Application of a hybrid biofilter column for the removal of Cr(VI) from aqueous solution using an indigenous bacterial strain Pseudomonas taiwanensis

In the present study, a laboratory-scale biofilter column was designed and fabricated. It was packed with a mixture of coal and compost as a packing medium. The column was enriched with an indigenous bacterial strain Pseudomonas taiwanensis isolated from aerobic mixed culture of Sewage Treatment Plant, BITS-Pilani, Pilani campus. The removal of hexavalent chromium [Cr(VI)] from aqueous solution was investigated in the biofilter column. The entire biofiltration operation was divided into five phases (I to V) for a period of 63 days. Biofilter column was subjected to shock loading conditions for 20 days immediately after 63 days of operation. The maximum removal efficiency of 89.4% was obtained during phase V for Cr(VI) inlet concentration of 40 mg L^?1. During shock loading, maximum removal efficiency was obtained as 90% for 48.5–50 mg L^?1 of initial Cr(VI) concentration. Kinetic parameters of biofiltration process for Cr(VI) removal were also determined by fitting Michaelis-Menten kinetic model with experimental data. The Michaelis-Menten kinetic constants were obtained as 0.258 mg L^?1 min^?1 and 26.83 mg L^?1. It was found that Ottengraf-Van den Oever model with zero-order diffusion limitation fit the experimental data quite well for phases III, IV, and V with coefficient of determination (R²) values .97, .99, and .984, respectively. A possible method for safe disposal of packing medium was also presented in this study.     

Two-dimensional gel-based alkaline proteome of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.     

Functional characterization of constituent enzyme fractions of mycobacillin synthetase.

The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.     

Molecular Cloning, Bacterial Overexpression and Characterization of L-myo- inositol 1- Phosphate Synthase from a Monocotyledonous Resurrection Plant, Xerophyta viscosa Baker

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS), an evolutionarily conserved enzyme-protein, catalyses the first and rate limiting step of inositol biosynthesis. Inositol and its derivatives play important roles in biological kingdom like growth regulation, membrane biogenesis, signal transduction and also acts as an osmolyte or osmoprotectant in abiotic stress tolerance. Here we report the cloning, sequencing and the characterization of the INO1 gene from Xerophyta viscosa (XINO1), a monocotyledonous resurrection plant. Nucleotide sequences of XINO1 show striking homology (70–99%) with a number of INO1 genes from plant sources particularly with the monocots. The gene is functionally identified through genetic complementation using a yeast inositol auxotrophic strain FY250. The gene is expressed in E. coli BL21, recombinant protein purified to homogeneity, biochemically characterized and compared with Oryza INO1 (RINO1) gene product. The XINO1 gene product is catalytically active in a broader range of lower temperature (between 10–40 °C) than the RINO1 gene- product. This is the first report of MIPS gene from any resurrection plant.     

Salt-induced abnormalities on root tip mitotic cells of Allium cepa: prevention by inositol pretreatment

Salt-induced growth reduction of plants is a well-known phenomenon which poses major problem in crop productivity in places where vast majority of land plants are affected by salt. In this report, studies were carried out to reveal the effect of salt injury on the cell division pattern in roots and the role of myo-inositol in preventing the salt-induced ion disequilibrium on the chromosome and DNA degradation in roots. Present study revealed induction of various chromosomal abnormalities on the root tip mitotic cells of Allium cepa by treatment with different concentrations of NaCl (0–500 mM) for 24 h as also the amelioration of such effect by prior treatment of the roots with different concentration of myo-inositol (0–300 mM). Results showed that a narrow albeit definite range of extracellular myo-inositol (100–150 mM) is effective in preventing internucleosomal fragmentation which is the early response in roots under salt stress. Transgenic tobacco plants overexpressing Oryza (OsINO1) as well as Porteresia (PcINO1) cytosolic l-myo-inositol-1-phosphate synthase coding genes can withstand and retain their chromosomal and DNA integrity in 100 mM NaCl solution and can subsequently prevent DNA fragmentation, caused by intracellular endonuclease activity at this salt concentration.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Drosophila MUS312 interacts with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers

MEI-9 is the Drosophila homolog of the human structure-specific DNA endonuclease XPF. Like XPF, MEI-9 functions in nucleotide excision repair and interstrand crosslink repair. MEI-9 is also required to generate meiotic crossovers, in a function thought to be associated with resolution of Holliday junction intermediates. We report here the identification of MUS312, a protein that physically interacts with MEI-9. We show that mutations in mus312 elicit a meiotic phenotype identical to that of mei-9 mutants. A missense mutation in mei-9 that disrupts the MEI-9-MUS312 interaction abolishes the meiotic function of mei-9 but does not affect the DNA repair functions of mei-9. We propose that MUS312 facilitates resolution of meiotic Holliday junction intermediates by MEI-9.     

Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.     

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2+-ATPase isolated from goat spermatozoa

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg²⁺-independent Ca²⁺-ATPase without any significant effect on Mg²⁺-dependent Ca²⁺-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.