An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

Novel microbial nitrogen removal processes

The present-day wastewater treatment practices can be significantly improved through the introduction of new microbial treatment technologies. Recently, several new processes for nitrogen removal have been developed. These new nitrogen removal technologies provide practicable options for treating nitrogen-laden wastewaters. The new processes are based on partial nitrification of ammonium to nitrite combined with anaerobic ammonium oxidation. These processes include the single reactor system for high ammonia removal over nitrite (SHARON) process, which involves part conversion of ammonium to nitrite; the anaerobic ammonium oxidation (ANAMMOX) process, which involves anaerobic ammonium oxidation; and the completely autographic nitrogen removal over nitrite (CANON) process, which involves nitrogen removal within one reactor under oxygen-limited conditions. These new processes target the removal of nitrogen from wastewaters containing significant quantities of ammonium.     

Regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.     

Binding and dissociation kinetics using fractals: an analysis of electrostatic effects and randomly coupled and oriented coupled receptors on biosensor surfaces

A fractal analysis is used to analyze the influence of: (a) electrostatic interactions on binding and dissociation rate coefficients for antibodies HH8, HH10, and HH26 in solution to hen egg-white lysozyme (HEL) immobilized on a sensor chip surface [Biophys. J. 83 (2002) 2946]; and (b) the binding and dissociation of recombinant Fab in solution to random NHS-coupled Cys-HEL and oriented thiol-coupled Cys-HEL immobilized on a sensor chip surface [Methods 20 (2000) 310]. Single- and dual-fractal models were employed to fit the data. Values of the binding and the dissociation rate coefficient(s) and the fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro 8.0 (Corel Corporation Limited, Ottawa, Canada. 1997). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. It is of interest to compare the results obtained by the fractal analysis with that of the original analysis [Biophys. J. 83 (2002) 2946]. For example, as one goes from the binding of 21 nM HH10/HEL to the binding of 640 nM HH10/HEL(K97A), Sinha et al. [Biophys. J. 83 (2002) 29461 indicate that the enhancement of diffusional encounter rates may be due to 'electrostatic steering' (a long-range interaction). Our analysis indicates that there is an increase in the value of the fractal dimension, Df1 by a factor of 1.12 from a value of 2.133-2.385. This increase in the degree of heterogeneity on the surface leads to an increase in the binding rate coefficient, k1 by a factor of 1.59 from 12.92 to 20.57. The fractal analysis of binding and dissociation of recombinant Fab in solution to random NHS-coupled Cys-HEL and oriented thiol-coupled Cys-HEL immobilized on a sensor chip [Methods 20 (2000) 310] surface are consistent with the degree of heterogeneity present on the sensor chip surface for the random and the oriented case. As expected, the random case will exhibit a higher degree of heterogeneity than the oriented case, leading to subsequently a higher binding rate coefficient.     

Synthesis, absolute stereochemistry and molecular design of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201

The absolute stereochemistry of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201 has been established by achieving the total synthesis of the product. A series of analogues have also been synthesized by changing the side chain and their bioactivity assessed against different microbial strains. Among them, 1e (R = C8H17) was found to be the most potent with MIC of 8 microg/mL against Mycobacterium tuberculosis, 12 microg/mL against Escherichia coli and 16 microg/mL against Bacillus subtilis 6 microg/mL against Proteus vulgaris. This was followed by 1b (R = C5H11) with MIC of 10-20 microg/mL range and 1d (R = C7H15) with MIC of 14-24 g/mL, whereas 1a (R = C4H9) and 1f (R = C18H35) were found to be completely inactive. Besides, 1c (R = C6H13) showed certain extent of antibacterial activity in the range of 24-50 microg/mL. Mycobacterium tuberculosis was very sensitive to 1e (R = C8H17) with MIC of 8 microg/mL. Antifungal activity of analogues 1d (R = C7H15) and 1e, (R = C8H17) against Fusarium oxysporum and Rhizoctonia solani were found promising with MFCs in the 15-18 microg/mL range.     

Bile acid amides derived from chiral amino alcohols: novel antimicrobials and antifungals

Cholic and deoxycholic acid amides 10-17 have been synthesised from (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, (1S,2S)-1-phenyl-2-amino-1,3-propanediol 4, (1R,2R)-1-para-nitrophenyl-2-amino-1,3-propanediol 3, (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5. Amide 12 derived from N-succinimidyl ester 9 of deoxycholic acid and (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, found to be active against Cryptococcus neoformans and the amide 17 obtained from N-succinimidyl ester 9 of deoxycholic acid and (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5, is found to be potent against various gram-positive bacteria.     

Pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates. Synthesis, DNA binding and cytotoxicity

New pyrrolobenzodiazepine-anthraquinone hybrids have been designed and synthesized, found to effectively bind to DNA and also exhibit cytotoxicity against many cancer cell lines     

HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

QSAR study on phenolic activity: need of positive hydrophobic term (log P) in QSAR

The phenolic activity (log 1/C) of a large series of phenols against L1210 leukaemia cells was modelled using physicochemical parameters other than conventional electronic and steric parameters. Attempts have also been made to examine need or otherwise of the hydrophobic parameter, log P, in such studies. The results have shown that contribution of log P in modelling log 1/C is favourable.