Parathyroid-Specific Deletion of Klotho Unravels a Novel Calcineurin-Dependent FGF23 Signaling Pathway That Regulates PTH Secretion

Klotho acts as a co-receptor for and dictates tissue specificity of circulating FGF23. FGF23 inhibits PTH secretion, and reduced Klotho abundance is considered a pathogenic factor in renal secondary hyperparathyroidism. To dissect the role of parathyroid gland resident Klotho in health and disease, we generated mice with a parathyroid-specific Klotho deletion (PTH-KL^−/−). PTH-KL^−/− mice had a normal gross phenotype and survival; normal serum PTH and calcium; unaltered expression of the PTH gene in parathyroid tissue; and preserved PTH response and sensitivity to acute changes in serum calcium. Their PTH response to intravenous FGF23 delivery or renal failure did not differ compared to their wild-type littermates despite disrupted FGF23-induced activation of the MAPK/ERK pathway. Importantly, calcineurin-NFAT signaling, defined by increased MCIP1 level and nuclear localization of NFATC2, was constitutively activated in PTH-KL^−/− mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression in PTH-KL^−/− mice whereas wild-type mice remained responsive. Similar results were observed in thyro-parathyroid explants ex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action.     

Genome size and ploidy levels in highly fragmented habitats: the case of western Mediterranean Juniperus (Cupressaceae) with special emphasis on J. thurifera L.

Mediterranean junipers are of special ecological importance as key components of resource islands in semi-arid mountain ecosystems of the Mediterranean basin. The fragmentation of their habitat, which was primarily natural and driven by climatic drought conditions, is currently being aggravated by anthropogenic pressure. In the framework of this concern, the present work aims to contribute establishing a genomic profile of Juniperus in its western Mediterranean range, with a special emphasis placed on J. thurifera. DNA contents were assessed by flow cytometry in 43 populations of nine taxa within their Mediterranean range (first reports for J. navicularis, J. thurifera subsp. africana and J. thurifera subsp. thurifera). Chromosome numbers were determined by orcein staining in eight taxa (first counts for J. oxycedrus subsp. badia, J. phoenicea subsp. phoenicea, J. phoenicea subsp. turbinata, of 2n?=?2x?=?22, and for J. thurifera subsp. thurifera, of 2n?=?4x?=?44). Tetraploid cytotypes have been the only ones found in the 19 populations of J. thurifera studied, this being the first report of a Juniperus species exclusively polyploid. In J. thurifera, C-value does not respond to habitat fragmentation, in the same way that genetic diversity within populations was previously shown to be unaltered, suggesting that this factor has not had, at least to date, a significant impact on populations at genomic and genetic levels. Habitat fragmentation leads to deeply age-biased populations with a male-biased imbalanced sex ratio (lack of females), indicating an urgent need to improve regeneration within the populations of this species.     

Health evaluation of wild gentoo penguins (Pygoscelis papua) in the Antarctic Peninsula

Historically wildlife conservation was based on habitat protection and exploitation control. Only recently have diseases been considered an important issue. However, pathogens are usually described during or after disease outbreaks, but to determine which pathogens may be emerging, surveys of wildlife health are critical in a given time. This study deals with the health status of gentoo penguins Pygoscelis papua in two localities at the Antarctica Peninsula and one at Ardley Island off the South Shetland Islands. Cloacal swaps, fresh fecal samples, ectoparasites, and blood smears were collected. We examined and dissected 14 penguin corpses found dead. Fecal samples were positive for Campylobacter, Escherichia coli and in the carcasses four endoparasitic species were found: Diphyllobothrium sp. and Parorchites zederi, Corynosoma shackletoni and Stegophorus adeliae. The tick Ixodes uriae occurred in five of the examined penguins, and the louse Austrogoniodes gressitti on six birds. From the colony grounds, we collected 1,184 I. uriae. We recorded antibiotic-resistant bacteria, such as E. coli, in ecosystems where gentoo penguins breed. Cloacal samples (300) were negative for Chlamydia, as well as for Salmonella, Campylobacter, E. coli, Newcastle and Influenza viruses.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.     

