Studies on the Interactions between Arachidonic Acid Metabolism and Calcium in Regulating Vasopressin-Induced Water Permeability in the Toad Urinary Bladder

The toad urinary bladder and epithelial cells isolated from it were found to synthesize prostaglandin E (PGE) and thromboxane A2 (TXA2). The syntheses of both of these compounds were found to be stimulated by arginine-vasopressin and by its non-pressor antidiuretic analog I-deamino-8-D-arginine-vasopressin. cAMP, a putative second messenger for vasopressin in the toad bladder, when added to the incubation media, did not affect TXA2 synthesis. However, in the isolated cells both TXA2 and PGE syntheses were found to be stimulated by calcium. In studies using the isolated toad bladder, inhibition of TXA2 synthesis with imidazole (1 mM) and 7-(1-imidazolyl)-heptanoic acid (50-100 µM) was found to inhibit vasopressin-stimulated water flow to a maximum of 30%. Similarly, an antagonist of TXA2 action, trans-13-azaprostanoic acid (50-300 µM) inhibited vasopressin-stimulated water flow in a dose-dependent fashion to a maximum of 35%, while the biologically inactive cis isomer was without effect. Several compounds which exhibit TXA2-like effects in other systems, (15Z)-hydroxy-9α,11α-(epoxymethano)prosta-5Z,13E dienoic acid (U44069), its 9α,11α-(methanoepoxy) isomer (U46619), and TXB2 , mimicked the hydroosmotic effect of vasopressin. This action was inhibited in a dose-dependent fashion by trans-13-azaprostanoic acid while cis-13-azaprostanoic acid was without effect. Vasopressin was found to elicit an enhancement in 45Ca efflux from prelabelled toad bladder epithelial cells. Compartmental analysis of 45Ca efflux from prelabelled cells revealed three components. Studies using EGTA, lanthanum, and mitochondrial inhibitors suggested that the first component of efflux (S1) represented 45Ca bound to heterogeneous sites on the extracellular surface of the plasma membrane, the second component (S2) represented 45Ca bound to some intracellular site(s), and the third component (S3) was composed of two sites, a slowly exchanging site near the plasma membrane, and mitochondria. Vasopressin reduced the sizes of calcium pools S1 and S3. Exogenous cAMP also reduced the size of S3, but increased the size of S2. The TXA2 mimetic, U46619, acted similarly to cAMP; it decreased the size of pool S3 and increased the size of pool S2. The TXA2 synthesis inhibitor 7-(1-imidazolyl)-heptanoic acid blocked the effect of vasopressin to reduce the size of S3. The TXA2 antagonist trans-13- azaprostanoic acid, in concentrations not lethal to the isolated cells, inhibited vasopressin-stimulated water flow only slightly (17%) and did not significantly alter vasopressin\u27s effects on 45Ca kinetics. However, trans-13-azaprostanoic acid significantly blocked the effect of U46619 to decrease the size of S3. Exogenous PGE 1, 1 µM, a concentration which completely inhibits vasopressin-stimulated water flow, enhanced both the efflux and influx of 45Ca. However, net cellular 45Ca was increased. The increased 45Ca was located entirely in pool S3. These results are consistent with a hypothesis wherein vasopressin causes a net release of calcium from an intracellular storage site, perhaps mitochondria. PGE and TXA2 syntheses are stimulated, perhaps by the altered calcium fluxes. TXA2, like vasopressin, reduces the size of a putative mitochondrial calcium compartment, and inhibition of TXA2 synthesis, or antagonism of TXA2 action, inhibits vasopressin-stimulated water flow. PGE enhances 45Ca uptake into the epithelial cells and increases the size of pool S3, resulting in inhibition of vasopressin-stimulated water flow

Thromboxane and Stable Prostaglandin Endoperoxide Analogs Stimulate Water Permeability in the Toad Urinary Bladder

The effects of thromboxane B2 and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9α - 1 lα - (epoxymethano)prosta - 5Z,13E – dienoic acid (U44069) and (15Z)-hydroxy-1 lα,9α-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A2 synthesis, thromboxane B2 stimulated water flow in a dose-dependent manner. U44069 (1 µM) stimulated water flow from 3.6±0.8 to 12.4±1.2 mg/min per 10 cm2 hemibladder surface area, while U46619 (1 µM) stimulated water flow from 2.8± 1.0 to 21.8±2.0 mg/min per 10 cm2. The prostaglandin endoperoxide/thromboxane A2 antagonist trans-13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B2- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 µM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P \u3c 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A2-like compounds stimulate water flow in the toad urinary bladder

Comparison of SIV and HIV-1 Genomic RNA Structures Reveals Impact of Sequence Evolution on Conserved and Non-Conserved Structural Motifs

