Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

Bioactive constituents from gum guggul (Commiphora wightii)

Bioactivity-directed fractionation and purification afforded cytotoxic components of Commiphora wightii. The exudates of C. wightii were extracted with EtOAc and the extract was subjected to repeated column chromatography. A fraction showing cytotoxic activity was characterized as a mixture of two ferulates with an unusual skeleton by spectral and chemical methods, including by NMR, GC-MS and chemical derivatization. This fraction also showed moderate scavenging effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.     

Activating mutation of GS alpha in McCune-Albright syndrome causes skin pigmentation by tyrosinase gene activation on affected melanocytes

McCune-Albright syndrome (MAS) is a sporadic disease characterized by café-au-lait spots, polyostotic fibrous dysplasia and hyperfunctional endocrinopathies. To elucidate the mechanism of skin pigmentation, melanocytes, keratinocytes and fibroblasts were primary cultured from the café-au-lait spot of a MAS patient. Then, mutational analysis and morphologic evaluation were performed. Also, cAMP level and tyrosinase gene expression in cultured cells were determined. Only Gsalpha mutation was found in affected melanocytes and the cAMP level in affected melanocytes was higher than that of normal melanocytes. The mRNA expression of tyrosinase gene was increased in the affected melanocytes. This study suggests that skin pigmentation of MAS results from activating mutation of Gsalpha in melanocytes and the mechanism involves the c-AMP-mediated tyrosinase gene activation.     

KpnBI is the prototype of a new family (IE) of bacterial type I restriction-modification system

KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5′ specific region and the adenine opposite the italicized thymine in the 3′ specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new ‘type IE’ family.     

Ligand modulates VDR-Ser/Thr protein phosphatase interaction and p70S6 kinase phosphorylation in a cell-context-dependent manner

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.     

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

A complete library of amino acid alterations at N304 in Streptomyces clavuligerus deacetoxycephalosporin C synthase elucidates the basis for enhanced penicillin analogue conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Effect of Cryptobia salmositica-induced anorexia on feeding behavior and immune response in juvenile rainbow trout Oncorhynchus mykiss

At 10 degrees C, rainbow trout Oncorhynchus mykiss (n = 13 per group) infected with Cryptobia salmositica Katz, 1951 became anorexic at 3 wk post-infection (w.p.i.), with feed-intake decreasing significantly from 1.33 to 0.94% body weight (b.w.). Anorexia was most severe at 4 w.p.i. (0.80% b.w.), coinciding with peak parasitemia (9.2 x 10(6) parasites ml blood(-1)) and anemia. At 8 w.p.i., fish had recovered their appetite although they still had contained detectable parasites (6.8 x 10(5) parasites ml(-1)) and were anemic (pack cell volume, PCV, of 24.4%). However at 5 degrees C, anorexia occurred at 5 w.p.i. (0.81% b.w.), and was most severe at 7 w.p.i. (0.40% b.w.). At 8 w.p.i. (0.43% b.w.), fish displayed high parasitemia (4.6 x 10(6) parasites ml(-1)) and low PCV (10.8%). Fish at 5 degrees C had lower gastric evacuation (GE) rates (GE48h) than 10 degrees C fish, however there were no differences between infected and naive fish at both temperatures. Before anorexia, there was no significant correlation between mean share of meal (MSM, a measure of how food was partitioned within a group) and coefficient of variation in feeding but this became significant during anorexia (p = 0.02 and p = 0.0002 at 10 and 5 degrees C respectively). Significant correlations were detected between b.w. and MSM before onset of anorexia at 10 degrees C (p = 0.005) and 5 degrees C (p = 0.02); this was maintained at 10 degrees C (p = 0.001) but not at 5 degrees C (p = 0.98). Fish on an anorexic diet (0.93% b.w.) responded well at 10 degrees C to a live C. salmositica vaccine; this could partly be due to constant antigenic stimulation by the live vaccine.