Activity Of Ca2 Research Articles

The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives.

Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO2 to HCO3− and H+. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences.

Calcium-activated chloride current determines action potential morphology during calcium alternans in atrial myocytes.

Cardiac alternans--periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic calcium transient (CaT) amplitude--is a high risk indicator for cardiac arrhythmias and sudden cardiac death. However, it remains an unresolved issue whether beat-to-beat alternations in intracellular Ca(2+) ([Ca(2+)]i ) or AP morphology are the primary cause of pro-arrhythmic alternans. Here we show that in atria AP alternans occurs secondary to CaT alternans. CaT alternans leads to complex beat-to-beat changes in Ca(2+)-regulated ion currents that determine alternans of AP morphology. We report the novel finding that alternans of AP morphology is largely sustained by the activity of Ca(2+)-activated Cl(-) channels (CaCCs). Suppression of the CaCCs significantly reduces AP alternans, while CaT alternans remains unaffected. The demonstration of a major role of CaCCs in the development of AP alternans opens new possibilities for atrial alternans and arrhythmia prevention. Cardiac alternans, described as periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias and sudden cardiac death. We investigated mechanisms of cardiac alternans in single rabbit atrial myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. Beat-to-beat alternations of AP morphology and CaT amplitude revealed a strong quantitative correlation. Application of voltage clamp protocols in the form of pre-recorded APs (AP-clamp) during pacing-induced CaT alternans revealed a Ca(2+)-dependent current consisting of a large outward component (4.78 ± 0.58 pA pF(-1) in amplitude) coinciding with AP phases 1 and 2 that was followed by an inward current (-0.42 ± 0.03 pA pF(-1); n = 21) during AP repolarization. Approximately 90% of the initial outward current was blocked by substitution of Cl(-) ions or application of the Cl(-) channel blocker DIDS identifying it as a Ca(2+)-activated Cl(-) current (ICaCC). The prominent AP prolongation at action potential duration at 30% repolarization level during the small alternans CaT was due to reduced ICaCC. Inhibition of Cl(-) currents abolished AP alternans, but failed to affect CaT alternans, indicating that disturbances in Ca(2+) signalling were the primary event leading to alternans, and ICaCC played a decisive role in shaping the beat-to-beat alternations in AP morphology observed during alternans.

Development of certain new 2-substituted-quinazolin-4-yl-aminobenzenesulfonamide as potential antitumor agents

Carbonic anhydrases (CA I, II, IX and XII) are known to be highly expressed in various human malignancies. CA IX is overexpressed in colorectal cancer specifically in hereditary nonpolyposis colorectal cancer. Inhibition of CA activity by small molecular CA inhibitor like sulphonamides, sulphonamide derivative (SU.D2) or HIF1a inhibitor Chetomin leads to inhibition of tumorigenesis. Eighteen new quinazolin-4-sulfonamide derivatives were prepared and characterized by means of IR, NMR and mass spectra. Certain selected derivatives were tested for their ability to inhibit four isoforms of the metalloemzyme CA, namely, CA I, CA II, CA IX and CA XII. Compound 3c was found to be highly effective in inhibiting the cancer cell proliferation. 3c decreased cell viability of human HT-29 cells in dose and time dependent manner and with IC50 of 5.45 μM. Moreover, it was tested on metastatic colon cancer cell SW-620 where it was found to be equally effective on human SW-620 cells. This novel compound inhibited the CA IX and CA XII protein expression in HT-29 cells without affecting CA I and CA II expression. These findings indicate that 3c inhibits cellular proliferation in two human colon cancer cells by specifically targeting the CA IX and CA XII expression.

Cell Penetrating Peptide-Mediated Caveolae-Dependent Activation of Lung Endothelial Nitric Oxide Synthase.

