In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.Freshwater fish absorb Cazecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs  and 7 isoforms of NCXs  were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of 2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.The present study provides molecular evidence for the specific isoforms of Ca In terrestrial vertebrates, the whole-body Ca2+ balance is mainly achieved by intestinal absorption and kidney reabsorption. Ca2+ uptake mechanisms in the mammalian kidney have been most extensively studied, while fish gills, a specialized organ in non-mammalian vertebrates, is another model for studying Ca2+ uptake mechanisms [2+ uptake from the aquatic environment to maintain the Ca2+ balance [2+ regulation mechanisms in aquatic animals are probably more complicated and challenging because of the dramatic fluctuations in ambient Ca2+ concentrations which can occur .Ca balance . Compare2+ transport is carried out as a 3-step process [2+ through apical epithelial Ca2+ channels , Ca2+ is bound to calbindins that facilitate diffusion to the basolateral membrane, and then it is extruded via the plasma membrane Ca2+-ATPase (PMCA) and/or Na+/Ca2+ exchanger (NCX). In this way, net transepithelial Ca2+ absorption from the luminal compartment (or environment) to the plasma is accomplished. A model of the capability of physiological regulation has also been similarly proposed in fish gills [2+ uptake. The ecac gene has recently been cloned and sequenced in fugu, zebrafish, and trout [2+ fresh water causes upregulation of the whole-body Ca2+ influx and zECaC expression in mitochondrion-rich (MR) cells of both gills and skin, providing molecular evidence for the role of ECaC in fish Ca2+ uptake [2+ uptake [2+ uptake mechanisms.According to the current model in mammals, active and transcellular Ca process . Followind trout ,7,8. In + uptake . On the + uptake ,10. Neve2+ from cells for maintaining a gradient across the plasma membrane [2+ homeostasis [3 modulates Ca2+ balance by upregulating NCX1 expression [2+ uptake mechanisms.Three NCX genes  and 4 PMCA genes  have been identified in mammals so far. The fundamental work of PMCA is the highly regulated active extrusion of Camembrane . PMCA bemembrane . In mammeostasis . Howevereostasis . Knock-oeostasis , while mpression . Recentlpression ,18. Howe2+ transport [2+ uptake mechanisms in teleost gills. Therefore, using a functional genomic approach to investigate all isoforms of PMCA and NCX in a species may provide both physiological and genomic insights into this issue.Because of a theoretical process of whole-genome duplication (WGD) from ancestor vertebrates ,20, the ransport ,23. Howe2+ environments were also examined by quantitative real-time PCR analysis. The present study provides molecular evidence to support the current Ca2+ uptake model in fish gill cells [In the present study, a strategy of a whole-genome survey was used to uncover any unidentified PMCA and NCX isoforms in zebrafish, and then these candidates were refined with the EST database and cloned. Six isoforms of PMCAs  and 7 isoforms of NCXs  were identified. Moreover, we report that some duplication of PMCA and NCX occurred, and the tissue distributions and expression patterns of these isoforms were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and triple fluorescence labeling, respectively. The gene expressions in gills of zebrafish acclimated to high- or low-Call cells .In this whole-genome survey, 6 distinct PMCAs Figure  and 7 di2+ binding and transporting, and calmodulin-regulating functions [th amino acid including from 1 to 3 small exons (less than 100 bp each in those cases), and 3' amino acid tails that usually stretch to the 3' UTR and alter the location of stop codons. On the other hand, a region of alternative splicing sites was found in zNCX1b and zNCX3 around the 610th amino acid, which is a part of the intracellular loop.Based on the topological structures of PMCA and NCX summarized in mammals ,26, zebrunctions ,27; the unctions ,29 was lzPMCAs share 63%~86% identities with each other, while zNCXs exhibit higher variety among isoforms . According to the phylogenetic analysis, zebrafish and 2 pufferfish species so far all have 6 PMCA and 7 NCX isoforms Figure . Comparezpmca2 and zpmca4 were ubiquitously expressed in various tissues compared with zpmca1a, zpmca1b, zpmca3a, and zpmca3b .Fluorescence in situ hybridizations were used for zecac and zpmca2 , while only 40.3 +/- 1.35% (n = 3) of zpmca2-positive cells showed zecac signals , while zncx1b signals could be detected in all zecac-positive cells . This functional substitution of paralogues found in Ca2+ transporters is not a unique example. In zebrafish, the expression profiles suggest the hypothesis that zebrafish hoxB1a and hoxB1b are functional equivalents of mouse Hoxb1 and Hoxa1, respectively. On the contrary, the zebrafish orthologue of mouse Hoxa1, zebrafish hoxA1a, is not expressed in a similar pattern [2+ uptake pathway in fish gills. NCX1a and NCX1b from teleosts including zebrafish and pufferfish were not clustered within an inner group on the phylogenetic tree  is attenuated during the high-temperature treatments used for in situ hybridization. All zecac-expressing cells co-expressed zncx1b, and about half of the zecac-expressing cells co-expressed zpmca2, indicating that about 50% of zecac-expressing cells co-expressed both zncx1b and zpmca2. Taking all these findings into account, only about 45% of the entire population of MR cells co-express zecac, zpmca2, and zncx1b, and this subset of ionocytes may specifically carry out the function of transepithelial Ca2+ uptake in zebrafish gills. It was noted that some gill cells express only 1 or 2  of the 3 major Ca2+ transporters. These cells may be in the process of terminal differentiation as Hsiao et al. [2+ transporter(s) expressed in those cells may be involved in intracellular Ca2+ homeostasis or other cellular events [Double o et al.  reportedr events ,40. This2D3 in mammals and chicken [2-vitamin D3 was shown to have a hypercalcemic effect on both freshwater and saltwater teleosts [2+ transport [2+ at the apical membrane [2+ transport [2+ environment, which would stimulate Ca2+ uptake capacity in fish [2+ extrusion mechanisms by PMCA and NCX is far below their maximum capacity in fish gills based on the kinetic properties of the 2 enzymes.ECaC is a vitamin D-sensitive transporter in mammals , and the chicken ,41,42. 1teleosts ,43, wherteleosts . Stannioransport , but quimembrane ,46. It hransport . The pre in fish ,49, caus in fish , who fou2+ environment stimulated the mRNA expression of ecac in gills, but not those of pmcas or ncxs; and (4) only a portion of gill MR cells co-expressed zecac, zpmca2, and zncx1b mRNAs.The major findings of the present study were that (1) 6 PMCAs and 7 NCXs were identified from zebrafish tissues; (2) differential expressions of these genes were found in various tissues including gills; (3) acclimation to low-CaDanio rerio) brood stocks at the Institute of Cellular and Organismic Biology, Academia Sinica were kept in fresh water  at 28.5°C under a 14-h:10-h light: dark photoperiod. Embryos were collected within 30 min after fertilization and incubated in a Petri dish until the desired developmental stages. For whole-mount in situ hybridization, PTU (1-phenyl 2-thiourea) at a final concentration of 0.003% was added to prevent melanogenesis. Fish were anesthetized with buffered MS222 before sampling following the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee .Zebrafish  and low-Ca2+ (0.02 mM) artificial fresh waters were prepared with double-deionized water  supplemented with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. Nominal Ca2+ concentrations of the high- and low-Ca2+ media were 2.00 and 0.02 mM, respectively, but the other ion concentrations of the media were the same  as those in the local tap water. Variations in the ion concentrations were maintained within 10% of the predicted values by examination with an atomic absorption spectrophotometer . Zebrafish were transferred to high- and low-Ca2+ media, respectively, for 2 wk. At the end of acclimation, fish gills were sampled for quantitative real-time PCR analysis.Following a previous process in our laboratory , high-CaFugu rubripes), and tetradon (Tetraodon nigroviridis). The putative full-length or partial open reading frames of PMCAs and NCXs obtained were confirmed using the EST database, and/or were used to design primers for cloning and the RT-PCR analysis. PCR products thus obtained were subcloned into a pGEM-T Easy vector , and the nucleotide sequences were determined with an ABI 377 sequencer . Sequence analysis was conducted with a BLASTx program (NCBI). The specific primers of 5' and 3'-rapid amplification of cDNA ends (RACE) were designed from the partial sequences obtained from the PCR with degenerate primers. The RACE PCR program followed a commercial protocol , and the RACE PCR products were also subcloned into pGEM-T Easy vectors and sequenced. The full-length deduced amino-acid sequences were aligned with ClustalX and then phylogenetically analyzed with Mega3.1 [The peptide sequences from other species (teleost had a higher priority) were used to BLAST the genome databases (of NCBI and Ensembl) for zebrafish, pufferfish  for 1.5 h at 42°C, followed by a 15-min incubation at 70°C. For the PCR amplification, 1 μL cDNA was used as a template in a 25-μL final reaction volume containing 0.25 mM dNTP, 1.25 units Gen-Taq polymerase , and 0.2 μM of each primer. The primer sets for