Learning from Alfred Wegener's pioneering field observations in West Greenland after a century of climate change.

The cryosphere in Greenland is currently undergoing strong changes. While remote sensing improves our understanding of spatial and temporal changes across scales, particularly our knowledge of conditions during the pre-satellite era is fragmented. Therefore, high-quality field data from that period can be particularly valuable to better understand changes of the cryosphere in Greenland at climate time scales. At Graz University, the last work-place of Alfred Wegener we have access to the extensive expedition results from their epic 1929-1931 expedition to Greenland. The expedition coincides with the warmest phase of the Arctic early twentieth century warm period. We present an overview of the main findings of the Wegener expedition archive and set it into context with further monitoring activities that occurred since, as well as the results from reanalysis products and satellite imagery. We find that firn temperatures have increased significantly, while snow and firn densities and have remained similar or decreased since. Local conditions at the Qaamarujup Sermia have changed strongly, with a reduction in length of more than 2 km, in thickness by up to 120 m and a rise in terminus position of approximately 300 m. The elevation of the snow line of the years 1929 and 1930 was similar to the one from the extreme years 2012 and 2019. Compared to the satellite era, we find that during the time of the Wegener expedition fjord ice extent was smaller in early spring and larger in late spring. We demonstrate that a well-documented snapshot of archival data can provide a local and regional context for contemporary climate change and that it can serve as the basis for process-based studies on the atmospheric drivers of glacier changes.

The pi-helix formation between Asp369 and Thr375 as a key factor in E1-E2 conformational change of Na+/K+-ATPase.

Molecular modeling of the H4-H5-loop of the alpha2 isoform of Na+/K+-ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short pi-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7 degrees and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+-ATPase activity.

Disentangling Drivers of Meteorological Droughts in the European Greater Alpine Region During the Last Two Centuries.

This study investigates the atmospheric drivers of severe precipitation deficits in the Greater Alpine Region during the last 210 years utilizing a daily atmospheric circulation type reconstruction. Precipitation deficit tends to be higher during periods with more frequent anticyclonic (dry) and less frequent cyclonic (wet) circulation types, as would be expected. However, circulation characteristics are not the main drivers of summer precipitation deficit. Dry soils in the warm season tend to limit precipitation, which is particularly the case for circulation types that are sensitive to a soil moisture-precipitation feedback. This mechanism is of specific relevance in explaining the major drought decades of the 1860s and 1940s. Both episodes show large negative precipitation anomalies in spring followed by increasing frequencies of circulation types sensitive to soil moisture precipitation feedbacks. The dry springs of the 1860s were likely caused by circulation characteristics that were quite different from those of recent decades as a consequence of the large spatial extent of Arctic sea ice at the end of the Little Ice Age. On the other hand, the dry springs of the 1940s developed under a persistent positive pressure anomaly across Western and Central Europe, triggered by positive sea surface temperatures in the western subtropical Atlantic.

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs.

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase.

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.

Chick embryo heart cells with high and low intracellular calcium concentrations respond differently to ouabain.

In cell cultures of 10-day-old chick embryo hearts, we found two cell populations, one with high intracellular calcium concentration ([Ca2+]i) of 116 +/- 34 nM (S.E., high [Ca2+]i cells, n = 154) and another one with low [Ca2+]i of 46 +/- 14 nM [Ca2+]i (S.E., low [Ca2+]i cells, n = 171), as revealed by fura-2 digital imaging fluorescence microscopy. The proportion of the high [Ca2+]i cells varied as a function of the cell density from 10-60% of all cells. Histochemical staining of the cells showed that cells with high and low [Ca2+]i did not represent differences between muscle and non-muscle cells. When the cells were exposed to different concentrations of ouabain, the high [Ca2+]i cells showed a half maximal effect at 2.10(-9) M ouabain, but only a small increase in [Ca2+]i of 30%. The low [Ca2+]i cells reached their half maximal increase in [Ca2+]i at 4.10(-8) M ouabain. A second increase in [Ca2+]i in this cell type was observed between 10(-6) and 10(-5) M ouabain. Toxic concentrations of ouabain produced an excessive increase in [Ca2+]i in low [Ca2+]i cells, whereas high [Ca2+]i cells showed morphological degeneration due to their higher sensitivity to ouabain. In conclusion, we demonstrate that chick embryo heart contains cells with high and low [Ca2+]i which show differences in the sensitivity of their sodium pumps to cardiac glycosides.

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex.

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.

Cultured chick-embryo heart cells respond differently to ouabain as measured by the increase in their intracellular Na+ concentration.

Using digital imaging microscopy with the sodium-sensitive fluorescent indicator sodium-binding benzofuran isophtalate (SBFI), we examined the cytosolic free sodium ion concentration ([Na+]i) in single chick-embryo heart cells. The distribution of the [Na+]i was homogeneous within one cell, but we found a wide cell to cell variation in the range of 3 to 18 mM [Na+]i. In contrast to former experiments showing a heterogeneity of chick-embryo heart cells with respect to their [Ca2+]i (Ahlemeyer et al. (1992) Eur. J. Biochem. 205, 269-275), we could not distinguish cell populations with different [Na+]i. We found a lognormal distribution of the resting [Na+]i with a median value of 8.8 mM with a standard deviation of 4.5 mM (n = 90). After the addition of varying concentrations of ouabain, we found a biphasic dose-response curve as measured by the increase in [Na+]i. Ouabain showed its half-maximal effect on the [Na+]i between 10(-9) M and 10(-8) M and at 4.3.10(-6) M under steady-state conditions. The finding of a heterogeneity of chick-embryo heart cells with respect to their ouabain-induced increase in [Na+]i is consistent with our previous observations of cells differing in their [Ca2+]i and in the sensitivity of their sodium pumps to cardiac glycosides.

