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Retailing consists of the activities involved in selling directly to the ultimate consumer for personal, non-business use. At present, organized and unorganized retail stores act as an important part of every human life. It helps the people to purchase all the items in one place. The consumer cannot act as same at the time of purchasing goods. Some consumer act as the prospective buyer to choose the right brand based on his/her needs. Some consumer are not with awareness to purchase quality product at reasonable price. In the competitive market, consumers should be prepared to purchase goods. [ABSTRACT FROM AUTHOR]

In India, stab injury is not uncommon, but identifying potential life threatening conditions in the emergency room (ER) and initiating prompt treatment are challenging. This is a case report of a young patient who presented to the ER with assault injury to the chest and shock; timely extended focused assessment with sonography for trauma helped to fast-track the patient to the operating room. A brief review of diagnosis and management of penetrating cardiac injury is presented herewith. [ABSTRACT FROM AUTHOR]

Genomic and transcriptomic data have been generated across a wide range of prostate cancer (PCa) study cohorts. These data can be used to better characterize the molecular features associated with clinical outcomes and to test hypotheses across multiple, independent patient cohorts. In addition, derived features, such as estimates of cell composition, risk scores, and androgen receptor (AR) scores, can be used to develop novel hypotheses leveraging existing multi-omic datasets. The full potential of such data is yet to be realized as independent datasets exist in different repositories, have been processed using different pipelines, and derived and clinical features are often not provided or  not standardized. Here, we present the curatedPCaData R package, a harmonized data resource representing >2900 primary tumor, >200 normal tissue, and >500 metastatic PCa samples across 19 datasets processed using standardized pipelines with updated gene annotations. We show that meta-analysis across harmonized studies has great potential for robust and clinically meaningful insights. curatedPCaData is an open and accessible community resource with code made available for reproducibility., (© 2023. The Author(s).)

Genomic and transcriptomic data have been generated across a wide range of prostate cancer (PCa) study cohorts. These data can be used to better characterize the molecular features associated with clinical outcomes and to test hypotheses across multiple, independent patient cohorts. In addition, derived features, such as estimates of cell composition, risk scores, and androgen receptor (AR) scores, can be used to develop novel hypotheses leveraging existing multi-omic datasets. The full potential of such data is yet to be realized as independent datasets exist in different repositories, have been processed using different pipelines, and derived and clinical features are often not provided or unstandardized. Here, we present the curatedPCaData R package, a harmonized data resource representing >2900 primary tumor, >200 normal tissue, and >500 metastatic PCa samples across 19 datasets processed using standardized pipelines with updated gene annotations. We show that meta-analysis across harmonized studies has great potential for robust and clinically meaningful insights. curatedPCaData is an open and accessible community resource with code made available for reproducibility., Competing Interests: COMPETING INTERESTS The authors declare the following competing interests: J.C.C. is co-founder of PrecisionProfile and OncoRX Insights. All other authors declare no competing interests.

Antimicrobial treatments for extensively drug-resistant Acinetobacter baumannii (XDR-AB) infections have proven lackluster, while dosing challenges in patients receiving continuous renal replacement therapy continue. We describe a patient receiving cefiderocol, ampicillin/sulbactam, and tigecycline for XDR-AB while undergoing continuous venovenous hemodiafiltration. The clinical course, cefiderocol and sulbactam pharmacokinetics, and synergy assessments are described., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Detection of copy number variations (CNVs) from high-throughput next-generation whole-genome sequencing (WGS) data has become a widely used research method during the recent years. However, only a little is known about the applicability of the developed algorithms to ultra-low-coverage (0.0005-0.8×) data that is used in various research and clinical applications, such as digital karyotyping and single-cell CNV detection., Result: Here, the performance of six popular read-depth based CNV detection algorithms (BIC-seq2, Canvas, CNVnator, FREEC, HMMcopy, and QDNAseq) was studied using ultra-low-coverage WGS data. Real-world array- and karyotyping kit-based validation were used as a benchmark in the evaluation. Additionally, ultra-low-coverage WGS data was simulated to investigate the ability of the algorithms to identify CNVs in the sex chromosomes and the theoretical minimum coverage at which these tools can accurately function. Our results suggest that while all the methods were able to detect large CNVs, many methods were susceptible to producing false positives when smaller CNVs (< 2 Mbp) were detected. There was also significant variability in their ability to identify CNVs in the sex chromosomes. Overall, BIC-seq2 was found to be the best method in terms of statistical performance. However, its significant drawback was by far the slowest runtime among the methods (> 3 h) compared with FREEC (~ 3 min), which we considered the second-best method., Conclusions: Our comparative analysis demonstrates that CNV detection from ultra-low-coverage WGS data can be a highly accurate method for the detection of large copy number variations when their length is in millions of base pairs. These findings facilitate applications that utilize ultra-low-coverage CNV detection.

