Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in FEBS Journal 273 (2006): 2779-2788, doi:10.1111/j.1742-4658.2006.05355_1.x.The cone snail is the only invertebrate system in which the vitamin K dependent carboxylase (or γ-carboxylase) and its product γ-carboxyglutamic acid (Gla)1 have been identified. It remains the sole source of structural information of invertebrate γ-carboxylase subtrates. Four novel γ- carboxyglutamic acid (Gla)1 containing peptides were purified from the venom of Conus textile and characterized by biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have 6 Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L-bromotryptophan. Gla(2)-TxVI/A and Gla(2)- TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids –46 to –27) were nearly identical and highly hydrophobic. The predicted propeptide region (–20 to –1) that contains the γ-carboxylation recognition site (γ-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus γ-carboxylase and synthetic peptide substrates localized the γ-CRS of Gla(1)-TxVI to the region –14 to –1 of the polypeptide precursor: the Km was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 μM when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km 10-fold.This work was supported by grants K2001-03X-04487-27A and K2001- 03GX-04487-27, 08647, 13147 from the Swedish Medical Research Council, the European Union Cono-Euro-Pain (QLK3-CT-2000-00204), the Swedish Foundation for Strategic Research, the Kock Foundation, the Påhlsson Foundation and the Foundation of University Hospital, Malmö

Arterial hypertension and remodeling of the right ventricle

Background: In case of long-term and physiological loads (e.g. during pregnancy or regular athletics training), reversible morphological changes occur in the heart - cardiomyocytes undergo hypertrophy, however, this is not accompanied by impairment of left ventricular function or myocyte metabolism. However, in the course of various pathological processes, as time goes by, gradually permanent morphological changes occur. These changes are referred to as remodeling of the heart muscle, which, regardless of the primary cause, can lead to the development of chronic heart failure. Materials and methods: The study was performed on post-mortem material of 35 human hearts obtained from forensic sections and anatomopathological sections of people who died of non-cardiac causes (mainly traffic accidents, suicide attempts, strokes, acute infections); material was fixed in a 4% formalin solution. The hearts were subjected to macro- and microscopic assessment. During microscopic assessment the features of remodeling were evaluated. Results and conclusions: In vivo and echocardiographic tests, as well as macroscopic evaluation of post-mortem material, suggest the presence of some kind of right ventricular muscle remodeling, however classic microscopic observations, presented in this study do not provide such unambiguous evidence. Thus, the question arises: why and how the right ventricular function is disturbed, sometimes at early stages of arterial hypertension

Multicenter, open-label, extension trial to evaluate the long-term efficacy and safety of early versus delayed treatment with tolvaptan in autosomal dominant polycystic kidney disease:The TEMPO 4:4 Trial

Background.: In TEMPO 3:4, the vasopressin V2 receptor antagonist tolvaptan slowed total kidney volume (TKV) growth and estimated glomerular filtration rate (eGFR) decline relative to placebo. Methods.: TEMPO 4:4 was designed to provide an additional 2 years of data on the long-term safety and efficacy of tolvaptan in subjects completing TEMPO 3:4. The objective was to assess the disease-modifying effects of tolvaptan on TKV and eGFR end-points including change from baseline over the combined duration of TEMPO 3:4 and TEMPO 4:4, and non-inferiority of slopes during TEMPO 4:4. Results.: Of the 1445 subjects randomized to TEMPO 3:4, 871 (60.3%) enrolled in TEMPO 4:4. Percent changes in TKV from TEMPO 3:4 baseline to TEMPO 4:4 Month 24 were 29.9% and 31.6% (prior tolvaptan versus prior placebo, P = 0.38). Adjusting for baseline covariates improved the TKV treatment difference at Month 24 in TEMPO 4:4 from -1.70% to - 4.15% between the groups (P = 0.04). Slopes of TKV growth during TEMPO 4:4 were higher in early- versus delayed-treatment groups (6.16% versus 4.96% per year, P = 0.05). Analysis of secondary eGFR endpoints demonstrated a persistent effect on eGFR (3.15 mL/min/1.73 m 2 , P < 0.001), and non-inferiority in eGFR slopes. The safety profile on exposure to tolvaptan in TEMPO 4:4 was similar to that in TEMPO 3:4. Conclusions.: The results of TEMPO 4:4 support a sustained disease-modifying effect of tolvaptan on eGFR. The lack of a sustained treatment difference on TKV may be accounted for by limitations of the trial design, including loss of randomization and baseline imbalances ensuing TEMPO 3:4. The safety profile was similar to that observed in TEMPO 3:4

Clinical Pattern of Tolvaptan-Associated Liver Injury in Subjects with Autosomal Dominant Polycystic Kidney Disease: Analysis of Clinical Trials Database

