Cardiac-restricted angiotensin-converting enzyme overexpression causes conduction defects and connexin dysregulation

Renin-angiotensin (RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrio-Hisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na(+) channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca(2+) channel, Kv4.2 K(+) channel alpha-subunit, and Cx45 mRNA abundances and the peak ventricular Na(+) current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.     

Incidence of deep vein thrombosis related to peripherally inserted central catheters in children and adolescents

Background

Peripherally inserted central catheters (PICC) in children and adolescents are being used with increasing frequency. We sought to determine the incidence and characterize risk factors of deep vein thrombosis associated with peripherally inserted central catheters in a pediatric population.

Methods

We conducted a prospective study involving consecutive patients referred to the radiology department of a tertiary care university-affiliated hospital for insertion of a peripherally inserted central catheter. We included patients aged 18 years or less who weighed more than 2.5 kg and had a peripherally inserted central catheter successfully inserted in his or her arm between June 2004 and November 2005. The primary outcome was the occurrence of partial or complete deep vein thrombosis evaluated by clinical examination, ultrasonography and venous angiography.

Results

A total of 214 patients (101 girls, 113 boys) were included in the study. Partial or complete deep vein thrombosis occurred in 20 patients, for an incidence of 93.5 per 1000 patients and 3.85 per 1000 catheter-days. Only 1 of the cases was symptomatic. In the univariable analyses, the only variable significantly associated with deep vein thrombosis was the presence of factor II mutation G20210A (odds ratio 7.08, 95% confidence interval 1.11–45.15, p = 0.04), a genetic mutation that increases the risk of a blood clot and that was present in 5 (2.3%) of the 214 patients.

Interpretation

The incidence of deep vein thrombosis related to peripherally inserted central catheters in our study was lower than the incidence related to centrally inserted venous catheters described in the pediatric literature (11%–50%).Most data available in the adult and pediatric literature on the incidence of deep vein thrombosis concern centrally inserted venous catheters, which are inserted directly in a central vein (jugular, subclavian or femoral). Typical symptoms of deep vein thrombosis are frequently absent in children and adolescents. Although the diagnosis of deep vein thrombosis is more reliable when based on Doppler ultrasonography or venous angiography,^1–10 in most studies these diagnostic tests were performed only when patients presented clinical symptoms of deep vein thrombosis or catheter dysfunction. In studies focused on the pediatric population, the frequency of deep vein thrombosis related to centrally inserted venous catheters has varied from 11% to 50%.^1,5,6,8,11In the past 10 years, peripherally inserted central catheters (PICC) have been used with increasing frequency in children and adolescents. The catheter is inserted percutaneously via a peripheral vein, with its tip residing in the superior vena cava. The main indications for this type of catheter insertion are difficult venous access, home intravenous antibiotic therapy, administration of chemotherapy or other hyperosmolar solution and long-term parenteral nutrition. The risk of deep vein thrombosis related to peripherally inserted central catheters could be greater among children and adolescents than among adults, given the size of the veins. Several studies have published complications related to peripherally inserted central catheters,^12–20 but few focused on the pediatric population.^13–21 Furthermore, in all of these studies, screening for deep vein thrombosis was not systematic. The real incidence of deep vein thrombosis related to peripherally inserted central catheters and their complications in the pediatric population are therefore unknown. We conducted this study to determine the incidence and characterize the risk factors of deep vein thrombosis related to peripherally inserted central catheters in children and adolescents in our institution.     

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.     