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve

The AQUA-FONTIS study: protocol of a multidisciplinary, cross-sectional and prospective longitudinal study for developing standardized diagnostics and classification of non-thyroidal illness syndrome

<p>Abstract</p> <p>Background</p> <p>Non-thyroidal illness syndrome (NTIS) is a characteristic functional constellation of thyrotropic feedback control that frequently occurs in critically ill patients. Although this condition is associated with significantly increased morbidity and mortality, there is still controversy on whether NTIS is caused by artefacts, is a form of beneficial adaptation, or is a disorder requiring treatment. Trials investigating substitution therapy of NTIS revealed contradictory results. The comparison of heterogeneous patient cohorts may be the cause for those inconsistencies.</p> <p>Objectives</p> <p>Primary objective of this study is the identification and differentiation of different functional states of thyrotropic feedback control in order to define relevant evaluation criteria for the prognosis of affected patients. Furthermore, we intend to assess the significance of an innovative physiological index approach (SPINA) in differential diagnosis between NTIS and latent (so-called "sub-clinical") thyrotoxicosis.</p> <p>Secondary objective is observation of variables that quantify distinct components of NTIS in the context of independent predictors of evolution, survival or pathophysiological condition and influencing or disturbing factors like medication.</p> <p>Design</p> <p>The <b>a</b>pproach to a <b>qua</b>ntitative <b>f</b>ollow-up <b>o</b>f <b>n</b>on-<b>t</b>hyroidal <b>i</b>llness <b>s</b>yndrome (AQUA FONTIS study) is designed as both a cross-sectional and prospective longitudinal observation trial in critically ill patients. Patients are observed in at least two evaluation points with consecutive assessments of thyroid status, physiological and clinical data in additional weekly observations up to discharge. A second part of the study investigates the neuropsychological impact of NTIS and medium-term outcomes.</p> <p>The study design incorporates a two-module structure that covers a reduced protocol in form of an observation trial before patients give informed consent. Additional investigations are performed if and after patients agree in participation.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NCT00591032</p

Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung

<p>Abstract</p> <p>Background</p> <p>Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.</p> <p>Methods</p> <p>The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-<it>ras</it>, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp^-/-/rag2^-/-mice. Sensitivity towards chemotherapy was analysed by MTT assay.</p> <p>Results</p> <p>PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp^-/-/rag2^-/-mice resulted in formation of primary tumors and spontaneous lung metastasis.</p> <p>Conclusion</p> <p>The established PaCa 5061 cell line and its injection into pfp^-/-/rag2^-/-mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.</p

Management of intra-abdominal infections : recommendations by the WSES 2016 consensus conference

This paper reports on the consensus conference on the management of intra-abdominal infections (IAIs) which was held on July 23, 2016, in Dublin, Ireland, as a part of the annual World Society of Emergency Surgery (WSES) meeting. This document covers all aspects of the management of IAIs. The Grading of Recommendations Assessment, Development and Evaluation recommendation is used, and this document represents the executive summary of the consensus conference findings.Peer reviewe

Food Consumption and its Impact on Cardiovascular Disease: Importance of Solutions Focused on the Globalized Food System A Report From the Workshop Convened by the World Heart Federation

Major scholars in the field, based on a 3-day consensus, created an in-depth review of current knowledge on the role of diet in CVD, the changing global food system and global dietary patterns, and potential policy solutions. Evidence from different countries, age/race/ethnicity/socioeconomic groups suggest the health effects studies of foods, macronutrients, and dietary patterns on CVD appear to be far more consistent though regional knowledge gaps are highlighted. There are large gaps in knowledge about the association of macronutrients to CVD in low- and middle-income countries (LMIC), particularly linked with dietary patterns are reviewed. Our understanding of foods and macronutrients in relationship to CVD is broadly clear; however major gaps exist both in dietary pattern research and ways to change diets and food systems. Based on the current evidence, the traditional Mediterranean-type diet, including plant foods/emphasizing plant protein sources, provides a well-tested healthy dietary pattern to reduce CV

Thromboxane and Stable Prostaglandin Endoperoxide Analogs Stimulate Water Permeability in the Toad Urinary Bladder

The effects of thromboxane B(2) and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9α - 11α - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11α,9α-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A(2) synthesis, thromboxane B(2) stimulated water flow in a dose-dependent manner. U44069 (1 μM) stimulated water flow from 3.6±0.8 to 12.4±1.2 mg/min per 10 cm(2) hemibladder surface area, while U46619 (1 μM) stimulated water flow from 2.8±1.0 to 21.8±2.0 mg/min per 10 cm(2). The prostaglandin endoperoxide/thromboxane A(2) antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B(2)- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 μM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A(2)-like compounds stimulate water flow in the toad urinary bladder