the PCR of tissue scans were zPMCA1a (428-bp fragment) forward 5'-AGACAGGGTGGATAGAAGGT-3', reverse 5'-CCCCAATAATGTGAAGATGA-3'; zPMCA1b (556-bp fragment), forward 5'-AGCCCCTTATTTCCCGCAC-3', reverse 5'-GCCCCCTTCTCAGCTCCATT-3'; zPMCA2 (444-bp fragment) forward 5'-GGGATCGGGATGAGATGGTA-3', reverse 5'-GCGTGTGTTTGTCTGTGGGT-3'; zPMCA3a (457-bp fragment) forward 5'-TTGCTGGTACAGATGTGGC-3', reverse 5'-GGAGGAGAGTGAAGCGGAG-3'; zPMCA3b (614-bp fragment) forward 5'-GGTCTGCTGGGTTTCCTACT-3', reverse 5'-TCCTGCTCAATCTCTCCTTT-3'; zPMCA4 (388-bp fragment) forward 5'-GTCAAGGCTGTGATGTGGGC-3', reverse 5'-TGGTTGGGAGTGGAGAAGGG-3'; zNCX1b (358-bp fragment) forward 5'-AGAGACGAGGAGAAGGAGGT-3', reverse 5'-GCACGAAAGCAAAGAGAACT-3'; zNCX2a (276-bp fragment) forward 5'-TGATGAAGAAGGAGGTGAAC-3', reverse 5'-CTTGCGAATGTGTCTGGTAT-3'; zNCX2b (477-bp fragment) forward 5'-GCAGCCGCATTTCCTTCG-3', reverse 5'-CGCCAGAGTTTCGGACCAC-3'; zNCX3 (244-bp fragment) forward 5'-GAGGAAGCAAGAAGAATAGC-3', reverse 5'-AGTCAAAACAAGATGGCAGA-3'; zNCX4a (462-bp fragment) forward 5'-GGAAGAGAGCGGAGAAGAGC-3', reverse 5'- ATGGTGAAGAGGGTGACGGA-3'; and zNCX4b (350-bp fragment) forward 5'-ATGCAGCAGCAGAAGAGC-3', reverse 5'-ACATTCTCGCCAGGTTTG-3'. A 514-bp fragment of zebrafish beta-actin was used as an internal control to evaluate the relative amounts of complementary DNAs (cDNAs), and the design of the primer pairs followed Hsiao et al. [An appropriate amount of zebrafish adult tissues was collected for the total RNA preparation with TRIZol reagent . The amount and quality of total RNA were determined by measuring the absorbances at 260 and 280 nm with a spectrophotometer (Hitachi U-2000) and RNA denatured gels. For cDNA synthesis, 5 μg of total RNA was reverse-transcribed in a final volume of 20 μL containing 0.5 mM dNTPs, 2.5 μM oligo (dT)o et al. . The ampin vitro transcription with T7 and SP6 RNA polymerase . The qualities of the probes were examined using RNA gels, and the concentrations were determined by a dot-blot assay with standard DIG-labeled RNA (100 ng/μl) (Roche). Excised gills were fixed with 4% paraformaldehyde overnight at 4°C and then washed several times with PBS. For the cryosections, fixed samples were immersed in PBS containing 30% sucrose overnight, and embedded in Optimal Cutting Temperature (OCT) compound embedding medium  at -20°C, and 10-μm frozen cross-sections were cut with a CM 1900 rapid sectioning cryostat  and attached to poly-L-lysine coated slides . Prepared samples from either cryosections or methanol-dehydrated whole gills were washed several times with phosphate-buffered saline with 0.1% tween-20 (PBST). After a brief washing with PBST, samples were incubated with hybridization buffer (HyB) containing 50% formamide, 5× SSC, and 0.1% Tween-20 for 5 min at 65°C. Prehybridization was performed for 2 h at 65°C with HyB+, which is the hybridization buffer supplemented with 500 ng/mL yeast tRNA and 50 μg/mL heparin. For hybridization, samples were incubated in 100 ng of the RNA probe in 200 μL HyB+ at 65°C overnight. Then, slides were washed at 65°C for 10 min in 75% HyB and 25% 2× SSC, 10 min in 50% HyB and 50% 2× SSC, 10 min in 25% HyB and 75% 2× SSC, 10 min in 2× SSC, and 30 min for 2 times in 0.2× SSC at 70°C. Further washes were performed at room temperature for 5 min in 75% 0.2× SSC and 25% PBST, 5 min in 50% 0.2× SSC and 50% PBST, 5 min in 25% 0.2× SSC and 75% PBST, and 5 min in PBST. Fluorescence staining was conducted with a commercial kit, TSA Plus Fluorescence Systems (Perkin-Elmer). The hybridization signals detected by the DIG-labeled RNA probes were amplified through fluorescein-tyramide signal amplification (TSA), while cyanine 3-TSA was used for the DNP-labeled probes. For triple labeling, samples were first subjected to in situ hybridizations of the 2 transporter genes and then to Na+/K+-ATPase immunocytochemistry (see below).Fragments of the target genes obtained by PCR were inserted into pGEM-T Eeasy vectors. Digoxigenin- (Dig)  or dinitrophenol (DNP)-labeled  RNA probes were synthesized by +/K+-ATPase α subunit  monoclonal immunoglobulin G (IgG)  at room temperature for 2 h. Samples were washed twice in PBS for 10 min each, and then incubated with 1:200 PBS-diluted goat anti-mouse IgG conjugated with Cy5  for 2 h at room temperature. Images were acquired with a confocal laser scanning microscope .Zebrafish embryos and gills were fixed in 4% paraformaldehyde for 10 min at 4°C. After washing in PBS, fixed embryos were treated with 100% ethanol for 10 min at -20°C and subsequently subjected to blocking with 3% BSA at room temperature for 30 min. Embryos were then incubated with 1:200 PBS-diluted mouse anti-chicken Na2+ transporters  was carried out using a SYBR green dye (Applied Biosystems)-based assay with an ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Primers targeting Cat-test.Values are presented as the mean ± SE and were compared by the 2-sample 2+ channel; EST, expressed sequence tag; FW, freshwater; HCa, high Ca2+; HyB, hybridization buffer; LCa, low Ca2+; MR, mitochondrion-rich; NCX, Na+/Ca2+ exchanger; OCT, Optimal Cutting Temperature; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.1% tween-20; PMCA, plasma membrane Ca2+-ATPase; PNA, peanut lectin agglutinin; qPCR, Quantitative real-time PCR; RACE, rapid amplification of cDNA ends; RT-PCR, reverse-transcriptase polymerase chain reaction; SE, standard error; SERCA, sarco-endoplasmic reticulum calcium ATPase; SSC, SSC, saline-sodium citrate; TRP, transient receptor potential cation channel; TSA, fluorescein-tyramide signal amplification; UTR, untranslated regions; WGD, whole-genome duplication.BSA, bovine serum albumin; Dig, digoxigenin; DNP, dinitrophenol; ECaC, epithelium CaPPH, the correspondence author, organized the whole project and manuscript. BKL designed and conducted the experiments and the data analysis; AND performed molecular cloning experiments; SCC, and MYC performed other biochemical experiments. All authors have read and approved the final manuscript.2+-ATPase (PMCA) and Na+/Ca2+ exchanger (NCX). The chromosome number and loci were obtained by the SSAHA program from the Ensembl database (zebrafish genome assemble version 6).Table S1: Primers for quantitative real-time PCR and the chromosome loci of zebrafish plasma membrane CaClick here for file


Hepatic stellate cells, which reside in the space of Disse in close contact with both sinusoidal endothelial cells and hepatocytes, play multiple roles in the pathophysiology of the liver . QuiesceThe genome project will finish by 2003 and will reveal the sequence of all genes encoded in the genome for a total of 3,000 Mb ,15. Thisprotein + genome), or proteomics, refers to the total protein profile of a given cell or tissue type. Advantage of proteomics is referred to more accurate picture of the genome's plan by measuring proteins directly because i) there exists difference between the rates of degradation of individual mRNAs and proteins and ii) proteins are received post-translational modification, e.g. phosphorylation and glycation. Disadvantage of proteomics may be i) requirement of "skilled staff" for 2-dimensional PAGE, ii) low sensitivity, iii) failure of the detection of some of the most interesting proteins, such as membrane receptor and low-abundance signal molecules, and iv) unavailability of amplification method for proteins like PCR. Recent advancement of technology related to proteomics will overcome these disadvantages in the near future.The proteome  . This teIn practice, samples were prepared from quiescent or activated stellate cells, intact or fibrotic liver, and conditioned medium. They were dissolved in a lysis buffer composed of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) ampholine, and 1% DTT and applied overnight to Immobilone DryStrip . After IEF, two-dimensional SDS-PAGE was performed in 9–18% acrylamide gradient gels. Proteins were visualized by silver staining. The protein spots of interest were excised from the 2-D gels and digested overnight at 37–C in a buffer containing trypsin. After a brief purification, they were applied to Q-TOF mass spectrometer. The MS mode was used to scan samples for detectable peptides. Subsequently, the MS/MS mode was used to fragment individual peptides, and from the resulting MS/MS spectra the amino acid sequence was deduced for each peptide. Finally, the proteins were identified after searching the obtained amino acid sequences against the SwissProt and GenBank database.2+/Zn2+ superoxide dismutase, plasminogen activator inhibitor 1, ubiquitin, vimentin, and several heat-shock proteins. Several proteins whose expression levels were up- or down-regulated in the course of stellate cell activation were identified. These include actin-binding proteins, such as calcyclin, calgizzarin and F-actin capping protein, proteases, such as cathepsin D, gamma enolase, and serine protease, and neural cell adhesion molecule (N-CAM). Interesting was that we detected the difference in protein level between "in vitro" activated stellate cells and "in vivo" activated stellate cells. For instance, FK506 binding protein 6, heat shock protein 90-beta and oestronectin were detected in "in vitro", but not "in vivo" activated stellate cells. In addition, protein level of coffilin, destrin and proteasome delta chain was much higher in "in vitro" activated stellate cells than in "in vivo" ones [A total of 308 protein spots from stellate cells have been successfully identified. These include 225 protein spots derived from stellate cell lysate and 83 spots of secreted proteins. Among the identified proteins were Cuvo" ones . Thus, wstap) [in