Mcl1 is a primary member of the Bcl-2 family-anti-apoptotic proteins (AAP)-that is overexpressed in several cancer pathologies. The apoptotic regulation is mediated through the binding of pro-apoptotic peptides (PAPs) (e.g., Bak and Bid) at the canonical hydrophobic binding groove (CBG) of Mcl1. Although all PAPs form amphipathic α-helices, their amino acid sequences vary to different degree. This sequence variation exhibits a central role in the binding partner selectivity towards different AAPs. Thus, constructing a novel peptide or small organic molecule with the ability to mimic the natural regulatory process of PAP is essential to inhibit various AAPs. Previously reported experimental binding free energies (BFEs) were utilized in the current investigation aimed to understand the mechanistic basis of different PAPs targeted to mMcl1. Molecular dynamics (MD) simulations used to estimate BFEs between mMcl1-PAP complexes using Molecular Mechanics-Generalized Born Solvent Accessible (MMGBSA) approach with multiple parameters. Predicted BFE values showed an excellent agreement with the experiment ( R 2 = 0.92). The van-der Waals (ΔG vdw ) and electrostatic (ΔG ele ) energy terms found to be the main energy components that drive heterodimerization of mMcl1-PAP complexes. Finally, the dynamic network analysis predicted the allosteric signal transmission pathway involves more favorable energy contributing residues. In total, the results obtained from the current investigation may provide valuable insights for the synthesis of a novel peptide or small organic inhibitor targeting Mcl1.

Myeloid cell leukemia 1 (Mcl1), is an antiapoptotic member of the Bcl-2 family proteins, has gained considerable importance due to its overexpression activity prevents the oncogenic cells to undergo apoptosis. This overexpression activity of Mcl1 eventually develops strong resistance to a wide variety of anticancer agents. Therefore, designing novel inhibitors with potentials to elicit higher binding affinity and specificity to inhibit Mcl1 activity is of greater importance. Thus, Mcl1 acts as an attractive cancer target. Despite recent experimental advancement in the identification and characterization of benzothiophene and benzofuran scaffold-merged compounds, the molecular mechanisms of their binding to Mcl1 are yet to be explored. The current study demonstrates an integrated approach - pharmacophore-based 3D-QSAR, docking, molecular dynamics (MD) simulation and free-energy estimation - to access the precise and comprehensive effects of current inhibitors targeting Mcl1 together with its known activity values. The pharmacophore - ANRRR.240 - based 3D-QSAR model from the current study provided high confidence (R 2 =0.9154, Q 2 =0.8736 and RMSE = 0.3533) values. Furthermore, the docking correctly predicted the binding mode of highly active compound 42. Additionally, the MD simulation for docked complex under explicit-solvent conditions together with free-energy estimation exhibited stable interaction and binding strength over the time period. Also, the decomposition analysis revealed potential energy contributing residues - M231, M250, V253, R265, L267 and F270 - to the complex stability. Overall, the current investigation might serve as a valuable insight, either to (i) improve the binding affinity of the current compounds or (ii) discover new generation anticancer agents that can effectively downregulate Mcl1 activity. Communicated by Ramaswamy H. Sarma.

