A human topoisomerase II alpha heterodimer with only one ATP binding site can go through successive catalytic cycles

Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.     

Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties

Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-β-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones.     

Discussion of the role of the extracellular signal-regulated kinase-phospholipase A2 pathway in production of reactive oxygen species in Alzheimer's disease

In this paper we show that exposure of a rat brain synaptosome fraction to the amyloid beta peptide fragment A(25-35), but not the inverted peptide A(35-25), stimulated production of reactive oxygen species (ROS) in a concentration- and time-dependent manner. The ROS formation was attenuated by the tyrosine kinase inhibitor genistein, the mitogen-activated protein kinase inhibitor U0126, and the phospholipase A₂ (PLA₂) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid. This strongly suggests that A(25-35) stimulated ROS production through an extracellular signal-regulated kinase-PLA₂-dependent pathway. The interaction between these enzymes and their possible involvement in free radical formation in Alzheimer's disease are discussed.     

Characterization of strains of Vibrio splendidus and V. tapetis isolated from corkwing wrasse Symphodus melops suffering vibriosis

Two vibrio bacteria pathogenic to the corkwing wrasse Symphodus melops were isolated. Vibriosis-inducing strain LP1 was isolated as the dominanting bacterium in kidney samples of dead and moribund wrasse from a population suffering vibriosis and high daily mortality in 1998 on the Norwegian west coast. The other vibriosis-inducing strain, LP2, was isolated from wrasse captured the following year. Re-infection experiments have confirmed that these strains cause vibriosis in corkwing wrasse. Both strains were typical vibrios sharing the traits of fermentative Gram-negative curved rods with motility and a positive oxidase reaction. Detailed biochemical and genetic characterisation revealed a close affiliation to known species of the marine environment. The first isolate, LP1, is a form of the ubiquitous seawater organism Vibrio splendidus, while the second isolate, LP2, is closely related to V. tapetis (previously only known as the brown ring disease agent in clams). Identification of the new wrasse pathogens V. splendidus LP1 and V. tapetis LP2 is facilitated by break points observed in this study.     

Arbuscular mycorrhizal communities in tropical forests are affected by host tree species and environment

Arbuscular mycorrhizal (AM) fungi are mutualists with plant roots that are proposed to enhance plant community diversity. Models indicate that AM fungal communities could maintain plant diversity in forests if functionally different communities are spatially separated. In this study we assess the spatial and temporal distribution of the AM fungal community in a wet tropical rainforest in Costa Rica. We test whether distinct fungal communities correlate with variation in tree life history characteristics, with host tree species, and the relative importance of soil type, seasonality and rainfall. Host tree species differ in their associated AM fungal communities, but differences in the AM community between hosts could not be generalized over life history groupings of hosts. Changes in the relative abundance of a few common AM fungal species were the cause of differences in AM fungal communities for different host tree species instead of differences in the presence and absence of AM fungal species. Thus, AM fungal communities are spatially distinguishable in the forest, even though all species are widespread. Soil fertility ranging between 5 and 9 Mg/ha phosphorus did not affect composition of AM fungal communities, although sporulation was more abundant in lower fertility soils. Sampling soils over seasons revealed that some AM fungal species sporulate profusely in the dry season compared to the rainy season. On one host tree species sampled at two sites with vastly different rainfall, relative abundance of spores from Acaulospora was lower and that of Glomus was relatively higher at the site with lower and more seasonal rainfall.     

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.     

Phenotypic characterisation of porcine counterparts of human NK cell populations--implications for pre-clinical studies

The phenotype of cryopreserved crossbred porcine PBMC, with special emphasis on NK cell related surface markers, was characterised. The phenotype expressed prior to stimulation with rHu IL2 and rHu IL12 was related to the in vitro cytotoxic capacity estimated in a 4-h or 21-h 51Cr-release assay. PBMC incubated in growth medium without addition of cytokines were used to investigate the spontaneous cytotoxic capacity. The results of this study suggest that crossbred porcine PBMC comprise a specific subset expressing the phenotype CD2+CD3-CD4-CD8+SWC3-CD16+CD21-. No spontaneous cytotoxicity of the PBMC could be estimated, but the expression of CD16 seems to be a basic marker of the cytokine induced cytotoxic activity. This study suggests that cryopreserved porcine PBMC can be used when possible influences on the porcine NK cell system are investigated in relation to disease models conducted experimentally in crossbred pigs.     

Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.     

Characterization of rhesus macaque natural killer activity against a rhesus-derived target cell line at the single-cell level

Natural killer (NK) cells and NK cell activities in the rhesus macaque have been incompletely characterized. Using a recently developed rhesus NK target cell line with down-regulated MHC-I (B116Lo) as stimulators and FACS-sorted cells as effectors in a 4-h [51Cr]-release assay we showed that the CD3-CD8lo subpopulation is the primary effector population for NK cell-mediated cytolysis. The majority of these cells co-express CD16, CD11b, NKG2D, and NKp46. To evaluate functional activity at the individual cell level, we employed intracellular cytokine staining and a flow cytometric assay for degranulation, based on cell surface CD107a expression. Flow cytometric analysis revealed that a greater proportion of NK cells degranulated than produced cytokines in response to B116Lo stimulation; the frequency of CD107a-expressing cells within the total NK cell population ranging from 5 to 39%. Somewhat surprisingly, we did not find a significant correlation between lysis, measured by [51Cr]-release assay, and the size of the degranulating NK cell population, implying that additional mechanisms may regulate lytic activity. Use of these approaches should facilitate an improved understanding of NK activity in the rhesus macaque.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.