Cell penetrating peptides can be used as therapeutic agents via modulation of selective cell functions. Nitric oxide (NO) generated by vascular endothelial NO synthase (eNOS) plays a critical role in the NO/ cyclic guanosine 5'-monophosphate (cGMP)-mediated pulmonary vascular function. Here we examined whether internalization of a fifteen amino acid (KRFNSISCSSWRRKR) synthetic peptide (P3) enhances the catalytic activity of eNOS via caveolae/eNOS dissociation leading to NO release and increased cGMP production in pulmonary artery endothelial cells (EC). ECs were treated with varying concentrations of P3 and used to monitor internalization, isolation of caveolae-enriched fraction, the catalytic activity of eNOS, NO/cGMP production, and intracellular Ca(2+) release. Confocal images show timedependent internalization of P3 in EC. Treatment of EC with P3, but not scrambled P3, increased the catalytic activity of eNOS in a dose-dependent manner without change in eNOS expression or phosphorylation. Treatment of EC with P3 stimulated intracellular Ca(2+) release, increased the catalytic activity of phospatidylinsositide 3 kinase (PI3K) and resulted in eNOS/caveolae-1 (Cav-1) dissociation leading to translocation of eNOS to intracellular compartment in EC. P3- mediated activation of eNOS was abolished by intracellular Ca(2+) chelator 1,2-bis(2-aminophenooxy)ethane-N,N,N',N'- tertraacetic acid-AM (BAPTA-AM), PI3K inhibition, or by siRNA-mediated Cav-1 suppression. These results demonstrate that exogenous peptide consisting of cationic amino acids can internalize and enhance the catalytic activity of eNOS via modulation of caveolar signaling and intracellular Ca(2+) release in EC.

The Relationship of Oxidation Sensitivity of Red Blood Cells and Carbonic Anhydrase Activity in Stored Human Blood: Effect of Certain Phenolic Compounds

It has been reported that many modifications occur with the increase of oxidative stress during storage in erythrocytes. In order to delay these negative changes, we evaluated whether the addition of substances likely to protect antioxidant capacity in stored blood would be useful. Therefore, we investigated the effects of resveratrol, tannic acid, and caffeic acid in lipid peroxidation and antioxidant capacity of erythrocytes in stored blood. Donated blood was taken into four CPD containing blood bags. One bag was used as the control, and the others were supplemented with caffeic acid (30 μg/mL), resveratrol (30 μg/mL), and tannic acid (15 μg/mL), respectively. Erythrocyte lipid peroxidation, sensitivity to oxidation, glutathione levels and carbonic anhydrase, glutathione peroxidase, and catalase activities were measured on days 0, 7, 14, 21, and 28. In the control group, erythrocyte malondialdehyde levels and sensitivity to oxidation were increased whereas glutathione, glutathione peroxidase, and catalase levels were decreased (p < 0.05). Resveratrol and caffeic acid prevented malondialdehyde accumulation and preserved glutathione, glutathione peroxidase, and catalase activities in erythrocytes. We demonstrated that resveratrol, caffeic acid, and tannic acid in stored blood could decrease the sensitivity to oxidation of erythrocytes in vitro but did not exhibit such effects on CA activity.

Alterations in Caveolin-1 Expression and Receptor-Operated Ca2+ Entry in the Aortas of Rats with Pulmonary Hypertension

Background/Aims: Alterations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlie the pathogenesis of various cardiovascular diseases. Caveolin-1 (Cav-1) is the primary functional protein associated with caveolae, which are invaginations in the plasma membrane, and is a regulator of [Ca²⁺]_i signaling. Caveolae and Cav-1 increase the activity of store-operated Ca²⁺ channels (SOCC) in rat pulmonary arterial smooth muscle cells (PASMCs), and these enhancing effects were more pronounced in rats with pulmonary hypertension (PH). Classical transient receptor potential (TRPC) proteins are highly expressed in vascular smooth muscle cells, and these proteins form functional receptor-operated Ca²⁺ channels (ROCC) and SOCC in PASMCs. Previous studies suggested that functional and structural changes in aortas might occur during the pathological process of PH. Our data demonstrated that Cav-1 and TRPC were also abundant in the aorta smooth muscle cells (AoSMCs) of PH rats. However, previous PH research primarily focused on Ca²⁺ channels in pulmonary arteries, but not functional changes in Ca²⁺ channels in aortas. The contribution of Cav-1 of AoSMCs to alterations of Ca²⁺ signaling in aortic functions during the pathological process of PH has not been fully characterized. Therefore, this study investigated alterations in Cav-1 expression and the relationship of these changes to Ca²⁺ channels in AoSMCs of PH rats. Methods: The present study examined physiological caveolae and Cav-1 expression and characterized the function of altered Cav-1 expression in rat aortas with PH. Results: The appearance of caveolae with Cav-1 expression increased significantly in the aortas of rats with PH, but TRPC1 and TRPC6 expression was not altered. In vitro experiments demonstrated that caveolae contributed to phenylephrine, endothelin-1, and 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced aortic vasoreactivity, but KCl and cyclopiazonic acid had no effect, which suggests the vital ability of Cav-1 to regulate ROCC activity. The introduction of Cav-1 scaffolding domain peptide enhanced OAG-induced ROCC function in primary AoSMCs. Conclusion: Cav-1 is specifically associated with ROCC in aortas and plays a vital role in altering vasoreactivity, which affects cardiovascular diseases pathology. Caveolae and Cav-1 up-regulation may affect the function of ROCC in rat models of PH.

