the breast and lung, mouse mammary carcinoma and solid JB-1 tumor. The method was further optimized for the JB-1 ascites tumour. The resulting two modified techniques are described. Differences in the staining of leucocytes with the analogues ethidium bromide and propidium iodide were demonstrated.     

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.