The Bcl-2 family proteins are the central regulators of apoptosis. Due to its predominant role in cancer progression, the Bcl-2 family proteins act as attractive therapeutic targets. Recently, molecular series of Benzothiazole Hydrazone (BH) inhibitors that exhibits drug-likeness characteristics, which selectively targets Bcl-xL have been reported. In the present study, docking was used to explore the plausible binding mode of the highly active BH inhibitor with Bcl-xL; and Molecular Dynamics (MD) simulation was applied to investigate the stability of predicted conformation over time. Furthermore, the molecular properties of the series of BH inhibitors were extensively investigated by pharmacophore based 3D-QSAR model. The docking correctly predicted the binding mode of the inhibitor inside the Bcl-xL hydrophobic groove, whereas the MD-based free energy calculation exhibited the binding strength of the complex over the time period. Furthermore, the residue decomposition analysis revealed the major energy contributing residues - F105, L108, L130, N136, and R139 - involved in complex stability. Additionally, a six-featured pharmacophore model - AAADHR.89 - was developed using the series of BH inhibitors that exhibited high survival score. The statistically significant 3D-QSAR model exhibited high correlation co-efficient (R 2  = .9666) and cross validation co-efficient (Q 2  = .9015) values obtained from PLS regression analysis. The results obtained from the current investigation might provide valuable insights for rational drug design of Bcl-xL inhibitor synthesis.

B-cell lymphoma 2 (Bcl-2) family proteins are the central regulators of apoptosis, functioning via mitochondrial outer membrane permeabilization. The family members are involved in several stages of apoptosis regulation. The overexpression of the anti-apoptotic proteins leads to several cancer pathological conditions. This overexpression is modulated or inhibited by heterodimerization of pro-apoptotic BH3 domain or BH3-only peptides to the hydrophobic groove present at the surface of anti-apoptotic proteins. Additionally, the heterodimerization displayed differences in binding affinity profile among the pro-apoptotic peptides binding to anti-apoptotic proteins. In light of discovering the novel peptide/drug molecules that contain the potential to inhibit specific anti-apoptotic protein, it is necessary to understand the molecular basis of recognition between the protein and its binding partner (peptide or ligand) along with its binding energies. Therefore, the present work focused on deciphering the molecular basis of recognition between pro-apoptotic Bak peptide binding to different anti-apoptotic (Bcl-xL, Bfl-1, Bcl-W, Mcl-1, and Bcl-2) proteins using advanced Molecular Dynamics (MD) approach such as Molecular Mechanics-Generalized Born Solvent Accessible. The results from our investigation revealed that the predicted binding free energies showed excellent correlation with the experimental values (r 2  = .95). The electrostatic (ΔG ele ) contributions are the major component that drives the interaction between Bak peptides and different anti-apoptotic peptides. Additionally, van der Waals (ΔG vdw ) energies also play an indispensible role in determining the binding free energy. Furthermore, the decomposition analysis highlighted the comprehensive information about the energy contributions of hotspot residues involved in stabilizing the interaction between Bak peptide and different anti-apoptotic proteins.

