Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

Large conductance Ca²⁺-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.     

GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja,Plays Differential Roles in Plant Responses to Bicarbonate,Salt and Osmotic Stresses

Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca²⁺ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na⁺, K⁺) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca²⁺-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.     

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.     

Long-Term Safety of Repeated Blood-Brain Barrier Opening via Focused Ultrasound with Microbubbles in Non-Human Primates Performing a Cognitive Task

Focused Ultrasound (FUS) coupled with intravenous administration of microbubbles (MB) is a non-invasive technique that has been shown to reliably open (increase the permeability of) the blood-brain barrier (BBB) in multiple in vivo models including non-human primates (NHP). This procedure has shown promise for clinical and basic science applications, yet the safety and potential neurological effects of long term application in NHP requires further investigation under parameters shown to be efficacious in that species (500kHz, 200–400 kPa, 4–5μm MB, 2 minute sonication). In this study, we repeatedly opened the BBB in the caudate and putamen regions of the basal ganglia of 4 NHP using FUS with systemically-administered MB over 4–20 months. We assessed the safety of the FUS with MB procedure using MRI to detect edema or hemorrhaging in the brain. Contrast enhanced T1-weighted MRI sequences showed a 98% success rate for openings in the targeted regions. T2-weighted and SWI sequences indicated a lack edema in the majority of the cases. We investigated potential neurological effects of the FUS with MB procedure through quantitative cognitive testing of’ visual, cognitive, motivational, and motor function using a random dot motion task with reward magnitude bias presented on a touchpanel display. Reaction times during the task significantly increased on the day of the FUS with MB procedure. This increase returned to baseline within 4–5 days after the procedure. Visual motion discrimination thresholds were unaffected. Our results indicate FUS with MB can be a safe method for repeated opening of the BBB at the basal ganglia in NHP for up to 20 months without any long-term negative physiological or neurological effects with the parameters used.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto-oncogene expression and the behaviour of myeloid leukemia cells in vitro

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c-fms, c-fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c-fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.     

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Summary Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.