Autumn-winter diet overlap of fallow, red, and roe deer in forest ecosystems, Southern Poland

The wild population of fallow deer in Central Europe has grown considerably over the last decade. However, information on feeding habits of this alien species in relation to the indigenous red deer or roe deer, in areas of their co-occurrence, is scarce. A prevailing view maintains that their food-niches are distinct, although direct comparative studies have not been carried out. Therefore, the aim of the research was to compare the diets of fallow, red, and roe deer feeding in the same habitat. Research was based on the rumen contents of 242 animals hunted in the autumn-winter season in the forests of Southern Poland. The analyses demonstrated that fallow deer are moderate grazers in such conditions and eat more graminoids in comparison to red or roe deer (36.4% vs. 16.1% or 5.5%, respectively). On the other hand, it feeds on less browse (17.2% vs. 41.4%) or dwarf shrubs (8.4% vs. 19.0%) than red deer, and on less bramble (10.9% vs. 34.6%) or forbs (4.0% vs. 7.6%) in comparison to roe deer (P=0.05). Although the diets of the three deer species differ in terms of the proportion of each food type in their diet, overlapping of their food-niches is high (52.6%).     

Gut Peptide GLP-1 and Its Analogue,Exendin-4, Decrease Alcohol Intake and Reward

Glucagon-like-peptide-1 (GLP-1) is a gut- and neuro-peptide with an important role in the regulation of food intake and glucose metabolism. Interestingly, GLP-1 receptors (GLP-1R) are expressed in key mesolimbic reward areas (including the ventral tegmental area, VTA), innervated by hindbrain GLP-1 neurons. Recently GLP-1 has emerged as a potential regulator of food reward behavior, an effect driven by the mesolimbic GLP-1Rs. Its role in other reward behaviors remains largely unexplored. Since a considerable overlap has been suggested for circuitry controlling reward behavior derived from food and alcohol we hypothesized that GLP-1 and GLP-1Rs could regulate alcohol intake and alcohol reward. We sought to determine whether GLP-1 or its clinically safe stable analogue, Exendin-4, reduce alcohol intake and reward. To determine the potential role of the endogenous GLP-1 in alcohol intake we evaluated whether GLP-1R antagonist, Exendin 9-39, can increase alcohol intake. Furthermore, we set out to evaluate whether VTA GLP-1R activation is sufficient to reduce alcohol intake. Male Wistar rats injected peripherally with GLP-1 or Exendin-4 reduced their alcohol intake in an intermittent access two bottle free choice drinking model. Importantly, a contribution of endogenously released GLP-1 is highlighted by our observation that blockade of GLP-1 receptors alone resulted in an increased alcohol intake. Furthermore, GLP-1 injection reduced alcohol reward in the alcohol conditioned place preference test in mice. To evaluate the neuroanatomical substrate linking GLP-1 with alcohol intake/reward, we selectively microinjected GLP-1 or Exendin 4 into the VTA. This direct stimulation of the VTA GLP-1 receptors potently reduced alcohol intake. Our findings implicate GLP-1R signaling as a novel modulator of alcohol intake and reward. We show for the first time that VTA GLP-1R stimulation leads to reduced alcohol intake. Considering that GLP-1 analogues are already approved for clinical use, this places the GLP system as an exciting new potential therapeutic target for alcohol use disorders.     

Zebularine Induces Long-Term Survival of Pancreatic Islet Allotransplants in Streptozotocin Treated Diabetic Rats

Background

Coping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed.

Methodology/Principal Findings

The DNA demethylating agent Zebularine, previously demonstrated to induce expression of the genes for the immunosuppressive enzymes indolamine-2,3-deoxygenase-1 (IDO1) and kynureninase of the kynurenine pathway, is tested for capacity to suppress rejection of allotransplants. Allogeneic pancreatic islets from Lewis rats were transplanted under the kidney capsule of Fischer rats previously made diabetic by a streptozotocin injection (40 mg/kg). One group was treated with Zebularine (225 mg/kg) daily for 14 days from day 6 or 8 after transplantation, and a control group received no further treatment. Survival of the transplants was monitored by blood sugar measurements. Rats, normoglycemic for 90 days after allografting, were subjected to transplant removal by nephrectomy to confirm whether normoglycemia was indeed due to a surviving insulin producing transplant, or alternatively was a result of recovery of pancreatic insulin production in some toxin-treated rats. Of 9 Zebularine treated rats, 4 were still normoglycemic after 90 days and became hyperglycemic after nephrectomy. The mean length of normoglycemia in the Zebularine group was 67±8 days as compared to 14±3 days in 9 controls. Seven rats (2 controls and 5 Zebularine treated) were normoglycemic at 90 days due to pancreatic recovery as demonstrated by failure of nephrectomy to induce hyperglycemia.

