Nucleobindin co-localizes and associates with cyclooxygenase (COX)-2 in human neutrophils

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca(2+)-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2) biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.     

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.     

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Rangeomorphs,Thectardis (Porifera?) and dissolved organic carbon in the Ediacaran oceans

The mid-Ediacaran Mistaken Point biota of Newfoundland represents the first morphologically complex organisms in the fossil record. At the classic Mistaken Point localities the biota is dominated by the enigmatic group of "fractally" branching organisms called rangeomorphs. One of the few exceptions to the rangeomorph body plan is the fossil Thectardis avalonensis, which has been reconstructed as an upright, open cone with its apex in the sediment. No biological affinity has been suggested for this fossil, but here we show that its body plan is consistent with the hydrodynamics of the sponge water-canal system. Further, given the habitat of Thectardis beneath the photic zone, and the apparent absence of an archenteron, movement, or a fractally designed body plan, we suggest that it is a sponge. The recognition of sponges in the Mistaken Point biota provides some of the earliest body fossil evidence for this group, which must have ranged through the Ediacaran based on biomarkers, molecular clocks, and their position on the metazoan tree of life, in spite of their sparse macroscopic fossil record. Should our interpretation be correct, it would imply that the paleoecology of the Mistaken Point biota was dominated by sponges and rangeomorphs, organisms that are either known or hypothesized to feed in large part on dissolved organic carbon (DOC). The biology of these two clades gives insight into the structure of the Ediacaran ocean, and indicates that a non-uniformitarian mechanism delivered labile DOC to the Mistaken Point seafloor.     

Germ-line DNA copy number variation frequencies in a large North American population

Genomic copy number variation (CNV) is a recently identified form of global genetic variation in the human genome. The Affymetrix GeneChip 100 and 500 K SNP genotyping platforms were used to perform a large-scale population-based study of CNV frequency. We constructed a genomic map of 578 CNV regions, covering approximately 220 Mb (7.3%) of the human genome, identifying 183 previously unknown intervals. Copy number changes were observed to occur infrequently (<1%) in the majority (>93%) of these genomic regions, but encompass hundreds of genes and disease loci. This North American population-based map will be a useful resource for future genetic studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.     

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

AIMS: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. METHODS AND RESULTS: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. CONCLUSIONS: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. SIGNIFICANCE AND IMPACT OF THE STUDY: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.