Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells

Fe₃O₄/Ag core/shell nanoparticles functionalized with the free amino (NH₂) functional groups (Fe₃O₄/Ag-NH₂) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3′-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD – Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the V_NP:V_AB ratio, where V_NP and V_AB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when V_NP:V_AB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the V_NP:V_AB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe₃O₄/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

Activation of the contractile system of insect fibrillar muscle at very low concentrations of Mg2+ and Ca2+

The mechanical properties of single fibres and fibre bundles of glycerinated dorsal longitudinal muscle from lethocerus maximus were investigated in ATP-salt solutions containing only trace concentrations of free Ca²⁺ (pCa>9). A reduction in the magnesium concentration (pMg 7) resulted in an increase in the instantaneous stiffness of glycerinated insect flight muscle fibres, though very little accompanying tension was developed. Stiffness was measured either using small amplitude sinusoidal length changes of high frequency (1 kHz) or rapid rectangular form length changes. The ratio of stiffness to tension in solutions free of added magnesium and calcium was equal to or greater than that obtained from the tissue in the rigor state, and much larger than that obtained in the presence of both magnesium and calcium. Extrapolation of the linear part of the change-tension relationship (obtained during rapid length changes completed within 0.3 ms) back to zero tension indicated that the elastic elements of attached crossbridges were less extended under conditions of Mg²⁺-deprivation than during Ca-activation in Mg²⁺-rich solution. Following a quick stretch a delayed tension development similar to that obtained in the presence of magnesium and calcium ions was observed. The rise in tension was delayed with respect to the accompanying rise in stiffness and reached a peak value after about 2 s. Similar tension transients followed a subsequent release. The possibility that an unusually slow corss-bridge cycle might be responsible for these slow transients was suggested by the finding that the fibres showed a very low ATPase activity under these conditions which could be slightly activated by stretches. On increasing the free Ca²⁺ concentration during magnesium deprivation, the time course of the stretch induced tension transients became faster, while stiffness and the steady state tension rose to reach a high tension state at aboutpCa 6.5.This work was supported by a grant from the Deutsche Forschungs-gemeinschaft (RU 154/12). One of us (P.J.G.) was supported by the Royal Society European Science Exchange Programme     

Facile synthesis of magnetic-/pH-responsive hydrogel beads based on Fe3O4 nanoparticles and chitosan hydrogel as MTX carriers for controlled drug release

In the present study, methotrexate (MTX)-encapsulated magnetic-/pH-responsive hydrogel beads based on Fe₃O₄ nanoparticles and chitosan were successfully prepared through a one-step gelation process, which is a very facile, economic and environmentally friendly route. The developed hydrogel beads exhibited homogeneous porous structure and super-paramagnetic responsibility. MTX can be successfully encapsulated into magnetic chitosan hydrogel beads, and the drug encapsulation efficiency (%) and encapsulation content (%) were 93.8 and 6.28%, respectively. In addition, the drug release studies in vitro indicated that the MTX-encapsulated magnetic chitosan hydrogel beads had excellent pH-sensitivity, 90.6% MTX was released from the magnetic chitosan hydrogel beads within 48 h at pH 4.0. WST-1 assays in human liver hepatocellular carcinoma cells (HepG2) demonstrated that the MTX-encapsulated magnetic chitosan hydrogel beads had good cytocompatibility and high anti-tumor activity. Therefore, our results revealed that the MTX-encapsulated magnetic chitosan hydrogel beads would be a competitive candidate for controlled drug release in the area of targeted cancer therapy in the near future.     

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP ₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP ₃-sensitive as well as InsP ₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP ₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP ₃ can be clearly documented using this experimental approach.     

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse

We compared the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), as measured with the fluorescent Ca²⁺ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K²⁺ depolarization and by agonists. Resting [Ca²⁺]_i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca²⁺ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca²⁺]_i increase, the agonist stimulation induced a higher force development than the K⁺ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca²⁺]_o. Our results indicate that the Ca²⁺ threshold for force development was about 150% of the resting [Ca²⁺]_i in both cell types. The resting [Ca²⁺]_i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca²⁺ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca²⁺ sensitivity during contraction. The desensitization to Ca²⁺ was specific for phasic muscle, in which it occurred as an early, timeand Ca²⁺-dependent process that was partially reversible.     