Conclusions/Significance

Zebularine treatment in vivo induces a long-lasting suppression of the immune destruction of allogeneic pancreatic islets resulting in protection of allograft function for more than 10 weeks after end of treatment.     

Temperature- and hydration-dependent protein dynamics in photosystem II of green plants studied by quasielastic neutron scattering

Protein dynamics in hydrated and vacuum-dried photosystem II (PS II) membrane fragments from spinach has been investigated by quasielastic neutron scattering (QENS) in the temperature range between 5 and 300 K. Three distinct temperature ranges can be clearly distinguished by active type(s) of protein dynamics: (A) At low temperatures (T < 120 K), the protein dynamics of both dry and hydrated PS II is characterized by harmonic vibrational motions. (B) In the intermediate temperature range (120 < T < 240 K), the total mean square displacement total slightly deviates from the predicted linear behavior. The QENS data indicate that this deviation, which is virtually independent of the extent of hydration, is due to a partial onset of diffusive protein motions. (C) At temperatures above 240 K, the protein flexibility drastically changes because of the onset of diffusive (large-amplitude) protein motions. This dynamical transition is clearly hydration-dependent since it is strongly suppressed in dry PS II. The thermally activated onset of protein flexibility as monitored by QENS is found to be strictly correlated with the temperature-dependent increase of the electron transport efficiency from Q(A)(-) to QB (Garbers et al. (1998) Biochemistry 37, 11399-11404). Analogously, the freezing of protein mobility by dehydration in dry PS II appears to be responsible for the blockage of Q(A)(-) reoxidation by Q(B) at hydration values lower than 45% r.h. (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). Similar effects were observed for reactions of the water-oxidizing complex as outlined in the Discussion section.     

Intracellular expression of the proliferative marker Ki-67 and viral proteins (NS3, NS5A and C) in chronic, long lasting hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) continues to represent the main causative agent of the hepatitis, which leads to chronic transformation of the process in 60-80% individuals. It remains unclear how far cellular expression of HCV proteins in vivo may represent an index of progression of the disease and of proliferative activity in the liver in chronic hepatitis C. Aim of the studies included detection and subcellular localization of three HCV proteins (NS3, NS5A and C) in liver biopsies from adults (n=19) with chronic, long lasting hepatitis C as related to hepatocyte proliferative activity. The immunocytochemical ABC (avidin biotin-peroxidase complex) technique was applied, alone or associated with the ImmunoMax technique. Results of the immunocytochemical tests were compared to histological alterations in liver biopsies, proliferation index and with selected clinical data. A significantly higher expression of NS3 protein was noted, as compared to expressions of NS5A and C proteins. In all the patients, cytoplasmic localization of all proteins dominated over nuclear localization (p0.05). At the level of electron microscopy, protein localization in endoplasmic reticulum (ER) membranes, mitochondria, perinuclear region and/or in hepatocyte cell nucleus was observed. No direct relationships could be demonstrated between expressions of HCV proteins and of Ki-67 antigen. No correlations could also be demonstrated between cellular expression of any HCV protein on one hand and grading or staging, alanine transaminase (ALT), serum level of HCV RNA or alpha-fetoprotein (AFP) on the other. However, positive correlations were disclosed between proliferative activity of hepatocytes on one hand and patient's age, grading and staging on the other. Advanced hepatic fibrosis correlated also with serum levels of AFP. The studies were supplemented with data on subcellular localization of HCV proteins. Moreover, they indicated that in HCV infection grading and staging, proliferative activity of hepatocytes and serum AFP level represent more valuable indices of the disease progress than those provided by cellular expression of three potentially oncogenic HCV proteins in vivo.