Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.     

Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia‐oxidizing bacteria

There is increasing evidence showing that ammonia‐oxidizing bacteria (AOB) are major contributors to N₂O emissions from wastewater treatment plants (WWTPs). Although the fundamental metabolic pathways for N₂O production by AOB are now coming to light, the mechanisms responsible for N₂O production by AOB in WWTP are not fully understood. Mathematical modeling provides a means for testing hypotheses related to mechanisms and triggers for N₂O emissions in WWTP, and can then also become a tool to support the development of mitigation strategies. This study examined the ability of four mathematical model structures to describe two distinct mechanisms of N₂O production by AOB. The production mechanisms evaluated are (1) N₂O as the final product of nitrifier denitrification with NO as the terminal electron acceptor and (2) N₂O as a byproduct of incomplete oxidation of hydroxylamine (NH₂OH) to NO. The four models were compared based on their ability to predict N₂O dynamics observed in three mixed culture studies. Short‐term batch experimental data were employed to examine model assumptions related to the effects of (1) NH concentration variations, (2) dissolved oxygen (DO) variations, (3) NO accumulations and (4) NH₂OH as an externally provided substrate. The modeling results demonstrate that all these models can generally describe the NH, NO, and NO data. However, none of these models were able to reproduce all measured N₂O data. The results suggest that both the denitrification and NH₂OH pathways may be involved in N₂O production and could be kinetically linked by a competition for intracellular reducing equivalents. A unified model capturing both mechanisms and their potential interactions needs to be developed with consideration of physiological complexity. Biotechnol. Bioeng. 2013; 110: 153–163. © 2012 Wiley Periodicals, Inc.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Ground‐ and excited‐state proton transfer and antioxidant activity of 3‐hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited‐state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3‐hydroxyflavone (3‐HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3‐HF against lipid peroxidation in this membrane system. When 3‐HF molecules are partitioned into EYPC liposomes, a weak long‐wavelength absorption band with λ ～410 nm appears in addition to the principal absorption at ～λ = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (λ～460 nm) of the ground‐state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (λ = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground‐state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3‐HF binding, suggesting that 3‐HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes. Copyright © 2008 John Wiley & Sons, Ltd.     

Lymphocyte CFTR promotes epithelial bicarbonate secretion for bacterial killing

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in lymphocytes has been reported for nearly two decades; however, its physiological role remains elusive. Here, we report that co‐culture of lymphocytes with lung epithelial cell line, Calu‐3, promotes epithelial HCO production/secretion with up‐regulated expression of carbonic anhydrase 2 and 4 (CA‐2, CA‐4) and enhanced bacterial killing capability. The lymphocyte‐enhanced epithelial HCO secretion and bacterial killing activity was abolished when Calu3 cells were co‐cultured with lymphocytes from CFTR knockout mice, or significantly reduced by interfering with E‐cadherin, a putative binding partner of CFTR. Bacterial lipopolysaccharide (LPS)‐induced E‐cadherin and CA‐4 expression in the challenged lung was also found to be impaired in CFTR knockout mice compared to that of the wild‐type. These results suggest that the interaction between lymphocytes and epithelial cells may induce a previously unsuspected innate host defense mechanism against bacterial infection by stimulating epithelial HCO production/secretion, which requires CFTR expression in lymphocytes. J. Cell. Physiol. 227: 3887–3894, 2012. © 2012 Wiley Periodicals, Inc.     

Performance of a pilot-scale packed bed reactor for perchlorate reduction using a sulfur oxidizing bacterial consortium

A novel sulfur‐utilizing perchlorate reducing bacterial consortium successfully treated perchlorate (ClO) in prior batch and bench‐scale packed bed reactor (PBR) studies. This study examined the scale up of this process for treatment of water from a ClO and RDX contaminated aquifer in Cape Cod Massachusetts. A pilot‐scale upflow PBR (～250‐L) was constructed with elemental sulfur and crushed oyster shell packing media. The reactor was inoculated with sulfur oxidizing ClO reducing cultures enriched from a wastewater seed. Sodium sulfite provided a good method of dissolved oxygen removal in batch cultures, but was found to promote the growth of bacteria that carry out sulfur disproportionation and sulfate reduction, which inhibited ClO reduction in the pilot system. After terminating sulfite addition, the PBR successfully removed 96% of the influent ClO in the groundwater at an empty bed contact time (EBCT) of 12 h (effluent ClO of 4.2 µg L^?1). Simultaneous ClO and NO reduction was observed in the lower half of the reactor before reactions shifted to sulfur disproportionation and sulfate reduction. Analyses of water quality profiles were supported by molecular analysis, which showed distinct groupings of ClO and NO degrading organisms at the inlet of the PBR, while sulfur disproportionation was the primary biological process occurring in the top potion of the reactor. Biotechnol. Bioeng. 2012; 109:637–646. © 2011 Wiley Periodicals, Inc.     

Hydrobiological Studies on Suraj Kund and Chandrama Kund,Hot Springs of Rajgir,Bihar, India

The hot water of Rajgir springs is used for drinking and bathing purposes by tourists. Certain physico-chemical characteristics (temperature, pH, NO, PO, etc.) of the water along with phycological parameters viz. community composition, species diversity, standing crop etc. were measured from June 1986 to April 1987. The water was deficient in Na, NO and PO ions. The hot springs were mainly dominated by algae belonging to Cyanophyceae and Bacillariophyceae. The algal community comprised 18 species, with dominance of Cyanophyceae over Bacillariophyceae. While Mastigocladus laminosus and species of Phormidium were dominant in Suraj Kund, species of Oscillaroria and Synechococcus dominated in Chandrama Kund. Diatoms comprised about 10 % of the algal community. Though there was a considerable seasonal change in species diversity of the algal community the total biomass (chlorophyll a extracted per unit area from the algal mat) remained constant.     