Calorimetric measurement of free energy utilization by Nitrosomonas and Nitrobacter

Summary Calorimetric estimates of the utilization efficiency of the free-energy derived from substrate oxidation by cell suspensions of two nitrifying bacteria, Nitrosomonas and Nitrobacter, provided two ranges of values: 11 to 27% and 15 to 51%, respectively. About 15 to 30% of the utilized free-energy is used for driving endergonic reactions other than CO₂ fixation, probably the synthesis of polyphosphates.The molar heat of substrate oxidation does not seem to be influenced by the age of cells harvested during growth or by the length of the incubation period during which cells have been kept in a buffer suspension in a starved condition. The loss of respiratory activity measured either by oxygen uptake or heat evolution in the presence of the specific substrate, nitrite or ammonium, decreases according to kinetics which are influenced by the aerobiosis of the suspension. The viability of the starved cells decreases in a way which is similar to that of the respiratory activity. It seemed impossible to obtain cells which had lost their viability but kept the ability to oxidize their substrate.Two inhibitors of the respiratory chain, quinacrine and cyanide, are without effect on the molar heat of substrate oxidation and consequently on the free-energy utilization efficiency. 2.4 dinitrophenol did decrease the rate of heat evolution during substrate oxidation at concentrations at which the rate of oxygen uptake was not depressed, with the consequences that free-energy efficiency was apparently increased.     

Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ∼90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO₄) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO₄ induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 × 10⁵ counts/s after 72 h of induction. Induction with 700 μM of CuSO₄ performed at the exponential phase of the S2MtEGFP culture (10⁶ cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO₄ induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.     

Effect of exogenous melatonin on viability, ingestion capacity, and free-radical scavenging in heterophils from young and old ringdoves (Streptopelia risoria)

The decrease of melatonin production with aging contributes to the decline in immune function as organisms age. Treatment with the exogenously administered indoleamine restores the reduced immunological functions. Therefore, we investigated the effect of melatonin on viability, phagocyte ingestion capacity, and free radical generation levels of heterophils from young and old ringdove (Streptopelia risoria) aged 3–4 and 11–13 years, respectively. Animals received a single oral dose of melatonin 1 h before lights off for three consecutive days. Experiments were performed at the acrophases and nadirs of melatonin. Melatonin treatment significantly increased serum melatonin levels at the acrophases, but not at the nadirs of the two age groups. In both young and old animals there was increased heterophil viability at acrophases with respect to nadirs, and also increased cell resistance to oxidative stress in the old animals after the melatonin treatment. At acrophases, the index, percentage and efficiency of phagocytosis all increased significantly, and superoxide anion levels decreased significantly with respect to the nadir values of vehicle and melatonin-treated animals, the effect being greater in young than in old ringdoves. At the nadirs, no change was observed in any parameter analyzed. In both young and old animals, phagocytosis and melatonin were positively correlated, while superoxide anion levels and melatonin were negatively correlated. In conclusion, exogenous melatonin enhanced heterophil viability in old animals as well as increasing phagocytosis and free-radical scavenging in both age groups during the nocturnal period, accompanied by an increase in the levels of the indoleamine.     

Pregnancy rate of Nelore females inseminated with male-sexed semen

Pregnancy rates of Nelore females inseminated with male-sexed semen and conventional semen from the same bulls were evaluated. The females included 433 heifers (2 years old) and 230 non-suckling cows, totaling 663 animals. Average body condition score was 3.5 (1-5 scale). Estrus was induced with prostaglandin F2α. The total pregnancy rate of females inseminated with male-sexed semen of bulls A, B and C was 38.8% (131/338) less (P<0.0001) than the total pregnancy rate observed for females inseminated with conventional semen from the same bulls (57.9% [188/325]). Pregnancy rates of non-suckling cows inseminated with male-sexed semen was 43.3% (49/113), which was similar (P≥0.05) to the values found for heifers inseminated with male-sexed semen from the same bulls (36.4% [82/225]). The pregnancy rate of females inseminated with male-sexed semen was less compared with females inseminated with conventional semen. In addition, there was no significant difference in the pregnancy rate of heifers versus non-suckling cows.     

Candida albicans induces selective development of macrophages and monocyte derived dendritic cells by a TLR2 dependent signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-)), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2(-/-) mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2(-/-) mice correlating with their higher fungal burden. Accordingly, emigration of Ly6C(high) monocytes and neutrophils to spleen was increased in TLR2(-/-) mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2(-/-) infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin(-) cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.