vivo and in culture. Induction of STAP protein coincided with the expression of another activation markers of the cells, such as PDGFR-beta, smooth muscle –-actin and N-CAM. STAP expression was dramatically augmented in a fibrotic liver model of rats induced by thioacetamide, indicating a profound role of STAP in the rat hepatic pathophysiology including fibrosis. Further information obtained by immunohistochemistry of whole body organs of rats revealed that STAP would be a promising marker for vitamin A-storing cells in the extra-hepatic organs; STAP was detected in pancreatic stellate cells and renal mesangial cells as well as mesenchymal cells in the submucosal layer of the stomach and the intestines where the presence of vitamin A-storing cells was confirmed previously.In the course of the proteomics approach, we successfully isolated a novel protein dabbed STAP after stellate cell activation-associated protein and cloned its cDNA (stap) . Rat STAstap cDNA were found in human genome DNA database. By using the data, we designed primers and performed RT-PCR to amplify human stap cDNA by using cDNA derived from mRNA of human cultured stellate cells as a template. As a result, a 658 bp cDNA was obtained. The deduced amino acid sequence of the cDNA had 94 and 97% identity to rat stap and unknown mouse full-length cDNA (accession number AK019410), respectively. Thus, we concluded this clone as human stap. Two histidine residues were found in human STAP as in rat STAP at 81 and 113 from the N-terminus that might be expected to be involved in heme binding sites from the similarity with amino acid sequences of hemoglobin and myoglobin. BLAST search identified that human stap locates on chromosome 17 between sialyltransferase and hypothetical protein FLJ22341 and is composed of 4 exons, spanning 111140 to 102176 in RP11-666A8 clone [Homologous sequences to rat STAP mRNA was expressed in human various organs and Hep G2 cells. Immunohistochemistry revealed that STAP-positive cells in the liver were stellate cells. STAP expression was not augmented around inflammatory necrotic areas where myofibroblasts were accumulated. As rat STAP, recombinant human STAP exhibited the characteristics of a heme protein with the activity of myeloperoxidase and lipid hydroperoxide dehydrogenase, but not glutathione peroxidase. These results indicate that human STAP is a novel human heme protein with peroxidase activity and can be used as a marker for stellate cells in human liver tissues.Very recently, another laboratories reported homologous cDNAs to STAP from mouse and human and named them as cytoglobin and histoglobin, respectively. In accordance with our results, both cytoglobin and histoglobin are hemoprotein and are expressed in multiple organs. Taken together, STAP consists of a new family of globin other than hemoglobin and myoglobin.Overexpression of STAP in NIH 3T3 cells revealed that STAP may regulate cell growth via reducing p21 expression. This experiment further showed that STAP may up-regulate mRNA expression of type I collagen and TGF-beta, indicating a profound role of STAP in fibrotic gene expression.Although molecular function of STAP is beginning to be uncovered, the data obtained to date indicate the profound role of STAP in fibrotic potential and vitamin A metabolism of stellate cells in the liver as well as in the extra-hepatic organs.in vitro and in vivo. Such a global catalogue of protein expression will greatly increase understanding of stellate cell biology and the molecular mechanism of liver fibrosis.Thus, the proteomics approach is highly useful to analyze protein/gene expression of stellate cells in response to their activation in Increasing numbers of studies have shown a variety of therapeutic approaches for liver fibrosis based on the molecular inhibition of stellate cell activation.Among them, interferon (IFN) has a clinical potential to treat liver fibrosis by eliminating HCV virus from patients . IFN-GamIn animal models, liver fibrosis could be inhibited by a soluble receptor consisting of a chimeric IgG at the extracellular portion of the TGF-beta type II receptor and by the adenovirus-mediated local expression of a dominant-negative type II TGF-beta receptor (AdCATb-TR) ,11.We reported that N-acetyl-L-cysteine triggers the degradation of PDGF receptor beta mediated by cathepsin B. N-acetyl-L-cysteine inhibits PDGF signaling, PDGF-dependent DNA synthesis, and in addition, affected the expression of TGF-beta receptor type II. N-acetyl-L-cysteine dramatically attenuated liver fibrosis in rat models .HGF gene therapy is promising for the treatment of liver fibrosis. Ueki et al. reported that repeated transfections of the human HGF gene into skeletal muscle suppressed the increase of TGF-beta 1, inhibited fibrogenesis and hepatocyte apoptosis, and resolved completely fibrosis in the cirrhotic liver .Thiazolidinediones are clinically used as antidiabetic agents, which are ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Thiazolidinediones and the other PPARgamma ligands, such as 15-J-PGJ2, have been reported to inhibit proliferation and smooth muscle –-actin expression in cultured stellate cells -29. A mo


In Acute Myeloid Leukemia (AML), malignant clones of immature myeloid cells (primarily blasts) proliferate, replace bone marrow, circulate in blood and invade other tissues. The unique presentation of bilateral proptosis and bilateral temporal swelling in AML is being reported.A 6-year-old girl presented with low-grade fever, progressively increasing bitemporal swelling and bilateral proptosis. Contrast Enhanced Computed Tomographic (CECT) images revealed enhancing infiltrates occupying the lateral orbital wall, causing proptosis. The infiltrate extended toward the bilateral temporal fossae beneath the temporalis muscle and extradurally beneath the frontal and temporal bones. A high total leucocytic count with immature and deformed cells and, Fine Needle Aspiration Cytology (FNAC) from the temporal swelling, the bone marrow aspirate and biopsy showing leukemic blast cells confirmed the diagnosis of AML. Chemotherapy brought about remission of the disease.To the best of the authors’ knowledge, simultaneous presence of both bilateral proptosis and bitemporal swellings have not been previously reported in AML. A peripheral blood smear with bone marrow aspirate and biopsy help in the early detection of AML. Institution of early intervention in this potentially fatal disease is often associated with gratifying survival rates. In Acute Myeloid Leukemia (AML), malignant clones of immature myeloid cells (primarily blasts) proliferate and eventually replace the bone marrow, circulate in blood and invade other tissues of the body. The usual manifestations are due to the suppression of normal hematopoiesis by leukemia. In this report, the unique presentation of bilateral proptosis resulting from orbital infiltration as well as bilateral temporal swelling by AML is being reported.3. Her differential leucocytic count showed 5% neutrophils, 14% lymphocytes and 1% reticulocytes. The rest of the cells were immature and deformed cells. The FNAC from the bitemporal swelling showed clusters of atypical cells with a high nuclear–cytoplasmic ratio. The cells contained an irregularly shaped nuclei, with two to three nucleoli and scanty cytoplasm. The cytology was suggestive of a leukemic infiltrate. The bone marrow aspirate and biopsy showed hypercellular marrow smears with proliferation of blasts (approximately 25–30%). The blasts were positive for myeloperoxidase; erythroid cells and megakaryocytes were reduced. The findings were suggestive of AML [Figures A 6-year-old girl presented with low-grade fever for 1 month, with progressive increasing bitemporal swelling and bilateral proptosis (right greater than left) . On exam Figures .2) and doxorubicin (30 mg/m2) in a 7-day cycle at her local hospital, with consequent remission of the disease. She did not report for a subsequent follow-up at our center.Following diagnosis, she undertook two cycles of intensive chemotherapy, including cytosine arabinoside (100 mg/mAML accounts for approximately 15% of all leukemias in children. This is Leukemic cells originate in the bone marrow. In the h2She also had bitemporal swelling that was firm, not tender and had molded into the inner and outer surfaces of bilateral orbital walls without causing any destruction. Although unilateral temporal swelling with AML has also been described in the literature, to the bPeripheral smear is an invaluable tool in diagnosing the systemic form of AML showing immature blast cells with a high total leukocyte count and relative neutropenia. Leukemic proptosis, however, may not always be associated with leukocytosis or immature cells in the peripheral smear. Doing a peripheral smear along with bone marrow aspirate and biopsy in all patients of AML manifesting with proptosis in the pediatric age is, therefore, justified. Immunohistochemistry and immunocytochemistry are used to detect the antibody against myeloperoxidase. On CT scan, the extramedullary leukemia within the orbits may appear as a well defined lesion, isodense to muscles and enha13Chemotherapy is the mainstay of treatment.6 ChemothAML should be kept in the differential diagnosis of a child presenting with proptosis or orbital mass with or without skull lesions. A peripheral blood smear should be performed in all cases along with bone marrow aspirate and biopsy for an early detection of AML. Institution of early intervention in this potentially fatal disease is often associated with gratifying 5-year survival rates.