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 μM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG‐treated cells. PG induced apoptosis in A549 cells, as evidenced by sub‐G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (Δ Ψ_m). The intracellular ROS (reactive oxygen species) level including O increased in PG‐treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:36–42, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20263     

Characterization of calcium signaling pathways in human preadipocytes

Intracellular free Ca²⁺ (Ca) is an important regulator of many cellular activities; however, Ca²⁺ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca²⁺ signal pathways using a confocal scanning technique and RT‐PCR. It was found that spontaneous Ca oscillations were observed in 12.1% preadipocytes, and number of cells with Ca²⁺ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry mainly via stored‐operated Ca²⁺ (SOC) entry. They were suppressed by the SOC entry channel blocker La³⁺, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2‐amino‐ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca²⁺ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca oscillations. In addition, the plasma membrane Ca²⁺ pump (PMCA) inhibitor carboxyeosin and Na⁺–Ca²⁺ exchanger (NCX) inhibitor Ni²⁺ both suppressed Ca²⁺ oscillations. RT‐PCR revealed that the mRNAs for IP3R1‐3, SERCA1,2, NCX3 and PMCA1,3,4, Ca_V1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca²⁺ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca²⁺ signals regulate biological and physiological activities of human preadipocytes. J. Cell. Physiol. 220: 765–770, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Heat capacity changes and partial molal heat capacities of some di- and tripeptides in water

Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with C_p2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.     

Enantioselective interactions of inversion-labile trigonal iron(II) complexes upon binding to DNA

We studied the interactions of the substitution-inert inversion-labile complexes Fe(bipy) and Fe(phen) [and the inversion-stable complex Ru(bipy)] with DNA. The association of these complexes to DNA is mainly electrostatic, and Fe(phen) shows a more effective binding to DNA than the two bipyridyl complexes, possibly owing to a different binding mode. The interactions are enantioselective, leading to a Pfeiffer shift in the diastereomeric inversion equilibria and an excess of the Δ-enantiomer of Fe(phen) and Fe(bipy), which is directly monitorable through CD. The partition constants for the inversion equilibrium range from 1.3 to 2.0 for Fe(bipy) and Fe(phen), depending on ionic conditions. From flow LD information about the orientation of the complexes on DNA was obtained: it is consistent with a fit of the Δ-enantiomer in the major groove of the right-handed DNA helix. The mechanisms of interaction are discussed against equilibrium, spectroscopic, and kinetic data.     

Apparent molar volumes of some amino acids and peptides in aqueous urea solutions

Densities of solutions of several α-amino acids and peptides in 3 and 6m aqueous urea solvents have been determined at 298.15 K. These data have been used to evaluate the infinite-dilution apparent molar volumes of the solutes and the volume changes due to transfer (V ) of the α-amino acids and peptides at infinite dilution from water to aqueous urea solutions. The sign and magnitude of the V values have been rationalized in the framework of Friedman's cosphere-overlap model. The V values for the glycyl group (? CH₂CONH? ) and alkyl side chains have been estimated.     

Conformation of poly(L-arginine). I. Effects of anions

The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P₂O, P₃O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH^?, Cl^?, F^?, H₂PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).     

Application of the nearest-neighbor Ising model to the helix-coil transition of polypeptides in mixed organic solvents.

Using the formalism of nearest-neighbor Ising model and assuming that the allowed states for a monomeric unity of a polypeptide chain in solutions containing strong acids are E (helix), C (coil), and CS (solvent-bonded coil), the partition function of the system was deduced analytically. Equations were obtained which permitted the prediction of the characteristic thermodynamic behavior of the helix–coil transition under these conditions. These equations were used to examine critically the possible correlations between experimental data obtained using different techniques. Particular attention was devoted to quantities called “transition enthalpies,” obtained from the slope of the transition curves at the point where the helix fraction is one-half (ΔH), or for measurements of the heat of solution of the polymer over the total range of solvent composition (ΔH), or from heat capacity measurements taken at various temperatures (ΔH). Literature data of ΔH(j = opt, sol, cal) for the system poly-γ-benzyl-L -glutamate in mixtures of dichloroacetic acid and 1,2-dichloroethane were carefully analyzed.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Gas balance method for testing of microbial growth efficiency after carbon substrate pulse

The present paper deals with the analysis of the amount of oxygen utilized for oxidation of a small dose of carbon substrate in carbon limited Brevibacterium flavum culture. The ratio of the measured oxygen consumption (mo₂) to the amount of added carbon substrate (m_s) gives a stoichiometric coefficient of the biological oxidation equation. A linear relationship between mo₂ and m_s was observed. To compare the efficiency of different carbon substrate utilization there has been introduced a normalized value β = m/m. There exists a simple relationship between β and the thermodynamical growth efficiency η The theoretical considerations are proved by experimental results with β, η and Y_x/s in a chemostat culture at various medium flow rates.     

Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell‐signaling pathways. Despite this, high‐throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO₂)‐based 3‐D LC (strong cationic exchange/TiO₂/C18)‐MS³‐linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low‐abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho‐biomarkers including plectin‐1(phopho‐Ser‐4253) and alpha‐HS‐glycoprotein (phospho‐Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO₂‐based online‐3‐D LC‐MS³‐linear ion trap system with high‐throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.