Counteracting regulation of chromatin remodeling at a fission yeast cAMP response element-related recombination hotspot by stress-activated protein kinase, cAMP-dependent kinase and meiosis regulators.

In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.

A family of cAMP-response-element-related DNA sequences with meiotic recombination hotspot activity in Schizosaccharomyces pombe.

The heptamer sequence ATGACGT is essential for activity of the M26 meiotic recombination hotspot in the ade6 gene of Schizosaccharomyces pombe. Hotspot activity is associated with binding of the heterodimeric transcription factor Atf1.Pcr1 to M26. We have found that the sequences (C/T/G) TGACGT also bound Atf1.Pcr1 and acted as meiotic hotspots, but unlike M26 they must be followed by A or C for Atf1.Pcr1 binding and hotspot activity. The basis of the hotspot activity of CTGACGTA (ade6-3013) appears to be identical to that of M26: hotspot activity of both sequences was abolished in cells mutant for atf1, pcr1, spc1, or wis1 and was undetectable in mitotic recombination and in meiotic recombination when located on a plasmid. Both hotspot sequences were sites of micrococcal nuclease hypersensitivity in meiotic chromatin, suggesting that they create an open chromatin structure during meiosis at the site of the hotspots. The newly identified hotspot sequences (C/T/G)TGACGT(A/C) and M26 are closely related to the cAMP response element (CRE) consensus sequence for binding of cAMP-responsive transcription factors such as Atf1.Pcr1, suggesting a link between transcription and meiotic recombination. These results significantly expand the list of identified sequences with meiotic recombination hotspot activity in S. pombe from a single sequence to a family of CRE-related sequences.

Genetically modified clostridium for gene therapy of tumors.

Many murine and human tumors contain hypoxic or necrotic regions in which the oxygen tension is abnormally low. For example,>50% of primary tumors of the breast, cervix, and head and neck contain areas that are hypoxic. Because hypoxic regions are not present in normal tissue, this provides the potential for selectively targeting gene therapy to tumor cells.

Relationship between moderate intensity endurance training volume and natural killer cell cytolytic activity.

BACKGROUND: This study was designed to examine the relationship between endurance training volume and natural killer cell (NK) cytolytic activity. We hypothesized that a dose dependent relationship exists between forced treadmill training volume and training induced increases in NK cell cytolytic activity. EXPERIMENTAL DESIGN: female, Swiss Webster mice were assigned to treadmill control (TC) or treadmill trained groups (n = 10 per group). Trained mice ran at 12 m per min. (8 degrees grade) for: 15 (EX15), 30 (EX30), or 60 minutes (EX60) per day, five days per week for 11 weeks. Splenic NK cell activity was expressed as median lytic unit (LU), median LU per asialo GM1 (AsGM1+) cell, and median LU per spleen. RESULTS: Median NK activity was not significantly increased by training volume. A trend toward greater median LU per AsGM1+ cell was observed in EX30 group versus TC (p = 0.1). Training volumes less than or greater than this level produced smaller increases in NK cytolytic activity. CONCLUSIONS: These data provide preliminary evidence indicating that training induced increases in splenic NK cell cytolytic activity do not exhibit a dose dependent relationship with treadmill training volume.

Control of meiotic recombination in Schizosaccharomyces pombe.

Homologous recombination occurs at high frequency during meiosis and is essential for the proper segregation of chromosomes and the generation of genetic diversity. Meiotic recombination is controlled in numerous ways. In the fission yeast Schizosaccharomyces pombe nutritional starvation induces meiosis and high-level expression of many genes, including numerous recombination (rec) genes, whose products are required for recombination. Accompanying the two meiotic divisions are profound changes in nuclear and chromosomal structure and movement, which may play an important role in meiotic recombination. Although recombination occurs throughout the genome, it occurs at high frequency in some intervals (hotspots) and at low frequency in others (coldspots). The well-characterized hotspot M26 is activated by the Mts1/Mts2 protein; this site and its binding proteins interact with the local chromosomal structure to enhance recombination. A coldspot between the silent mating-type loci is repressed by identified proteins, which may also alter local chromatin. We discuss in detail the rec genes and the possible functions of their products, some but not all of which share homology with other identified proteins. Although some of the rec gene products are required for recombination throughout the genome, others demonstrate regional specificity and are required in certain genomic regions but not in others. Throughout the review contrasts are made with meiotic recombination in the more thoroughly studied budding yeast Saccharomyces cerevisiae.

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment.

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

A WD repeat protein, Rec14, essential for meiotic recombination in Schizosaccharomyces pombe.

Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differes from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103.

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26.

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5'-ATGACGT-3'. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.

Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia.

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

Subcellular localisation of the antitumour drug mitoxantrone and the induction of DNA damage in resistant and sensitive human colon carcinoma cells.

