A structural basis for the role of nucleotide specifying residues in regulating the Rv1625c adenylyl cyclase oligomerization of the from M-tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7 angstrom. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase. (c) 2005 Elsevier Ltd. All rights reserved.

Autophosphorylation of Ser(428) of EhC2PK Plays a Critical Role in Regulating Erythrophagocytosis in the Parasite Entamoeba histolytica

The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn2+-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KD Delta C) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KD Delta C proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the omega Subunit

The widely conserved omega subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of omega is known to be substituted by GroEL in omega-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of omega variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to omega, which is largely unstructured, omega mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, omega(6) bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that omega(6) binds to beta ` subunit in vitro with greater affinity than that of omega. The reconstituted RNAP holoenzyme in the presence of omega(6) in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant omega results in death of the cell. Furthermore, lethality of omega(6) is relieved in cells expressing the rpoC2112 allele encoding beta ` (2112), a variant beta ` bearing Y457S substitution, immediately adjacent to the beta ` catalytic center. Our results suggest that the enhanced omega(6)-beta ` interaction may perturb the plasticity of the RNAP active center, implicating a role for omega and its flexible state.

A Laboratory Analysis of Plasma Based Hybrid Techniques for Treating Engine Exhaust

A detailed study on the removal of pollutants (NOx, aldehydes and CO) from the exhaust of a stationary diesel engine is carried out using barrier discharge hybrid plasma techniques. The objective of the study is to make a comparative analysis. For this purpose, the exhaust treatment was carried out in two stages. In the first stage, the exhaust was treated with plasma process and plasma-adsorbent hybrid process. The effectiveness of the two processes with regard to NOx removal and by-product reduction was discussed. In the second stage, the exhaust was treated by plasma and plasma-catalyst hybrid process. The effectiveness of the two processes with regard to pollutants (NOx, CO) removal and by-product reduction was analyzed. Finally, a comprehensive comparison of different techniques has been made and feasible plasma based hybrid techniques for stationary and non-stationary engine exhaust treatments were proposed.

Flow cytometry analysis of human fetal osteoblast fate processes on spark plasma sintered hydroxyapatite-titanium biocomposites

Hydroxyapatite (HA)-based biocomposites have been widely investigated for a multitude of applications and these studies have been largely driven to improve mechanical properties (toughness and strength) without compromising cytocompatibility properties. Apart from routine cell viability/proliferation analysis, limited efforts have been made to quantify the fate processes (cell proliferation, cell cycle, and cell apoptosis) of human fetal osteoblast (hFOB) cells on HA-based composites, in vitro. In this work, the osteoblast cell fate process has been studied on a model hydroxyapatite-titanium (HA-Ti) system using the flow cytometry. In order to retain both HA and Ti, the novel processing technique, that is, spark plasma sintering, was suitably adopted. The cell fate processes of hFOBs, as evaluated using a flow cytometry, revealed statistically insignificant differences among HA-10 wt % Ti and HA and control (tissue culture polystyrene surface) in terms of osteoblast apoptosis, proliferation index as well as division index. For the first time, we provide quantified flow cytometry results to demonstrate that 10 wt % Ti additions to HA do not have any significant influence on the fate processes of human osteoblast-like cells, in vitro.

Thermal shock characteristics of plasma sprayed mullite coatings

Commercially available mullite (3Al(2)O(3). 2SiO(2)) powders containing oxides of calcium and iron as impurities, have been made suitable for plasma spraying by using an organic binder. Stainless steel substrates covered with Ni-22Cr-10Al-1.0Y bond coat were spray coated with mullite, The 425 mu m thick coatings were subjected to thermal shock cycling under burner rig conditions between 1000 and 1200 degrees C and less than 200 degrees C with holding times of 1, 5, and 30 min. While the coatings withstood as high as 1000 shock cycles without failure between 1000 and 200 degrees C, spallation occurred early at 120 cycles when shocked from 1200 degrees C, The coatings appeared to go through a process of self erosion at high temperatures resulting in loss of material. Also observed were changes attributable to melting of the silicate grains, which smooth down the surface. Oxidation of the bond coat did not appear to influence the failure, These observations were supported by detailed scanning electron microscopy and quantitative chemical composition analysis, differential thermal analysis, and surface roughness measurements.

