Carboxyl terminus structural requirements for glycosyl-phosphatidylinositol anchor addition to cell surface proteins.

Glycosyl phosphatidylinositol (GPI)-anchored proteins contain in their COOH-terminal region a peptide segment that is thought to direct glycolipid addition. This signal has been shown to require a pair of small amino acids positioned 10-12 residues upstream of an hydrophobic C-terminal domain. We analysed the contribution of the region separating the anchor acceptor site and the C-terminal hydrophobic segment by introducing amino acid deletions and substitutions in the spacer element of the GPI-anchored Thy-1 glycoprotein. Deletions of 7 amino acids in this region, as well as the introduction of 2 charged residues, prevented the glycolipid addition to Thy-1, suggesting that the length and the primary sequence of the spacer domain are important determinants in the signal directing GPI anchor transfer onto a newly synthesized polypeptide. Furthermore, we tested these rules by creating a truncated form of the normally transmembranous Herpes simplex virus I glycoprotein D (gDI) and demonstrating that when its C-terminal region displays all the features of a GPI-anchored protein, it is able to direct glycolipid addition onto another cell surface molecule.

Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

Development of a high throughput PCR to detect Coxiella burnetii and its application in a diagnostic laboratory over a 7-year period.

Q fever is a worldwide zoonotic infectious disease due to Coxiella burnetii. The clinical presentation may be acute (pneumonia and/or hepatitis) or chronic (most commonly endocarditis). Diagnosis mainly relies on serology and PCR. We therefore developed a quantitative real-time PCR. We first tested blindly its performance on various clinical samples and then, when thoroughly validated, we applied it during a 7-year period for the diagnosis of both acute and persistent C. burnetii infection. Analytical sensitivity (< 10 copies/PCR) was excellent. When tested blindly on 183 samples, the specificity of the PCR was 100% (142/142) and the sensitivity was 71% (29/41). The sensitivity was 88% (7/8) on valvular samples, 69% (20/29) on blood samples and 50% (2/4) on urine samples. This new quantitative PCR was then successfully applied for the diagnosis of acute Q fever and endovascular infection due to C. burnetii, allowing the diagnosis of Q fever in six patients over a 7-year period. During a local small cluster of cases, the PCR was also applied to blood from 1355 blood donors; all were negative confirming the high specificity of this test. In conclusion, we developed a highly specific method with excellent sensitivity, which may be used on sera for the diagnosis of acute Q fever and on various samples such as sera, valvular samples, aortic specimens, bone and liver, for the diagnosis of persistent C. burnetii infection.

Precise U-Pb age constraints for end-Triassic mass extinction, its correlation to volcanism and Hettangian post-extinction recovery

New precise zircon U-Pb ages are proposed for the Triassic-Jurassic (Rhetian-Hettangian) and the Hettangian-Sinemurian boundaries, The ages were obtained by ID-TIMS dating of single chemical-abraded zircons from volcanic ash layers within the Pucara Group, Aramachay Formation in the Utcubamba valley, northern Peru. Ash layers situated between last and first occurrences of boundary-defining ammonites yielded Pb-206/U-238 ages of 201.58 +/- 0.17/0.28 Ma (95% c.l., uncertainties without/with decay constant errors, respectively) for the Triassic-Jurassic and of 199.53 +/- 0.19/0.29 Ma for the Hettangian-Sinemurian boundaries. The former is established on a tuff located 1 m above the last local occurrence of the topmost Triassic genus Choristoceras, and 5 m below the Hettangian genus Psiloceras. The latter sample was obtained from a tuff collected within the Badouxia canadensis beds. Our new ages document total duration of the Hettagian of no more than c. 2 m.y., which has fundamental implications for the interpretation and significance of the ammonite recovery after the topmost Triassic extinction. The U-Pb age is about 0.8 +/- 0.5% older than Ar-40-Ar-39 dates determined on flood basalts of the Central Atlantic Magmatic Province (CAMP). Given the widely accepted hypothesis that inaccuracies in the K-40 decay constants or physical constants create a similar bias between the two dating methods, our new U-Pb zircon age determination for the T/J boundary corroborates the hypothesis that the CAMP was emplaced at the same time and may be responsible for a major climatic turnover and mass extinction. The zircon Pb-206/U-238 age for the T/J boundary is marginally older than the North Mountain Basalt (Newark Supergroup, Nova Scotia, Canada), which has been dated at 201.27 +/- 0.06 Ma [Schoene et al., 2006. Geochim. Cosmochim. Acta 70, 426-445]. It will be important to look for older eruptions of the CAMP and date them precisely by U-Pb techniques while addressing all sources of systematic uncertainty to further test the hypothesis of volcanic induced climate change leading to extinction. Such high-precision, high-accuracy data will be instrumental for constraining the contemporaneity of geological events at a 100 kyr level. (C) 2007 Elsevier B.V. All rights reserved.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

