Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.     

Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission

The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.     

Carapichea guianensis: the correct name for Psychotria carapichea (Rubiaceae), with a correction on the authority of P. carapichea, and a new combination in Carapichea

The correct name forPsychotria carapichea isCarapichea guianensis. Also, the authority ofPsychotria carapichea was previously incorrectly cited, because of an oversight of the illegitimacy ofTapogomea carapichea Poir. The complex taxonomic history of this species is analyzed, and the authority of the binomial is correctly cited. In addition,P. ligularis, which was treated as closely related toC. guianensis in previous publications, is transferred toCarapichea and it is now known asCarapichea ligularis.     

A new synthetic approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one(J-113397), the first non-peptide ORL-1 receptor antagonist..

An efficient approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one (J-113397) 1, the first non-peptide ORL-1 receptor antagonist described in literature, is outlined. After construction of the piperidine framework through Dieckmann cyclization of the Michael adduct 8 of cyclooctylmethylamine to methyl acrylate, condensation with o-phenylendiamine produced the beta-enamino ester 2, which has been conveniently used to construct the benzimidazolone substituent at C-4. Catalytic hydrogenation of intermediate 11 followed by base-promoted cis--trans isomerization of the key compound 12 led to the formation of ester 13, which was converted to the racemic title compound by LiAlH(4) reduction. The pure enantiomers were obtained by chiral preparative HPLC separation using a derivatized cellulose-based stationary phase.     

The impact of demographic changes on the epidemiology of herpes zoster: Spain as a case study

Varicella zoster virus (VZV) causes varicella upon first exposure and may reactivate later in life into herpes zoster (HZ), with a risk that is thought to be reduced by re-exposures to VZV. Given the decades-long time scales of reactivation and its dependence on the accumulation of re-exposure episodes, adopting a long-term perspective may be useful to correctly interpret current epidemiological trends of VZV. In this study, we investigate the possible impact of demographic changes on varicella and HZ in Spain, using an age-structured mathematical model informed with historical demographic data and calibrated against age-specific profiles of varicella seroprevalence and HZ incidence data. The model qualitatively reproduces the remarkable growth of HZ incidence observed in Spain between 1997 and 2004, before the introduction of varicella vaccination programmes. We demonstrate that this growth may be partially ascribed to the reduction of varicella circulation that followed the overall decline of the birth rate in the twentieth century. Model predictions further suggest that, even under the most optimistic projections, HZ incidence will continue its rise until at least 2040. Considering the effect of demographic changes can help interpreting variations in epidemiological trends of HZ, contributing to a more accurate evaluation of vaccination programmes against VZV.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

A complex species complex: The controversial role of ecology and biogeography in the evolutionary history of Syllis gracilis Grube, 1840 (Annelida,Syllidae)

The cryptic diversity in the polychaete Syllis gracilis Grube, 1840, in the Mediterranean Sea was examined with an integrative morpho-molecular approach. Individuals of S. gracilis were collected at eleven Mediterranean localities to provide an insight into the role of brackish environments in inducing cryptic speciation. The examination of morphological features combined with a molecular genetic analysis based on a partial sequence of the 16S rRNA gene highlighted discrepancies between morphological and molecular diversity. Morphological data allowed to identify a morphotype with short appendages occurring in coralline algae communities and another one with long appendages observed in brackish-water environments and Sabellaria reefs. Multivariate analyses showed that sampling localities were the greatest source of morphological divergence, suggesting that phenotypic plasticity may play a role in local adaptations of S. gracilis populations. Molecular data showed the occurrence of four divergent lineages not corresponding to morphological clusters. Different species delimitation tests gave conflicting results, retrieving, however, at least four separated entities. Some lineages occurred in sympatry and were equally distributed in marine and brackish-water environments, excluding a biogeographic or ecological explanation of the observed pattern and suggesting instead ancient separation between lineages and secondary contact. The co-occurrence of different lineages hindered the identification of the lineage corresponding to S. gracilis sensu stricto. The discrepancy between morphological and molecular diversity suggests that different environmental and biogeographic features may interact in a complex and unpredictable way in shaping diversity patterns. An integrative approach is needed to provide a satisfactory insight on evolutionary processes in marine invertebrates.     

Neuropathological findings processed by artificial neural networks (ANNs) can perfectly distinguish Alzheimer's patients from controls in the Nun Study

Background  

Many reports have described that there are fewer differences in AD brain neuropathologic lesions between AD patients and control subjects aged 80 years and older, as compared with the considerable differences between younger persons with AD and controls. In fact some investigators have suggested that since neurofibrillary tangles (NFT) can be identified in the brains of non-demented elderly subjects they should be considered as a consequence of the aging process. At present, there are no universally accepted neuropathological criteria which can mathematically differentiate AD from healthy brain in the oldest old.     

PRMT5 silencing selectively affects MTAP-deleted mesothelioma: In vitro evidence of a novel promising approach

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)-deficient cancers, in which the accumulation of the substrate 5'-methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the MTAP gene is frequently codeleted with the adjacent cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock-down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP-deleted MM cells. We also observed that PRMT5 knock-down in MTAP-deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial-to-mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP-deleted MMs.