Geometry Of Proline And Hydroxyproline .1. An Analysis Of X-Ray Crystal-Structure Data

We have carried out an analysis of crystal structure data on prolyl and hydroxyprolyl moieties in small molecules. The flexibility of the pyrrolidine ring due to the pyramidal character of nitrogen has been defined in terms of two projection angles δ1 and δ2. The distribution of these parameters in the crystal structures is found to be consistent with results of the energy calculations carried out on prolyl moieties in our laboratory.

Bull sperm plasma and acrosomal membranes: Fluorescence studies of lipid phase fluidity

Bull sperm plasma and outer acrosomal membranes have been isolated by discontinuous sucrose density gradient centrifugation and Ca2+-ATPase activity has been determined for both the membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization studies show that the lipid phase of the sperm plasma membranes is more fluid than the lipids of the outer acrosomal membranes. Approximately, a three fold increase in the cholesterol content has been found in the outer acrosomal membranes as compared to that in the plasma membranes.

Regional differentiationnext term in previous termbull sperm plasma membranesnext term

previous termBull spermnext term heads and tails have been separated by proteolytic digestion (trypsin) and previous termplasma membranesnext term have been isolated, using discontinuous sucrose density gradient centrifugation. previous termPlasma membranenext term bound Ca2+-ATPase is shown to be associated mostly with the tail previous termmembranes.next term Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization experiments indicate a more fluid lipid phase in the tail region. Differences in surface charge distribution have been found, using 1-anilinonaphthalene-8-sulfonate and Tb3+ as fluorescent probes.

Regional differentiation in bull sperm plasma-membranes

Bull sperm heads and tails have been separated by proteolytic digestion (trypsin) and plasma membranes have been isolated, using discontinuous sucrose density gradient centrifugation. Plasma membrane bound Ca2+-ATPase is shown to be associated mostly with the tail membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization experiments indicate a more fluid lipid phase in the tail region. Differences in surface charge distribution have been found, using 1-anilinonaphthalene-8-sulfonate and Tb3+ as fluorescent probes.

Adriamycin affects plasma membrane redox functions

Adriamycin (Doxorubicin) stimulates NADH oxidase activity in liver plasma membrane, but does not cause NADH oxidase activity to appear where it is not initially present, as in erythrocyte membrane. NADH dehydrogenase from rat liver and erythrocyte plasma membranes shows similar adriamycin effects with other electron acceptors. Both NADH ferricyanide reductase and vanadate-stimulated NADH oxidation are inhibited by adriamycin, as is a cyanide insensitive ascorbate oxidase activity, whereas NADH cytochrome c reductase is not affected. The effects may contribute to the growth inhibitory (control) and/or deleterious effects of adriamycin. It is clear that adriamycin effects on the plasma membrane dehydrogenase involve more than a simple catalysis of superoxide formation.

Analysis of a Filter Bag Reactor for gaseous pollution abatement

The conveying zone and the filter bag zone of a Filter Bag Reactor have been analysed as individual reactors. The gas and solid particles flow almost in plug flow through the pneumatic conveying section. In the filter bag the height of the packed column varies with time, a cell model has been used to calculate the concentration of outgoing stream. The total conversion obtained is the sum of conversions in each section of the reactor.

Mammalian brain plasma membranes: Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na+-K+) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.

Orbit perturbation theory of beam-plasma instability

The nonlinear theory of the instability caused by an electron beam-plasma interaction is studied. A nonlinear analysis has been carried out using many-body methods. A general formula for a neutral collisionless plasma, without external fields, is derived. This could be used for calculating the saturation levels of other instabilities. The effect of orbit perturbation theory on the beam-plasma instability is briefly reviewed.

Korteweg-De Vries Equation for Non-Linear Ion-Acoustic Waves in a Plasma with Negative Ions

The theoretical analysis, based on the perturbation technique, of ion-acoustic waves in the vicinity of a Korteweg-de Vries (K-dV) equation derived in a plasma with some negative ions has been made. The investigation shows that the negative ions in plasma with isothermal electrons introduced a critical concentration at which the ion-acoustic wave plays an important role of wave-breaking and forming a precursor while the plasma with non-isothermal electrons has no such singular behaviour of the wave. These two distinct features of ion waves lead to an overall different approach of present study of ion-waves. A distinct feature of non-uniform transition from the nonisothermal case to isothermal case has been shown. Few particular plasma models have been chosen to show the characteristics behaviour of the ion-waves existing in different cases