Myeloid cell leukemia 1 (Mcl1) is an antiapoptotic protein that plays central role in apoptosis regulation. Also, Mcl1 has the potency to resist apoptotic cues resulting in up-regulation and cancer cell protection. A molecular probe that has the potential to specifically target Mcl1 and thereby provoke its down-regulatory activity is very essential. The aim of the current study is to probe the internal conformational dynamics of protein motions and potential binding mechanism in response to a series of picomolar range Mcl1 inhibitors using explicit-solvent molecular dynamics (MD) simulations. Subsequently, domain cross-correlation and principal component analysis was performed on the snapshots obtained from the MD simulations. Our results showed significant differences in the internal conformational dynamics of Mcl1 with respect to binding affinity values of inhibitors. Further, the binding free energy estimation, using three different samples, was performed on the MD simulations and revealed that the predicted energies (ΔG mmgbsa ) were in good correlation with the experimental values (ΔG expt ). Also, the energies obtained using all sampling models were efficiently ranked. Subsequently, the decomposition energy analysis highlighted the major energy-contributing residues at the Mcl1 binding pocket. Computational alanine scanning performed on high energy-contributing residues predicted the hot spot residues. The dihedral angle analysis using MD snapshots on the predicted hot spot residue exhibited consistency in side chain conformational motion that ultimately led to strong binding affinity values. The findings from the present study might provide valuable guidelines for the design of novel Mcl1 inhibitors that might significantly improve the specificity for new-generation chemotherapeutic agents.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a promising antimicrobial target involved in the folate biosynthesis pathway. Although, the results from crystallographic studies of HPPK have attracted a great interest in the design of novel HPPK inhibitors, the mechanism of action of HPPK due to inhibitor binding remains questionable. Recently, mercaptoguanine derivatives were reported to inhibit the pyrophosphoryl transfer mechanism of Staphylococcus aureus HPPK (SaHPPK). The present study is an attempt to understand the SaHPPK-inhibitors binding mechanism and to highlight the key residues that possibly involve in the complex formation. To decipher these questions, we used the state-of-the-art advanced insilico approach such as molecular docking, molecular dynamics (MD), molecular mechanics-generalized Born surface area approach. Domain cross correlation and principle component analysis were applied to the snapshots obtained from MD revealed that the compounds with high binding affinity stabilize the conformational dynamics of SaHPPK. The binding free energy estimation showed that the van der Waals and electrostatic interactions played a vital role for the binding mechanism. Additionally, the predicted binding free energy was in good agreement with the experimental values (R 2  = .78). Moreover, the free energy decomposition on per-residue confirms the key residues that significantly contribute to the complex formation. These results are expected to be useful for rational design of novel SaHPPK inhibitors.

The immunoglobulin heavy chain locus (Igh) features higher-order chromosomal interactions to facilitate stage-specific assembly of the Ig molecule. Cohesin, a ring-like protein complex required for sister chromatid cohesion, shapes chromosome architecture and chromatin interactions important for transcriptional regulation and often acts together with CTCF. Cohesin is likely involved in B cell activation and Ig class switch recombination. Hence, binding profiles of cohesin in resting mature murine splenic B lymphocytes and at two stages after cell activation were elucidated by chromatin immunoprecipitation and deep sequencing. Comparative genomic analysis revealed cohesin extensively changes its binding to transcriptional control elements after 48 h of stimulation with LPS/IL-4. Cohesin was clearly underrepresented at switch regions regardless of their activation status, suggesting that switch regions need to be cohesin-poor. Specific binding changes of cohesin at B-cell specific gene loci Pax5 and Blimp-1 indicate new cohesin-dependent regulatory pathways. Together with conserved cohesin/CTCF sites at the Igh 3'RR, a prominent cohesin/CTCF binding site was revealed near the 3' end of Cα where PolII localizes to 3' enhancers. Our study shows that cohesin likely regulates B cell activation and maturation, including Ig class switching.

The main objective of this study was to increase the shelf life of fruits through vacuum packing. Papaya fruits were pretreated with waxing, oil application, purafil packets, tissue paper wrapping given along with control and were packed in 150 gauge thickness polyethylene film bags under vacuum and another set of these samples under without vacuum. The fruits were then stored at room and refrigerated temperature and analyzed for chemical changes. Results showed that the shelf life of the fruits increased under vacuum packing with room and refrigeration temperatures for one and four weeks respectively. The fruits packed without any pretreatment in 150 gauge polyethylene bags maintained the quality with minimum changes followed by waxing, purafil and oil application. During storage moisture, acidity, Vitamin C and total sugar decreased whereas reducing sugar and total soluble solids (TSS) increased during storage. The sensory qualities declined with the period of storage and temperature differences. The study concluded that vacuum packaging and refrigeration conditions increased the shelf life of the papaya fruits.

