Activity Of Ca2 Research Articles

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8–10, expressed their maximum activities at pH10 and temperature range of 30–50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51–97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

RNA‐seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): Evidence for modulation by calcium pumps and apyrase activity

Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

Antioxidant and Inhibitory Properties of Essential Oil of Ocimum Gratissimum Against Extracellular Protease of Escherichia Coli

The oil of Ocimum gratissimum extracted by hydrodistillation was tested against the partally purified extracellular protease of E.coli The kinetics of the extracellular protease showed that the inhibition was competitive and that the extracellular protease has a km of 0.056 mg/ml in the presence of the inhibitor (oil of Ocimum gratissimum) and 0.08 mg/ml in its absence and with a Vmax of 1.66 vol (μmol/min)×10. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.53% and 0.72% respectively. The volatile oil of Ocimum gratissimum showed good antioxidant activity when used in scavenging DPPH radicals as compared to the BHT control. The oil has a mean activity value of 66.983 compared with 53.290 of BHT. The minimum percentage scavenging activity against DPPH is 50% for Ocimum gratissimum volatile oil as compared to 30% for BPH. The enzyme had an optimal pH of 6.5 and optimum temperature of 40C. Fe, Ca, Mn and Feare metal activators of the extracellular protease of E.coli.

The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration

Carbonic anhydrase IX is a hypoxia-induced transmembrane enzyme linked with solid tumors. It catalyzes the reversible hydration of CO2 providing bicarbonate ions for intracellular neutralization and protons for extracellular acidosis, thereby supporting tumor cell survival and invasiveness. CA IX is the only human CA isoform containing the proteoglycan (PG) domain in its extracellular part. The PG domain appears to enhance the catalytic activity of CA IX and mediate its binding to the extracellular matrix. Moreover, manipulation of the CA IX level by siRNA or overexpression modulates cell adhesion pathway so that in the presence of CA IX, cells display an increased rate of adhesion and spreading. Here we show that deletion of the PG domain as well as treatment with the PG-binding monoclonal antibody M75 can impair this CA IX effect. Accordingly, CA IX-expressing cells show more prominent and elongated maturing paxillin-stained focal contacts (FC) than CA IX-negative controls, proving the role of CA IX in cell spreading. However, during active cell movement, CA IX is relocalized to lamellipodia and improves migration via its catalytic domain. Thus, we examined the influence of CA IX on FC turnover in these structures. While the lamellipodial regions lacking CA IX display dash-like adhesions, the CA IX-enriched neighboring regions exhibit dynamic dot-like FCs. These results suggest that CA IX can promote initial adhesion through its PG domain, but at the same time it facilitates formation of nascent adhesions at the leading edge of moving cells. Thereby it may allow for transmission of large forces and enhanced migration rate, presumably through catalytic activity and impact of pHe on FC dynamics. Thus, we provide the first evidence that CA IX protein localizes directly in focal adhesion (FA) structures and propose its functional relationship with the proteins involved in the regulation of FC turnover and maturation.

The Calmodulin Regulator Protein, PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca(2+) binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca(2+) binding activity, which may modulate Ca(2+) binding to CaM by stabilizing an initial Ca(2+)-CaM complex or by electrostatically steering Ca(2+) to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca(2+) dynamics, we tested the hypothesis that it influences ligand-dependent Ca(2+) release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca(2+) release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca(2+) oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca(2+) binding to CaM greatly inhibit its effect on ATP-induced Ca(2+) release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca(2+) mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.

Synthesis, Crystal Structure and Antibacterial Activity of Ca(II) Complex with 2-(Phenylsulfonamido)acetic Acid

Synthesis, Crystal Structure and Antibacterial Activity of Ca(II) Complex with 2-(Phenylsulfonamido)acetic Acid

Porous calcium–manganese oxide microspheres for electrocatalytic oxygen reduction with high activity

A series of calcium–manganese oxides (Ca–Mn–O) were prepared through thermal decomposition of carbonate solid–solution precursors and investigated as electrocatalysts for oxygen reduction reaction (ORR). The synthesized crystalline Ca–Mn–O compounds, including perovskite-type CaMnO3, layered structured Ca2Mn3O8, post-spinel CaMn2O4 and CaMn3O6, presented similar morphologies of porous microspheres with agglomerated nanoparticles. Electrochemical results, surface analysis, and computational studies revealed that the catalytic activities of Ca–Mn–O oxides, in terms of onset potential, reduction current, and transferred electron number, depended strongly on both the surface Mn oxidation state and the crystallographic structures. Remarkably, the as-synthesized CaMnO3 and CaMn3O6 exhibited considerable activity and enabled an apparent quasi 4-electron oxygen reduction with low yield of peroxide species in alkaline solutions, suggesting their potential applications as cheap and abundant ORR catalysts.

