The evolving story of the omega subunit of bacterial RNA polymerase

Omega (omega) is the smallest subunit of bacterial RNA polymerase (RNAP). Although identified early in RNAP research, its function remained ambiguous and shrouded by controversy for a considerable period. It has subsequently been shown that the protein has a structural role in maintenance of the conformation of the largest subunit, beta', and recruitment of beta' to the enzyme assembly. Conservation of this function across all forms of life indicates the importance of its role. Several recent observations have suggested additional functional roles for this protein and have settled some long-standing controversies surrounding it. In this context, revisiting the omega subunit story is especially interesting; here, we review the progress of omega research since its discovery and highlight the importance of these recent observations.     

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.     

Markerless multiple-gene-deletion system for Streptococcus mutans

Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.     

A guanine-linked end-effect is a sensitive reporter of charge flow through DNA and RNA double helices

The property of charge (electron hole) flow in DNA duplexes has been the subject of intensive study. RNA-DNA heteroduplexes have also been investigated; however, little information exists on the conductive properties of purely RNA duplexes. In investigating the relative conductive properties of a three molecule DNA-DNA duplex design, using piperidine and aniline to break strands at modified bases, we observed that duplexes with guanine-rich termini generated a large oxidative end-effect, which could serve as a highly sensitive reporter of charge flow through the duplexes. The end-effect was found faithfully to report attenuations in charge flow due to certain single-base mismatches within a duplex. Comparative charge flow experiments on DNA-DNA and RNA-RNA duplexes found large end-effects from both, suggesting that the A and B family of double helices conduct charge comparably. The sheer magnitude of the end-effect, and its high sensitivity to helical imperfections, suggest that it may be exploited as a sensitive reporter for DNA mismatches, as well as a versatile device for studying the structure, folding, and dynamics of complexly folded RNAs and DNAs.     

Aryl sulfones as novel bradykinin B1 receptor antagonists for treatment of chronic pain

We report the development of aryl sulfones as Bradykinin B1 receptor antagonists. Variation of the linker region identified diol 23 as a potent B1 antagonist, while modifications of the aryl moiety led to compound 26, both of which were efficacious in rabbit biochemical challenge and pain models.     

Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 5-(4-hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole and its methanesulfonyl analog: synthesis, biological evaluation and nitric oxide release studies

A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs 12a-b) wherein an O(2)-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (11, O(2)-acetoxymethyl PROLI/NO) NO-donor moiety was covalently coupled to the bromomethyl group of 5-(4-bromomethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (9a), and its methanesulfonyl analog (9b), were synthesized. The diazen-1-ium-1,2-diolate compounds 12a-b released a low amount of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (6.1-8.2% range). In comparison, the percentage NO released was significantly higher (76-77% of the theoretical maximal release of two molecules of NO/molecule of the parent hybrid ester prodrug) when the diazen-1-ium-1,2-diolate ester prodrugs 12a-b were incubated in the presence of rat serum. These incubation studies suggest that both NO and the anti-inflammatory 5-(4-hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (10a), and its methanesulfonyl analog (10b), would be released from the parent NONO-coxib 12a or 12b upon in vivo cleavage by non-specific serum esterases. The hydroxymethyl compounds 10a-b were weak inhibitors of the cyclooxygenase-1 (COX-1) and COX-2 isozymes (IC(50)=3.7-10.5 microM range). However, the hydroxymethyl compounds 10a-b and the parent NONO-coxibs 12a-b exhibited good AI activities (ED(50)=76.7-111.6 micromol/kg po range) that were greater than that exhibited by the reference drugs aspirin (ED(50)=710 micromol/kg po) and ibuprofen (ED(50)=327 micromol/kg po), but less than that of celecoxib (ED(50)=30.9mumol/kg po). These studies indicate hybrid ester AI/NO-donor prodrugs (NONO-coxibs) constitutes a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.     

A role for microwave processing in the dry preservation of mammalian cells

Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre-incubated in full complement media containing 50 mM trehalose, for 18-h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45-min recovery period using Calcein-AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH(2)O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post- rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (P > 0.05). Brightfield and fluorescence images of microwave-dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave-processing successfully facilitated rapid and uniform dehydration of cell-based samples.     

A distinctive physiological role for IkappaBbeta in the propagation of mitochondrial respiratory stress signaling

The NFkappaBs regulate an array of physiological and pathological processes, including propagation of mitochondrial respiratory stress signaling in mammalian cells. We showed previously that mitochondrial stress activates NFkappaB using a novel calcineurin-requiring pathway that is different from canonical or non-canonical pathways. This study shows that IkappaBbeta is essential for the propagation of mitochondrial stress signaling. Knock down of IkappaBbeta, but not IkappaBalpha, mRNA reduced the mitochondrial stress-mediated activation and nuclear translocation of cRel:p50, inhibiting expression of nuclear target genes RyR1 and cathepsin L. IkappaBbeta mRNA knock down also reduced resistance to staurosporine-induced apoptosis and decreased in vitro invasiveness. Induced receptor switching to insulin-like growth factor-1 receptor and increased glucose uptake are hallmarks of mitochondrial stress. IkappaBbeta mRNA knock down selectively abrogated the receptor switch and altered tubulin cytoskeletal organization. These results show that mitochondrial stress signaling uses an IkappaBbeta-initiated NFkappaB pathway that is distinct from the other known NFkappaB pathways. Furthermore, our results demonstrate the distinctive physiological roles of the two inhibitory proteins IkappaBbeta and IkappaBalpha.     

Dynamic gait stability index based on plantar pressures and fuzzy logic

Stability during locomotion, or dynamic stability, is critical to ensure safe locomotion and a high quality of life. A dynamic stability measure should be easily applied in a clinical setting and must provide a quantitative index that can be used for comparisons over a range of tasks and environments. Plantar foot pressure data acquired by shoe-insole sensors have potential to provide such a measure. To generate a quantitative dynamic gait stability index, six gait parameters were extracted from a commercial plantar pressure measurement system (F-Scan): anterior–posterior (A/P) center of force (CoF) motion, medial–lateral (M/L) CoF motion, maximum lateral position, cell triggering, stride time (ST), and double support time (DST). A fuzzy logic controller combined these six parameters and generated the index. To validate the stability index, 15 healthy subjects performed four tasks intended to induce increasing levels of instability. Fifty-seven gait parameter combinations were assessed to determine the most effective index. A combination of A/P motion, M/L motion, maximum lateral position, and cell triggering parameters was the most consistently effective index across all subjects. However, small changes in ST and DST for able-bodied subjects may have reduced the effectiveness of these measures in the index calculation. The index combining all six parameters should be investigated further with populations with disabilities or pathological gait.