Soil spectral library and its use in soil classification

Soil science has sought to develop better techniques for the classification of soils, one of which is the use of remote sensing applications. The use of ground sensors to obtain soil spectral data has enabled the characterization of these data and the advancement of techniques for the quantification of soil attributes. In order to do this, the creation of a soil spectral library is necessary. A spectral library should be representative of the variability of the soils in a region. The objective of this study was to create a spectral library of distinct soils from several agricultural regions of Brazil. Spectral data were collected (using a Fieldspec sensor, 350-2,500 nm) for the horizons of 223 soil profiles from the regions of Matão, Paraguaçu Paulista, Andradina, Ipaussu, Mirandópolis, Piracicaba, São Carlos, Araraquara, Guararapes, Valparaíso (SP); Naviraí, Maracajú, Rio Brilhante, Três Lagoas (MS); Goianésia (GO); and Uberaba and Lagoa da Prata (MG). A Principal Component Analysis (PCA) of the data was then performed and a graphic representation of the spectral curve was created for each profile. The reflectance intensity of the curves was principally influenced by the levels of Fe2O3, clay, organic matter and the presence of opaque minerals. There was no change in the spectral curves in the horizons of the Latossolos, Nitossolos, and Neossolos Quartzarênicos. Argissolos had superficial horizon curves with the greatest intensity of reflection above 2,200 nm. Cambissolos and Neossolos Litólicos had curves with greater reflectance intensity in poorly developed horizons. Gleisols showed a convex curve in the region of 350-400 nm. The PCA was able to separate different data collection areas according to the region of source material. Principal component one (PC1) was correlated with the intensity of reflectance samples and PC2 with the slope between the visible and infrared samples. The use of the Spectral Library as an indicator of possible soil classes proved to be an important tool in profile classification.

Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain--an immunohistochemical study.

The aim of this work was to study the distribution and cellular localization of GLUT2 in the rat brain by light and electron microscopic immunohistochemistry, whereas our ultrastructural observations will be reported in a second paper. Confirming previous results, we show that GLUT2-immunoreactive profiles are present throughout the brain, especially in the limbic areas and related nuclei, whereas they appear most concentrated in the ventral and medial regions close to the midline. Using cresyl violet counterstaining and double immunohistochemical staining for glial or neuronal markers (GFAp, CAII and NeuN), we show that two limited populations of oligodendrocytes and astrocytes cell bodies and processes are immunoreactive for GLUT2, whereas a cross-reaction with GLUT1 cannot be ruled out. In addition, we report that the nerve cell bodies clearly immunostained for GLUT2 were scarce (although numerous in the dentate gyrus granular layer in particular), whereas the periphery of numerous nerve cells appeared labeled for this transporter. The latter were clustered in the dorsal endopiriform nucleus and neighboring temporal and perirhinal cortex, in the dorsal amygdaloid region, and in the paraventricular and reuniens thalamic nuclei, whereas they were only a few in the hypothalamus. Moreover, a group of GLUT2-immunoreactive nerve cell bodies was localized in the dorsal medulla oblongata while some large multipolar nerve cell bodies peripherally labeled for GLUT2 were scattered in the caudal ventral reticular formation. This anatomical localization of GLUT2 appears characteristic and different from that reported for the neuronal transporter GLUT3 and GLUT4. Indeed, the possibility that GLUT2 may be localized in the sub-plasmalemnal region of neurones and/or in afferent nerve fibres remains to be confirmed by ultrastructural observations. Because of the neuronal localization of GLUT2, and of its distribution relatively similar to glucokinase, it may be hypothesized that this transporter is, at least partially, involved in cerebral glucose sensing.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

Nedd4-2 ubiquitin ligase: a contributor to experimental neuropathic pain?

