Nonbonding interaction analyses on PVDF/[BMIM][BF4] complex system in gas and solution phase.

The present study provides a detailed quantum chemical description of the physicochemical interactions between poly-vinylidene fluoride (PVDF) and 1-butyl-3-methyl-imidazolium tetrafluoro borate ([BMIM][BF4]) ionic liquid (IL). Geometry optimization and frequency calculations are carried out for four monomer units of α- and ß-PVDF, [BMIM][BF4], and PVDF/[BMIM][BF4] using dispersion corrected density functional theory. The effects of solvation on the systems under study are demonstrated for three polar aprotic solvents, namely tetra-hydrofuran (THF), acetone, and n,n-dimethyl formamide (DMF) using the integral equation formalism polarizable continuum model (IEFPCM). Calculated negative solvation free energy values suggest solution phase stability of the systems under study. Binding and interaction energies for ß-PVDF/IL are found higher in magnitude than those for α-PVDF/IL. The nonbonding interaction phenomenon of ß-PVDF/[BMIM][BF4] is elucidated on the basis of natural bond orbital (NBO), Bader's quantum theory of atoms in molecules (QTAIM), delocalization indices, Hirshfeld surface, and reduced density gradient (RDG) analyses. Both anions and cations of ionic liquids are found to show weak van der Waals interaction with PVDF molecule but the anion ([BF4]-)/PVDF interaction is found to be stronger than cation ([BMIM]+)/PVDF interaction. Inter-unit C-H⋯F type hydrogen bonds are found to show improper (causing blue shifts in vibrational frequencies) nature. Frontier molecular orbital analysis is carried out, and different chemical parameters like electronegativity, chemical potential, chemical hardness and softness, and electrophilicity index are calculated using Koopmans' theorem. Thermochemical calculations are also performed, and the variation in different standard thermodynamic parameters with temperature is formulated. Graphical abstract (a) Hirshfeld surface mapped onto electron density and (b) NCI isosurfaces showing inter-unit interactions of ß-PVDF/[BMIM][BF4].

Poisoning, stings and bites in children-- what is new? An experience from a tertiary care hospital in Kolkata.

Poisonings, stings and bites continue to be important cause of pediatric morbidity and hospitalization. The toxic product involved in the poisoning varies in different geographical areas and in same area over time. A retrospective study was conducted amongst the children of the age group up to 12 years admitted to a tertiary care hospital in Kolkata from January 2005 to December 2008. Total number of admissions was 17019 and that for accidental poisoning was 451 (2.65%). Kerosene constituted the largest group (54.55%). Mosquito coil and refill liquid were the new additions to the list of poisons and their ingestion was cause for admission of 15 (3.33%) children. The number of admissions due to stings and bites was 108 (0.63% of all admissions) during the above period. Of all the cases, 9 (1.83%) cases of accidental poisoning and 4 (3.7%) cases of stings and bites died.

Profile of pediatric dengue cases from a tertiary care hospital in Kolkata.

A study was conducted on the 52 serologically positive cases of dengue, admitted to the Dept. of Paediatrics, R.G. Kar Medical College & Hospital, from an outbreak in Kolkata. The most unusual feature observed in this study was that the rash in some cases was urticarial and intensely pruritic. The shock appeared early in the course of the disease and it was less commonly associated with bleeding (22%). One out of three dengue cases was a severe disease. It was not possible to predict a severe disease from the early symptomatology.

Atomistic simulation studies of magnetite surface structures and adsorption behavior in the presence of molecular and dissociated water and formic acid.

Static energy minimization techniques have been used to elucidate the surface structures of magnetite crystals in pure and hydroxylated forms. Adsorption energy values in the presence of molecular water, dissociate water and simple carboxylic group molecule (formic acid) are calculated and we found that the carboxylic group do not adsorb strongly in most of the pure and hydroxylated surfaces in comparison to water. Since the associated calcium minerals are floated from magnetite using fatty acid collector, our calculations corroborate the flotation practice of removing these impurity minerals from magnetite.

Competitive adsorption on wollastonite: an atomistic simulation approach.

Atomistic simulation techniques are used to simulate surface structure and adsorption behavior of scarcely floatable wollastonite mineral in the presence of molecular and dissociated water, methanoic acid, and methylamine. The latter two additives represent the two widely used collector head-group molecules. The static energy minimization code METADISE was used to perform the simulation to obtain pure surface energy and adsorption energy in the presence of added molecule. The hydroxylation was performed on those surfaces where low-coordinated silicon was made to saturate by bonding with hydroxyl group, and the subsequent charge neutralization was maintained by adding proton on single-coordinated surface oxygen. A comparison of surface energies revealed that all the surfaces become stabilized in the presence of added molecules; however, the presence of methylamine decreased the surface energy to lower values. Adsorption of dissociated water is preferred by the {100} and {102} surfaces, whereas the {001} surface preferred methylamine adsorption, because these show highly negative adsorption energies. In terms of molecular adsorption, the preferred adsorption sequence for all the surfaces is methylamine > methanoic acid > water without considering coadsorption. For the {100} and {102} surfaces, the adsorption energy values of carboxylic acid and amine are more negative than that of water and therefore we conclude that both carboxyl and amine head-group molecules adsorb preferably on wollastonite. Our simulation verify usability of carboxylic acid head group as widely used collectors for wollastonite flotation and, at the same time, it predicts the use of amine head-group collectors as possible modifiers, which corresponds well with our experimental findings.

