Alkalinity and dissolved oxygen as controlling parameters for ammonia removal through partial nitritation and ANAMMOX in a single-stage bioreactor

The oxidation of ammonia to dinitrogen through partial nitritation and anaerobic ammonium oxidation (ANAMMOX) in a single-stage bioreactor is based on suppressing the nitratation process. The single-stage process operated on a laboratory-scale fixed film bioreactor achieved ammonia removal of 0.7 kg NH₄-N/(m³ day) at 4 h hydraulic retention time (HRT) by controlling the nitratation process through a ‘three-way control mechanism’ comprising control of electron donor (nitrite), electron acceptor (oxygen) and carbon source (bicarbonate). The control of alkalinity and dissolved oxygen (DO) concentrations in feed to maintain an alkalinity to ammonia ratio of less than 8 and DO loading of less than 0.06 mg O/(mg N day), respectively, was necessary for inhibiting nitratation and enhancing partial nitritation and ANAMMOX. Therefore, feed alkalinity along with DO concentrations are critical controlling parameters in a single-stage biological process for nitrogen removal.     

Bombesin marine toxin conjugates inhibit the growth of lung cancer cells

Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively. Addition of Hem-LA-BA1 and Hem-LB-BA1 to Fura-2 AM loaded cells containing BB2R, caused elevated cytosolic Ca2+. In a growth assay, Hem-LA-BA1 and Hem-LB-BA1 inhibited the proliferation of NCI-H1299 cells. Dol-succinamide (Dols)-LD-BA1 and Dols-LE-BA1 bound with high affinity to NCI-H1299 cells and elevated cytosolic Ca2+, but did not inhibit the proliferation of NCI-H1299 cells. Also, Hem-LA-BA1 inhibited 125I-DTyr-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 (BA2) binding to Balb/3T3 cells transfected with BB1R or BB2R as well as with BRS-3 with IC50 values of 130, 8, and 540 nM, respectively. These results show that Hem-BB conjugates are cytotoxic for cancer cells containing BB2R.     

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K_d ～20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.     

Anti-apoptotic potential of gymnemic acid phospholipid complex pretreatment in Wistar rats with experimental cardiomyopathy

Cardiomyocyte apoptosis in heart failure has been the topic of research in many recent studies. In the present investigation, the potential cardioprotective effect of gymnemic acid phospholipid complex (GPC) on myocardial apoptosis and cardiac function was studied in doxorubicin (DOX; 30 mg/kg/ip/single dose)-induced cardiomyopathy model in rats. Doxorubicin induced cardiomyopathy was evidenced by significant hemodynamic changes (increased systolic, diastolic, mean arterial pressure and heart rate), decreased heart weight to body weight ratio, increase in serum lactate dehydrogenase (LDH) and Ca2+ levels and decrease in myocardial Na+/K+ ATPase levels along with caspase-3 activation. A marked reduction in glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase and catalase levels along with increase in the levels of thiobarbituric acids (TBARS) were also observed in rat myocardium. In addition, DNA laddering observed on agarose gel electrophoresis and cardiac histopathology study further supplemented myocardial apoptosis. Pre-treatment with GPC significantly reduced DOX-induced cardiac toxicity, including improvement of hemodynamic variables and heart weight to body weight ratio, decreased serum Ca2+ level and LDH levels, myocardial caspase-3 levels, increased Na+/K+ ATPase levels and decreased myocardial TBARS levels and elevated antioxidant enzymes as compared to pathogenic control group. Further, the anti-apoptotic effect of GPC was verified by prevention of internucleosomal DNA laddering on agarose gel electrophoresis and attenuation of histopathological perturbations by doxorubicin. These observations demonstrate that GPC might serve as a cardioprotective formulation in DOX-induced cardiomyopathy in rats.     

