Identification and Quantification by NMR Spectroscopy of the 22 and 22 Epimers in Budesonide Pharmaceutical Forms.

The authors developed four variants of the qNMR technique (H or C nucleus, DMSO-d6 or CDCl solvent) for identification and quantification by NMR of 22 and 22 epimers in budesonide active pharmaceutical ingredient and budesonide drugs (sprays, capsules, tablets). The choice of the qNMR technique version depends on the drug excipients. The correlation of H and C spectra signals to molecules of different budesonide epimers was carried out on the basis of a comprehensive analysis of experimental spectral NMR data (H-H gCOSY, H-C gHSQC, H-C gHMBC, H-H ROESY).

Acute NelfA knockdown restricts compensatory gene expression and precipitates ventricular dysfunction during cardiac hypertrophy.

Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy.

Glucocorticoid Receptor-Binding and Transcriptome Signature in Cardiomyocytes.

Background An increase in serum cortisol has been identified as a risk factor for cardiac failure, which highlights the impact of glucocorticoid signaling in cardiomyocytes and its influence in the progression of failure. Dexamethasone, a synthetic glucocorticoid, is sufficient for induction of cardiomyocyte hypertrophy, but little is known of the glucocorticoid receptor (GR) genome-binding and -dependent transcriptional changes that mediate this phenotype. Methods and Results In this study using high-resolution sequencing, we identified genomic targets of GR and associated change in the transcriptome after 1 and 24 hours of dexamethasone treatment.

Unique synergistic formulation of curcumin, epicatechin gallate and resveratrol, tricurin, suppresses HPV E6, eliminates HPV+ cancer cells, and inhibits tumor progression.

Curcumin (from curry) (C) is highly potent against cervical cancer cells (CCC), but poor bioavailability has limited its clinical use. Similar natural polyphenols resveratrol (from grapes) (R), and epicatechin gallate (from green tea) (E) also display activity against CCC. By treating CCC (HeLa) with C, E, or R, or combinations of these compounds, we computed combination indices and observed a strong synergism among C, E, and R at the unique molar ratio 4:1:12.

Localization of the Escherichia coli RNA polymerase beta' subunit residue phosphorylated by bacteriophage T7 kinase Gp0.7.

During bacteriophage T7 infection, the Escherichia coli RNA polymerase beta' subunit is phosphorylated by the phage-encoded kinase Gp0.7. Here, we used proteolytic degradation and mutational analysis to localize the phosphorylation site to a single amino acid, Thr(1068), in the evolutionarily hypervariable segment of beta'.

Genome sequence and gene expression of Bacillus anthracis bacteriophage Fah.

Fah, a lytic bacteriophage of Bacillus anthracis, is used widely in the former Soviet Union to identify anthrax bacteria. Here, we present the analysis of a 37,974 bp sequence of the Fah genome and examine gene expression of the phage in a model host, Bacillus cereus. Half of the Fah genome contains genes coding for structural proteins and host lysis functions in an arrangement typical of Syphoviridae.

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation.

Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA.

The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, sigma 70, with high affinity and inhibits its transcription activity. AsiA binds to sigma 70 primarily through an interaction with sigma 70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the -35 consensus promoter element.

Disruption of Escherichia coli hepA, an RNA polymerase-associated protein, causes UV sensitivity.

During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of the hepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein.

Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit.

We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma 70.

Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase.

The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E. coli RNA polymerase is described.

The beta subunit Rif-cluster I is only angstroms away from the active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were cross-linked to the beta subunit. Using limited proteolysis and chemical degradation, the cross-link was mapped to a segment of beta between amino acids Val516 and Arg540. This region (Rif-cluster I) is known to harbor many rifampicin-resistant (RifR) mutations.

Three-dimensional structure of E. coli core RNA polymerase: promoter binding and elongation conformations of the enzyme.

The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E.

Streptolydigin-resistant mutants in an evolutionarily conserved region of the beta' subunit of Escherichia coli RNA polymerase.

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the beta subunit (Heisler, L. M., Suzuki, H.

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro.

The sigma subunit conserved region 3 is part of "5'-face" of active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits. Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3. This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex.

A non-essential domain of Escherichia coli RNA polymerase required for the action of the termination factor Alc.

An evolutionarily nonconserved region of approximately 250 amino acids can be deleted from the amino-terminal part of the beta subunit of Escherichia coli RNA polymerase without effect on the enzyme's basic function. The non-essential segment is located between two highly conserved motifs and is flanked by sequences participating in the rifampicin-binding site. The results define the second non-essential domain in the beta subunit, in addition to the more distal dispensable segment identified previously.