Many inhibitors could prevent calpain-induced proteolysis of α- and β-crystallins in rodent cataracts. Evaluating natural sources with antioxidant property and subsequent prevention of calpain activation may lead to the development of safer and more effective agents against cataractogenesis. There are no reports on the protective role of bioactive components against calpain-mediated proteolysis and subsequent cataractogenesis. The purpose of the study was to evaluate the role of Drevogenin D, a triterpenoid aglycone, isolated from Dregea volubilis in preventing selenite-induced, calcium-activated, calpain-mediated proteolysis in cultured rat lenses.Selenite-induced cataractogenesis is mediated by oxidative stress, accumulation of calcium and activation of lenticular calpains. Calpains are a super family of Ca2+ ATPase, concentration of Ca2+, levels of sulfhydryl content, and thiobarbituric acid reacting substances (TBARS) were determined. Changes to casein zymography for calpains, immunoblot for Lp82, and SDS-PAGE of lens water soluble protein fraction (WSF) were also done.Lenses were extracted from Sprague-Dawley strain rats at the age of one month and were organ cultured in M-199 medium with HEPES buffer. The lenses were divided into three groups with eight lenses in each group as follows: lenses cultured in a normal medium (GI), lenses cultured in a sodium selenite supplemented medium (GII), and lenses cultured in a medium supplemented with sodium selenite and Drevogenin D-treated (GIII). Changes to transparency and opacity formation of lenses were monitored under microscopic observation. At the end of the experiment, biochemical parameters such as activity of lens superoxide dismutase (SOD), lens Ca2+ATPase, SOD, and SDS-PAGE profile of water soluble proteins was normalized following treatment with Drevogenin D.Microscopic evaluation of lens morphology showed that Drevogenin D prevented the opacification in G-III. Drevogenin D inhibited the accumulation of calcium, the activation of calpain system, and lipid peroxidation. Activity of Ca2+ ATPase, resulting in the accumulation of calcium and the subsequent activation of lenticular calpains. The results obtained indicated that Drevogenin D treatment was effective in protecting the lens proteins by controlling stress-induced protein oxidation, maintenance of Ca2+ ATPase activity, calcium accumulation, lipid peroxidation, and prevention of calpain activation. Hence, Drevogenin D can be used as a potential therapeutic agent against oxidative stress-induced cataract.Selenite-induced cataractogenesis is mediated by oxidative stress leading to a decrease in the activity of Ca Cataract remains the major cause of curable blindness, accounting for more than half of the total blind  and its 2+ homeostasis has been implicated in most types of cataract [2+ are maintained in the sub-micromolar range in the cytoplasm by membrane Ca2+ pumps [+/Ca2+ exchangers [2+ pumps [2+ uptake and is highest in the nucleus [2+ homeostasis are responsible for the activation of lens calpains, which results in proteolytic precipitation and aggregation of soluble proteins to insoluble proteins. Naturally occurring compounds like flavonoids, tannins, anthocyanins, terpenoids etc. are known to exhibit antioxidant activity and hence might be of potential therapeutic value [Emilia sonchifolia to exhibit good antioxidant and anticataractogenic potential [Various experimental models have been used to study the etiology of cataract and to evaluate therapeutic effects. Selenite-induced cataract has been proven to be an extremely rapid and convenient model for nuclear cataracts. Cataract is induced in suckling rat pups by an overdose of the essential trace element selenium . Severalcataract ,6. Level2+ pumps , plasma changers , and end2+ pumps . Selenit nucleus . An impo nucleus . Calpain nucleus ,12. Stud nucleus . Lp82 is nucleus ,15. Thusic value ,17. Prevotential .Dregea volubilis Benth. ex. Hook. f. belongs to the family Asclepiedaceae and is a tall woody climber growing wild in hotter parts of India. The whole plant extract has been traditionally used to treat several diseases including eye ailments [D. volubilis has been subjected to phytochemical analysis and found to contain several triterpenoid glycosides. Many aglycones including Drevogenin D, an aglycone of volubiloside A have been isolated from D. volubilis [ailments . D. voluolubilis . Our preM-199 (TC 199) culture medium, fetal bovine serum (FBS), antibiotic-antimycotic solution, sodium selenite, anti-Lp82 antibody, all chemicals for SDS-PAGE, and buffer salts for immunoblot were purchased from Sigma Chemical Company, St. Louis, MO. Alkaline phosphatase-conjugated secondary antibody, BCIP/NBT (5-bromo-4-chloro-3- indolyl phosphate/Nitro blue tetrazolium) substrate, and Tween 20 were bought from Genei, Bangalore, India. Nitrocellulose membrane (0.45 μm), Mini-Transblot, and Mini-Protean electrophoresis apparatus were from BioRad, Hercules, CA. Culture plates were acquired from Axygen Scientific, Union City, CA. All other chemicals and solvents were from SRL, Mumbai, India.D. volubilis were collected locally during early summer, and the specimens were stored for reference. The specimen was verified with the herbarium of Tropical Botanical Garden & Research Institute (TBGRI), Thiruvananthapuram, India (Accession No.16591 P.S Jyothish) and authenticated by an expert . Specimen herbarium was submitted to the Department of Botany, University of Kerala and specimen voucher number obtained (KUBH-5582-Biju. P.G).Fresh leaves of D. volubilis using the method of Yashimura et al. [21H34O5 and answered the Libermann-Burchard reactions for terpenoids. The compound was identified as Drevogenin D  from its physical and spectral data and by direct comparison with an authentic sample using mixed melting point (m.m.p), combined infra red spectrum (co-IR), and combined thin layer chromatography (co-TLC) analysis.Drevogenin D was isolated from a et al.  with modLenses were extracted through a posterior approach from the eyes of one-month-old female Sprague-Dawley strain rats under deep anesthesia. Rats used for the study were obtained from the animal house stock of the host department and handled in accordance with the guidelines as per the host's "Institutional Animal Ethical Committee" and CPCSEA  rules.2 at 37 °C in a CO2 incubator. Selenite medium was prepared by adding sodium selenite to the medium to give a final concentration of 0.1 mM. Lenses were maintained in a 24 well culture plate with 2 ml medium/well and one lens/well for five days. Lenses developing opacification in the first 24 h were discarded.Lenses were organ cultured in M-199 medium with HEPES buffer, supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin under 5% COAssay concentration of Drevogenin D was prepared by dissolving 5 mg/ml dimethyl sulphoxide (DMSO) and given at a concentration of 50 μg/ml medium. Lenses were maintained in normal and selenite-supplemented medium and were grouped as follows with eight lenses in each group: G-I; lenses cultured in a normal medium, G-II; lenses cultured in a selenite-supplemented medium (0.1 mM), and G-III; lenses cultured in a medium supplemented with sodium selenite (0.1 mM) + Drevogenin D-treated (50 μg/ml). Drevogenin D treatment to G-III was from the second to the fifth day while selenite administration to G-II and G-III were done on the third day.The activity of superoxide dismutase (SOD) in the lens samples was measured by the method of Kakkar et al . The ass2+ ATPase in the lens samples was measured by the method of Rorive and Kleinzeller [The activity of Cainzeller . To the The levels of calcium ions in the lenses were estimated as follows. Individual lenses were weighed and digested in concentrated nitric acid:perchloric acid (5:1). After complete digestion, the samples were dried, diluted with 1% nitric acid, and made up to 50 ml in a standard flask. The samples were analyzed by flame photometry and the results were expressed as μM/g wet tissue.The sulfhydryl content of lens proteins was determined using the Ellman's procedure as modified by Altomare et al . AliquotThe concentration of thiobarbituric acid reactive substance (TBARS) in the lens samples was estimated by the method of Niehaus and Samuelsson . BrieflyCalpain activity was studied zymographically by the method of Raser et al. . Eight-pWater soluble proteins of rat lens (40 μg/well) were separated on a 12% SDS-gel. The proteins were electroblotted onto a nitrocellulose membrane  under ice-cold temperature for one h at 100 V. The membrane was blocked overnight at 4 °C with 2% BSA . After rinsing the membrane with Tris buffered saline Tween (TBST), it was incubated for two h with 1:1000 diluted rabbit anti-Lp82 primary antibody. The membrane was washed three times (15 min each) with TBST and incubated with 1:5000 diluted goat anti-rabbit IgG secondary antibody coupled to alkaline phosphatase. The membrane was again washed three times (15 min each) with TBST buffer and developed with the BCIP/NBT substrate that developed into purple blue insoluble precipitates indicating the presence of Lp82. β-Actin was used as a loading control with rabbit anti-β-actin primary antibody at 1:1000 dilution.Lenses were homogenized on ice in 1.0 ml of ice cold PBS (pH 7.4). Each homogenate was centrifuged at 10,000x g for 20 min at 4 °C and the precipitate was washed three times with the same buffer. The supernatant obtained was taken as the water soluble protein fraction.Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis of water soluble protein (SDS-PAGE) was carried out in Hoeffer Slab gel apparatus using the method of Laemmli (1970) with some modifications . A 12% gThe protein content of the samples was determined by the method of Lowry et al  using boAll statistical calculations were carried out with the Statistical Package for Social Sciences (SPSS) software program (version 10.0 for Windows). The values are expressed as the mean±SD. The data were statistically analyzed using one-way analysis of variance (ANOVA) and significant difference of means was determined using Duncan's multiple range tests at the level of p<0.05 .All the lenses in G-I were found to be transparent. Twenty-four h of incubation in the presence of sodium selenite produced a dense cortical vacuolization and opacification. The addition of Drevogenin D  to the cThe activity of superoxide dismutase (SOD) was decreased following selenite administration, while treatment with Drevogenin D was found to maintain significantly higher levels of enzyme activity .2+ ATPase, was found to be decreased significantly following selenite induction whereas, treatment with Drevogenin D was found to maintain activity close to the normal levels  showed the normal protein profile of the WSF. Two bands in G-II (Lane-2) were found to have lower expression intensity in WSF and correspond to 22.0 kDa and 30.5 kDa, respectively. These bands in G-III (Lane-2) showed expression near to normal levels found in G-I .2+ ATPase activity and accumulation of Ca2+, which in turn leads to the activation of lens calpains and subsequent proteolytic degradation of lens soluble proteins in selenite cataract.Oxidative stress, which occurs due to the formation of free radicals in the cell as a consequence of both enzymatic and non-enzymatic reactions, has been established as a major factor involved in the development of most types of cataract . SelenitPrevious reports from India have shown that phytochemicals play an important role in the prevention of oxidative stress-induced cataract ,32. Our 2+ in the lens is maintained at submicromolar range and is lower than that of the aqueous humor [2+ have been implicated in cataractogenesis [2+ ATPase was significantly decreased by selenite administration with a corresponding increase in Ca2+. Treatment with Drevogenin D was observed to maintain the activity of Ca2+ ATPase and levels of Ca2+ close to normal range (2+ is maintained by Ca2+ ATPase, which counteracts the inward passive diffusion of Ca2+ [2+ ATPase is a major factor involved in maintaining lenticular Ca2+ levels and loss of its activity could explain the rise in Ca2+. Earlier reports have shown that Ca2+ ATPases are particularly sensitive to oxidation and that oxidative damage leads to decreased activity of these enzymes, resulting in elevated levels of Ca2+ in the lens [2+ ATPases are also reported to be sensitive to membrane lipid order and their decreased activity may be related to structural changes in membrane lipids [2+ and any alteration may decrease its capacity, which results in elevated levels of Ca2+ [2+ ATPase, and accumulation of Ca2+ in the lens. The lower levels of Ca2+, higher activity of Ca2+ ATPase, and decreased levels of lipid peroxidation in the lens of the Drevogenin D-treated group could be attributed to its antioxidant protection against selenite-induced oxidative stress.Calcium is essential for various lens fiber cell processes including its differentiation . The levus humor . It has ogenesis . In thisal range . The lev of Ca2+ . Ca2+ ATthe lens . Ca2+ ATe lipids . Studies of Ca2+ ,39. Thes2+-activated neutral proteases found in all tissues. In spite of their important regulatory functions under normal physiological conditions, accumulated levels of Ca2+ are observed to result in the proteolysis of crystallins associated with cataractogenesis. Though not found in humans, Lp82 are of significant importance in rodent cataractous models [2+ as described above (2+ observed. Hence, it could be inferred that the near normal levels of Ca2+ and Ca2+ ATPase activity following Drevogenin D treatment has a direct bearing on the observed decrease in the activation of Lp82 and calpain 2. The immunoblot of Lp82 was carried out to assess the level of its protein in relation to calpain activation. Major loss of Lp82 protein has been reported during selenite-induced cataract, which might be explained by the process of auto-proteolysis [Calpains are Cas models . In the ed above . Treatmeteolysis . The levCrystallins, the major structural proteins of the lens, are excellent substrates for lenticular calpains . The ele2+ and also the source of ATP in cells. Oxidative damage of mitochondria has been shown to increase cellular Ca2+ levels [2+ level normal and preventing calpain activation.Oxidative stress induced by selenite and its mitigation by Drevogenin D treatment was assessed by measuring the activity of SOD , levels + levels ,49 and c+ levels . The pro2+ ATPase activity, preventing an accumulation of calcium thus calpain activation, and prevention of protein oxidation and lipid peroxidation, which are all results of selenite induction, are supportive of the anticataractogenic potential of the compound. In our study Drevogenin D acts as an antioxidant to prevent critical sulfhydryl oxidation of Ca2+ ATPase and thereby, controlling the levels of calcium. Our results are found to be in agreement with the reported data [These results suggest that Drevogenin D treatment is effective in protecting the lens proteins by maintaining Cated data . The ant


Recently, several other hydroxylated BDE-47 metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood.Brominated flame retardants, including the widely used polybrominated diphenyl ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neurotransmitter release by releasing calcium ions (Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells.To investigate the neurotoxic potential of other environmentally relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47, BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47—6-OH-BDE-47 , 6′-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and 4′-OH-BDE-49—on intracellular Ca2+ release from intracellular stores, although with different lowest observed effect concentrations (LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum  or both ER and mitochondria . When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we observed lower LOECs for 6-OH-BDE-47 and 4′-OH-BDE-49, as well as for BDE-47.In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced Ca2+]i. Importantly, shielding of the OH group on both sides with bromine atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE metabolites.The present findings demonstrate that hydroxylated metabolites of BDE-47 cause disturbance of the [Ca Brominated flame retardants are added to a wide array of consumer products. Adverse effects of these compounds on the developing nervous system give cause for concern , BDE-100 , and BDE-153 , the main constituents of the commercial DE-71 pentaBDE mixture , as wellp-dioxins and dibenzofurans were removed from the PBDEs with a charcoal column as described by PBDEs  i were measured using the Ca2+-sensitive fluorescence ratio dye Fura-2 as described previously .Changes in i during exposure were determined per cell to investigate effects of PBDEs on Ca2+ homeostasis. The standard deviation during baseline [Ca2+]i recording ranged from 2% to 74% of average [Ca2+]i . To prevent registration of false-positive effects, an increase in [Ca2+]i to > 175% of baseline was used to determine no observed effect concentrations (NOECs) and lowest observed effect concentrations (LOECs). A transient increase within 0–10 min from the start of exposure is referred to as an “initial increase,” whereas additional increases are referred to as “late increases” . Categorical and continuous data were compared using Fisher’s exact test and Student’s t-test, respectively, paired or unpaired where applicable. Analysis of variance (ANOVA) and post hoc t-tests were performed to investigate possible dose–response relationships. To investigate structure–activity relationships, we investigated the possible influence of hydroxylation position and/or shielding of the OH group by adjacent atomic groups on the efficacy to increase [Ca2+]i. To this aim, we performed a multifactorial ANOVA using the mean increase in basal [Ca2+]i induced by 20 μM of the different OH-PBDEs as the dependent variable. Hydroxylation position  and the presence of either one or two shielding atomic groups (phenyl ring and/or Br atom) adjacent to the OH group were used as fixed variables ]. However, the frequency and duration of fluctuations in [Ca2+]i increased during exposure to 20 μM BDE-47. At 2 μM BDE-47, similar effects were observed . No effects could be detected on the frequency, duration, or amplitude of fluctuations during exposure to 20 μM BDE-49, BDE-99, BDE-100, or BDE-153 .Exposure to the solvent control [dimethyl sulfoxide (DMSO)] or to 20 μM of BDE-47, BDE-49, BDE-99, BDE-100, or BDE-153 did not decrease cell viability (data not shown) or increase the average or amplitude of i were observed during exposure to 6-OH-BDE-47, 6′-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, or 4′-OH-BDE-49 . Exposure to OH-PBDEs resulted in a dose-dependent increase in average and amplitude of [Ca2+]i with varying LOECs. LOECs are determined by the amplitude of the increase in [Ca2+]i .For the OH-PBDEs with an effect at 2 μM (6-OH-BDE-47 and 5-OH-BDE-47), we also tested lower concentrations , showing2+]i during exposure to 20 μM of the hydroxylated metabolites was independent of the position  of the OH group on the PBDE molecule . However, OH-PBDEs in which the OH group was shielded on only one side, with either the other phenyl ring or a Br atom, induce significantly higher increases in [Ca2+]i compared with OH-PBDEs in which the OH group was shielded on both sides . However, this influence of the shielding of the OH group (on one compared with two sides) was independent of its position  on the PBDE molecule .The mean amplitude of [Ca2+]i induced by 6-OH-BDE-47, 5-OH-BDE-47, and, to a lesser extent, 4′-OH-BDE-49 were reduced in Ca2+-free conditions, increases were still observed for all of the OH-PBDEs  or both ER and mitochondrial Ca2+ stores were depleted by either 1 μM thapsigargin (TG) or 1 μM TG plus 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). TG and TG/FCCP pretreatment, respectively, caused a transient increase in [Ca2+]i of 321 ± 11% (n = 191) and 393 ± 11% of baseline (n = 195), after which [Ca2+]i stabilized and the OH-PBDE was applied.Although initial increases in i less than when the OH group was less shielded . Also, the OH-PBDEs with only one shielded side of the OH group induced release of Ca2+ from ER at the lowest concentrations (6-OH-BDE-47 and 5-OH-BDE-47) or with the highest amplitude in Ca2+-free conditions (4′-OH-BDE-49). Thus, the toxicity of OH-PBDEs appears attenuated by shielding of the OH group on both sides by either the other phenyl ring and/or Br atoms.None of the parent PBDEs except BDE-47 showed any effects on Cal levels . Becausel levels . The meain vivo  and highest (899.1 ng/g lipids) concentrations of total OH-PBDEs observed in fetal plasma correspond to approximately 0.4 and 17.4 nM, respectively, in blood . Thus, the highest concentration observed in human blood is only two orders of magnitude lower than the LOEC for Ca2+ release from intracellular stores by OH-PBDEs (1 μM). Moreover, the LOEC for increased Ca2+ fluctuations is even lower (0.2 μM), meaning that the margin of exposure is insufficient in some individual exposure situations. Also, because OH-PBDEs are not associated with lipids—as are the parent PBDEs—but have a high affinity for plasma proteins (2+]i could result in functional or even adverse effects in vivo.All five OH-PBDEs investigated in the present study caused Caproteins , the estin vivo (in vitro (2+]i by release from intracellular stores appears to be a common mechanism for OH-PBDEs and ortho-PCBs (for review, see 2+]i, which not only is a trigger for neurotransmitter release but affects many cellular processes (Because exposure to organohalogen compounds within the time frame of rapid brain development can result in behavioral defects in mice (for review, see in vivo  and in vin vitro . Concernrocesses , is not Because very high concentrations of PBDEs are occasionally measured in humans, the voluntary and legislative measures to reduce the release of PBDEs into the environment appear justified. Also, hydroxylated metabolites of PBDEs, which were recently found to bioaccumulate in humans , either