Synthesis and unusual electron paramagnetic resonance spectrum of metastable nanoclusters of ZnO semiconductor crystallites.

Metastable nanoclusters of ZnO semiconductor crystallites, 20 to 30 nm diameter, are synthesized by a reconstructive decomposition of a polymer precursor of dispersed Zn2+ cations in poly vinyl alcohol (PVA) polymer molecules. They have EPR (electron paramagnetic resonance) spectrum of distinct excitonic features. Multiple EPR bands appear in prominent intensities in oxygen vacancies VO+ and singly ionized Oi- and Zn(i)+ interstitials. A paramagnetic VO+ vacancy derives from usual diamagnetic O2- vacancy of VO++ (behaves as if doubly charged compared to the lattice) by addition of one electron. The results demonstrate the existence of a surface-interface or surface barrier layer in free-carrier depletion at the crystallite surface in the clusters and its effects on the Oi- and Zn(i)+ ionization states (determine green photoluminescence). Both VO+ and Zn(i)+ are curable by a thermal annealing in O2 gas. A cured sample of equilibrium structure achieved by heating at approximately 550 degrees C has a single EPR in Oi- at g = 1.990. The results are useful in understanding their correlation with EPR and optical properties in ZnO semiconductors and devices.

Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo.

GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.

p300-mediated acetylation of human transcriptional coactivator PC4 is inhibited by phosphorylation.

The human positive coactivator 4 (PC4) acts as a general coactivator for activator-dependent transcription, the activity of which is regulated negatively by phosphorylation. We report here that PC4 can be acetylated specifically by another coactivator, p300. Interestingly, phosphorylation of PC4 by casein kinase II inhibits the p300-mediated acetylation. Mass spectral analysis revealed that there are at least two lysine residues acetylated in PC4, as a result of which its DNA binding activity is stimulated.

Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300.

The transcriptional coactivator p300 shows physical and functional interactions with a diverse group of activators and contains an intrinsic acetyltransferase activity whose exact coactivator functions in the acetylation of nucleosomal histones versus other factors are poorly documented. Here, we show that p300 mediates acetyl-CoA-dependent transcription by GAL4-VP16 from a nucleosomal array template, that this involves p300 targeting by GAL4-VP16 and promoter-proximal histone acetylation prior to transcription, and that the affinities of different activators for p300 roughly correlate with corresponding levels of p300-dependent transcription. These results indicate that activators recruit p300 to nucleosomal templates by direct interactions and that bound p300 stimulates transcription, at least in part, by localized histone acetylation.

HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.

Histone acetyltransferases (HATs) play important roles in the regulation of gene expression. In this report, we describe the design, synthesis, and application of peptide CoA conjugates as selective HAT inhibitors for the transcriptional coactivators p300 and PCAF. Two inhibitors (Lys-CoA for p300 and H3-CoA-20 for PCAF) were found to be potent (IC(50) approximately = 0.5 microM) and selective (approximately 200-fold) in blocking p300 and PCAF HAT activities. These inhibitors were used to probe enzymatic and transcriptional features of HAT function in several assay systems. These compounds should be broadly useful as biological tools for evaluating the roles of HATs in transcriptional studies and may serve as lead agents for the development of novel antineoplastic therapeutics.

The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity.

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.

CpG islands in chromatin organization and gene expression.

CpG islands are stretches of DNA sequence that are enriched in the (CpG)n repeat and are present in close association with all housekeeping genes as well as some tissue-specific genes in the mammalian genome. Methylation of CpG islands strongly influences both structural organization and function of chromatin. The presence of a CpG island in a given chromosomal domain can, by itself, give rise to relatively open and active chromatin. Recently, several histone acetyltransferases, histone deacetylases, and chromatin remodeling factors have been found to be part of the transcription machinery. It is becoming increasingly clear that CpG islands and their methylation status may influence the function or recruitment of these newly discovered chromatin remodeling factors, especially the histone deacetylases. In addition, CpG islands may also play a significant role in the reorganization of chromatin during mammalian spermiogenesis.

Human TFIIIC relieves chromatin-mediated repression of RNA polymerase III transcription and contains an intrinsic histone acetyltransferase activity.

