Calcium-sensing receptor and aquaporin 2 interplay in hypercalciuria-associated renal concentrating defect in humans. An in vivo and in vitro study

One mechanism proposed for reducing the risk of calcium renal stones is activation of the calcium-sensing receptor (CaR) on the apical membranes of collecting duct principal cells by high luminal calcium. This would reduce the abundance of aquaporin-2 (AQP2) and in turn the rate of water reabsorption. While evidence in cells and in hypercalciuric animal models supports this hypothesis, the relevance of the interplay between the CaR and AQP2 in humans is not clear. This paper reports for the first time a detailed correlation between urinary AQP2 excretion under acute vasopressin action (DDAVP treatment) in hypercalciuric subjects and in parallel analyzes AQP2-CaR crosstalk in a mouse collecting duct cell line (MCD4) expressing endogenous and functional CaR. In normocalciurics, DDAVP administration resulted in a significant increase in AQP2 excretion paralleled by an increase in urinary osmolality indicating a physiological response to DDAVP. In contrast, in hypercalciurics, baseline AQP2 excretion was high and did not significantly increase after DDAVP. Moreover DDAVP treatment was accompanied by a less pronounced increase in urinary osmolality. These data indicate reduced urinary concentrating ability in response to vasopressin in hypercalciurics. Consistent with these results, biotinylation experiments in MCD4 cells revealed that membrane AQP2 expression in unstimulated cells exposed to CaR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin (FK). Interestingly, we found that CaR activation by specific agonists reduced the increase in cAMP and prevented any reduction in Rho activity in response to FK, two crucial pathways for AQP2 translocation. These data support the hypothesis that CaR-AQP2 interplay represents an internal renal defense to mitigate the effects of hypercalciuria on the risk of calcium precipitation during antidiuresis. This mechanism and possibly reduced medulla tonicity may explain the lower concentrating ability observed in hypercalciuric patients.     

The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.     

Modelling the effect of environmental variables on the reproductive success of Griffon Vulture (Gyps fulvus) in Sardinia,Italy

Old World vultures are experiencing dramatic population declines and now are among the species most threatened with extinction. Understanding the environmental variables that can influence the reproductive indexes of vulture populations can facilitate both habitat and species management. The aim of this study was to identify which environmental variables primarily affect the breeding successes of the Griffon Vulture Gyps fulvus in northern Sardinia by applying a Bayesian hierarchical model. A unique dataset of reproductive records (197 nests monitored over 39 years for a total of 992 breeding records) was used. Eight environmental and topographical variables describing the habitat at the nesting sites were considered as potential predictors of breeding success. These included mean annual temperature, mean annual precipitation, isothermality, elevation, the normalized difference vegetation index, wind speed, and the aspect and slope of the land surface. In addition, we also considered the effect of human disturbance and the type of nest. According to our best model, the probability of successfully raising a chick in Griffon Vultures was higher in nests exposed to a high wind speed, not covered by natural shelters, where the vegetation was mostly represented by shrub and pastures, with low human disturbance and in years with low rainfall. This model will be useful for management of the breeding habitat and to identify the area most suitable for Griffon Vulture reproduction. This information is crucial for programming conservation measures aimed at enlarging the area of occupancy of the species.     

Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes – the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue , is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Δ mdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.     

Different sealing materials for petri dishes strongly affect shoot regeneration and development from leaf explants of quince ‘BA 29’

