Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR

The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply the magic-angle spinning (MAS) solid-state NMR (SSNMR) technique to study a uniformly ¹³C and ¹⁵N labeled wild-type T7 phage. We describe the details of the large-scale preparation and purification of an isotopically enriched phage sample under fully hydrated conditions, and show a complete ¹³C and a near-complete ¹⁵N nucleotide-type specific assignment of the sugar and base moieties in the 40 kbp dsDNA of T7 using two-dimensional ¹³C–¹³C and ¹⁵N–¹³C correlation experiments. The chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS SSNMR was found to be extremely useful in determining the structures of proteins in native-like environments, its application to nucleic acids has lagged behind, leaving a missing ¹³C and ¹⁵N chemical shift database. This work therefore expands the ¹³C and ¹⁵N database of real B-form DNA systems, and opens routes to characterize more complex nucleic acid systems by SSNMR.     

Settlement and early survival of red coral on artificial substrates in different geographic areas: some clues for demography and restoration

The red coral Corallium rubrum (L 1758) is a long-lived, slow-growing gorgonian, endemic to Mediterranean rocky bottoms. Because of its high economic value, red coral has long been harvested, and most populations have been depleted. In the present study, 54 marble tiles were placed in June 2003 within red coral populations over 3 different geographic areas (Calafuria–Livorno and Elba MPA in Italy and Medes Islets MPA, in Spain), on vertical cliffs between 25 and 35 m. In each area 2 different sites were randomly selected. Tiles were subsequently sampled photographically. Between July and August 2003 red coral recruits settled on tiles in all the geographic areas and sites, exhibiting wide variability in their density. On the basis of a 2-factors nested ANOVA a significant variability between different sites at a few hundred metres distance occurred, indicating high variations in the recruitment process within the same red coral population. Mortality, measured in June 2004, widely varied between different geographic areas.     

Clinical Patterns and Treatment Outcome in Patients with Melancholic,Atypical and Non-Melancholic Depressions

Objective

To assess sociodemographic, clinical and treatment factors as well as depression outcome in a large representative clinical sample of psychiatric depressive outpatients and to determine if melancholic and atypical depression can be differentiated from residual non-melancholic depressive conditions.

Subjects/Materials and Method

A prospective, naturalistic, multicentre, nationwide epidemiological study of 1455 depressive outpatients was undertaken. Severity of depressive symptoms was assessed by the Hamilton Depression Rating Scale (HDRS) and the Self Rated Inventory of Depressive Symptomatology (IDS-SR₃₀). IDS-SR₃₀ defines melancholic and atypical depression according to DSM-IV criteria. Assessments were carried out after 6–8 weeks of antidepressant treatment and after 14–20 weeks of continuation treatment.

Results

Melancholic patients (16.2%) were more severely depressed, had more depressive episodes and shorter episode duration than atypical (24.7%) and non-melancholic patients. Atypical depressive patients showed higher rates of co-morbid anxiety disorders and substance abuse. Melancholic patients showed lower rates of remission.

Conclusion

Our study supports a different clinical pattern and treatment outcome for melancholic and atypical depression subtypes.     

USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma

In advanced cancer, including glioblastoma, the transforming growth factor β (TGF-β) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-β signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-β receptor (TβR-I), leading to an enhanced TGF-β signal. High expression of USP15 correlates with high TGF-β activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-β activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-β signaling. Our results show that USP15 regulates the TGF-β pathway and is a key factor in glioblastoma pathogenesis.     

Amplified Fragment Length Polymorphism (AFLP) Reveals Species-Specific Markers in the Daphnia Galeata–HyalinaSpecies Complex

Daphnia often occur in species complexes that consist of two or more co-occurring species and their hybrids. Hybrid individuals are often capable of sexual reproduction and so backcrossing with introgression occurs. To better understand hybridization and backcrossing frequency, we sought to develop PCR-based, species-specific markers in the Daphnia galeata–hyalina species complex using amplified fragment length polymorphism (AFLP). This technique produces large numbers of reproducible markers for assessing diversity across the nuclear genome and provides several advantages over mtDNA and microsatellite approaches. We examined 28 clones of D. galeata, D. hyalina, and their hybrids isolated from Lake Constance on the Swiss-German border. Using a single AFLP primer combination we found five potential species-specific markers, defined as bands that occurred in >80% of one parental species and <20% of the other. Two bands appeared to be co-dominant and were present (homozygous) in D. galeata, absent in D. hyalina, and heterozygous in the hybrid. We conclude AFLP could provide enough PCR-based, species-specific markers to identify species, hybrids, and backcrosses from even small amounts of tissue (i.e. resting eggs).     

