Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: A subcellular correlate to ischemic preconditioning?

Background: There is increasing evidence that mitochondria – owning a high degree of autonomy within the cell – might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K _ATP) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia.Methods and Results: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K _ATP opener diazoxide (30 μmol/l) and was completely inhibited by the K _ATP blocker 5-hydroxydecanoic acid (300 μmol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia.Conclusion: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K_ATP but is independent of ATP preservation.     

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. ₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation     

The chemical composition of throughfall beneath oak, birch and pine canopies in Northwest Germany

The chemical composition of rainwater is altered upon its passage through tree canopies. In order to investigate how rainwater chemistry is affected by canopy-dependent processes in characteristic forest types of Northwest German sandy lowland regions – oak–birch-forests, Betula pubescens Ehrh. swamp forests, and stands of Pinus sylvestris L. – comparative studies on the chemical composition of throughfall were carried out at seven forest sites, situated in close proximity within a nature reserve. Additionally, rainwater was sampled at three heathland sites for analysis of open-field precipitation and at three sites along an oak–birch-forest edge. Throughfall concentrations of most of the parameters analysed were significantly higher than open-field concentrations, especially with regard to electric conductivity, NH₄-N, K⁺, and KMnO₄-index. Ion concentrations in throughfall were the lowest in a 10-year-old stand of Betula pendula Roth. and Pinus sylvestris and in a Betula pubescens swamp forest and were highest beneath a stand of Pinus sylvestris. Except for Na⁺, Cl⁻, and NO₃⁻, ion concentrations in both throughfall and open-field precipitation increased during the growing season (May–October). In throughfall, Ca²⁺, Mg²⁺, K⁺, and Mn²⁺ were strongly correlated. Enrichment ratios between throughfall and open-field deposition varied among sites and elements and were the highest for K^‰+, Mg^2‰+, and Mn^2‰+. Estimates of canopy leaching indicated high leaching rates of K^‰+ and Mn^2‰+ and moderate leaching of Mg^2‰+. The contribution of foliar leaching to throughfall deposition was higher at the deciduous than at the coniferous stands.     

Genetic manipulation of cardiac Na+/Ca2+ exchange expression

The Na+/Ca2+ exchanger (NCX) is the primary Ca2+ extrusion mechanism in cardiomyocytes. To further investigate the role of NCX in excitation-contraction coupling and Ca2+ homeostasis, we created murine models with altered expression levels of NCX. Homozygous overexpression of NCX resulted in mild cardiac hypertrophy. Decline of the Ca2+ transient and relaxation of contraction were increased and the reverse mode of NCX was augmented. Overexpression also led to a higher susceptibility to ischemia-reperfusion injury and to a greater ability of NCX to trigger Ca2+-induced Ca2+ release. Furthermore, an increase in peak L-type Ca2+ current was observed suggesting a direct influence of NCX on L-type Ca2+ current. Whereas global knockout of NCX led to prenatal death, a recently generated cardiac-specific NCX knockout mouse was viable with surprisingly normal contractile properties. Expression levels of other Ca2+-handling proteins were not altered. Ca2+ influx in these animals is limited by a decrease of peak L-type Ca2+ current. An alternative Ca2+ efflux mechanism, presumably the plasma membrane Ca2+-ATPase, is sufficient to maintain Ca2+-homeostasis in the NCX knockout mice.     

Middle cerebral artery blood velocity during a valsalva maneuver in the standing position.

Occasionally, lifting of a heavy weight leads to dizziness and even to fainting, suggesting that, especially in the standing position, expiratory straining compromises cerebral perfusion. In 10 subjects, the middle cerebral artery mean blood velocity (V(mean)) was evaluated during a Valsalva maneuver (mouth pressure 40 mmHg for 15 s) both in the supine and in the standing position. During standing, cardiac output decreased by 16 +/- 4 (SE) % (P < 0.05), and at the level of the brain mean arterial pressure (MAP) decreased from 89 +/- 2 to 78 +/- 3 mmHg (P < 0.05), as did V(mean) from 73 +/- 4 to 62 +/- 5 cm/s (P < 0.05). In both postures, the Valsalva maneuver increased central venous pressure by approximately 40 mmHg with a nadir in MAP and cardiac output that was most pronounced during standing (MAP: 65 +/- 6 vs. 87 +/- 3 mmHg; cardiac output: 37 +/- 3 vs. 57 +/- 4% of the resting value; P < 0.05). Also, V(mean) was lowest during the standing Valsalva maneuver (39 +/- 5 vs. 47 +/- 4 cm/s; P < 0.05). In healthy individuals, orthostasis induces an approximately 15% reduction in middle cerebral artery V(mean) that is exaggerated by a Valsalva maneuver performed with 40-mmHg mouth pressure to approximately 50% of supine rest.     

Single-cell ATAC-seq: strength in numbers

Single-cell ATAC-seq detects open chromatin in individual cells. Currently data are sparse, but combining information from many single cells can identify determinants of cell-to-cell chromatin variation.     

Sincronologia e sincorologia dei boschi di faggio (Fagetalia sylvaticae) nell'Europa centrale

Abstract

From Preboreal to Late Atlantic times the landscape of Central Europe was covered by dense primeval woodlands, with the final stages of the natural succession differing from site to site, according to the climatic and soil conditions. There is evidence today, that the potential natural state of vegetation exists under the prevailing environmental conditions. In the same way, the Late- and Postglacial vegetation types and stages are supposed to have had floristical differences, or at least regional differences in the dominance or composition of their characteristic species. According to that, the periodic development phases of broad-leaved woodland with the successive immigration of the respective trees till the middle of the Atlantic period (until about 6000 yr. B.P.; POTT 1988) reflect the ecological balance under different climatic and edaphic conditions and are exclusively due to natural factors. In conifer-woods of Boreal and Pre-Atlantic fires, insects and diseases were the most important natural factors influencing vegetation in the past, and they also affected the virgin forests.So, during the long-lasting Postglacial process of woodland development - even before the Neolithic landnam periods - the changes in the appearance and the composition of plant communities resulted from habitat patterns and were modified by edaphic or biological factors. The vegetation of the EMW (oak-mixed Atlantic forests) varied in space and time with Quercus, Ulmus, Tilia, Fraxinus and Alnus as predominant species. The broad-leaved forests of the Atlantic period are often reflected in pollen diagrams derived from the Pleistocene loess and sand accumulations for example of the Northwest German lowlands. The abundance of Tilia, Quercus and Ulmus on loess soils and their restricted occurrence with dominance of Alnus on sandy soils with high ground-water levels is very significant (BAKELS 1978, 1982; POTT 1982).     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.     

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells

Background

Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P₂ in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane_. For controlled and reversible depletion of PtdIns(4,5)P₂, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.

Methods and Results

Expression and correct targeting of ECFP-PH-PLCδ1_, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P₂ in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K⁺ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P₂ was identified.

Conclusions

Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.