Sulfocoumarins (1,2-Benzoxathiine-2,2-dioxides): A Class of Potent and Isoform-Selective Inhibitors of Tumor-Associated Carbonic Anhydrases

Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The sulfocoumarins were hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids, which thereafter bind to the enzyme in a region rarely occupied by other classes of inhibitors. The X-ray structure of one of these compounds in adduct with a modified CA II enzyme possessing two amino acid residues from the CA IX active site, allowed us to decipher the inhibition mechanism. The sulfonic acid was observed anchored to the zinc-coordinated water molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating substituted-1,2,3-triazole moieties were prepared by using click chemistry and showed low nanomolar inhibitory action against the tumor-associated isoforms CA IX and XII, being less effective against the cytosolic CA I and II.

Adoption Potential for Conservation Agriculture in Africa: A Newly Developed Assessment Approach (QAToCA) Applied in Kenya and Tanzania

AbstractConservation agriculture (CA) is often promoted as a set of cropping practices to reduce soil erosion and maintain soil fertility, while decreasing production costs and increasing crop yields. However, CA adoption is extremely low in Africa. Most investigations on constraints of its adoption leave out (i) the characteristics of CA as an emerging innovation and (ii) the wider institutional context. A comprehensive self‐assessment tool for a systematic evaluation of factors influencing the CA adoption process at the field, farm and regional scale in a variety of regional contexts in Africa is still lacking. In an attempt to fill this knowledge gap, this article presents the motivation, development and testing of a Qualitative expert Assessment Tool for CA adoption in Africa (QAToCA) and its application. QAToCA is directed to regional experts, research teams and managers of development projects with a focus on CA, and allows them to assess their CA activities along a systematic, expert‐based list of questions and criteria. Specifically, it aims at assessing the adoption potential of CA under the varied agro‐ecological, socio‐economic, cultural and institutional conditions of Africa as well as the specific supporting and hindering factors influencing this process.As an example, its application in Kenya and Tanzania identified a relatively high CA adoption potential. The following factors, however, are noticed to require further improvement: accessibility of markets for CA products and inputs; adaptation of machinery and seeds to the CA practices; introduction of quality implementation measures; and a renewed motivation (interest) among CA service providers. Copyright © 2012 John Wiley & Sons, Ltd.

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium

Calcium (Ca(2+)) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca(2+) level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca(2+) triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca(2+) agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U-2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell-cycle distribution, apoptotic cells in sub-G1 phase, reactive oxygen species (ROS), Ca(2+) levels, and mitochondrial membrane potential (ΔΨm ). Comet assay and 4'-6-diamidino-2-phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis-associated protein levels in U-2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U-2 OS cells. CECF-stimulated activities of caspase-8, caspase-9, and caspase-3, ROS, and Ca(2+) production, decreased ΔΨm levels of in U-2 OS cells. CECF increased protein levels of caspase-3, caspase-9, Bax, cytochrome c, GRP78, AIF, ATF-6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell-cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U-2 OS cells via ROS-modulated caspase-dependent and -independent pathways.

Spontaneous calcium waves in Bergman glia increase with age and hypoxia and may reduce tissue oxygen.

Glial calcium (Ca(2+)) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca(2+) waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O(2) tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca(2+) activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology.

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI-induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca2+-pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca2+-stimulated ATPase activity and α-myosin heavy chain mRNA levels were depressed, whereas β-myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca2+-release activity and mRNA levels for SR Ca2+-pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca2+-release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions.

PIP2 hydrolysis stimulates the electrogenic Na+–bicarbonate cotransporter NBCe1‐B and ‐C variants expressed in Xenopus laevis oocytes

Electrogenic Na(+)-bicarbonate cotransporter NBCe1 variants contribute to pH(i) regulation, and promote ion reabsorption or secretion by many epithelia. Most Na(+)-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP(2) on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP(2) (10 μM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO(3)(-)-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP(2) hydrolysis and endoplasmic reticulum (ER) Ca(2+) release. Stimulation by PIP(2) injection was mimicked by injecting IP(3), but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca(2+) with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca(2+) channels (SOCCs) to trigger a Ca(2+) influx mimicked the PIP(2)/IP(3) stimulation of the B and C variants. Activating the endogenous G(q) protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca(2+)-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH(i) studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH(i)-recovery rate from a CO(2)-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP(3)-induced stimulation of NBCe1-C. In summary, injecting PIP(2) stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP(3)/Ca(2+) that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP(2) regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP(2) on at least NBCe1-A, as well as an indirect stimulatory effect of IP(3)/Ca(2+) on the B and C variants.

