Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

The unfolding story of polypeptide release factors

Two recent cryo-EM reconstructions of the ribosome-bound release factor RF2 reveal an open, tri-lobed shape of RF2, in contrast to the comma-shaped molecule seen in the crystal structure. This indicates that RF2 undergoes a conformational change upon binding to the ribosome. Moreover, RF2 does not seem to be a molecular mimic of tRNA as is the case for elongation factor G.     

Multiplex real-time PCR for monitoring Heterobasidion annosum colonization in Norway spruce clones that differ in disease resistance

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.     

Bi- and tricyclic nucleoside derivatives restricted in S-type conformations and obtained by RCM-reactions

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.     

Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer was induced by administration of four weekly doses of AOM (15 mg/kg of body weight per week) separated by a one-week break between the second and third injections. As the histopathologic characteristics of this model resemble those of the human counterpart, further characterization of the genetic changes was undertaken. The animals were euthanized 28 to 29 weeks after the first AOM injection, and tumor specimens were taken for histologic and DNA analyses. Since microsatellite variation was found in only a few (< 2%) specimens, the model can be considered as having stable microsatellites. This result is in accordance with those of similar studies in other rat models and with most human colorectal cancers. Immunohistochemical analyses of beta-catenin did not reveal loss of gene activity, nor did the sequencing of Ki-ras reveal mutations. These results are in contrast to most findings in comparable rat studies. The deviations may be due to differences in exposure to the carcinogen or difference in strain and/or age. The lack of beta-catenin and Ki-ras alterations in this colon cancer model is unlike human sporadic colorectal cancers where these genetic changes are common findings.     

The Use of Fluorogenic Substrates To Measure Fungal Presence and Activity in Soil

Our objective was to determine if 4-methylumbelliferyl-labelled enzyme substrates could be used to detect and quantify specific components of chitinase and cellulase activities as specific indicators of the presence and activity of fungal biomass. The fluorogenic substrates 4-methylumbelliferyl (MUF) N-acetyl-β-d-glucosaminide and MUF β-d-lactoside were used for the detection and quantification of β-N-acetylglucosaminidase (EC 3.2.1.30) (NAGase) and endo 1,4-β-glucanase (EC 3.2.1.4)/cellobiohydrolase (EC 3.2.1.91) (CELase), respectively. Culture screenings on solid media showed a widespread ability to produce NAGase among a taxonomically diverse selection of fungi on media with and without added chitin. NAGase activity was expressed only in a limited number of bacteria and on media supplemented with chitin. The CELase activity was observed only in a limited number of fungi and bacteria. Bacterial CELase activity was expressed on agar media containing a cellulose-derived substrate. In soil samples, NAGase activity was significantly correlated with estimates of fungal biomass, based on the content of two fungus-specific indicator molecules, 18:2ω6 phospholipid fatty acid (PLFA) and ergosterol. CELase activity was significantly correlated with the PLFA-based estimate of fungal biomass in the soil, but no correlation was found with ergosterol-based estimates of fungal biomass.The determination of enzyme activities is a simple approach to the study of microbially mediated processes within the soil environment. Thus, soil enzyme activities have been interpreted as indirect measures of microbial biomass, rhizosphere effects, soil productivity, and mineralization potential of naturally occurring substrates or xenobiotics (4). However, few studies have attempted to correlate soil enzyme activities with the presence and activities of specific components of the microbial community. The ability of soil-inhabiting fungi to produce a range of enzymes capable of degrading complex litter substances could make the use of an enzymatic approach to study soil fungal populations possible. These enzymes must be specific for fungal presence and activity. In one study of chitinase in soil (24), chitinase activity and the number of fungal propagules in chitin-amended soils were strongly correlated. The same correlation was not found for actinomycetes or bacteria. Thus, chitinase activity appears to be a suitable indicator of actively growing fungi in the soil. The hydrolysis of cellulose requires the interaction of a number of hydrolases produced by cellulolytic microorganisms. A major role is played by the cellulase system, which consists of several distinct enzymes that are produced by a large number of microorganisms, including fungi, actinomycetes, and bacteria. However, fungi have been suggested as the predominant source of β-d-glucosidase (EC 3.2.1.21) (16, 17) and endo 1,4-β-glucanase (EC 3.2.1.4) (23) activity in soils.Fluorogenic 4-methylumbelliferyl (MUF)-labelled enzyme substrates have been introduced for process-oriented studies in aquatic systems (3, 18) and, more recently, in peatlands (11). MUF substrates have been used to assay cell-bound activities in pure cultures of fungi, as the soluble substrate can enter the cell wall, making periplasmic enzyme activity detectable (15). These substrates have been used to detect fungal chitinolytic activities (17a) and cellulases (6) in vitro. The substrates may be added to environmental samples and, when hydrolyzed, release 4-methyl-umbelliferone (4-MU), which fluoresces and can be quantified in nanomolar concentrations (3).A variety of methods to quantify fungi in soil have been described. The techniques include direct microscopic observation and extraction of fungus-specific indicator molecules such as glucosamine or ergosterol (9). More recently, the phospholipid fatty acid (PLFA) 18:2ω6 has been proposed as an indicator of fungal biomass (7, 12). Our objectives in the present study were to determine if (i) components of chitinase and cellulase activities could be used as indicators of the presence and activity of fungal biomass and (ii) enzyme activities detected with specific MUF substrates in soil samples were correlated with the content of the fungus-specific indicator molecules 18:2ω6 PLFA and ergosterol.     

Out of hours service in Denmark: evaluation five years after reform

Objective: Five years after its introduction, to evaluate the 1992 reform in the out of hours service in Denmark. Design: Comparison of data before and after reform. Data were collected from published reports, Danish national health statistics, and the Danish trade union for general practitioners. Setting: Denmark. Main outcome measures: Number of out of hours services; workload of general practitioners; cost of the service; patient satisfaction. Results: Five years after the reform, the percentage of telephone consultations had almost doubled, to 48%. Consultations in doctors’ surgeries were relatively unchanged, but home visits were much reduced, to 18%. The percentage of doctors who worked 5 hours or more out of hours per week dropped from about 70% to about 50%. Overall patient satisfaction in 1995 was high (72%). Conclusion: The organisation of the out of hours service, with a fully trained general practitioner in a telephone triage function, is working satisfactorily. Many calls that previously would have required home visits are now dealt with by telephone or through consultations. The out of hours workload for general practitioners has decreased considerably.

Key messages

• The out of hours reform in Denmark has resulted in an organisation with a fully trained general practitioner performing the telephone triage function
• Hours on call for general practitioners have decreased considerably
• Home visits have largely been replaced by telephone consultations
• Patient satisfaction has declined slightly

     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.