The global epidemiology of fungal infections is changing. While overall, Candida albicans remains the most common pathogen; several institutions in Europe, Asia and South America have reported the rapid emergence to predominance of Candida parapsilosis. This mini-review examines the impact of gene deletions achieved in C. parapsilosis that have been published to date. The molecular approaches to gene disruption in C. parapsilosis and the molecularly characterized genes to date are reviewed. Similar to C. albicans, factors influencing virulence in C. parapsilosis include adherence, biofilm formation, lipid metabolism, and secretion of hydrolytic enzymes such as lipases, phospholipases and secreted aspartyl proteinases. Development of a targeted gene deletion method has enabled the identification of several unique aspects of C. parapsilosis genes that play a role in host-pathogen interactions - CpLIP1, CpLIP2, SAPP1a, SAPP1b, BCR1, RBT1, CpFAS2, OLE1, FIT-2. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012)., (Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) has great potential for elucidating transcriptional networks, by measuring genome-wide binding of transcription factors (TFs) at high resolution. Despite the precision of these experiments, identification of genes directly regulated by a TF (target genes) is not trivial. Numerous target gene scoring methods have been used in the past. However, their suitability for the task and their performance remain unclear, because a thorough comparative assessment of these methods is still lacking. Here we present a systematic evaluation of computational methods for defining TF targets based on ChIP-seq data. We validated predictions based on 68 ChIP-seq studies using a wide range of genomic expression data and functional information. We demonstrate that peak-to-gene assignment is the most crucial step for correct target gene prediction and propose a parameter-free method performing most consistently across the evaluation tests.

The role of p53 in inducing apoptosis following acute kidney injury is well-established; however, the molecular mechanisms remain largely unknown. We report here that the p53 proapoptotic target Siva and its receptor CD27, a member of the tumor necrosis factor receptor family, are upregulated following renal ischemia-reperfusion injury (IRI). Inhibition of Siva using antisense oligonucleotides conferred functional and morphological protection, and it prevented apoptosis postrenal IRI in mice. Renal IRI in CD27-deficient mice displayed functional protection and partial inhibition of apoptosis, suggesting an incomplete role for CD27 in Siva-mediated apoptosis. To further elucidate mechanisms by which Siva elicits apoptosis, in vitro studies were performed. In Siva-transfected LLC-PK(1)cells, Siva is persistently expressed in the nucleus at 3 h onwards and its translocation to mitochondria and the plasma membrane occurred at 6 h. Moreover, Siva overexpression induced mitochondrial permeability, cytochrome c release, caspase-8 and -9 activation, translocation of apoptosis-inducing factor (AIF) to the nucleus, and apoptosis. Inhibition of Siva in ischemic kidneys prevented mitochondrial release of cytochrome c and AIF. These data indicate that Siva function is pivotal in regulating apoptosis in the pathology of renal IRI. Targeting Siva may offer a potential therapeutic strategy for renal IRI.

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.

The p53 tumor suppressor gene plays a crucial role in mediating apoptotic cell death in renal ischemia-reperfusion injury (IRI). To further elucidate the p53-dependent pathway, we investigated the role of the p53 apoptosis effector related to PMP-22 (PERP), an apoptosis-associated p53 transcriptional target. PERP mRNA and protein are highly induced in the outer medullary proximal tubular cells (PTC) of ischemic kidneys postreperfusion at 3, 12, and 24 h in a p53-dependent manner. In PTC, overexpression of PERP augmented the rate of apoptosis following hypoxia by inducing mitochondrial permeability and subsequent release of cytochrome c, apoptosis-inducing factor (AIF), and caspase 9 activation. In addition, silencing of the PERP gene with short hairpin RNA prevented apoptosis in hypoxia-mediated injury by precluding mitochondrial dysfunction and consequent cytochrome c and AIF translocation. These data suggest that PERP is a key effector of p53-mediated apoptotic pathways and is a potential therapeutic target for renal IRI.

