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A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

Doxorubicin (DXR) is a widely used and efficient anticancer drug. However, its application is limited by the risk of severe cardiotoxicity. Impairment of cardiac high-energy phosphate homeostasis is an important manifestation of both acute and chronic DXR cardiotoxic action. Using the Langendorff model of the perfused rat heart, we characterized the acute effects of 1-h perfusion with 2 or 20 [micro]M DXR on two key kinases in cardiac energy metabolism, creatine kinase (CK) and AMP-activated protein kinase (AMPK), and related them to functional responses of the perfused heart and structural integrity of the contractile apparatus as well as drug accumulation in cardiomyocytes. DXR-induced changes in CK were dependent on the isoenzyme, with a shift in protein levels of cytosolic isoenzymes from muscle-type CK to brain-type CK, and a destabilization of octamers of the mitochondrial isoenzyme (sarcometric mitochondiral CK) accompanied by drug accumulation in mitochondria. Interestingly, DXR rapidly reduced the protein level and phosphorylation of AMPK as well as phosphorylation of its target, acetyl-CoA-carboxylase. AMPK was strongly affected already at 2 p,M DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate but became significant at 20 p,M DXR. Only at 2 [micro]M DXR did upregulation of brain-type CK compensate for inactivation of other isoenzymes. These results suggest that an impairment of kinase systems regulating cellular energy homeostasis is involved in the development of DXR cardiotoxicity. creatine kinase; adenosine 5'-monophosphate-activated protein kinase; anthracyclines; cardiac energetics; cardiotoxicity

The standard Äkta Explorer high-performance liquid chromatography (HPLC) system has limitations for the automation of multidimensional protein purification. Here, we describe simple modifications that allow for automated multidimensional purification protocols to extend the possibilities of the Äkta three-dimensional purification kit in terms of column number, flexibility of volumes stocked for re-injection of samples, and available choice of buffers. These modifications do not preclude the use of standard one-dimensional purification protocols. Additionally, we demonstrate a technology for encrypted full remote control of the machine over the Internet by cost-effective use of standard asymmetric digital subscriber line (ADSL) that enables direct remote interaction with the machine without preventing local control. A 4-column purification scheme, including equilibration and cleaning in place (CIP) procedures, was implemented on such a system. It significantly increased reproducibility and shortened processing time by 85%, as compared with manual operation, thus allowing for automated protein purification overnight.

• Little is known about the level of climate change to which societies can adapt to. • Limits to adaptation are more likely to be reached and breached in the future. • Limits can be viewed from a justice informed perspective and coupled with modelling. • Assessing limits is central to national and international adaptation policymaking. Anthropogenic climate change is already causing dangerous and widespread disruptions in global ecological and social systems and affects the lives of billions of people around the world. Even with scaled-up risk management and adaptation, the limits of adaptation will often be reached. Currently, very little is known about the degree to which societies can adapt to climate change, and where and when limits to adaptation will be reached. In this paper, we conceptualize adaptation limits through a novel methodological framework, assess adaptation limits along adaptation pathways, and propose a research strategy for empirical and model-based limits assessments based on biophysical and socio-economic data. Assessing limits is central to national and international adaptation policymaking. More efficient adaptation can also help climate mitigation efforts. [ABSTRACT FROM AUTHOR]

The backbone conformation of a peptide or protein defines its circular dischroism spectrum in the range 190-240 nm, providing a uniquely convenient tool for assigning secondary structure. A two-state helix-coil model underlies all calculation all calculation of fractional helicities FH from CD spectra of helical polypeptides.

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CRT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog [beta]-guanidinopropionic acid ([beta]-GPA). Creatine transport in control hearts followed saturation kinetics with a [K.sub.m] of 70 [+ or -] 13 mM and a [V.sub.max] of 3.7 [+ or -] 0.07 nmol * [min.sup.-1] * g wet [wt.sup.-1]. Creatine supplementation significantly decreased the [V.sub.max] of the CrT (2.7 [+ or -] 0.17 nmol * [min.sup.-1] * g wet [wt.sup.-1]). This was matched by an ~35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed [beta]-GPA exhibited a >80% decrease in tissue creatine and increase in [beta]-GP[A.sub.total]. The [V.sub.max] of the CrT was increased (6.0 [+ or -] 0.25 nmol * [min.sup.-1] * g wet [wt.sup.-1]) and the [K.sub.m] decreased (39.8 [+ or -] 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool. creatine transport(er); creatine feeding; creatine depletion; isolated perfused heart; cell-surface biotinylation

The availability of attractive complementary products offered by a nearby company positively affects other companies in the value network of a winter sports destination. We aim to illustrate empirically the positive effects of the campaign of a local ski lift company in Switzerland on other companies in the same value network. For the first time in tourism research, the synthetic control method is used to investigate the impact of a new pricing strategy. In this case, the company's practice of radically discounting the prices of its seasonal lift passes had a positive impact on accommodation businesses in the same winter sports destination. The impact amounts to about 35% additional overnight stays by domestic tourists per winter season that is a seasonal increase of 32,000 overnight stays. However, the ski lift company concluded that the campaign had been unsuccessful. We therefore discuss the difficulties of such a new pricing strategy and emphasize the importance of cooperation between independent companies in the wider destination area. [ABSTRACT FROM AUTHOR]

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 [+ or -] 9.4 [micro] M and maximal velocity value 17.3 [+ or -] 3.1 pmol * [min.sup.-1] * mg vesicle [protein.sup.-1]), which cotransports Cr into skeletal muscle together with [Na.sup.+] and [Cl.sup.-] ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and N[H.sub.2]-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of ~58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [[sup.14]C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies. plasma membrane giant vesicles; sarcolemma; mitochondria; subcellular compartmentation of creatine; bioenergetics