Mechanism of suppression of physiological functions in hypothermia and means for their stimulation without body warming

Cold-suppressed thermoregulatory reactions and respiration in rats in deep hypothermia (rectal body temperature (25–22°C) were shown to be stimulated by injecting disodium ethylenediaminetetraacetic acid (EDTA) solution into the blood stream of cold rats at a dose of 16.5 mg/100 g (0.0045 mmol/100 g). EDTA binds Ca²⁺ ions in the blood, forming complexes. Increases in cold shivering and pulmonary respiration (by 5 min after the start of administration) coincided with a reduction in the blood Ca²⁺ concentration by 42–45% of normal. By 15 min after the start of the EDTA injection, the blood Ca²⁺ concentration returned to the normal level present in cold rats before EDTA treatment. This was accompanied by suppression of cold shivering and pulmonary respiration. Repeated injection of EDTA into the blood stream produced a new drop in blood Ca²⁺ and repeated stimulation of cold shivering and pulmonary respiration. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 11, pp. 1373–1381, November, 2006.     

TrkB inhibition as a therapeutic target for CNS-related disorders

The interaction of brain-derived neurotrophic factor (BDNF) with its tropomyosin-related kinase receptor B (TrkB) is involved in fundamental cellular processes including neuronal proliferation, differentiation and survival as well as neurotransmitter release and synaptic plasticity. TrkB signaling has been widely associated with beneficial, trophic effects and many commonly used psychotropic drugs aim to increase BDNF levels in the brain. However, it is likely that a prolonged increased TrkB activation is observed in many pathological conditions, which may underlie the development and course of clinical symptoms. Interestingly, genetic and pharmacological studies aiming at decreasing TrkB activation in rodent models mimicking human pathology have demonstrated a promising therapeutic landscape for TrkB inhibitors in the treatment of various diseases, e.g. central nervous system (CNS) disorders and several types of cancer. Up to date, only a few selective and potent TrkB inhibitors have been developed. As such, the use of crystallography and in silico approaches to model BDNF-TrkB interaction and to generate relevant pharmacophores represent powerful tools to develop novel compounds targeting the TrkB receptor.     

Modification of HSP proteins and Ca2+ are responsible for the NO-derived peroxynitrite mediated neurological damage in PC12 cell

Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca²⁺]_i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca²⁺]_i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca²⁺ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge.     

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.     

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca²⁺ and Mg²⁺ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca²⁺ and Mg²⁺ transport were investigated. Transepithelial voltages (PD_te) and transepithelial ion net fluxes (J _Na, J _Cl, J _K, J _Ca, J _Mg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca²⁺ and Mg²⁺ reabsorption when active NaCl reabsorption, and thus PD_te, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca²⁺ and Mg²⁺ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net reabsorption. In view of the observed small effect of PTH on passive Ca²⁺ and Mg²⁺ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca²⁺ and Mg²⁺ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on J _Ca and J _Mg measured. In the absence of PD_te and under symmetrical conditions, no Ca²⁺ and Mg²⁺ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca²⁺ and Mg²⁺ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased J _Ca and J _Mg, whereas only a small increase in PD_te was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca²⁺ and Mg²⁺ transport in the mouse cTAL by increasing the transepithelial driving force for Ca²⁺ and Mg²⁺ reabsorption through hormone-mediated PD_te alterations and by modifying the passive permeability for Ca²⁺ and Mg²⁺ of the epithelium, very probably at the level of the paracellular shunt pathway.     