The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-μs rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions (∼100 nM) to approximately 1 μM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O 2+ in a cooperative manner and plays an important role in shaping Ca2+ signals in various cells. We used two models, a widely tested one and a novel, mathematically simplified one, to resolve the dynamics of a cooperative binding process. The cooperative nature of Ca2+ binding to calretinin results in accelerated binding as calretinin binds more Ca2+. This behavior constitutes an important new insight into the regulation of intracellular Ca2+ that cannot be matched by noncooperative artificial Ca2+ buffers. Our simple mathematical model can be used as a tool in determining the kinetics of other biologically important molecular interactions.The binding of a ligand to a protein is one of the most important steps in determining the function of these two interactive biological partners. In many cases, successive binding steps occur at multiple sites such that binding at one site influences ligand binding at other sites. This concept is called cooperative binding, and constitutes one of the most fundamental properties of biological interactions. The functional consequences of cooperativity can be accurately resolved when reactions are at equilibrium, but mathematical complexity has prevented insights into the dynamics of the interactions. We studied the protein calretinin, which binds Ca 2+-buffering kinetics due to cooperative binding in calretinin.A novel and relatively simple mathematical model for the kinetics of cooperative binding reveals, the tuning of Ca To achieve its role as messenger, the intracellular Ca2+ concentration ([Ca2+]) is very tightly regulated in time, space, and magnitude. The spatiotemporal characteristics of short-lived and often highly localized changes in intracellular [Ca2+] result from a complex interplay between Ca2+ influx/extrusion systems, mobile/stationary Ca2+-binding proteins (CaBPs), and intracellular sequestering mechanisms. Understanding the kinetics of cellular Ca2+ transients and its influence on Ca2+-regulated processes requires a precise knowledge of the Ca2+ sensitivities and binding properties of all the components involved, including the binding dynamics to buffering and signaling CaBPs. However, uncertainties in current models studying intracellular Ca2+ signaling arise mostly from the lack of accurate data on the binding properties of specific molecules involved in Ca2+ handling, considerably limiting the value of such modeling  with the fluorescent Ca2+ indicator dye Oregon Green BAPTA-5N (OGB-5N) as previously described [2+] ensued from a resting concentration of approximately 2.4 μM to 11 μM. At this initial [Ca2+], approximately 99.5% of the DMn is in the Ca2+-bound form, ensuring that (1) virtually every uncaged DMn molecule will release Ca2+, and (2) the amount of free DMn capable of rebinding uncaged Ca2+ ions [2+] in the absence of CR, leading to an almost step-wise increase in [Ca2+] . The aim was to find a mathematical description for the Ca2+-binding properties of CR that best fits the experimental fluorescence traces generated under various conditions.We determined the kinetics of Caescribed . Changesa2+ ions ,26 is con [Ca2+] A. The pr2+ binding, we relied on steady-state Ca2+-binding properties determined previously. With the same human recombinant CR, selected Hummel-Dryer experiments yielded a Hill coefficient of 1.3 for the four binding sites, with a Kd of 1.5 μM  decay with a set of exponential functions. However, rebinding of Ca2+ to free DMn affects the decay kinetics. In addition, the changing properties of the binding sites that underlie cooperativity are expected to cause a shift in the kinetic properties of binding during the decay phase. As a consequence, the relative contribution of multiple decay time constants is continuously shifting. Although fitting the [Ca2+] decay with exponentials might result in time constants for a given trace, this does not allow accurate deduction of the Ca2+-binding kinetics of CR. A mathematical model simultaneously describing all processes taking place in the recording chamber, including a “total” description of the cooperative and noncooperative binding of Ca2+ to CR is expected to yield more reliable information on the kinetic properties of CR. To model the cooperativity, we started out by including an allosteric influence between the binding sites of the pairs. This was achieved by setting two states (R and T) for a particular binding site, with each having its own set of rate constants. A binding site is in the “tensed” state (T), with a low affinity for Ca2+, when no Ca2+ is bound to the other site in the pair, whereas a binding site is in the “relaxed” state (R), with a high affinity for Ca2+, when the other site already has a Ca2+ ion bound. We assumed that binding of Ca2+ to one site always leads to a rapid transition T→R in the other site and that an unbinding of Ca2+ from one site always leads to a rapid transition R→T in the other site. This allowed us to incorporate the transition rates between states R and T in the binding and unbinding rate constants, further simplifying the model (The most straightforward approach to determine the kinetics of a system would consist of fitting the  , total [CR], total [DMn], as well as on the lot number of OGB-5N, with each lot having slightly different properties  and kon(V) for the independent site. However, to aid the choice of starting values, the cooperative part of the model was set up such that kon(R), kon(T), the apparent Kd (Kd(app)) for the pairs, and the Hill number (nH) could be fitted. This was achieved by adding a calculation step that determined Kd(R) and Kd(T) from the latter two parameters ,kon(V),kon(R), kon(T), Kd(app), and nH) were never constrained or fixed to a certain value. The atypical fit results were probably caused by local minima in the error function of the fit routine. Such local minima are expected, based on the fairly large number of degrees of freedom where the parameters are not completely independent. Deviations in one parameter can be partially compensated by “shifting” other parameters. Initially, we used a model that did not include cooperativity and found that most of the 38 individual sets could be fit reasonably well. For a given individual dataset, the quality of the fits were similar between a model with a Hill coefficient of either 1 or 1.9. But when comparing the fit results of all 38 sets, most of the fitted parameters showed strong deviations  when using nH = 1. The high variability of the binding parameters found when assuming nH = 1 is shown in 2+-binding properties without cooperativity, in line with previous steady-state findings of nH values between 1.3 and 2.4 , when Ca2+-free binding sites of any affinity in any state are abundant  Kd of 28 μM with a kon of 1.8×106 M−1s−1 and a koff of 53 s−1 that will dramatically change to a (R state) Kd of 68 nM with a kon of 3.1 × 108 M−1s−1 and a koff of 20 s−1 once the cooperative partner site has already bound Ca2+. The Kd(app) for the cooperative sites is 1.4 μM with an nH of 1.9.All 38 results for the fitted values were plotted as a log-normal cumulative probability distribution because they have a log-normal distribution  and 5, etributed E. These L, which is also dependent on the number of Ca2+ ions bound  suffices, as described in the original MWC model  (2.0–5.3 μM), dictated by the constraints that most (>99%) of the DMn should be in the Ca2+-bound form to obtain valuable data. At lower resting [Ca2+], the unbound DMn, present at higher concentrations than the free [CR], would rapidly rebind most of the released Ca2+ [2+] decay small and difficult to distinguish. Because buffering kinetics depend on both the on-rates and the concentration of free buffer, the overall Ca2+ binding speed to DMn would be much higher than that to CR, thus masking Ca2+ binding to CR. With much less Ca2+-bound DMn, the changes in [Ca2+] would be quite small and difficult to detect. Such technical constraints do not allow performing the experiments over the whole “physiological” range of [Ca2+], e.g., from 10 nM to 100 μM. Thus, we could not exclude that the two models describe systems with different kinetic properties outside the boundaries of our experiments. This possibility was tested by examining the behavior of each model as a filtering system for Ca2+ signals regulated by resting levels of [Ca2+]. The filtering properties of both models were determined over a range of conditions covering the whole physiological range that CR is expected to encounter. CR (500 μM) was subjected to a wide frequency range (0.3 Hz to 10 kHz) of small (1 nM) sinusoidal perturbations of the [Ca2+] at a wide range of starting [Ca2+] (1 nM to 100 μM). The resulting Ca2+ “waves” were close to sinusoidal. We used their amplitude as output of the filter to determine the transfer function of CR  or when CR becomes fully occupied as the starting [Ca2+] gets higher. Remarkably, both models show a similar strong increase in attenuation at the lower frequencies, when the Ca2+ concentration gets close to the apparent Kd value of the cooperative sites, i.e., approximately 1.5 μM. The similarity of the transfer function of CR using either model indicates that they quantify the kinetics of Ca2+ binding by CR in a similar way (but via different mechanisms) over the