Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Which comes first: Renal inflammation or oxidative stress in spontaneously hypertensive rats?

The present study was undertaken to identify whether inflammation or oxidative stress is the primary abnormality in the kidney in spontaneously hypertensive rats (SHR). Renal inflammation and oxidative stress were evaluated in 2- and 3-week-old prehypertensive SHR and age-matched genetically normotensive control Wistar-Kyoto (WKY) rats. Blood pressure was similar in WKY and SHR rats at 2 and 3 weeks, of age. Renal inflammation (macrophage and nuclear factor-κB) was elevated in SHR at 3 weeks, but not at 2 weeks, of age compared with age-matched WKY rats. Renal oxidative stress (nitrotyrosine, 8-hydroxy-2′-deoxyguanosine and p47phox) was also clearly elevated in 3-week-old SHR compared with age-matched WKY rats. Additionally, NADPH oxidase subunit p47phox was found elevated in 2-week-old SHR compared to age-matched WKY rats. Moreover, antioxidant (N-acetyl-l-cysteine and Tempol) treatment reduced renal inflammation in prehypertensive SHR. We therefore conclude that the oxidative stress appears before inflammation as a primary abnormality in the kidney in prehypertensive SHR.     

Conserved arginines on the rim of Hfq catalyze base pair formation and exchange

The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq’s chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq’s chaperone function.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

TNF-α induced altered signaling mechanism in human neutrophil

In the present study we investigated the TNF- induced signal transduction mechanism in human neutrophil. Exogenously added TNF- affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF- induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF- dose dependently enhances the expression of -PKC isotype but not the -PKC. Morphology and cell cytotoxicity are studied in TNF- treated neutrophil to understand the TNF- induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF- induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent -PKC. These observations provide an insight towards understanding the function of -PKC in apoptotic pathway.     

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C. While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C. The CIts391 showed almost no binding at 42 degrees C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C. Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C. All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.     

QSAR study on some pyridoacridine ascididemin analogues as anti-tumor agents

Pyridoacridine ascididemin analogues have been reported as anticancer agents for their interesting antitumor activity against human cancer cells. A quantitative structure–activity relationship (QSAR) analysis of ascididemin analogues was attempted using the physicochemical parameters and the electrotopological state atom (ETSA) indices. This study indicates that the electron withdrawing substituents with higher MR (molar refractivity) value at R₁ position favor the anti-tumor activity and the presence of NHR (R is hydrogen or alkyl group) at the R₃ position has contribution to the anti-tumor activity. ETSA indices have been incorporated as independent variable in the QSAR model with physicochemical parameters. It clearly suggests the importance of atoms 2, 3, 4, 5, 6 and 7 to the anti-tumor activity.     

Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (Delta YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, Delta YRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.