IntroductionSubjects with autosomal dominant polycystic kidney disease (ADPKD) who were taking tolvaptan experienced aminotransferase elevations more frequently than those on placebo in the TEMPO 3:4 (Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and its Outcomes) clinical trial.MethodsAn independent, blinded, expert Hepatic Adjudication Committee re-examined data from TEMPO 3:4 and its open-label extension TEMPO 4:4, as well as from long-term (>14months) non-ADPKD tolvaptan trials, using the 5-point Drug-Induced Liver Injury Network classification.ResultsIn TEMPO 3:4, 1445 subjects were randomized 2:1 (tolvaptan vs. placebo) and 1441 had post-baseline assessments of hepatic injury. Sixteen patients on tolvaptan and one on placebo had significant aminotransferase elevations judged to be at least probably related to study drug. No association with dose or systemic exposure was found. Two of 957 subjects taking tolvaptan (0.2%) and zero of 484 taking placebo met the definition of a Hy’s Law case. One additional Hy’s Law case was identified in a TEMPO 4:4 subject who had received placebo in the lead study. The onset of a hepatocellular injury occurred between 3 and 18months after starting tolvaptan, with gradual resolution over the subsequent 1–4months. None of the events were associated with liver failure or chronic liver injury/dysfunction. No imbalance in hepatic events was observed between tolvaptan and placebo in lower-dose clinical trials of patients with hyponatremia, heart failure, or cirrhosis.ConclusionsAlthough hepatocellular injury following long-term tolvaptan treatment in ADPKD subjects was infrequent and reversible, the potential for serious irreversible injury exists. Regular monitoring of transaminase levels is warranted in this patient population

Characterization of the Conus bullatus genome and its venom-duct transcriptome

<p>Abstract</p> <p>Background</p> <p>The venomous marine gastropods, cone snails (genus <it>Conus</it>), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. <it>Conus bullatus </it>belongs to a clade of <it>Conus </it>species called <it>Textilia</it>, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic <it>Textilia </it>clade.</p> <p>Results</p> <p>We have carried out a sequencing survey of the <it>Conus bullatus </it>genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an <it>in silico </it>pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities.</p> <p>Conclusions</p> <p>Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of <it>Conus bullatus </it>venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of <it>C. bullatus </it>transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.</p

Fluid challenges in intensive care: the FENICE study A global inception cohort study

Fluid challenges (FCs) are one of the most commonly used therapies in critically ill patients and represent the cornerstone of hemodynamic management in intensive care units. There are clear benefits and harms from fluid therapy. Limited data on the indication, type, amount and rate of an FC in critically ill patients exist in the literature. The primary aim was to evaluate how physicians conduct FCs in terms of type, volume, and rate of given fluid; the secondary aim was to evaluate variables used to trigger an FC and to compare the proportion of patients receiving further fluid administration based on the response to the FC.This was an observational study conducted in ICUs around the world. Each participating unit entered a maximum of 20 patients with one FC.2213 patients were enrolled and analyzed in the study. The median [interquartile range] amount of fluid given during an FC was 500 ml (500-1000). The median time was 24 min (40-60 min), and the median rate of FC was 1000 [500-1333] ml/h. The main indication for FC was hypotension in 1211 (59 %, CI 57-61 %). In 43 % (CI 41-45 %) of the cases no hemodynamic variable was used. Static markers of preload were used in 785 of 2213 cases (36 %, CI 34-37 %). Dynamic indices of preload responsiveness were used in 483 of 2213 cases (22 %, CI 20-24 %). No safety variable for the FC was used in 72 % (CI 70-74 %) of the cases. There was no statistically significant difference in the proportion of patients who received further fluids after the FC between those with a positive, with an uncertain or with a negatively judged response.The current practice and evaluation of FC in critically ill patients are highly variable. Prediction of fluid responsiveness is not used routinely, safety limits are rarely used, and information from previous failed FCs is not always taken into account

Clinical Pattern of Tolvaptan-Associated Liver Injury in Subjects with Autosomal Dominant Polycystic Kidney Disease: Analysis of Clinical Trials Database

INTRODUCTION: Subjects with autosomal dominant polycystic kidney disease (ADPKD) who were taking tolvaptan experienced aminotransferase elevations more frequently than those on placebo in the TEMPO 3:4 (Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and its Outcomes) clinical trial. METHODS: An independent, blinded, expert Hepatic Adjudication Committee re-examined data from TEMPO 3:4 and its open-label extension TEMPO 4:4, as well as from long-term (>14 months) non-ADPKD tolvaptan trials, using the 5-point Drug-Induced Liver Injury Network classification. RESULTS: In TEMPO 3:4, 1445 subjects were randomized 2:1 (tolvaptan vs. placebo) and 1441 had post-baseline assessments of hepatic injury. Sixteen patients on tolvaptan and one on placebo had significant aminotransferase elevations judged to be at least probably related to study drug. No association with dose or systemic exposure was found. Two of 957 subjects taking tolvaptan (0.2 %) and zero of 484 taking placebo met the definition of a Hy’s Law case. One additional Hy’s Law case was identified in a TEMPO 4:4 subject who had received placebo in the lead study. The onset of a hepatocellular injury occurred between 3 and 18 months after starting tolvaptan, with gradual resolution over the subsequent 1–4 months. None of the events were associated with liver failure or chronic liver injury/dysfunction. No imbalance in hepatic events was observed between tolvaptan and placebo in lower-dose clinical trials of patients with hyponatremia, heart failure, or cirrhosis. CONCLUSIONS: Although hepatocellular injury following long-term tolvaptan treatment in ADPKD subjects was infrequent and reversible, the potential for serious irreversible injury exists. Regular monitoring of transaminase levels is warranted in this patient population