whole physiological range of conditions.Both models accurately fitted the data in a quantitative manner; based on the quality of the fits, they were indistinguishable, and technically can both be used to quantify the kinetic properties of Cadentical . For the Kd and E; nH. Osed Ca2+ , making on of CR . The att2+-binding kinetics of CR. However, we consider the new model to facilitate the “intuitive” understanding of how the kinetic properties of the cooperative sites relate to the binding kinetics at the level of the whole protein or at the macroscopic level. The Adair-Klotz model is the most general description of equilibria in terms of stochiometric binding. It describes the steady-state equilibrium using the constants  for the successive binding (or macroscopic) steps, but not as the affinity constants of the individual (or microscopic) binding sites:ν) of a protein P is described by the Adair-Klotz equation:Both our new model, which is closely linked to the Adair-Klotz model ,7, and tK values are denoted, which is sufficient for steady-state descriptions. The equilibrium constants for the new model (KT and KR) and the MWC model  can often rather easily be translated into macroscopic K values  around the apparent Kd for the cooperative binding sites, CR will more effectively buffer perturbations at lower frequencies  from which the [Ca2+] step was induced. As the resting [Ca2+] increases, the [Ca2+] decay kinetics in the presence of either one of the noncooperative chelators BAPTA and EGTA is slowed down . The amount of BAPTA necessary to mimic CR is on the order of 500 μM when the initial [Ca2+] is approximately 1–10 μM  (2+] (20–100 nM) typically seen in neurons and more BAPTA-like at the higher [Ca2+] observed during bouts of activity.As described above, at a starting  from a resting [Ca2+] of 100 nM in the presence of 100 μM CR, the initial ratio for [unoccupied T sites]/[unoccupied R sites] is 395 μM/1.4 μM  will not be sufficient, and most of the bindings to the R site will be time-limited by initial binding to T sites. However, at a resting [Ca2+] of 1 μM, the initial ratio of unoccupied T/R sites is 250 μM/9 μM. In this case, 5 μM Ca2+ will bind to T sites, 4.8 μM will bind to R sites, and 0.15 μM to site V. Thus, enough sites (9 μM) in the fast R state will be available and allow for fast buffering, not necessitating the slower stepwise change from the T to the R state. Model simulations of these experiments indicate that the surplus of sites in the R state will, within approximately 1 ms, bind up to 0.7 μM Ca2+ more, which at a later time shifts to a CR molecule in the T state. By acting as a temporary substitute, the free sites in the R state can even “speed up” the eventual buffering of binding to sites in the T state. Albeit the initial amount of free sites in the R state is relatively small, it will significantly contribute to the buffering speed at initial [Ca2+] around Kd(app). Also, the low-affinity independent site, which will hardly play a role in the steady-state buffering, initially binds up to 1.8 μM Ca2+ which is later “transferred” to sites in the T state and will contribute to the overall buffering speed. However, this contribution is virtually identical when starting from either 100 nM or 1 μM Ca2+.Since the second step in this process is governed by a high-affinity site, a considerable number of the cooperative pairs will be fully occupied. The combination of these reaction steps gives rise to the intermediate apparent 2+ buffers BAPTA and EGTA. Since BAPTA and EGTA have significantly different on-rates , it is generally assumed that a process influenced by BAPTA, but not by EGTA, must have Ca2+-binding on-rates comparable to ones of BAPTA. So far, two studies ), at least 40% of either EGTA or BAPTA are occupied by Ca2+ ions and do not add to the buffering speed. Our findings show that if BAPTA (or EGTA) can replicate a cellular buffering process under certain experimental conditions defined by, e.g., initial resting [Ca2+], step size, and geometry of Ca2+ influx, it does necessarily mean that the cellular buffering is “comparable to BAPTA or EGTA” and therefore “fast” or “slow,” respectively.It was thus surmised that CR must have one or several binding sites with fast on-rate(s) . The con2+ hotspots, as suggested for calmodulin around the L-type Ca2+ channel  together with its suggested mobility  can never fully replicate certain functional aspects of CaBPs, because the complexity of Ca2+ binding to “real” CaBPs such as CR cannot be mimicked by small synthetic Ca2+ buffers lacking cooperativity.The exact intracellular distribution of CR is not well understood ; althoug channel . Such lo CaBP CB ,35), supficiency ,36. Acco2+ sensors such as calmodulin , a CaBP with two Ca2+ binding sites and minimal cooperativity , when Ca2+ elevations were small. Under these conditions, CR caused a leftward shift in the concentration-response relationship as observed with the slow buffer PV. Under the same conditions, CR produced localized Ca2+ transients or “puffs,” a phenomenon never observed in the presence of BAPTA  in our experimental solutions were calculated, and the corrected values were used in our analyses. Experiments were carried out at room temperature (∼25 °C). All chemicals were obtained from Sigma-Aldrich, unless otherwise mentioned. Values are expressed as mean ± the standard error of the mean.All experiments were performed in solutions containing 120 mM KCl, 40 mM HEPES (pH set at 7.30), 100 μM Oregon Green BAPTA-5N , a varying amount (4–17 mM) of DM-nitrophen -1,2-diaminoethane-N,N,N′,N′-tetrasodium salt; Calbiochem) and CaCl for DMn . For the2+] of each solution was titrated to be between around 2 μM or 8 μM (indicated in Results), using custom-made Ca2+-selective electrodes [Kd of the Ca2+ buffer in standard solutions was regulated by changing the pH [2+]) standard solutions with an ionic strength of 120 mM and pH around 7.3 (see http://www.stanford.edu/~cpatton/maxc.html). These standard solutions were used for calibrations; the standard pCa 6 solution was used as a reference solution in the Ca2+ electrodes.The initial free  was measured with the Ca2+-sensitive electrodes. This procedure was repeated until the sought concentration was reached. All [Ca2+] were verified in the uncaging experiments by comparing baseline fluorescence (Frest) with the maximal possible fluorescence (Fmax) for that solution. Fmax was determined by repetitive uncaging of Ca2+ in some of the samples until fluorescence did not increase any further (so that [Ca2+] ≫Kd(OGB-5N)). The [Ca2+] was calculated using the following equilibrium formula:To obtain the experimental solutions, Ca2+ binding to CR with high temporal resolution, we used UV-flash photolysis of DMn. We used a setup that was described earlier [2+] changes. The [Ca2+] transients changed substantially only when the illumination spot was moved towards the outer edge of the optical fiber, i.e., to the edge where uncaging took place. This indicated that diffusion artifacts between areas of UV illumination (uncaging) and no UV illumination (no uncaging) on the timescale of our measurements occur only very close to the edge of the UV-illuminated area and not in the area where data were collected. The fluorescence of OGB-5N was measured with a photodiode  in the focal plane of the microscope. The small diameter of the photodiode (200 μm) minimized errors caused by diffusion in the z-axis. Despite using appropriate optical filters and excitation spectrum of OGB-5N peaks at 494 nm, the high-energy UV flashes still induced brief, but large, optical transients that saturated the detection system. To avoid these artifacts, a patch clamp amplifier  with an integrating headstage was used to measure the currents generated by the photodiode. The feedback capacitor of the headstage was short-circuited (reset), and its readout was blanked exactly at the time of the UV flash so that there was no signal measured at the instant of the flash. The analog signal was low-pass filtered with the eight-pole low-pass Bessel filter of the amplifier at 10 kHz, digitized at 50 kHz , and sampled on a PC with a custom-made program (EVAN) written in LabView . A pulse generator  was used to trigger the sampling, UV laser, headstage reset and blanking, and the shutter for the OGB-5N excitation light.To measure the dynamics of Ca earlier ,42,43. BFor a typical experiment, an approximately10-μl droplet of DMn solution was placed in the recording chamber. During each flash, only 0.05% to 1.5% of the DMn in the spot directly in front of the optical fiber was uncaged , we measured a Kd of 36.1 μM, a koff of 8.67 × 103 s−1, a kon of 2.4 × 108 M−1s−1, and an Fmin/Fmax ratio of 40.0. These values were used in the mathematical model (see below) to describe the properties of the two batches of OGB-5N used in the various experiments. We have no explanation for the variability between these two batches other than the fact that specific contaminations might occur in different batches from the supplier .We used the low-affinity dye OGB-5N because of its fast kinetics of Caescribed . For oneFor each group of experiments, we determined the properties of DMn independently by uncaging experiments with no protein present, as described before . These pEscherichia coli and purified with a series of chromatographic steps as described before ). The results for the MWC model (blue symbols in the paper) are omitted for clarity. The black symbols are the fit results when a model for CR was used in which the four cooperative sites are simulated when nH = 1 (no cooperativity). Although fitting the uncaging curves with such a model gives reasonable fits, the results (black symbols) of most of the fitted parameters showed strong deviations when using nH = 1 for the four cooperative sites. This indicated that there is no unique solution to describe CR's Ca2+-binding properties without cooperativity, in line with previous steady-state findings of nH values.The figures shown here are identical to (298 KB PDF)Click here for additional data file.Protocol S1(1) Supplemental 2+-sensistive electrodes and to determine the Kd's of various OGB-5N lots.(2) Supplemental (3) Complete description of the mathematical model (the Berkeley Madonna model) describing the reactions occurring in the experiments represented in a schematic and a mathematical way.(4) Description of the mathematical relationship between the Hill equation and the new and the MWC models via the Adair-Klotz equation for two binding sites.(624 KB DOC)Click here for additional data file.