Bone marrow mesenchymal stem (stromal) cells (MSCs) are shown to differentiate into different renal lineages in in vivo injury models. Nevertheless, the in vitro differentiation of MSCs into a renal tubular epithelial lineage has not been investigated. We hypothesize that the injured renal epithelial cells express renotypic factors that may influence the differentiation of MSCs into a renal tubular epithelial lineage. MSCs were cocultured for up to seven days with injured or uninjured murine cortical tubular renal epithelial cells (MCTs), which are separated by a physical barrier; following the coculture, MSCs were examined for the expression of two renal tubular epithelial-specific markers, kidney-specific cadherin (Ksp-cadherin) and aquaporin-1 (AQP1). MSCs differentiated into a tubular epithelial-like phenotype, as shown by the appearance of Ksp-cadherin and AQP1 by day 7 when cocultured with injured MCTs. Further, MSCs showed tubulogenic characteristics when cocultured in a three-dimensional matrix. Nonetheless, MSCs cultured with the conditioned medium from injured MCTs, cocultured with ureteric bud cells, or treated with nephrogenic factors did not differentiate into renal epithelial cells. Based on our findings, we conclude that MSCs can differentiate into a renal epithelial lineage independent of cell fusion when cocultured with injured renal cells.

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

Store-operated Ca(2+) entry (SOCE) has been extensively studied in non-neuronal cells, such as glial cells and smooth muscle cells, in which Ca(2+)-independent phospholipase A(2) (iPLA(2)) has been shown to play a key role in the regulation of SOCE channels. In the present study, we have investigated the role of iPLA(2) for store-operated Ca(2+) entry in rat cerebellar granule neurons in acute brain slices using confocal Ca(2+) imaging. Depletion of Ca(2+) stores by cyclopiazonic acid (CPA) induced a Ca(2+) influx, which could be inhibited by SOCE channel blockers 2-aminoethoxy-diphenylborate (2-APB) and 3,5-bistrifluoromethyl pyrazole derivative (BTP2), but not by the voltage-operated Ca(2+) channel blocker diltiazem and by the Na+ channel blocker tetrodotoxin. The inhibitors of iPLA(2), bromoenol lactone (BEL) and 1,1,1-trifluoro-2-heptadecanone, and the selective suppression of iPLA(2) expression by antisense oligodeoxynucleotides, inhibited CPA-induced Ca(2+) influx. Calmidazolium, which relieves the block of inhibitory calmodulin from iPLA(2), elicited a Ca(2+) influx similar to CPA-induced Ca(2+) entry. The product of iPLA(2), lysophosphatidylinositol, elicited a 2-APB- and BTP2-sensitive, but BEL-insensitive, Ca(2+) influx. Spontaneous Ca(2+) oscillations in granule cells in acute brain slices were reduced after inhibiting iPLA(2) activity or by blocking SOCE channels. The results suggest that depletion of Ca(2+) stores activates iPLA(2) to trigger Ca(2+) influx by the formation of lysophospholipids in these neurons.

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.

We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 microM) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 microM). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 microM) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 microM) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 microM) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA2, lysophosphatidylcholine (250 nM) and lysophosphatidylinositol (250 nM), but not lysophosphatidic acid (250 nM), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.

Acute Renal Failure (ARF) is the most costly kidney disease in hospitalized patients and remains as a serious problem in clinical medicine. The mortality rate among ARF patients remains around 50% and no pharmaceutical agents are currently available to improve its clinical outcome. Although several successful therapeutic approaches have been developed in animal models of the disease, translation of the results to clinical ARF remains elusive. Understanding the cellular and molecular mechanisms of vascular and tubular dysfunction in ARF is important for developing acceptable therapeutic interventions. Following an ischemic episode, cells of the affected nephron undergo necrotic and/or apoptotic cell death. Necrotic cell death is widely considered to be a futile process that cannot be modulated by pharmacological means as opposed to apoptosis. However, recent reports from various laboratories including ours indicate that inhibition or absence of poly(ADP)-ribose polymerase (PARP), one of the molecules involved in cell death, provides remarkable protection in disease models such as stroke, myocardial infarction and renal ischemia which are characterized predominantly by necrotic type of cell death. Overactivation of PARP in conditions such as ischemic renal injury leads to cellular depletion of its substrate NAD+ and consequently ATP. The severely compromised cellular energetic state induces acute cell injury and diminishes renal functions. PARP activation also enhances the expression of proinflammatory agents and adhesion molecules in ischemic kidneys. Pharmacological inhibition and gene ablation of PARP-1 decreased energy depletion, inflammatory response and improved renal functions in the setting renal ischemia/reperfusion injury. The biochemical pathways and the cellular and molecular mechanisms mediated by PARP-1 activation in eliciting the energy depletion and inflammatory responses in ischemic kidney are not fully elucidated. Dissecting the molecular mechanisms by which PARP activation contributes to oxidant-induced cell death will provide new strategies to interfere in those pathways to modulate cell death in renal ischemia. The current review evaluates the experimental evidences in animal and cell culture models implicating PARP as a pathophysiological modulator of acute renal failure with particular emphasis on ischemic renal injury.

