Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation

Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E₂ significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 ^waf1 and p27 ^Kip1, associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients.     

Effects of ajugarins and related neoclerodane diterpenoids on feeding behaviour of Leptinotarsa decemlineata and Spodoptera exigua larvae

Three naturally occurring ajugarins and seven semisynthetic derivatives of them, possessing different functionalities in the decalin part, together with two natural furoneoclerodane diterpenes, have been assessed as feeding behavior modifying agents of larvae of the generalist Spodoptera exigua and a specialist like Leptinotarsa decemlineata. Ajugarin I and some of its derivatives exhibited a significant antifeedant activity against larvae of S. exigua in both choice and no-choice assays. Conversely, the furoneoclerodane diterpenes only presented antifeedant activity against larvae of L. decemlineata. These results indicate that the biological action of the tested substances is strongly modulated by minimal structural variations, which are also responsible for the specificity of action.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion

The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 microg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart's exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation.     

Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.     

Bipolar anastomosis technique with removable instruments: an easy, fast, and reliable technique for vascular anastomosis

The interrupted suture technique is most commonly used for microsurgical vascular anastomosis. For several reasons (e.g., exposure of suture material to blood, time needed), many attempts have been made to find other solutions. This article describes a new means of performing a microsurgical vascular anastomosis. The aim of this study was to show the feasibility and possible advantages of this new technique. The basic components at work here are a modified cuff and electrically generated heat used to unite the vessel walls. In this way, both endothelial layers are adapted without manipulating the inside of the vessel or leaving behind foreign matter. Various energy/coagulation time settings were used to perform arterial anastomoses (n = 42) in an isogeneic abdominal aorta interposition model in the rat. The quality of anastomosis was evaluated at days 1, 10, 21, and 120. Immediately after the welding process all anastomoses (n = 42) were patent. No stenosis was found at any observation time. Anastomosis time ranged from 3 to 18 minutes (average, 11 minutes). This new technique permits a vascular anastomosis to be performed easily and reliably with a high patency rate. With this technique, the authors are convinced that a skilled surgeon can create a high-quality anastomosis in a fraction of the time needed to sew an anastomosis.