BK Channels in the Vascular System.

Autoregulation of blood flow is essential for the preservation of organ function to ensure continuous supply of oxygen and essential nutrients and removal of metabolic waste. This is achieved by controlling the diameter of muscular arteries and arterioles that exhibit a myogenic response to changes in arterial blood pressure, nerve activity and tissue metabolism. Large-conductance voltage and Ca(2+)-dependent K(+) channels (BK channels), expressed exclusively in smooth muscle cells (SMCs) in the vascular wall of healthy arteries, play a critical role in regulating the myogenic response. Activation of BK channels by intracellular, local, and transient ryanodine receptor-mediated "Ca(2+) sparks," provides a hyperpolarizing influence on the SMC membrane potential thereby decreasing the activity of voltage-dependent Ca(2+) channels and limiting Ca(2+) influx to promote SMC relaxation and vasodilation. The BK channel α subunit, a large tetrameric protein with each monomer consisting of seven-transmembrane domains, a long intracellular C-terminal tail and an extracellular N-terminus, associates with the β1 and γ subunits in vascular SMCs. The BK channel is regulated by factors originating within the SMC or from the endothelium, perivascular nerves and circulating blood, that significantly alter channel gating properties, Ca(2+) sensitivity and expression of the α and/or β1 subunit. The BK channel thus serves as a central receiving dock that relays the effects of the changes in several such concomitant autocrine and paracrine factors and influences cardiovascular health. This chapter describes the primary mechanism of regulation of myogenic response by BK channels and the alterations to this mechanism wrought by different vasoactive mediators.

Phospholamban spontaneously reconstitutes into giant unilamellar vesicles where it generates a cation selective channel.

Phospholamban (PLN) is a small integral membrane protein, which modulates the activity of the Sarcoplasmic Reticulum Ca(2+)-ATPase (SERCA) of cardiac myocytes. PLN, as a monomer, can directly interact and tune SERCA activity, but the physiological function of the pentameric form is not yet fully understood and still debated. In this work, we reconstituted PLN in Giant Unilamellar Vesicles (GUVs), a simple and reliable experimental model system to monitor the activity of proteins in membranes. By Laser Scanning Confocal Microscopy (LSCM) and Fluorescence Correlation Spectroscopy (FCS) we verified a spontaneous reconstitution of PLN into the phospholipid bilayer. In parallel experiments, we measured with the patch clamp technique canonical ion channel fluctuations, which highlight a preference for Cs(+) over K(+) and do not conduct Ca(2+). The results prove that PLN forms, presumably in its pentameric form, a cation selective ion channel.

Evidence for the Inhibition by Temozolomide, an Imidazotetrazine Family Alkylator, of Intermediate-Conductance Ca2+-Activated K+ Channels in Glioma Cells