Short-and long-term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C

In Xenopus oocytes injected with rat brain RNA, serotonin (5HT) and acetylcholine (ACh) evoke membrane responses through a common biochemical cascade that includes activation of phospholipase C, production of inositol 1,4,5-trisphosphate (Ins1,4,5-P ₃), release of Ca²⁺ from intracellular stores, and opening of Ca-dependent Cl^– channels. The response is a Cl^– current composed of a transient component (5HT₁ or ACh₁) and a slow, long-lasting component (5HT₂ or ACh₂). Here we show that only the fast, but not the slow, component of the response is subject to desensitization that follows a previous application of the transmitter. The recovery of 5HT₁ from desensitization is biphasic, suggesting the existence of two types of desensitization: short-term desensitization (STD), which lasts for less than 0.5 h; and long-term desensitization (LTD) lasting for up to 4 h. The desensitization between 5HT and ACh is heterologous and long-lasting. We searched for (a) the molecular target and (b) the cause of desensitization.(a) Pre-exposure to 5HT does not reduce the response evoked by intracellular injection of Ca²⁺ and by Ca²⁺ influx. Cl^– current evoked by intracellular injection of Ins1,4,5-P ₃ was reduced shortly after application of 5HT, but fully recovered 30 min later. Thus, the Cl^– channel is not a target for desensitization. Neither Ins1,4,5-P ₃ receptor nor the Ca²⁺ store is a target of LTD but they may be the targets of STD. (b) Ca²⁺ injection did not inhibit the 5HT response, suggesting that Ca²⁺ is not a sole cause of STD or LTD. An activator of protein kinase C, -phorbol 12,13-dibutyrate (PhoOBt₂), is known to inhibit the 5HT response, but this inhibition had completely subsided 30 min after washout of PhoOBt₂. A protein kinase inhibitor H-7 did not prevent LTD. Thus, protein kinase C does not appear to be the cause of LTD, but its role in STD cannot be ruled out at present. Injection of Ins1,4,5-P ₃ caused a dose-dependent, long-lasting inhibition of subsequent Ins1,4,5-P ₃ and 5HT responses. Desensitization induced by Ins1,4,5-P ₃ affected both 5HT₁ and 5HT₂. Thus, Ins1,4,5-P ₃ is a possible cause of STD and LTD, but non-specific effects cannot be ruled out at present. The self-desensitization of Ins1,4,5-P ₃ response was reversed by PhoOBt₂ suggesting a role for protein kinase C in recovery from desensitization.D. Singer and R. Boton are to be considered as equal first authors of this paper     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

A mechanism for the inactivation of Ca2+/calmodulin-dependent protein kinase II during prolonged seizure activity and its consequence after the recovery from seizure activity in rats in vivo

Seizure is a form of excessive neuronal excitation and seizure-induced neuronal damage has profound effects on the prognosis of epilepsy. In various seizure models, the inactivation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) occurs during seizure activity preceding neuronal cell death. CaMKII is a multifunctional protein kinase enriched in the brain and involved in various ways the regulation of neuronal activity. CaMKII inactivation during seizure activity may modify neuronal cell survival after seizure. However, the mechanism for CaMKII inactivation and its consequence after seizure recovery remain to be elucidated yet. In the present study, we employed a prolonged seizure model by systemic injection of kainic acid into rats and biochemically examined the activity state of CaMKII. In status epilepticus induced by kainic acid, not only the inactivation of CaMKII in brain homogenate, but also a shift in the distribution of CaMKII protein from the soluble to particulate fraction occurred in both hippocampus and parietal cortex. The particulate CaMKII showed a large decrease in the specific activity and a concurrent large increase in the autophosphorylation ratio at Thr-286 (alpha) and at Thr-287 (beta). In contrast, the soluble CaMKII showed normal or rather decreased specific activity and autophosphorylation ratio. After 24 h of recovery from kainic acid-induced status epilepticus, all such changes had disappeared. On the other hand, the total amount of CaMKII was decreased by 35% in hippocampus and 20% in parietal cortex, but the existing CaMKII was indistinguishable from those of controls in terms of the autonomous activity ratio, specific activity and autophosphorylation ratio. Thus, CaMKII inactivation in kainic acid-induced status epilepticus seems to be derived not from simple degradation of the enzyme, but from the formation of the autophosphorylated, inactivated and sedimentable CaMKII. Such a form of CaMKII may be important during pathological conditions in vivo in preventing excessive CaMKII activation due to Ca2+ overload.     

Human epidermal growth factor receptor 2, Na+/H+ exchanger regulatory factor 1, and breast cancer susceptibility gene-1 as new biomarkers for familial breast cancers

