Biosorption of lead and copper from aqueous solutions by pre-treated crab and arca shell biomass

Sorption potential of pretreated crab and arca shell biomass for lead and copper from aqueous media was explored. The effects of pH, initial concentration, biosorbent dosage and contact time were studied in batch experiments. Effects of common ions like sodium, potassium, calcium and magnesium on the sorption capacity of pretreated crab and arca biomasses were also studied. At equilibrium, the maximum uptake by crab shell biomass was 19.83+/-0.29 and 38.62+/-1.27 mg/g for lead and copper, respectively. In case of arca shell biomass the maximum uptake capacity was 18.33+/-0.44 mg/g and 17.64+/-0.31 mg/g for lead and copper, respectively. Combined effect of all the common ions up to 50 microg/ml concentration was negligible for both the metals using both biomasses. Sorption isotherms were studied to explain the removal mechanism of both elements by fitting isotherms data into Lagergren, Freundlich and Langmuir equations.     

Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant

Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.     

Studies on the intestinal disaccharidases of the pigeon. II. Subcellular localization and solubilization

In the pigeon, 70-80% of the activities of maltase (alpha-D-glucoside glucohydrolase EC 3.2.1.20), sucrase (alpha-glucohydrolase, EC 3.2.1.48), isomaltase (dextran 6-alpha-D-glucan hydrolase, EC 3.2.1.10) and glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) were found to be localized in the brush-border membrane of intestinal epithelial cells. Of the total glycosidase activities in the mucosal homogenate, nearly 60 to 70% were recovered in the microsomal (105 000 X g) fraction, about 30% in the mitochondrial (22 000 X g) fraction and less than 5% from the cytosol (105 000 X g supernatant) fraction. The hydrolases were solubilized by digestion with papain but not with trypsin, and the phosphate ion had a protective effect in the solubilization. Amongst detergents, Triton X-100 but not sodium deoxycholate, was found to truly solubilize these enzymes.     

A Mass Spectrometric Study for Comparative Analysis and Evaluation of Metabolite Recovery from Plasma by Various Solvent Systems

A solvent system that extracts a maximum number of metabolites belonging to diverse chemical classes from complex biofluids, such as plasma, may offer useful inputs to understand the metabolic and physiological state of an individual. The present study compared seven solvent systems for extraction of metabolites from plasma. The extracts were analyzed by mass spectrometry (MS) and MS/MS (MS2) using a quadrupole time-of-flight liquid chromatography/MS system in positive and negative modes of ionization. Metabolites with molecular mass below 400 were identified using Human Metabolome Database MS2 and MS search interfaces. The acetone/isopropanol (2:1) system yielded promising results in positive ionization mode, as the maximum number of MS and MS2 features was detected in the extract. It was found to be superior in extraction of various classes of metabolites, especially organic acids, nucleosides and nucleoside derivatives, and heterocyclic molecules. Glycerophosphocholines in the mass range of 400–700 were found to be efficiently extracted by the methanol/chloroform/water (8:1:1) system. In negative mode as well, the maximum number of MS2 features was detected in methanol/chloroform/water and acetone/isopropanol extracts. The fingerprints of molecular features obtained in the negative and positive modes differed from each other to a significant extent.     

A Multistep, ATP-dependent Pathway for Assembly of Human Immunodeficiency Virus Capsids in a Cell-free System

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.