Urease-Encoding Genes in Ammonia-Oxidizing Bacteria

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the β-proteobacterium Nitrosospira sp. strain NpAV and the γ-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several β-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from β-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other β-proteobacteria. Instead, it appears that urease in β-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of γ- and β-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from β-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.     

Patterns of fish and sea urchin grazing on tropical Indo-Pacific seagrass beds

Thalassodendron ciliatum shoots were collected from natural populations of fished and unfished protected seagrass meadows to assess the herbivory of fish and sea urchins. Fish herbivory was restricted to unfished seagrass meadows, while sea urchin herbivory took place in fished as well as unfished areas where fish appeared not to be effective urchin predators. The results of this study confirm that grazing by sea urchins is important in tropical seagrass ecosystems and indicate that herbivorous fish graze, and probably consume, substantial amounts of seagrass production in fishing-protected habitats. The fact that much of the information on seagrass herbivory comes from heavily-fished meadows indicates that large-scale studies, which include unfished areas, are necessary to provide reliable spatial patterns of seagrass grazing distribution.     

Climatic and hydrological controls over the zoobenthos in a southern Baltic coastal lagoon

In a four-year-long (1985–1988) study of macro- and meiobenthos of the Szczecin Lagoon, a eutrophic and polluted coastal water body directly connected with the southern Baltic Sea, peculiar interannual dynamics of variables related to abundance was observed. The interannual component of variability was stronger than fine-scale seasonal dynamics. The interannual variations followed a trend whereby abundance of meio- and macrobenthos as well as biomass of the latter was high in 1985, dropped in 1986 and 1987, and rose again in 1988. As the trend could not be explained by local environmental data, explanation was sought by invoking wider-scale climatic and hydrological processes. Data for severity of winters preceding each year of study as well as trends in atmospheric circulation patterns, wind regimes, river runoff characteristics, and near-bottom conductance (used as a proxy for seawater intrusions into the lagoon) were compared to seasonal anomalies in meiobenthic abundance as well as in macrobenthic abundance and biomass. The seasonal mean meiobenthic log abundance anomalies produced no significant correlation with any of the variables tested. In contrast, the seasonal mean log macrobenthos abundance and biomass anomalies were significantly negatively correlated with winter severity proxies (mean winter temperature and cold sum) and flow rate determined with time lags generally longer than one month. The results provide indication of differences in responses of meio- and macrobenthic communities to climatic (winter severity) and hydrological changes, the long-term effects being visible primarily within the macrobenthos. However, establishing sound causal relationships requires longer time-series of data.     

Improvement of Fermentation Conditions for the Production of X-Prolyl-Dipeptidyl Aminopeptidase from Lactococcus Lactis

The quantitative effects of some fermentation conditions on the production of the enzyme X-prolyl-dipeptidyl aminopeptidase (PepXP)(EC 3.4.14.5) of Lactococcus lactis subsp. lactis and cremoris were studied. The PepXP activity was found both in the membrane and in the cytoplasm, suggesting the presence of multiple molecular forms. Both microorganisms showed higher PepXP activities when glucose (5 g/l) was used as the carbon source and the yeast extract in the culture medium was increased to 3.5 g/l. In these conditions, 226 mU/ml of PepXP activity were obtained with L. lactis subsp. lactis and 235 mU/ml with the subsp. cremoris after 6 h. The best fermentation temperature was in the 30–32 °C range. The enzyme activity remained stable even during the stationary phase.     

A screening system for the identification of refolding conditions for a model protein kinase, p38alpha

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38α, and applied this system to denatured p38α derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38α can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38α protein kinase.     

Direct analysis of urinary trans,trans-muconic acid by coupled column liquid chromatography and spectrophotometric ultraviolet detection: method applicability to human urine

A coupled column liquid chromatographic (LC–LC) method for the direct analysis in human urine of the ring opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. The method was tested using urine samples collected from five refinery workers exposed to low concentrations of airborne benzene (0.2–0.5 ppm), and from non-exposed volunteers. The analytical columns used were of 50×4.6 mm I.D. packed with 3 μm p.s. Microspher C₁₈ material as the first column (C-1), and a 100×4.6 mm I.D. column packed with 3 μm p.s. Hypersil ODS material as the second one (C-2). The mobile phases applied consisted, respectively, of methanol–0.074% trifluoroacetic acid (TFA) in water (4:96, v/v) on C-1, and of methanol–0.074% TFA in water (10:90, v/v) on C-2. Under these conditions t,t-MA eluted 15 min after injection. The present method, coupling the LC–LC technique with UV detection at 264 nm, permits the quantitation of t,t-MA directly in urine at levels as low as 0.05 mg/l. The determination is performed with a sample throughput of 2 h⁻¹ requiring only pH adjustment and centrifugation of the sample. Calibration plots of standard additions of t,t-MA to pooled urine taken from five non-exposed subjects were linear (r>0.999) over a wide concentration range (0.05, 0.1, 0.5, 1.0, 2.0 mg/l). The precision of the method (RSD) was in the range of 0.5 to 3.8%, and the within-session repeatability on workers urine samples (levels 0.06, 0.1, 0.2, 1.0 mg/l) was in the range of 3 to 8%. The present method improves the applicability of routine t,t-MA analysis, where it is most desirable that a large number of biological samples can be processed automatically or with minimal human labour, at low cost, and with a convenient turn-around time.     

Bone marrow microenvironment: The guardian of leukemia stem cells

Bone marrow microenvironment(BMM) is the main sanctuary of leukemic stem cells(LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease.Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.     

Regulation of RAS in human platelets. Evidence that activation of RAS is not sufficient to lead to ERK1-2 phosphorylation.

In this study, we show that the G protein-coupled receptor agonist thrombin, the glycoprotein VI agonist convulxin, and the cytokine receptor Mpl agonist thrombopoietin (TPO) are able to induce activation of RAS in human platelets. Recruitment of GRB2 by tyrosine-phosphorylated proteins in response to TPO and convulxin but not by thrombin occurred with a similar time-course to RAS activation, consistent with a causal relationship. On the other hand, activation of ERK2 by thrombin and convulxin is delayed and also inhibited by the protein kinase C inhibitor Ro-31 8220, whereas RAS activation is unaffected. Further evidence for differential regulation of RAS and ERK is provided by the observations that TPO, which activates RAS but not protein kinase C, does not activate ERK, and that the inhibitor of SRC kinases PP1 inhibits activation of RAS but not ERK2 in response to thrombin. Our results demonstrate that activation of RAS is not necessarily coupled to ERK in human platelets.     

Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6.

Adenines mismatched with guanines or 7,8-dihydro-8-oxo-deoxyguanines that arise through DNA replication errors can be repaired by either base excision repair or mismatch repair. The human MutY homolog (hMYH), a DNA glycosylase, removes adenines from these mismatches. Human MutS homologs, hMSH2/hMSH6 (hMutSalpha), bind to the mismatches and initiate the repair on the daughter DNA strands. Human MYH is physically associated with hMSH2/hMSH6 via the hMSH6 subunit. The interaction of hMutSalpha and hMYH is not observed in several mismatch repair-defective cell lines. The hMutSalpha binding site is mapped to amino acid residues 232-254 of hMYH, a region conserved in the MutY family. Moreover, the binding and glycosylase activities of hMYH with an A/7,8-dihydro-8-oxo-deoxyguanine mismatch are enhanced by hMutSalpha. These results suggest that protein-protein interactions may be a means by which hMYH repair and mismatch repair cooperate in reducing replicative errors caused by oxidized bases.