Quantitative study of a hippuritid rudist lithosome in a Santonian carbonate platform in the southern Central Pyrenees

The Santonian carbonate platform deposits of the Sant Corneli anticline in the southern Central Pyrenees contain distinctive lenticular to tabular lithosomes formed by congregations of slender hippuritid rudists. Here we make a quantitative study of one of the lithosomes in the Sant Pere de Vilanoveta Member, in the northern side of Sant Corneli. It is exposed over some 4 km, along a WNW–ESE section, and shows a remarkably persistent thickness up to a maximum of 5.15 m, though for the most part it is less than 3 m thick. In the lithosome, hippuritid shells are mostly preserved inclined about 30°–60° from the perpendicular with respect to bedding. The majority of inclined shells lean towards the southwestern to southeastern quadrants, the inferred principal downstream direction. Hippuritids are loosely clustered and supported in a fine bioclastic matrix. Percentage cover of hippuritids ranges from 19.8% at the base to 31.9% in the middle and top of the lithosome. The preliminary data on numerical densities (number of individuals per unit area) of hippuritids show some clustering around values of about 290–750 individuals per m². The inclined orientation of hippuritid shells seems original and the result of active growth by the animals. The high numerical density of hippuritids at the base of the lithosome may indicate that the hippuritid congregation grew rapidly. The congregation appears to have reached an optimal level of density by the middle of the lithosome and to have maintained it through time during the development of the rudist congregation. The lithosome lacks relief and has no evidence of any rigid framework.     

Rosgorgia inexspectata, new genus and species of Subergorgiidae (Cnidaria, Octocorallia) from off the Antarctic Peninsula

The family Subergorgiidae comprises two genera, Subergorgia with three species and Annella with two species. All the previously known species are distributed in tropical and subtropical waters from the northern Red Sea to the central Pacific. The presence of a subergorgiid in Antarctic waters could support the hypothesis that at least part of the Antarctic fauna has its origin in the Cretaceous period, when Antarctica was part of the Gondwana continent. The new genus Rosgorgia with the new species inexspectata is placed in the family Subergorgiidae by the presence of smooth, fusiform and often anastomosing needles in the axis, wart spindles in the coenenchyme, and coelenteric cavities of polyps only present in the coenenchyme. The genus Rosgorgia differs from Subergorgia and Annella by the presence of tuberculate rods in the axis. It is further distinguished from Annella by the absence of double-disk sclerites in the coenenchyme, and not anastomosing branches. Accepted: 25 June 2000     

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.     

Inhibition or knock out of Inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury

Background

In the present study, by comparing the responses in wild-type mice (WT) and mice lacking (KO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the development of on the lung injury caused by bleomycin administration. When compared to bleomycin-treated iNOSWT mice, iNOSKO mice, which had received bleomycin, exhibited a reduced degree of the (i) lost of body weight, (ii) mortality rate, (iii) infiltration of the lung with polymorphonuclear neutrophils (MPO activity), (iv) edema formation, (v) histological evidence of lung injury, (vi) lung collagen deposition and (vii) lung Transforming Growth Factor beta1 (TGF-β1) expression.

Methods

Mice subjected to intratracheal administration of bleomycin developed a significant lung injury. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from bleomycin-treated iNOSWT mice.

Results

The intensity and degree of nitrotyrosine staining was markedly reduced in tissue section from bleomycin-iNOSKO mice. Treatment of iNOSWT mice with of {"type":"entrez-nucleotide","attrs":{"text":"GW274150","term_id":"282552565","term_text":"GW274150"}}GW274150, a novel, potent and selective inhibitor of iNOS activity (5 mg/kg i.p.) also significantly attenuated all of the above indicators of lung damage and inflammation.

Conclusion

Taken together, our results clearly demonstrate that iNOS plays an important role in the lung injury induced by bleomycin in the mice.