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs) leading to peripheral nerve hyperexcitability. Interestingly, in cell expression systems, the ubiquitin ligase Nedd4-2 regulates the cell membrane density of the most abundant peripheral and pain-related VGSC, namely Nav1.7, and decreases its sodium current. Yet nothing is known about the involvement of Nedd4-2 in nociception and chronic pain. Therefore, the goal of this study is (i) to characterize Nedd4-2 and Nav1.7 expression in an experimental model of neuropathic pain (ii) to design by viral vector-mediated gene therapy an approach to depict the implication of Nedd4-2 in chronic pain. Methods: Western Blot and immunohistochemistry experiments detecting Nav1.7 and Nedd4-2 were performed in rodent DRGs 7 days after spared nerve injury (SNI). For the viral vector-mediated gene therapy, a recombinant Adeno-Associated Virus (rAAV2/6) was generated expressing the Nedd4-2 gene. Intrathecal injection of rAAV2/6 was followed 2 weeks after by the SNI surgery. Data are expressed in mean ± SEM, n = 4 in each condition. Results: Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 ± 1.2%) versus sham group (43.4 ± 3.5%; p <0.005), as well as an increase in positive Nav1.7 cells in SNI (50.1 ± 2.9%) versus Sham (41.6 ± 1.8%; p <0.05). The change of Nedd4-2 expression was confirmed by western-blot analysis. In addition, we show that Nedd4-2 and Nav1.7 are largely expressed in overlapping cell populations, chiefly colocalizing with markers of small nociceptive neurons. Furthermore, we report that intrathecal injection of rAAV is able to counteract the reduction of Nedd4-2 expression in SNI animals. Conclusion: Our results indicate that Nedd4-2 is mainly expressed in nociceptors and downregulated after nerve injury. Moreover, our data suggest that the reduction of Nedd4-2, after nerve injury, may modulate Nav1.7 activity and contribute to hyperexcitability in neuropathic pain. A normal level of Nedd4-2 can be restored using a viral vector and we will further assess its functional effect on pain sensitivity.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Carbon stock and its compartments in a subtropical oxisol under long-term tillage and crop rotation systems

Soil organic matter (SOM) plays a crucial role in soil quality and can act as an atmospheric C-CO2 sink under conservationist management systems. This study aimed to evaluate the long-term effects (19 years) of tillage (CT-conventional tillage and NT-no tillage) and crop rotations (R0-monoculture system, R1-winter crop rotation, and R2- intensive crop rotation) on total, particulate and mineral-associated organic carbon (C) stocks of an originally degraded Red Oxisol in Cruz Alta, RS, Southern Brazil. The climate is humid subtropical Cfa 2a (Köppen classification), the mean annual precipitation 1,774 mm and mean annual temperature 19.2 ºC. The plots were divided into four segments, of which each was sampled in the layers 0-0.05, 0.05-0.10, 0.10-0.20, and 0.20-0.30 m. Sampling was performed manually by opening small trenches. The SOM pools were determined by physical fractionation. Soil C stocks had a linear relationship with annual crop C inputs, regardless of the tillage systems. Thus, soil disturbance had a minor effect on SOM turnover. In the 0-0.30 m layer, soil C sequestration ranged from 0 to 0.51 Mg ha-1 yr-1, using the CT R0 treatment as base-line; crop rotation systems had more influence on soil stock C than tillage systems. The mean C sequestration rate of the cropping systems was 0.13 Mg ha-1 yr-1 higher in NT than CT. This result was associated to the higher C input by crops due to the improvement in soil quality under long-term no-tillage. The particulate C fraction was a sensitive indicator of soil management quality, while mineral-associated organic C was the main pool of atmospheric C fixed in this clayey Oxisol. The C retention in this stable SOM fraction accounts for 81 and 89 % of total C sequestration in the treatments NT R1 and NT R2, respectively, in relation to the same cropping systems under CT. The highest C management index was observed in NT R2, confirming the capacity of this soil management practice to improve the soil C stock qualitatively in relation to CT R0. The results highlighted the diversification of crop rotation with cover crops as a crucial strategy for atmospheric C-CO2 sequestration and SOM quality improvement in highly weathered subtropical Oxisols.

The sandstone-hosted Beverley uranium Deposit, Lake Frome Basin, South Australia: Mineralogy, geochemistry, and a time-constrained model for its genesis

The sandstone-hosted Beverley uranium deposit is located in terrestrial sediments in the Lake Frome basin in the North Flinders Ranges, South Australia. The deposit is 13 km from the U-rich Mesoproterozoic basement of the Mount Painter inlier, which is being uplifted 100 to 200 m above the basin by neotectonic activity that probably initiated in the early Pliocene. The mineralization was deposited mainly in organic matter-poor Miocene lacustrine sands and partly in the underlying reductive strata comprising organic matter-rich clays and silts. The bulk of the mineralization consists of coffinite and/or uraninite nodules, growing around Co-rich pyrite with an S isotope composition (delta S-34 = 1.0 +/- 0.3 parts per thousand), suggestive of an early diagenetic lacustrine origin. In contrast, authigenic sulfides in the bulk of the sediments have a negative S isotope signature (delta S-34 ranges from -26.2 to -35.5 parts per thousand), indicative of an origin via bacterially mediated sulfate reduction. Minor amounts of Zn-bearing native copper and native lead also support the presence of specific, reducing microenvironments in the ore zone. Small amounts of carnotite are associated with the coffinite ore and also occur beneath a paleosoil horizon overlying the uranium deposit. Provenance studies suggest that the host Miocene sediments were derived from the reworking of Early Cretaceous glacial or glaciolacustrine sediments ultimately derived from Paleozoic terranes in eastern Australia. In contrast, the overlying Pliocene strata were in part derived from the Mesoproterozoic basement inlier. Mass-balance and geochemical data confirm that granites of the Mount Painter domain were the ultimate source of U and BEE at Beverley. U-Pb dating of coffinite and carnotite suggest that the U mineralization is Pliocene (6.7-3.4 Ma). The suitability of the Beverley deposit for efficient mining via in situ leaching, and hence its economic value, are determined by the nature of the hosting sand unit, which provides the permeability and low reactivity required for high fluid flow and low chemical consumption. These favorable sedimentologic and geometrical features result from a complex conjunction of factors, including deposition in lacustrine shore environment, reworking of angular sands of glacial origin, deep Pliocene weathering, and proximity to an active fault exposing extremely U rich rocks.