Background: Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is used to treat glioma. Whether this drug has any ionic effects in glioma cells remains largely unclear. Methods: With the aid of patch-clamp technology, we investigated the effects of TMZ on the ionic currents in U373 glioma cells. The mRNA expression of KCNN4 (KCa3.1) in U373 glioma cells and TMZ's effect on K+ currents in these KCNN4 siRNA-transfected U373 cells were investigated. Results: In whole-cell recordings, TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373 cells. TMZ-induced IK inhibition was reversed by ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached configuration, TMZ concentration-dependently reduced the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels with an IC50 value of 9.2 µM. Chlorzoxazone or 1-EBIO counteracted the TMZ-induced inhibition of IKCa channels. Although TMZ was unable to modify single-channel conductance, its inhibition of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neitherlarge-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels were affected by TMZ. In current-clamp mode, TMZ depolarized the cell membrane and 1-EBIO reversed TMZ-induced depolarization. TMZ had no effect on IK in KCNN4 siRNA-transfected U373 cells. Conclusion: In addition to the DNA damage it does, its inhibitory effect on IKCa channels accompanied by membrane depolarization could be an important mechanism underlying TMZ-induced antineoplastic actions.

CO2 and HCO3- Permeability of the Rat Liver Mitochondrial Membrane

Background/Aims: Across the mitochondrial membrane an exceptionally intense exchange of O2 and CO2 occurs. We have asked, 1) whether the CO2 permeability, PM,CO2, of this membrane is also exceptionally high, and 2) whether the mitochondrial membrane is sufficiently permeable to HCO3- to make passage of this ion an alternative pathway for exit of metabolically produced CO2. Methods: The two permeabilities were measured using the previously published mass spectrometric 18O exchange technique to study suspensions of mitochondria freshly isolated from rat livers. The mitochondria were functionally and morphologically in excellent condition. Results: The intramitochondrial CA activity was exclusively localized in the matrix. PM,CO2 of the inner mitochondrial membrane was 0.33 (SD ± 0.03) cm/s, which is the highest value reported for any biological membrane, even two times higher than PM,CO2 of the red cell membrane. PM,HCO3- was 2· 10-6 (SD ± 2· 10-6) cm/s and thus extremely low, almost 3 orders of magnitude lower than PM,HCO3- of the red cell membrane. Conclusion: The inner mitochondrial membrane is almost impermeable to HCO3- but extremely permeable to CO2. Since gas channels are absent, this membrane constitutes a unique example of a membrane of very high gas permeability due to its extremely low content of cholesterol.

Mitochondria and carbon monoxide: cytoprotection and control of cell metabolism - a role for Ca(2+) ?

Carbon monoxide (CO) is an endogenously produced gasotransmitter with important biological functions: anti-inflammation, anti-apoptosis, vasomodulation and cell metabolism modulation. The most recognized cellular target for CO is the mitochondria. Physiological concentrations of CO generate mitochondrial reactive oxygen species (ROS), which are signalling molecules for CO-induced pathways. Indeed, small amounts of ROS promote cytoprotection by a preconditioning effect. Furthermore, CO prevents cell death by limiting mitochondrial membrane permeabilization, which inhibits the release of pro-apoptotic factors into the cytosol; both events are ROS dependent. CO also increases the ability of mitochondria to take up Ca(2+) . Mitochondrial metabolism is modulated by CO, namely by increasing TCA cycle rate, oxidative phosphorylation and mitochondrial biogenesis, which, in turn, increases ATP production. CO's modulation of metabolism might be important for cellular response to diseases, namely cancer and ischaemic diseases. Finally, another cytoprotective role of CO involves the control of Ca(2+) channels. By limiting the activity of T-type and L-type Ca(2+) channels, CO prevents excitotoxicity-induced cell death and modulates cell proliferation. Several questions concerning Ca(2+) signalling, mitochondria and CO can be asked, for instance whether CO modulation of cell metabolism would be dependent on the mitochondrial Ca(2+) uptake capacity, since small amounts of Ca(2+) can increase mitochondrial metabolism. Whether CO controls Ca(2+) communication between mitochondria and endoplasmic reticulum is another open field of research. In summary, CO emerges as a key gasotransmitter in the control of several cellular functions of mitochondria: metabolism, cell death and Ca(2+) signalling.