Human TFIIIC is a multisubunit factor that is essential for transcription by RNA polymerase III on tRNA and virus-associated RNA genes and initiates preinitiation complex assembly by direct recognition of promoter elements. We show that highly purified TFIIIC, at concentrations above those sufficient for transcription of naked DNA templates, effectively relieves nucleosome-mediated repression on an in vitro-reconstituted chromatin template. Highly purified TFIIIC alone can bind to the A and B boxes of a tRNA gene within a chromatin template and, further, displays a histone acetyltransferase activity that is intrinsic to at least one (and probably three) of its subunits. The possibility of a direct link between TFIIIC-dependent chromatin transcription and acetyltransferase activities is suggested by the partial loss of these activities, but not DNA transcription activity, following pretreatment of TFIIIC with p-hydroxymercuribenzoic acid.

A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.

In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivo either with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex.

Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes.

The rpoF gene of Escherichia coli codes for the RNA polymerase sigmaF (or sigma28) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigmaF and sigma70 (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigmaF to core RNA polymerase (E) is higher than that of sigma70, as measured by gel filtration high-pressure liquid chromatography. In an in vitro transcription system, the holoenzyme (E sigmaF) containing sigmaF selectively transcribed the flagellar and chemotaxis genes, all of which could not be transcribed by E sigma70. This strict promoter recognition property of sigmaF is similar to those of other stress response minor sigma subunits but different from those of the principal sigma subunits, sigma70 and sigma38. sigma70-dependent transcription in vitro is inhibited at high concentrations of all salts tested, showing maximum activity at 50 mM. In contrast, sigmaF-dependent transcription was maximum at 50 mM KCI and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that sigmaF alone is unable to bind DNA, but E sigmaF specifically recognizes -10 and -35 regions of the sigmaF-dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E sigmaF was suggested.

Zinc dependent recognition of a human CpG island sequence by the mammalian spermatidal protein TP2.

Rat spermatidal protein TP2 is a zinc metalloprotein with two atoms of zinc coordinated to cysteine and histidine residues and condenses alternating GC copolymer preferentially in a zinc dependent manner [Kundu, T. K., & Rao, M. R. S. (1995) Biochemistry 34,5143-5150]. In the present study, we have used a 40-mer oligonucleotide containing a human CpG island sequence to study its interaction with TP2 by gel mobility shift assays. A specific complex was observed in the presence of poly(dI).poly(dC). Preincubation of TP2 with 10 mM EDTA or 1 mM 1, 10-o-phenanthroline inhibited the complex formation by more than 90%. Competition experiments with various polynucleotides revealed the following order of efficiency: poly(dG-dC).poly(dG-dC) > cold homologous oligonucleotide > poly(dA-dT).poly(dA-dT). Homoduplexes poly(dG).poly(dC) and poly(dA).poly(dT) had no effect on the complex formation. Chromomycin A3, a GC minor groove binding drug, inhibited the complex formation. Methylation of the CpG doublet within the CpG island sequence by SssI methylase (CpG methylase) completely abolished the complex formation. Methylation of G at the N-7 position with dimethyl sulfate did not affect the recognition of CpG island by TP2. Thus, CpG islands, widely distributed in the mammalian genome, may serve as specific loci for initiation of chromatin condensation by TP2 during the later stages of spermiogenesis.

A method for in vivo [32P]phosphate labelling of testis proteins.

The direct intratesticular injection of [32P]phosphate resulted in 4-9 times more labelling of rat testis proteins compared to the conventional method of in vitro incubation. Moreover this is a simple technique requiring minimum (7-10 times less) radioactive phosphate and is less hazardous.

DNA condensation by the rat spermatidal protein TP2 shows GC-rich sequence preference and is zinc dependent.

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 microM ZnSO4), namely, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG).poly-(dC) and poly(dA).poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC).poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT).poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC).poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC).poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT).poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC).poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT).poly(dA-dT) more effectively than poly(dG-dC).poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

Characterization of the zinc-metalloprotein nature of rat spermatidal protein TP2.

Spermatidal transition protein, TP2, was purified from rat testes by Hg-affinity chromatography. The present study reports the details of the zinc-metalloprotein nature of TP2 by employing the 65 Zn-blotting technique. Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of 65Zn-binding to TP2. The 65Zinc-binding was localized to the V8 protease-derived N-terminal two-third polypeptide fragment. Circular dichroism spectroscopy studies of TP2 (zinc pre-incubated) and its V8 protease-derived polypeptide fragments revealed that the N-terminal fragment has a Type I-beta-turn spectrum, while the C-terminal fragment has a small but significant alpha-helical structure. EDTA altered the circular dichroism spectrum of TP2 and the N-terminal fragment (zinc binding domain) but not that of the C-terminal fragment.