Incidence of bullous pemphigoid and pemphigus in Switzerland: a 2-year prospective study.

BACKGROUND: Bullous pemphigoid (BP), pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune bullous diseases characterized by the presence of tissue-bound and circulating autoantibodies directed against disease-specific target antigens of the skin. Although rare, these diseases run a chronic course and are associated with significant morbidity and mortality. There are few prospective data on gender- and age-specific incidence of these disorders. OBJECTIVES: Our aims were: (i) to evaluate the incidence of BP and PV/PF in Swiss patients, as the primary endpoint; and (ii) to assess the profile of the patients, particularly for comorbidities and medications, as the secondary endpoint. METHODS: The protocol of the study was distributed to all dermatology clinics, immunopathology laboratories and practising dermatologists in Switzerland. All newly diagnosed cases of BP and pemphigus occurring between 1 January 2001 and 31 December 2002 were collected. In total, 168 patients (73 men and 95 women) with these autoimmune bullous diseases, with a diagnosis based on clinical, histological and immunopathological criteria, were finally included. RESULTS: BP showed a mean incidence of 12.1 new cases per million people per year. Its incidence increased significantly after the age of 70 years, with a maximal value after the age of 90 years. The female/male ratio was 1.3. The age-standardized incidence of BP using the European population as reference was, however, lower, with 6.8 new cases per million people per year, reflecting the ageing of the Swiss population. In contrast, both PV and PF were less frequent. Their combined mean incidence was 0.6 new cases per million people per year. CONCLUSIONS; This is the first comprehensive prospective study analysing the incidence of autoimmune bullous diseases in an entire country. Our patient cohort is large enough to establish BP as the most frequent autoimmune bullous disease. Its incidence rate appears higher compared with other previous studies, most likely because of the demographic characteristics of the Swiss population. Nevertheless, based on its potentially misleading presentations, it is possible that the real incidence rate of BP is still underestimated. Based on its significant incidence in the elderly population, BP should deserve more public health concern.

Manganese accumulation and its relation to "eucalyptus shoot blight in the Vale do Rio Doce"

Eucalyptus Shoot Blight in the Vale do Rio Doce (ESBVRD) is an anomaly that leads to reduced growth and, in more extreme cases, to death of eucalyptus plants. Initially diagnosed in plantations in the region of the Vale do Rio Doce, in the State of Minas Gerais, Brazil, this problem has also been found in plantations in other regions of the country and even in other countries. Although the symptoms of this anomaly are well-known, its causes are not yet understood. The aim of this study was to evaluate the cause-effect relationship between accumulation of manganese (Mn) in eucalyptus clones and ESBVRD. Characterization of the environment in areas of greater occurrence of this problem in regard to soil, climate and fluctuation of the water table was undertaken in eucalyptus plantations of the Celulose Nipo-brasileira S.A. (Cenibra) company in the region of the Vale do Rio Doce. Plant tissues were sampled in two situations. In the first situation, diagnosis occurred in the initial phase of the anomaly in clones with differentiated tolerance to the problem; in the second situation, diagnosis was made in a single clone, considered to be sensitive, in two time periods - in the phase with the strong presence of symptoms and in the recovery phase, in areas of occurrence and in areas of escape from the problem. The most ESBVRD-sensitive clone showed much higher (4.8 times higher) leaf Mn contents than more tolerant clones. In plants with the anomaly, Mn leaf contents were greater than 3,070 mg kg-1, much greater than the quantity found in those without the anomaly (734 mg kg-1). In the period in which the symptoms began to wane, there was a sharp decline in leaf Mn contents, from 2,194 to 847 mg kg-1. Manganese content in the above ground part and plant litter (44.4 g ha-1) in the area of occurrence of the anomaly was three times greater than that found in these same components (14.1 g ha-1) in the area of absence of the symptom. Based on the evidence found, such as the existence of environmental conditions favorable to high Mn availability to the plants in the areas of greatest incidence of ESBVRD, greater uptake of Mn in sensitive clones and in plants with symptoms, and a synchronism between the intensity of symptoms of ESBVRD and leaf Mn contents, it may be inferred that temporary excess of Mn in eucalyptus plants is closely related to ESBVRD.