Stathmin strongly increases the minus end catastrophe frequency and induces rapid treadmilling of bovine brain microtubules at steady state in vitro

Stathmin is a ubiquitous microtubule destabilizing protein that is believed to play an important role linking cell signaling to the regulation of microtubule dynamics. Here we show that stathmin strongly destabilizes microtubule minus ends in vitro at steady state, conditions in which the soluble tubulin and microtubule levels remain constant. Stathmin increased the minus end catastrophe frequency approximately 13-fold at a stathmin:tubulin molar ratio of 1:5. Stathmin steady-state catastrophe-promoting activity was considerably stronger at the minus ends than at the plus ends. Consistent with its ability to destabilize minus ends, stathmin strongly increased the treadmilling rate of bovine brain microtubules. By immunofluorescence microscopy, we also found that stathmin binds to purified microtubules along their lengths in vitro. Co-sedimentation of purified microtubules polymerized in the presence of a 1:5 initial molar ratio of stathmin to tubulin yielded a binding stoichiometry of 1 mol of stathmin per approximately 14.7 mol of tubulin in the microtubules. The results firmly establish that stathmin can increase the steady-state catastrophe frequency by a direct action on microtubules, and furthermore, they indicate that an important regulatory action of stathmin in cells may be to destabilize microtubule minus ends.     

Dinuclear metal phosphonates and -phosphates

The reaction of Cu(II) or Cd(II) salts with 2,4,6-iPr₃C₆H₂PO₃H₂, 2,4,6-iPr₃C₆H₂CH₂PO₃H₂ or 2,6-iPr₂C₆H₃OPO₃H₂ in the presence of strong chelating nitrogen ligands such as 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), 2-pyridylpyrazole (pypz) or 3,5-dimethyl pyrazole (dmpz) as the ancillary ligands afforded dinuclear copper or cadmium complexes [Cu₂(2,4,6-iPr₃C₆H₂PO₃H)₄(bpy)₂] (4), [Cu₂(2,6-iPr₂C₆H₃OPO₃H)₂(bpy)₂(OAc)₂(CH₃OH)₂]·(CH₃OH) (5), [Cd₂(2,6-iPr₂C₆H₃OPO₃H)₄ (bpy)₂(CH₃OH)₂]·2(CH₃OH) (6), [Cd₂(2,6-iPr₂C₆H₃OPO₃H)₄(phen)₂] (7), [Cu₂(2,6-iPr₂C₆H₃OPO₃H)₂(PyPz)₂(CH₃OH)₂] (8) and [Cu₂(2,4,6-iPr₃C₆H₂CH₂PO₃H)₂(DMPz)₂Cl₂]·(CH₃OH) (9) The molecular structures of 4-7 are grossly similar. The common structural features in these complexes are that the two metal centers are bridged by two bidentate [RPO₂(OH)]⁻ ligands generating a central eight-membered ring. Each of the metal centers also contains a chelating nitrogen ligand and a monodentate phosphonate or a phosphate ligand. In 5 and 6 other terminal ancillary ligands are also present. In compound 8, each of the two copper centers contains a monodentate [RPO₂(OH)]⁻ ligand along with a molecule of methanol. The two coppers are bridged by two monoanionic pyridylpyrazole ligands. The molecular structure of 9 is similar to that of 4-7. However, in 9 each of the two copper centers contain only terminal monodentate ligands in the form of two chlorides and a pyrazole. Magnetic studies on all of these copper complexes reveal an anti-ferromagnetic behavior at low temperatures. In addition, these complexes were found to be artificial nucleases and can convert supercoiled pBR322 DNA form I into nick form II in 1 min in the presence of an external oxidant through a hydrolytic and/or an oxidative pathway.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.^[1 Trivedi, R.N.; Agarwal, P.; Kumawat, M.; Pesingi, P.K.; Gupta, V.K.; Goswami, T.K.; Mahawar, M. Methionine Sulfoxide Reductase A (MsrA) Contributes to Salmonella Typhimurium Survival Against Oxidative Attack of Neutrophils. Immunobiology 2015, 220(12), 1322–1327.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]^] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26?kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.