Purification and properties of pyruvate kinase type M2 from rat lung.

(1) Pyruvate kinase type M2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of Mr = 224000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M2, except that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K0.5 value for phosphoenolpyruvate is 0.26 mM (nH = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (nH = 1.06). 1 microM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The Km value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The Km value for other nucleoside diphosphates increases in the order ADP less than GDP less than IDP less than UDP. (3) No evidence for an interconversion of pyruvate kinase type M2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047-1055) we suggest that the interconvertible form of pyruvate kinase type M2 may represent a separate form of the pyruvate kinase type M2 family.

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase.

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.

Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells.

Interactions between integral proteins of the plasma membrane and the cytoskeleton may be important for localizing certain membrane proteins in a nonrandom fashion at specialized domains of the cell surface. Here, we show that ankyrin, the key protein for the linkage of the erythrocyte anion exchanger (band 3) to the spectrin-based membrane cytoskeleton, is also present in kidney distal tubular cells where ankyrin is precisely colocalized with Na+,K+-ATPase. Both proteins are confined to the basolateral plasma membrane and are absent from the apical membrane, the junctional complex and the membrane surface that contacts the basal lamina. Purified Na+,K+-ATPase of sheep and pig kidney contains a binding site for erythrocyte ankyrin as demonstrated by immunoprecipitation experiments. A band 3-like binding site for ankyrin is likely, since binding of ankyrin to Na+,K+-ATPase could be inhibited in a competitive fashion by the isolated cytoplasmic domain of erythrocyte band 3.

Pulse pressure correlates in humans with a proscillaridin A immunoreactive compound.

Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.

Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.

The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.

Erythrosin 5'-isothiocyanate labels Cys549 as part of the low-affinity ATP binding site of Na+/K+-ATPase.

The high-affinity E1ATP site of Na+/K+-ATPase labeled with fluorescein 5'-isothiocyanate and its E2ATP site labeled with erythrosin 5'-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813-28821], reside on separate and adjacent catalytic alpha subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545-Leu-Gly-Phe-Cys549-His550. Hence, Cys549 is part of or close to the low-affinity E2ATP binding site of Na+/K+-ATPase.

Inactivation of phosphofructokinase in chicken hepatocytes by epinephrine.

The effects of epinephrine on phosphofructokinase activity in hepatocytes from fed chickens were studied in crude extracts and after partial purification. The mechanism of epinephrine action was elucidated by use of phentolamine and penbutulol. Phosphofructokinase activity was decreased in extracts of cells exposed to epinephrine when measured at sub-saturating as well as saturating concentrations of fructose 6-phosphate. This effect was still present after filtration through Sephadex G25 gel and a subsequent precipitation with ammonium sulfate. Half-maximal effects of epinephrine were found in a concentration range between 10(-8) and 10(-7) M epinephrine. The effect of epinephrine was unaffected by Ca2+ or the alpha-adrenergic antagonist phentolamine, and was abolished by the beta-adrenergic antagonist penbutulol. We assume that the effects of epinephrine on the activity of chicken liver phosphofructokinase are mediated by a cAMP-dependent mechanism which cannot be explained by concentration shifts of low-molecular-mass substances such as fructose 2,6-bisphosphate.

Endogenous ouabain and its binding globulin: effects of physical exercise and study on the globulin's tissue distribution.

Ouabain, that has been isolated from bovine adrenals and hypothalamus, is a new cardiotonic steroid hormone, which is either synthesized in the adrenals or stored there after it has absorbed from the diet. Little is known in vivo which events may lead to the release of ouabain into blood. Moreover, a binding protein for cardiotonic steroids exists in blood, which binds cardiac glycosides with high affinity. It may affect the action of endogenous ouabain on heart and circulation, but the physiological function of this protein is unclear. To realize, which physiological stimuli in vivo may affect blood concentrations of endogenous ouabain and which function the cardiotonic binding protein may have in modulating ouabain effects, the effect of physical exercise on endogenous ouabain was studied and the tissue distribution of its binding protein was investigated. We found that endogenous ouabain changes rapidly in blood upon physical exercise and behaves like expected for a hormone of circulation. The cardiotonic steroid binding globulin shows the highest concentration in the kidney, which suggests that sodium pumps of the kidney are protected against its inhibition by ouabain which would lead not only to natriuresis but also to a deleterious loss of glucose, amino acids and phosphate.

Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.

Ouabain, a new steroid hormone of adrenal gland and hypothalamus.

Ouabain has been isolated and identified as a constituent of human blood, bovine adrenal glands and hypothalamus. This water soluble inhibitor of the sodium pump (Na+/K+-ATPase) circulates in elevated concentrations in blood plasma of 50% of Caucasians with elevated blood pressure. It is released from adrenal cortical cells in tissue culture by angiotensin II. A ouabain antagonist, PST2238, lowers blood pressure in hypertensive rats. Hence, ouabain is most probably a new steroid hormone formed in adrenal glands and hypothalamus. Consistent therewith is the demonstration of a specific binding globulin for cardiac glycosides in blood plasma.