Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane

Cauda epididymal spermatozoa of the golden hamster were labelled with the thiol-alkylating reagent, monobromobimane (MB). Female hamsters underwent uterine insemination with labelled spermatozoa at laparotomy under metofane anesthesia. All 12 females examined between 5 and 54 h postinsemination yielded a total of 83/100 (83%) eggs in the process of fertilization or embryos. Under ultraviolet (UV) exposure all exhibited a fluorescent tail which, in the 4- and 8-cell embryos, could be seen to be fraying or disintegrating. As cleavage progressed, labelled tail components came to be restricted among the blastomeres such that at the 4- and 8-cell stage the tail could be seen in only one to three blastomeres. To study complete development and pregnancy another 12 females received uterine insemination. After recovery from anesthesia (approximately equal to 4 h) these females were mated with a vasectomized male bearing a dominant genetic marker (black eyes) to allow unequivocal determination of paternity in the fetuses and young produced. Seven became pregnant with one female losing her pregnancy about Day 7 of gestation. Two females sacrificed on Day 13 produced 17 normal fetuses and one resorption. Four females delivered 16 young, all normal at birth and in subsequent growth and fertility. In addition, insemination of female rabbits with MB-labelled spermatozoa yielded normal embryos (50/52 96%) from 3 does on Day 2 and (38/64 60%) from 4 does on Days 4 or 5. Two normal litters (9 bunnies) have delivered from 3 does allowed to carry to term.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of Comorbidities on Mortality in Patients with Idiopathic Pulmonary Fibrosis

Introduction

Comorbidities significantly influence the clinical course of idiopathic pulmonary fibrosis (IPF). However, their prognostic impact is not fully understood. We therefore aimed to determine the impact of comorbidities, as individual and as whole, on survival in IPF.

Methods

The database of a tertiary referral centre for interstitial lung diseases was reviewed for comorbidities, their treatments, their frequency and survival in IPF patients.

Results

272 patients were identified of which 12% had no, 58% 1–3 and 30% 4–7 comorbidities, mainly cardiovascular, pulmonary and oncologic comorbidities. Median survival according to the frequency of comorbidities differed significantly with 66 months for patients without comorbidities, 48 months when 1–3 comorbidities were reported and 35 months when 4–7 comorbidities were prevalent (p = 0.004). A multivariate Cox proportional hazard analyses identified other cardiac diseases and lung cancer as significant predictors of death, gastro-oesophageal reflux disease (GERD) and diastolic dysfunction had a significant positive impact on survival. A significant impact of comorbidities associated therapies on survival was not discovered. This included the use of proton pump inhibitors at baseline, which was not associated with a survival benefit (p = 0.718). We also established a predictive tool for highly prevalent comorbidities, termed IPF comorbidome which demonstrates a new relationship of IPF and comorbidities.

Conclusion

Comorbidities are frequent in IPF patients. Some comorbidities, especially lung cancer, mainly influence survival in IPF, while others such as GERD may inherit a more favourable effect. Moreover, their cumulative incidence impacts survival.     

Circulating Angiopoietin-1 Is Not a Biomarker of Disease Severity or Prognosis in Pulmonary Hypertension

BackgroundCirculating angiopoietin-1 (Ang-1) has been linked to pulmonary hypertension (PH) in experimental studies. However, the clinical relevance of Ang-1 as a biomarker in PH remains unknown. We aimed to investigate the prognostic and clinical significance of Ang-1 in PH using data from the prospectively recruiting Giessen PH Registry.MethodsPatients with suspected PH (without previous specific pulmonary arterial hypertension [PAH] therapy) who underwent initial right heart catheterization (RHC) in our national referral center between July 2003 and May 2012 and who agreed to optional biomarker analysis were included if they were diagnosed with idiopathic PAH, connective tissue disease-associated PAH (CTD-PAH), PH due to left heart disease (PH-LHD), or chronic thromboembolic PH (CTEPH), or if PH was excluded by RHC (non-PH controls). The association of Ang-1 levels with disease severity (6-minute walk distance and pulmonary hemodynamics) was assessed using linear regression, and the impact of Ang-1 levels on transplant-free survival (primary endpoint) and clinical worsening was assessed using Kaplan—Meier curves, receiver operating characteristic (ROC) analyses, and Cox regression.Results151 patients (39, 39, 32, and 41 with idiopathic PAH, CTD-PAH, PH-LHD, and CTEPH, respectively) and 41 non-PH controls were included. Ang-1 levels showed no significant difference between groups (p = 0.8), and no significant associations with disease severity in PH subgroups (p ≥ 0.07). In Kaplan—Meier analyses, Ang-1 levels (stratified by quartile) had no significant impact on transplant-free survival (p ≥ 0.27) or clinical worsening (p ≥ 0.51) in PH subgroups. Regression models found no significant association between Ang-1 levels and outcomes (p ≥ 0.31). ROC analyses found no significant cut-off that would maximize sensitivity and specificity.ConclusionsDespite a strong pathophysiological association in experimental studies, this first comprehensive analysis of Ang-1 in PH subgroups suggests that Ang-1 is not a predictive and clinically relevant biomarker in PH.     