Cellular uptake and subcellular localisation of the antitumour agent mitoxantrone were studied in a human colon-carcinoma cell line and a mitoxantrone-resistant subline showing features consistent with an atypical multidrug-resistance phenotype involving altered topoisomerase II. Flow cytometry indicated a reduced uptake of mitoxantrone in the resistant line. Confocal microscopy indicated that mitoxantrone-associated fluorescence was primarily found within discrete cytoplasmic inclusions and around the periphery of the nucleus, with low levels being observed within the nucleus. The frequency of cytoplasmic inclusions was reduced in mitoxantrone-resistant cells as compared with parental cells. Fluorescence in cytoplasmic inclusions persisted throughout a 24-h post-treatment period in both cell lines. The results suggest that the persistence of mitoxantrone in cells is a determinant for the continuous induction of DNA damage, perhaps through chronic topoisomerase II trapping, and that modified sequestration may contribute to clinically relevant moderate levels of non-classic multidrug resistance.

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.

Racial aspects of blood pressure in children and adolescents.

The incidence, prevalence, and severity of essential hypertension are higher in minority adult populations, especially black Americans. Studies have not uniformly shown that black children and adolescents have higher blood pressure values than whites of the same age. The goal of this article is to review data available comparing minority and white populations. A section discussing studies addressing racial differences in related topics is included to demonstrate the existing fields of research that may not be familiar to the general pediatrician.

Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage.

Flow cytometry and laser scanning confocal imaging have been used to analyze the uptake of the anticancer topoisomerase II poison mitoxantrone by intact mammalian cells and the results correlated with the induction of DNA damage. Unlike Adriamycin, mitoxantrone displays only minimal levels of red fluorescence when excited at 514 wavelength. However, using these excitation and emission conditions, flow cytometry could detect low levels of fluorescence in human transformed fibroblasts exposed to high concentrations (5-20 microM) of mitoxantrone for 1 h. Over this dose range whole cell fluorescence was a function of cell size and increased with drug concentration while drug-induced DNA-protein cross-linking showed saturation. Confocal microscopy revealed the time- and dose-dependent appearance of fluorescence, interpreted here as reflecting the disposition of drug molecules, preferentially within the cytoplasm, nuclear membrane, and nucleoli. This pattern contrasted with the intense intranuclear fluorescence observed in Adriamycin-treated human cells. Loss of the nuclear membrane during mitosis resulted in an apparent increase in chromatin-associated fluorescence. Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosomal, location for fluorescence from human cells exposed for 1 h to a low but cytotoxic concentration (0.1 microM, yielding approximately 90% cell kill) of mitoxantrone. At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNA-protein cross-linking. Murine cells, displaying mitoxantrone resistance as part of the P-glycoprotein-mediated multidrug resistance phenotype, showed specific extinction of mitoxantrone-associated fluorescence from inside nuclei but not from within extranuclear compartments. The study demonstrates the feasibility of high resolution studies on the intracellular distribution of mitoxantrone in intact living cells. We suggest a mechanism by which cytoplasmic sequestration of mitoxantrone may be important in determining the response of normal and multidrug-resistant cells as they attempt to progress through mitosis.

Effects of interdental osteotomies on the periodontal and osseous supporting tissues.

The effects of 40 interdental osteotomies on surrounding osseous and periodontal structures were evaluated clinically and radiographically. No statistically significant changes were noted in any of the clinical parameters except gingival height, which decreased slightly in the maxilla and increased in the mandible. These changes were found to be statistically significant, but not clinically significant, and are most likely explained by the direction of movement of the bony segment. Radiographic evaluations revealed no statistically significant changes other than an increase in interdental width in both the maxillary and mandibular sites. These measurements were made at the root level, and, although the crowns were moved closer, the roots may actually have flared apart because the anterior segment was tipped to upright the incisors. The lack of change in the other radiographic parameters indicated that there was no loss of alveolar bone support.

Mitoxantrone-DNA binding and the induction of topoisomerase II associated DNA damage in multi-drug resistant small cell lung cancer cells.

The cytotoxicity anti-tumour intercalating agents such as the anthraquinone mitoxantrone is thought to relate to DNA binding and the trapping of DNA topoisomerase II complexes on cellular DNA. We have studied the uptake, nuclear location, DNA binding mode and DNA damaging capacity of mitoxantrone in a small cell lung carcinoma cell line (NCI-H69) compared with an in vitro-derived variant subline (NCI-H69/LX4) that exhibits "classical" multi-drug resistance (MDR). Variant cells maintained under doxorubicin selection showed reduced RNA levels that returned to control values within 7 days of growth under non-selective conditions. Variant cells released from selection stress showed resistance to DNA cleavage by doxorubicin, mitoxantrone, 4'-epidoxorubicin, 4'-deoxy-doxorubicin but reduced resistance to aclacinomycin A and a 9-alkyl substituted anthracycline in broad agreement with the cross-resistance patterns for cytotoxicity. Mitoxantrone treated NCI-H69 cells were found to accumulate DNA-protein crosslinks during a 4 hr post-treatment incubation period whereas variant cells maintained depressed levels of crosslinking. There was no apparent abnormality in the availability or drug sensitivity of topoisomerase II assayed in crude nuclear extracts of NCI-H69/LX4 cells. Whole cell uptake of radiolabelled mitoxantrone was depressed (50%) in NCI-H69/LX4 compared with NCI-H69, whereas assessment of nuclear-bound drug in individual cells by a fluorescence quenching technique showed at least a 10-fold greater level of target protection. The quenching results provide evidence of a high affinity, saturable mode of drug binding, favoured at low drug concentrations, that correlated with DNA cleavage capacity. We propose that the cytotoxic action of mitoxantrone is dependent upon a restricted and persistent form of binding to DNA that favours the long-term or progressive trapping of topoisomerase II complexes.