Dyrk1A-ASF-CaMKIIδ Signaling Is Involved in Valsartan Inhibition of Cardiac Hypertrophy in Renovascular Hypertensive Rats

Objectives: It is known that the expression, activity and alternative splicing of Ca²⁺/calmodulin-dependent protein kinase IIδ (CaMKIIδ) are dysregulated in the cardiac remodeling process. Recently, we found a further signaling pathway, by which dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) regulates the alternative splicing of CaMKIIδ via the alternative splicing factor (ASF), i.e. Dyrk1A-ASF-CaMKIIδ. In this study, we aimed to investigate whether Dyrk1A-ASF-CaMKIIδ signaling was involved in valsartan inhibition of cardiac hypertrophy in renovascular hypertensive rats. Methods: Rats were subjected to two kidney-one clip (2K1C) surgery and then treated with valsartan (30 mg/kg/day) for 8 weeks. Hypertrophic parameter analysis was then performed. Western blot analysis was used to determine the protein expression of Dyrk1A and ASF and RT-PCR was used to analyze the alternative splicing of CaMKIIδ in the left ventricular (LV) sample. Results: Valsartan attenuated cardiac hypertrophy in 2K1C rats but without impairment of cardiac systolic function. Increased protein expression of Dyrk1A and decreased protein expression of ASF were observed in the LV sample of 2K1C rats. Treatment of 2K1C rats with valsartan reversed the changes in Dyrk1A and ASF expression in the LV sample. Valsartan adjusted the 2K1C-induced imbalance in alternative splicing of CaMKIIδ by upregulating the mRNA expression of CaMKIIδC and downregulating the mRNA expression of CaMKIIδA and CaMKIIδB. Conclusions: Valsartan inhibition of cardiac hypertrophy in renovascular hypertensive rats was mediated, at least partly, by Dyrk1A-ASF-CaMKIIδ signaling.

Temperature stability of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] in the form of a solution or in the lyophilized form during storage at −80 °C, 4 °C, 25 °C and 37 °C or pasteurization at 70 °C

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-min 30 mm Hg Mean Arterial Pressure (MAP) sustained hemorrhagic shock rat model shows that it is more effective than blood in the lowering of elevated intracellular pCO2, recovery of ST-elevation and histology of the heart and intestine. This paper is to analyze the storage and temperature stability. Allowable storage time for RBC is about 1 d at room temperature and 42 d at 4 °C. Also, RBC cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for 1 year even at room temperature. However, Poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. Results of storage stability show that lyophilization extends the storage time to 1 year at 4 °C and 40 d at room temperature (compared to respectively, 42 d and 1 d for RBC). After the freeze-dry process, the enzyme activities of Poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD and 93 ± 3.5% for CAT. After heat pasteurization at 70 °C for 2 h, lyophilized Poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA 97 ± 4%, SOD 100 ± 2.5% and CAT 63.8 ± 4%. More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of Poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving in suitable solutions that continues to have good storage stability though less than that for the lyophilized form. According to the P50 value, Poly-[SFHb-SOD-CAT-CA] retains its oxygen carrying ability before and after long-term storage.

PTH modulation of NCC activity regulates TRPV5 Ca2+ reabsorption.

Since parathyroid hormone (PTH) is known to increase transient receptor potential vanilloid (TRPV)5 activity and decrease Na(+)-Cl(-) cotransporter (NCC) activity, we hypothesized that decreased NCC-mediated Na(+) reabsorption contributes to the enhanced TRPV5 Ca(2+) reabsorption seen with PTH. To test this, we used mDCT15 cells expressing functional TRPV5 and ruthenium red-sensitive (45)Ca(2+) uptake. PTH increased (45)Ca(2+) uptake to 8.8 ± 0.7 nmol·mg(-1)·min(-1) (n = 4, P < 0.01) and decreased NCC activity from 75.4 ± 2.7 to 20.3 ± 1.3 nmol·mg(-1)·min(-1) (n = 4, P < 0.01). Knockdown of Ras guanyl-releasing protein (RasGRP)1 had no baseline effect on (45)Ca(2+) uptake but significantly attenuated the response to PTH from a 45% increase (6.0 ± 0.2 to 8.7 ± 0.4 nmol·mg(-1)·min(-1)) in control cells to only 20% in knockdown cells (6.1 ± 0.1 to 7.3 ± 0.2 nmol·mg(-1)·min(-1), n = 4, P < 0.01). Inhibition of PKC and PKA resulted in further attenuation of the PTH effect. RasGRP1 knockdown decreased the magnitude of the TRPV5 response to PTH (7.9 ± 0.1 nmol·mg(-1)·min(-1) for knockdown compared with 9.1 ± 0.1 nmol·mg(-1)·min(-1) in control), and the addition of thiazide eliminated this effect (a nearly identical 9.0 ± 0.1 nmol·mg(-1)·min(-1)). This indicates that functionally active NCC is required for RasGRP1 knockdown to impact the PTH effect on TRPV5 activity. Knockdown of with no lysine kinase (WNK)4 resulted in an attenuation of the increase in PTH-mediated TRPV5 activity. TRPV5 activity increased by 36% compared with 45% in control (n = 4, P < 0.01 between PTH-treated groups). PKC blockade further attenuated the PTH effect, whereas combined PKC and PKA blockade in WNK4KD cells abolished the effect. We conclude that modulation of NCC activity contributes to the response to PTH, implying a role for hormonal modulation of NCC activity in distal Ca(2+) handling.

Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

Biodeinking of old newspaper pulp using a cellulase-free xylanase preparation of Aspergillus niger DX-23

Old newspapers (ONP) are a significant source of raw materials for recycled paper industry. To obtain bright pulp for recycling, the ink from the ONP pulp is removed by chemical deinking process which is highly polluting and expensive. Due to high xylan content in ONP, the ink particles adhering on ONP pulp surface can be removed through detachment of ink/fiber bond by action of xylanases. In the present study, cellulase-free xylanases from fungi were specifically screened for deinking of ONP pulp. Among 16 cellulase-free xylanase producing isolates, strain DX-23 (identified as Aspergillus niger) produced maximum xylanase (48.9±0.02 U ml−1). The xylanase of A. niger DX-23 (50 U g−1 pulp), efficiently deinked the ONP pulp which exhibited 34.5% ISO brightness (22% higher than the untreated pulp). Improvement up to 78.8% ISO brightness in ONP pulp compared to untreated pulp was achieved after optimization of conditions for deinking by response surface methodology and subsequent mild H2O2 treatment of deinked pulp. ATR-FTIR spectra, X-ray diffraction analysis and scanning electron micrographs of pulps confirmed the removal of surface ink particles from ONP pulp. The xylanase of A. niger DX-23 possessed molecular weight of 15.0 kDa, showed maximum activity at 50 °C and pH 5.0. Additives such as Mn+2, Fe+2, and sodium dodecyl sulfate enhanced the activity of xylanase, whereas, Zn+2, Mg+2, Ca+2 and Hg+2 and EDTA completely inhibited the activity. The Km and Vmax for the xylanase of A. niger DX-23 for hydrolysis of Birchwood xylan was found to be 2.38 mg/ml and be 230.8 µM/min/mg, respectively.

Chronic imaging of movement-related Purkinje cell calcium activity in awake behaving mice.

Purkinje cells (PCs) are a major site of information integration and plasticity in the cerebellum, a brain region involved in motor task refinement. Thus PCs provide an ideal location for studying the mechanisms necessary for cerebellum-dependent motor learning. Increasingly, sophisticated behavior tasks, used in combination with genetic reporters and effectors of activity, have opened up the possibility of studying cerebellar circuits during voluntary movement at an unprecedented level of quantitation. However, current methods used to monitor PC activity do not take full advantage of these advances. For example, single-unit or multiunit electrode recordings, which provide excellent temporal information regarding electrical activity, only monitor a small population of cells and can be quite invasive. Bolus loading of cell-permeant calcium (Ca(2+)) indicators is short-lived, requiring same-day imaging immediately after surgery and/or indicator injection. Genetically encoded Ca(2+) indicators (GECIs) overcome many of these limits and have garnered considerable use in many neuron types but only limited use in PCs. Here we employed these indicators to monitor Ca(2+) activity in PCs over several weeks. We could repeatedly image from the same cerebellar regions across multiple days and observed stable activity. We used chronic imaging to monitor PC activity in crus II, an area previously linked to licking behavior, and identified a region of increased activity at the onset of licking. We then monitored this same region after training tasks to initiate voluntary licking behavior in response to different sensory stimuli. In all cases, PC Ca(2+) activity increased at the onset of rhythmic licking.