Genotypic differences in physiological characteristics in the tolerance to drought and salinity combined stress between Tibetan wild and cultivated barley

Greenhouse pot experiments were conducted to investigate genotypic differences in response to individual and combined stresses of drought and salinity between Tibetan wild barley genotypes (XZ5, drought-tolerant; XZ16, salinity/aluminum tolerant) and cv. CM72 (salinity-tolerant). Either drought (D) or salinity (S) alone and in combination (D + S) stresses significantly decreased plant growth, chlorophyll content, net photosynthetic rate (Pn), maximal photochemical efficiency of PSII (Fv/Fm), water potential and osmotic potential, with the largest suppression under combined stress, and two wild genotypes showing more tolerance than CM72. Water use efficiency (WUE) increased significantly in XZ5 and XZ16 after D + S, but no significant change in CM72. XZ5 and XZ16 showed 30.9% and 12.1% higher K(+) level and 30.5% and 24.1% lower Na(+)/K(+) ratio in plants, compared with CM72, with increased metal nutrients as Ca, Fe and Mn under D + S. The peak accumulation in proline and glycine-beatine was recorded in combined stress with larger accumulation in two wild genotypes. Moreover, larger increases in the level of ASA and GSH, and the activities of Ca(2+)Mg(2+)-ATPase, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), guaiacol peroxidase (POD) and glutathione reductase (GR) under D + S vs control were observed in XZ5 and XZ16 than CM72, with less accumulation of H(2)O(2) and malondialdehyde. These results suggest that high tolerance to D + S stress of XZ5 and XZ16 is closely related to lower Na(+)/K(+) ratio and enhanced Ca(2+)Mg(2+)-ATPase, proline, glycine-beatine and WUE, and improved capacity of antioxidative performance to scavenge reactive oxygen species and thus suppressed level of lipid peroxidation.

Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase

The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(β1)-Flag+AMPKγ1(R70Q)), or of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(β1)-Flag+AMPK(γ1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(α1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111mM KCl, 30mM NaHCO(3), 4.7mM NaCl, and 2.25g/dl BSA) at a rate of 500μl/h. As a result, coexpression of AMPK(γR70Q) but not of AMPK(αK45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(γR70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute and fractional Na(+) and K(+) excretion were higher in ampk ( -/- ) than in ampk ( +/+ ) mice. According to micropuncture following a K(+) load, delivery of Na(+) to the early distal tubule but not delivery of K(+) to late proximal and early distal tubules was increased in ampk (-/-) mice. The upregulation of renal ROMK1 protein expression by acute K(+) load was more pronounced in ampk (-/-) than in ampk ( +/+ ) mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K(+) following an acute K(+) load.

Enhanced Ca2+ Entry, Ceramide Formation, and Apoptotic Death of Erythrocytes Triggered by Plumbagin

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis include mitochondrial depolarization, inactivation of NF-κB, and altered expression of anti- and proapoptotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)]i) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 μM) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca(2+)]i and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of 1 (2 μM) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.

Distinct Sensitivity of Slo1 Channel Proteins to Ethanol

Ethanol levels reached in circulation during moderate-to-heavy alcohol intoxication (50-100 mM) modify Ca(2+)- and voltage-gated K(+) (BK) channel steady-state activity, eventually altering both physiology and behavior. Ethanol action on BK steady-state activity solely requires the channel-forming subunit slo1 within a bare lipid environment. To identify the protein regions that confer ethanol sensitivity to slo1, we tested the ethanol sensitivity of heterologously expressed slo1 and structurally related channels. Ethanol (50 mM) increased the steady-state activities of mslo1 and Ca(2+)-gated MthK, the latter after channel reconstitution into phospholipid bilayers. In contrast, 50-100 mM ethanol failed to alter the steady-state activities of Na(+)/Cl(-)-gated rslo2, H(+)-gated mslo3, and an mslo1/3 chimera engineered by joining the mslo1 region encompassing the N terminus to S6 with the mslo3 cytosolic tail domain (CTD). Collectively, data indicate that the slo family canonical design, which combines a transmembrane 6 (TM6) voltage-gated K(+) channel (K(V)) core with CTDs that empower the channel with ion-sensing, does not necessarily render ethanol sensitivity. In addition, the region encompassing the N terminus to the S0-S1 cytosolic loop (missing in MthK) is not necessary for ethanol action. Moreover, incorporation of both this region and an ion-sensing CTD to TM6 K(V) cores (a design common to mslo1, mslo3, and the mslo1/mslo3 chimera) is not sufficient for ethanol sensitivity. Rather, a CTD containing Ca(2+)-sensing regulator of conductance for K(+) domains seems to be critical to bestow K(V) structures, whether of TM2 (MthK) or TM6 (slo1), with sensitivity to intoxicating ethanol levels.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes.

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Synthesis and characterization of phenolic Mannich bases and effects of these compounds on human carbonic anhydrase isozymes I and II

Mannich bases 2a-f derived from 3,4-dimethylphenol (1), formaldehyde and different amines are prepared and subjected to spectral (IR, 1H and 13C NMR) and elemental analyses. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with 1 and synthesized Mannich bases 2a-f and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. In relation to hydratase and esterase activities of the half maximal inhibitory concentration (IC50) and the inhibition equilibrium constants (Ki)values were determined. Only two compounds (2a and 2e)exhibit weak hCA II inhibitory effects on esterase activity. IC50 and Ki values for 2a and 2e with respect to esterase activity of hCA II are0.88 × 103 and 6.3–7.6 µM and 0.44 × 103 and 19.0–96.4 µM,respectively. On the contrary, compounds 2b and 2d might be used as CA activators due to increasing esterase activity of hCA I and hCA II isozymes.