Long-term inhibition of DNA synthesis and the persistence of trapped topoisomerase II complexes in determining the toxicity of the antitumor DNA intercalators mAMSA and mitoxantrone.

The cytotoxic actions of several classes of antitumor DNA intercalators are thought to result from some disturbance to DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here we have studied topoisomerase II trapping and DNA synthesis patterns in relation to the acute cytotoxic actions of 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) or mitoxantrone on SV40 transformed human fibroblasts. These two DNA intercalators differed significantly in their cytotoxic potential, mitoxantrone being 24-fold more toxic than mAMSA when assayed by the inhibition of clonogenicity. Although both drugs induced G2 delay at cytotoxic concentrations, mAMSA-treated cells recovered normal cell cycle phase distributions within 24 h of removal of drug, while mitoxantrone-treated cells continued to accumulate in G2 up to 48 h following drug treatment with evidence of complete inhibition of entry into mitosis. Compared with mAMSA, mitoxantrone showed a similar capacity to induce cleavable complexes in cellular DNA, and only a 2-fold greater ability to inhibit DNA synthesis. Within a 4-h posttreatment period, mAMSA-treated cells recovered normal rates of DNA synthesis, whereas a continued depression of DNA synthesis was observed in mitoxantrone-treated cells. The recovery patterns of DNA synthesis correlated with the rapid disappearance of mAMSA-induced complexes (less than 27% lesions remaining 2 h after drug removal) and the persistence of mitoxantrone-induced complexes during a 4-h posttreatment period. This difference in complex longevity was observed in other human transformed fibroblast cell lines irrespective of differences in the absolute levels of complexes induced by either agent. We suggest that the results provide evidence that DNA intercalators may differ in the forms of complexes induced and that the comparatively high cytotoxicity of mitoxantrone relates to the ability of the drug to trap topoisomerase II complexes in a form which effects a long-term inhibition of DNA replication and G2 traverse.

Freshwater leeches (Hirudinea) as a screening tool for detecting organic contaminants in the environment.

In earlier work, we found that leeches from an industrially polluted creek bioaccumulated chlorophenols to much higher concentrations than other resident benthic invertebrates and fish. We suggested that leeches may have significant potential as biomonitors for these and other organic contaminants in the environment. In this study, we compared the bioaccumulation and depuration of 16 organic compounds, including eight chlorophenols (CPs), lindane, DDT and four derivatives, benzothiazole (BT) and 2-(Methylthio)benzothiazole (MMBT) for three species of leeches. Dina dubia had the highest bioaccumulation capacity for most contaminants, but residues persisted longest in Erpobdella punctata. Helobdella stagnalis appeared capable of degrading some compounds. Half lives of CPs, DDT and DDT derivatives were generally longer than one month. In contrast, half lives were only 1 day for lindane, 1-2.5 days for MMBT and 7 days for BT despite very high initial tissue concentrations of the latter two compounds. Bioconcentration factors for contaminants in leeches were higher than those reported for other aquatic organisms. Half lives for lindane, DDT and DDT derivatives were consistent with the literature for other organisms, but half lives for CPs were much longer. The results suggest that leeches would be excellent biomonitors of both continuous and intermittent contamination of a waterway with CPs and DDT, as they retain these compounds for long periods after exposure. Their usefulness as a screening tool for lindane and benzothiazoles would be limited to chronically contaminated environments.

Disappearance kinetics of 2,4- and 3,4-dichlorophenol in a fluvial system.

The disappearance rates of 2,4- and 3,4-dichlorophenol in a small stream were studied and were shown to be first order with respect to either distance or time of flow. Both chlorophenols disappeared at approximately the same rate with average half-lives in the stream of about 4 h. The absence of seasonal variability in the rate constants along with the observance of first order kinetics over several ecological zones of the stream led to the conclusion that the rate controlling factor was not biological. It is suggested that the disappearance was due to degradation within the biofilm covering the stream bed and that the rate is controlled by diffusion of the chlorophenols across the water-biofilm interface.