Soil quality index for evaluation of reclaimed coal mine spoil

Success in the remediation of mine spoil depends largely on the selection of appropriate tree species. The impacts of remediation on mine soil quality cannot be sufficiently assessed by individual soil properties. However, combination of soil properties into an integrated soil quality index provides a more holistic status of reclamation potentials of tree species. Remediation potentials of four tree species (Acacia auriculiformis, Cassia siamea, Dalbergia sissoo, and Leucaena leucocephala) were studied on reclaimed coal mine overburden dumps of Jharia coalfield, Dhanbad, India. Soil samples were collected under the canopies of the tree species. Comparative studies on the properties of soils in the reclaimed and the reference sites showed improvements in soil quality parameters of the reclaimed site: coarse fraction (−20.4%), bulk density (−12.8%), water holding capacity (+0.92%), pH (+25.4%), EC (+2.9%), cation exchange capacity (+46.6%), organic carbon (+91.5%), N (+60.6%), P (+113%), K (+19.9%), Ca (+49.6%), Mg (+12.2%), Na (+19.6%), S (+46.7%), total polycyclic aromatic hydrocarbons (−71.4%), dehydrogenase activity (+197%), and microbial biomass carbon (+115%). Principal component analysis (PCA) was used to identify key mine soil quality indicators to develop a soil quality index (SQI). Selected indicators include: coarse fraction, pH, EC, soil organic carbon, P, Ca, S, and dehydrogenase activity. The indicator values were converted into a unitless score (0–1.00) and integrated into SQI. The calculated SQI was significantly (P<0.001) correlated with tree biomass and canopy cover. Reclaimed site has 52–93% higher SQI compared to the reference site. Higher SQI values were obtained for sites reclaimed with D.sissoo (+93.1%) and C.siamea (+86.4%).

Photosynthetic variation and yield attributes of two mustard varieties against cadmium phytotoxicity

AbstractExcess cadmium accumulation in shoot decreases the photosynthetic attributes and the activity of carbonic anhydrase (CA; E.C. 4.2.1.1) thereby retarding plant growth metabolism, in a cultivar dependent manner. Two mustard (Brassica juncea) varieties were treated with increasing cadmium doses (0, 25, 50 or 100 mg CdCl2 kg−1 of soil) in the soil in a net house. The photosynthetic features, activity of CA, and the yield attributes were recorded in association with morphological characters. A clear-cut difference in these features was noted among the varieties, where Varuna excelled in its resistance to the cadmium toxicity with better growth and yield, at harvest.

Actions of KMUP-1, a xanthine and piperazine derivative, on voltage-gated Na(+) and Ca(2+) -activated K(+) currents in GH3 pituitary tumour cells.

7-[2-[4-(2-Chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) is a xanthine-based derivative. It has soluble GC activation and K(+) -channel opening activity. Effects of this compound on ion currents in pituitary GH3 cells were investigated in this study. The aim of this study was to evaluate effects of KMUP-1 on the amplitude and gating of voltage-gated Na(+) current (INa ) in pituitary GH3 cells and in HEKT293T cells expressing SCN5A. Both the amplitude of Ca(2+) -activated K(+) current and the activity of large-conductance Ca(2+) -activated K(+) (BKCa ) channels were also studied. KMUP-1 depressed the transient and late components of INa with different potencies. The IC50 values required for its inhibitory effect on transient and late INa were 22.5 and 1.8 μM respectively. KMUP-1 (3 μM) shifted the steady-state inactivation of INa to a hyperpolarized potential by -10 mV, despite inability to alter the recovery of INa from inactivation. In cell-attached configuration, KMUP-1 applied to bath increased BKCa -channel activity; however, in inside-out patches, this compound applied to the intracellular surface had no effect on it. It prolonged the latency in the generation of action currents elicited by triangular voltage ramps. Additionally, KMUP-1 decreased the peak INa with a concomitant increase of current inactivation in HEKT293T cells expressing SCN5A. Apart from activating BKCa channels, KMUP-1 preferentially suppresses late INa . The effects of KUMP-1 on ion currents presented here constitute an underlying ionic mechanism of its actions.