Summary The effect of different sealing materials [i.e., polyvinyl chloride (PVC) transparent film, and Parafilm (PARA) for Petri dishes was investigated on shoot regeneration from quince (Cydonia oblonga L.) ‘BA 29’ leaf explants. Leaves were excised from proliferating shoot cultures, transversally scored, and placed with the abaxial side down in 60-mm Petri dishes containing 10 ml of Murashige and Skoog modified medium, with 5.4 μM α-naphthaleneacetic acid, 4.5 μM thidiazuron, 200 mg l⁻¹ cefotaxime, and 0.25% (w/v) Phytagel (IM medium) for shoot bud induction, and cultured in darkness at 22±2°C for 28 d. Then the explants were transferred to standard conditions (16-h photoperiod at 30 μmol m⁻² s⁻¹ photosynthetically active radiation) on a medium similar to IM, except for lack of NAA, and with 0.65% (w/v) agar instead of Phytagel, for an additional 15–28 d. The sealing combinations PARA-PARA, PARA-PVC, PVC-PARA, and PVC-PVC (in the induction-expression phases) were compared during regeneration and for their carry-over effect on shoot development after transfer of explants to an elongation medium (0.9 μM 6-benzyladenine). Carbon dioxide accumulated at 27.2 mmol mol⁻¹ at the end of induction, and gradually decreased from 35.4 mmol mol⁻¹ on day 9 to 22.5 mmol mol⁻¹ on day 28 of the expression phase in PARA-sealed Petri dishes, being always much higher than after sealing with PVC (1–2 mmol mol⁻¹). Ethylene concentration was 0.1 and 0.04 μmol mol⁻¹ in the first part of the induction and expression phase, respectively, in PARA-sealed Petri dishes, and slightly decreased with duration of exposure to light during expression; while it was absent in most PVC-sealed dishes. The PARA-PARA and PVC-PVC (induction-expression) combinations gave, respectively, the worst and best results of regeneration and successive shoot development.     

Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.     

An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background

Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis.

Methodology/Principal Findings

Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results.

Conclusions/Significance

The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.     

Pneumococcal Carriage in Young Children One Year after Introduction of the 13-Valent Conjugate Vaccine in Italy

Background

In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced.

Methods

Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST).

Results

Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively.

Conclusions

Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.     

Diet Induced Mild Hypercholesterolemia in Pigs: Local and Systemic Inflammation,Effects on Vascular Injury – Rescue by High-Dose Statin Treatment

Objective

The aim of the present study was to comprehensively evaluate systemic and local inflammation as well as progression of vascular inflammation in normal and mechanically injured vessels in a large animal model of mild hypercholesterolemia. Our aim was also to test the effect of high-dose statin treatment on these processes.

Methods

Pigs were kept for 120 days on a standard diet (SD, n=7), high-cholesterol diet (HCD, n=7) or high-cholesterol diet with Atorvastatin starting after 50 days (STATIN, n=7). Left carotid artery balloon injury was conducted in all groups after 60 days of diet treatment. Biochemical analysis together with evaluation of blood and tissue markers of vascular injury and inflammation were performed in all groups at the end of experiment.

Results

HCD compared to SD induced systemic inflammation demonstrated by increased number of circulating monocytes and lymphocytes. HCD compared to SD induced also local inflammation demonstrated by adipocyte hypertrophy and infiltration of T-lymphocytes in abdominal white adipose tissue, activation of hepatic stellate cells with infiltration of T- and B-lymphocytes and macrophages in the liver and increased macrophage content in lung parenchyma. These changes were accompanied by increased Intima/Media thickness, stenosis, matrix deposition and activated T-cell infiltrates in injured but not in uninjured contralateral carotid artery as we previously reported. The treatment with high-dose statin attenuated all aspects of systemic and local inflammation as well as pathological changes in injured carotid artery.

Conclusions

Diet related mild hypercholesterolemia induce systemic and local inflammation in the liver, lung and adipose tissue that coincide with enhanced inflammation of injured vessel but is without deleterious effect on uninjured vessels. High dose statin attenuated systemic and local inflammation and protected injured vessels. However, finding exact role of reduced systemic and remote inflammation in vascular protection requires further studies.     

The molecular and functional interaction between ICln and HSPC038 proteins modulates the regulation of cell volume

Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a "new gene-finding tool" (Eichmüller, S., Vezzoli, V., Bazzini, C., Ritter, M., Fürst, J., Jakab, M., Ravasio, A., Chwatal, S., Dossena, S., Bottà, G., Meyer, G., Maier, B., Valenti, G., Lang, F., and Paulmichl, M. (2004) J. Biol. Chem. 279, 7136-7146) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In C. elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling-induced chloride current (ICl(swell)). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of ICl(swell). Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.