The aim of this study is to evaluate the analysis of markers related with progression, to further characterize familial breast cancers. Here, we investigated the expression of breast cancer susceptibility gene-1, hypoxia-inducible factor-1α, vascular endothelial growth factor receptor 1, and Na+/H+ exchanger regulatory factor 1 in 187 microarrayed breast carcinomas from 94 familial and 93 sporadic breast cancer patients by immunohistochemical staining. Furthermore, the expression levels of these biomarkers were compared with triple-negative phenotype. Familiarity was significantly associated with younger age (P < .000), higher tumor grade (P = .038), negative estrogen receptor hormonal status (P = .036), and high proliferative activity (P = .029). The familial cancers were immunonegative for membranous Na+/H+ exchanger regulatory factor 1 expression compared with sporadic cancers (P = .001); notably, vascular endothelial growth factor receptor 1 staining correlated with cytoplasmic Na+/H+ exchanger regulatory factor 1 expression in familial tumors (P = .009). In multivariate analysis, the "new biomarkers," including negative human epidermal growth factor receptor 2 status (odds ratio, 4.538; 95% confidence interval, 1.756-11.728), negative membranous Na+/H+ exchanger regulatory factor 1 expression (odds ratio, 7.686; 95% confidence interval, 1.876-31.483) and positive nuclear breast cancer susceptibility gene-1 (odds ratio, 0.3982; 95% confidence interval, 0.169-0.936), significantly correlated with family history of breast cancer. We hypothesize that the evaluation of human epidermal growth factor receptor 2, Na+/H+ exchanger regulatory factor 1, and breast cancer susceptibility gene-1 could be clinically useful to identify familial breast tumors and to select patients candidate to breast cancer susceptibility genes 1/2 gene sequencing.     

Adrenergic and cholinergic inhibition of Ca2+ channels mediated by different GTP-binding proteins in rat sympathetic neurones

Effects of acetylcholine (ACh) and noradrenaline (NA) on voltage-gated ion channels of sympathetic neurones acutely dissociated from rat superior cervical ganglion (SCG) were examined using the whole-cell voltage-clamp technique. Depolarizing voltage steps elicited two types of low- and high-voltage-activated (LVA and HVA) Ca²⁺ currents. Pressure applications of ACh and NA produced concentration-dependent inhibition of the HVA Ca²⁺ current without affecting the LVA Ca²⁺ current. The inhibitory action of ACh on the Ca²⁺ current was blocked by a muscarinic antagonist, atropine. The action of NA was suppressed by an ₂-adrenergic antagonist, yohimbine, but not by an ₁-adrenergic antagonist, prazosin. Delayed rectifying outward K⁺ currents and inward rectifying K⁺ current were not affected by either ACh or NA. Tetrodotoxin-sensitive and -insensitive Na⁺ currents also remained unaffected under actions of ACh and NA. When recorded with electrode containing guanosine-5-O-(3-thiotriphosphate) (GTP--S), the inhibitory actions of ACh and NA on Ca²⁺ currents became irreversible. After treatment of SCG neurones with pertussis toxin, the inhibitory action of ACh on the Ca²⁺ current was almost completely abolished, whereas the action of NA was only partially reduced. The results suggest that ACh and NA differentially inhibit the HVA Ca²⁺ current via different G proteins coupling muscarinic and ₂-adrenergic receptors to Ca²⁺ channels in rat SCG neurones.     

Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells

Abstract

Context: Metformin is widely used for treatment of type 2 diabetes and has a potential application on the treatment of inflammation and cancer. Phosphatase and tensin homolog (PTEN) plays a critical role in cancer cell growth and inflammation; however, precise mechanisms remain unclear.

Objective: We aimed to investigate the possible mechanisms of how PTEN regulates metformin against cell growth and inflammation.

Materials and methods: We established PTEN knockdown in RAW264.7 murine macrophages (shPTEN cells) to detect inflammatory mediators using commercial kits, production of reactive oxygen species (ROS) by flow cytometry, cell growth by MTT assay and phosphorylated levels of signal molecules by western blot.

Results: The shPTEN cells had a significant large amount of inflammatory mediators, such as inducible nitric oxide synthase (iNOS)/nitric oxide (NO) and cyclooxygenase-2 (COX-2)/prostaglandin E₂ (PGE₂); and also elevated the production of ROS and increased cell proliferation. These effects were accompanied with the activation of Akt and p38 mitogen-activated protein kinase (MAPK), and the inactivation of an AMP-activated protein kinase (AMPK) activator and extracellular signal-regulated kinase 1/2. Pretreatment with metformin not only blocked these inflammatory mediators, but also caused growth inhibition induced by significant apoptosis. Furthermore, inactivation of Akt, blockade of ROS generation and independence of activations of AMPK and MAPK by metformin were also observed.

Conclusion: Macrophages with PTEN deficiency developed a continuous inflammatory microenvironment, which further aggravated tumor cell growth. Moreover, metformin affected PTEN-deficient cells dependent of inhibition of ROS production and Akt activation against enlarged inflammatory mediators and/or cell growth in shPTEN cells.