Increased generation of reactive oxygen species (ROS) and the subsequent DNA damage and excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in the pathogenesis of ischemic injury. We previously demonstrated that pharmacological inhibition of PARP protects against ischemic renal injury (IRI) in rats (Martin DR, Lewington AJ, Hammerman MR, and Padanilam BJ. Am J Physiol Regul Integr Comp Physiol 279: R1834-R1840, 2000). To further define the role of PARP-1 in IRI, we tested whether genetic ablation of PARP-1 attenuates tissue injury after renal ischemia. Twenty-four hours after reperfusion following 37 min of bilateral renal pedicle occlusion, the effects of the injury on renal functions in PARP-/- and PARP+/+ mice were assessed by determining glomerular filtration rate (GFR) and the plasma levels of creatinine. The levels of plasma creatinine were decreased and GFR was augmented in PARP-/- mice. Morphological evaluation of the kidney tissues showed that the extent of damage due to the injury in PARP-/- mice was less compared with their wild-type counterparts. The levels of ROS and DNA damage were comparable in the injured kidneys of PARP+/+ and PARP-/- mice. PARP activity was induced in ischemic kidneys of PARP+/+ mice at 6-24 h postinjury. At 6, 12, and 24 h after injury, ATP levels in the PARP+/+ mice kidney declined to 28, 26, and 43%, respectively, whereas it was preserved close to normal levels in PARP-/- mice. The inflammatory cascade was attenuated in PARP-/- mice as evidenced by decreased neutrophil infiltration and attenuated expression of inflammatory molecules such as TNF-alpha, IL-1beta, and intercellular adhesion molecule-1. At 12 h postinjury, no apoptotic cell death was observed in PARP-/- mice kidneys. However, by 24 h postinjury, a comparable number of cells underwent apoptosis in both PARP-/- and PARP+/+ mice kidneys. Thus activation of PARP post-IRI contributes to cell death most likely by ATP depletion and augmentation of the inflammatory cascade in the mouse model. PARP ablation preserved ATP levels, renal functions, and attenuated inflammatory response in the setting of IRI in the mouse model. PARP inhibition may have clinical efficacy in preventing the progression of acute renal failure complications.

Background/aims: We have previously reported that ischemia/reperfusion injury (IRI) to the kidney leads to induced expression of RACK1 and changes in the level of expression and subcellular distribution of PKC isozymes alpha, betaII and zeta. In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or inhibition of the isozymes on cytotoxicity mediated by H(2)O(2) in LLCPK(1) cells., Methods: Cytotoxicity was analyzed by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis., Results: Western blot analysis showed that the expression of PKC-alpha was up-regulated in a triphasic pattern with the initial induction within the first 10 min of injury followed by higher levels of expression at 2 and 24 h postinjury. The expression of PKC-zeta was highly induced within the first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC-alpha and PKC-zeta translocated to the nucleus and perinuclear region during H(2)O(2) treatment. Following injury, PKC-alpha expression was localized to the nuclear membrane at earlier time points but a translocation to the nucleus occurred at later time points. PKC-zeta translocated to nucleus at 30 minutes post injury and relocated back to the nuclear membrane at later time points., Conclusion: These data suggest that activation of PKC-alpha and PKC-zeta is involved in the H(2)O(2) induced injury of LLCPK1 cells., (Copyright 2003 S. Karger AG, Basel)