The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels , and hyperpolarization-induced anion currents.The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca The cell-attached configuration of the patch-clamp technique respects the integrity of the intracellular milieu. It therefore reflects best the physiological condition of the currents recorded across the membrane patch trapped within the tip of the microelectrode. Patch-clamping of human red cells in this configuration is particularly difficult because of the extreme fragility of the cell membrane, which accounts for the scarcity of the literature on its application to study channel activity in intact red cells Although a negative pressure pulse of about 10 mmHg is usually applied to establish GigaOhm seals at the tip of the patch pipette, comparable good seals can be obtained without underpressure, albeit with a lower success rate. Once the seal is established, the membrane deformation induced by the glass pipette, regardless of the intensity of underpressure, is not under experimental control, and may vary from one cell to another.2+ in the bathing medium was essential for the transient current response.In our previous studies in intact red cells we seldom observed spontaneous channel activity in cell attached patches when the cells were bathed in physiological saline solution. Episodically though, we did detect transient activity immediately following seal formation, but only when contact was facilitated by underpressure. Intrigued by the systematic link between the negative pressure pulse and the transient current response, we explored some of the medium requirements in preliminary experiments. It soon became clear that the presence of Ca2+ and other ions has been documented for a number of physiological and pathological processes in the past, based mostly on experimentation with red blood cell suspensions. Physiological shear stress in the circulation has been claimed to cause a reversible increase in Ca2+ permeability2+ extrusion capacity of the plasma membrane Ca2+ pump, aided by minor episodes of increased Ca2+ permeability in the circulation +, K+, Ca2+ and Mg2+2+]i resulting from Psickle activation in turn activates the Ca2+-sensitive K+ channel of the red cell membrane 2+ associated with local dynamic membrane deformations was also suggested to be involved in the process of apical alignment of malaria merozoites, just before invasion The association between red cell membrane deformation and changes in membrane permeability affecting Ca2+ conductance (PCa) and spontaneous decay.There is a suggestive link between our observations on deformation-induced red cell currents and the body of research briefly reviewed above. The potential relevance of this link prompted the current investigation. The electrophysiological evidence presented here shows that seal formation, when associated with negative pressure, causes transient activation of Gardos channels and anion currents, and that this phenomenon results from activation of a permeability pathway with a finite CaIn the experimental series reported here, over 500 attempts were made to obtain a GigaOhm seal in cell attach configuration, among which 284 were successful. The mean duration of the high-resistance cell-attached seals was 25–35 min, allowing multiple sequences of 1 min recording. The noise level remained within the ±0.08 pA range, ensuring optimal signal to noise ratios in the detection and measurement of channel-mediated currents in the red cell membrane. Channel conductances were measured by applying alternating ramps of 1 min duration of hyperpolarizing or depolarizing voltages, but most other current measurements were performed at the physiological spontaneous membrane potential of RBCs (∼−10 mV).i.e. at pipette potential of zero mV (-Vp = 0 mV), whatever the degree of membrane deformation induced by underpressure. However, recordings made during the first ∼10 min following seal formation frequently displayed inward current at the spontaneous membrane potential (41 out of 56), corresponding to long channel openings followed by long silent intervals, as shown with two examples in Using the cell-attached configuration with solution A  in the pipette and bathing solutions (56 patches), RBC membranes seldom displayed channel activity when recordings were performed more than 10–15 min after seal formation (5 out of 56), at the spontaneous membrane potential, In contrast, when NaCl was replaced by KCl  in the pipette solution, channel activity was always observed immediately after seal formation as bursts of channel openings separated by brief closures, and all patches (n = 136) contained one or more active channels . The sizA protocol of hyperpolarizing and depolarizing voltage ramps was performed to characterize the conductance properties of the measured currents. Sixty seconds of current recordings were obtained for each imposed voltage, in the following order (in mV): 0, ±10, ±20, …, ±100. With this protocol we systematically obtained unusual V-shaped I/V curves  which coTo confirm this suspected time-decline and to investigate its modality, currents were recorded continuously for up to 16 min in 35 different patches . The resWhen the declining currents reached the 0.3–0.5 pA level their magnitude remained quite constant for a few minutes but their Po declined progressively before total loss of channel activity. The duration of this period varied considerably among cells. The currents recorded at different imposed potentials during this pre-extinction period are shown in In 27 out of 136 patches we could record very fast transitions in channel activity instead of progressive decline. + gradients (whilst chloride gradients remained constant) suggesting mediation by a K+-selective channel. The inset between 2+-dependence of the K+ currents it was important to reduce the Ca2+ concentration in the medium. With pCa = 7 in the extracellular media (bath and pipette), the success rate of gigaOhm seal formation was reduced by half. In these conditions (pCa = 7), channel activity was fully inhibited . The transient nature of the response may reflect a declining time-course of PCa, a delayed response of the plasma membrane Ca2+ pump (PMCA) slowly restoring [Ca2+]i to sub-activation levels of the K+ channels, or both. The pattern of change in open state probability documented here offers some clues. 2+ desaturation of the channels, the sustained Po levels prior to terminal desaturation indicate that the pump-leak flux-balance within the cells must have been with [Ca2+]i levels sufficiently high to elicit maximal, Ca2+-saturated channel activity. If the transient response is due to delayed pump activation then pump inhibition should lead to a sustained response. In the experiment of 2+]i and consequently reduces the duration of the Gardos-mediated currents, but pump inhibition does not suppress the declining pattern. It follows that the rate of decline in the transient currents measured in the absence of pump inhibition overestimates the true inactivation rate of the deformation-induced PCa.A main question arising from these results concerns the transient nature of the deformation-induced response. Instant Gardos channel activation after underpressure-aided seal formation suggests a rapid initial raise in i. The powerful, ATP-fueled plasma membrane Ca2+ pump maintains a physiological pump-leak balance with an intracellular Ca2+ concentration in the 20–50 nM range 2+ gradient, induces a new pump-leak balance with [Ca2+]i high enough to elicit maximal activation of the Gardos channels. The results showed that at the temperature of our experiments (35°C±2°C) the open state probability of the channels during the first ∼ten minutes was in the range 0.3 to 0.4 . Secondly, a relatively brief terminal period with a sharp decline in Po, reflecting Cavanadate , the dur2+ extrusion capacity by the pump (Vmax) among cells Vmax will be able to restore [Ca2+]i down to sub-activation levels for Gardos channels faster than an older cell.Another feature of the transient response was the variability in its duration, as apparent in a represents a [Ca2+]i threshold level for Gardos channel activation below which the channels remain silent. Line b represents the [Ca2+]i saturation level above which Gardos channel activity is maximal. Thus, while the Ca2+ content is above b Po remains constant at it maximal level. Between b and a, Po becomes progressively reduced following Ca2+ desaturation. With this representation, it is possible to explain the observed variability in the duration of the transients by the variable pump-mediated calcium extrusion capacity among the cells, young cells represented by curve 1, older cells or pump-inhibited cells , and the equilibrium potentials for K+ and Cl− . The effect of membrane deformation was simulated by a sudden and sustained increase in K+ permeability (PGK) to 10 h−1, a value representing the mean increase in K+ permeability measured in red blood cell suspensions with maximally activated Gardos channels The observed reduction in current amplitude without variation in open-state probability cannot be attributed to Gardos channel Ca decline . CurrentGK caused immediate and sustained hyperpolarization, from −12 mV to −60 mV . However, our results show that upon deformation-induced Gardos channel activation, anion-selective channels also become activated , N-methyl-D-glucamine chloride (NMDG) 5-Nitro-2-(3-phenylpropylamino)-benzoate (NPPB), ethylene glycol-bis(2-aminoethylether)-N,N,N, N,-tetraacetic acid (EGTA), were purchased from Sigma .2, 10 Hepes, 10 glucose, pH 7.4. For identification of K+ channels, in cell-attached or excised inside-out configuration, pipette and/or bathing solutions contained (in mM) 150 KCl, 5 NaCl, 1 MgCl2 10 Hepes, pH 7.4 (solution B).Basic pipette and bathing solutions for cell-attached configuration (solution A) contained (in mM): 150 NaCl, 5 KCl, 1 MgCl2+ buffer when the pCa (-Log[Ca2+]) was adjusted to pCa = 7. All solutions were filtered through 0.2 µm Millipore cellulose disks and equilibrated in air . Osmolality was 310±5 mosmolal.EGTA was used as CaVenous blood was drawn into heparinized vacutainers from healthy volunteers upon written informed consent. RBCs were washed thrice by centrifugation and resuspension in large volumes of culture RPMI medium (Gibco BRL). The buffy coat containing platelets and white cells was removed by suction after each wash. After the last wash, the cells were suspended at 50% hematocrit in RPMI and kept at 4°C. They were suspended at 10% hematocrit in solution A and incubated at 37°C for about 1 hour prior to experiments. Experiments were done at the temperature of 35±2°C.Patch pipettes (tip resistance 10–20 MOhm) were prepared from borosilicate glass capillaries , pulled and polished on a programmable puller . Doming patch-clamp seals (4 to 20 GOhm) were obtained by a suction pulse of 10 mmHg applied for less than 5 s, using a syringe connected to the patch pipette. Single channel currents were recorded using a RK400 patch-clamp amplifier , filtered at 0.3 or 1 kHz, digitized (48 kHz) and stored. For analysis, played back data was processed by the WinEDR V2.7.6 computer program .http://www.physiol.cam.ac.uk/staff/lew/index.htm.The sign of the clamped voltage (Vp) refers to the pipette solution with respect to the bath. Outward currents (positive charges flowing across the patch membrane into the pipette) are shown as upward deflections in the current traces. In the cell-attached configuration the imposed membrane potential (Vm) is referred to as -Vp. Current-voltage (I/V) curves were constructed by plotting the mean current amplitude for each clamped potential. Open probability (Po) was determined as the fraction of digitized points above a threshold set midway between the closed and open peaks of current-amplitude histograms. Each Po was determined from 60 seconds stable recordings. In these conditions, Po was defined as the ratio of the total time spent in the open state to the total time of the record. Analyses were confined to patches containing one channel event histogram. Data are given as mean values ± SEM. The Lew-Bookchin mathematical-computational model