Leukotriene C4 binding to rat lung membranes

A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.     

Asymmetric and Symmetric Dimethylarginine as Risk Markers for Total Mortality and Cardiovascular Outcomes: A Systematic Review and Meta-Analysis of Prospective Studies

BackgroundA growing number of studies linked elevated concentrations of circulating asymmetric (ADMA) and symmetric (SDMA) dimethylarginine to mortality and cardiovascular disease (CVD) events. To summarize the evidence, we conducted a systematic review and quantified associations of ADMA and SDMA with the risks of all-cause mortality and incident CVD in meta-analyses accounting for different populations and methodological approaches of the studies.MethodsRelevant studies were identified in PubMed until February 2015. We used random effect models to obtain summary relative risks (RR) and 95% confidence intervals (95%CIs), comparing top versus bottom tertiles. Dose-response relations were assessed by restricted cubic spline regression models and potential non-linearity was evaluated using a likelihood ratio test. Heterogeneity between subgroups was assessed by meta-regression analysis.ResultsFor ADMA, 34 studies (total n = 32,428) investigating associations with all-cause mortality (events = 5,035) and 30 studies (total n = 30,624) investigating the association with incident CVD (events = 3,396) were included. The summary RRs (95%CI) for all-cause mortality were 1.52 (1.37–1.68) and for CVD 1.33 (1.22–1.45), comparing high versus low ADMA concentrations. Slight differences were observed across study populations and methodological approaches, with the strongest association of ADMA being reported with all-cause mortality in critically ill patients. For SDMA, 17 studies (total n = 18,163) were included for all-cause mortality (events = 2,903), and 13 studies (total n = 16,807) for CVD (events = 1,534). High vs. low levels of SDMA, were associated with increased risk of all-cause mortality [summary RR (95%CI): 1.31 (1.18–1.46)] and CVD [summary RR (95%CI): 1.36 (1.10–1.68) Strongest associations were observed in general population samples.ConclusionsThe dimethylarginines ADMA and SDMA are independent risk markers for all-cause mortality and CVD across different populations and methodological approaches.     

Errata

Chromosomal proteins extracted from calf thymus by 0.35 M NaCl were insoluble at the low salt concentrations needed initially for ion-exchange chromatography. However, the addition of high-affinity carboxymethyl-dextran rendered the precipitated proteins soluble, permitting them to be fractionated by elution from DEAE-Sephacel. In this way, the separation of HMG-1 from HMG-2 was achieved, as well as a partial fractionation of the low mobility group proteins, under conditions that did not disturb the native conformation of the proteins.     

The Drosophila melanogaster sperm proteome-II (DmSP-II)

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.     

Hydrology and Diatom Phytoplankton of High Arctic Lakes and Ponds on Store Koldewey,Northeast Greenland

We document hydrological and phytoplankton characteristics of nine lakes and two ponds on Store Koldewey, a culturally undisturbed island off Northeast Greenland. The limnological survey included the recording of temperature, conductivity, oxygen concentration and saturation, pH, ionic composition, transparency, and the diatom phytoplankton community. In summer 2003, the lakes were cold, monomictic, thermally unstratified, alkaline and likely oligotrophic water bodies. Diatom phytoplankton was present in six lakes and consisted of four dominant species (Aulacoseira tethera, Cyclotella pseudostelligera, C. rossii, and Fragilaria tenera). The concentration of planktonic diatoms varied distinctly between the lakes. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)