Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

Fluocinolone acetonide intravitreal sustained release device--a new addition to the armamentarium of uveitic management

Uveitis is a potentially sight-threatening inflammatory eye disease caused by multiple infectious and non-infectious etiologies for which the standard of care involves corticosteroids or various immunomodulary therapy (IMT) drugs. These available treatments, although effective, may cause significant morbidity and sometimes mortality in uveitis patients due to their toxic side-effects and the necessity of long-term therapy to prevent recurrences. In order to avoid the systemic toxicity ofcorticosteroids and IMT or the repeated injections of local steroids necessary to control ocular inflammation, and to prevent development of cumulative damage resulting from recurrent episodes of inflammation, researchers have developed a number of local corticosteroid sustained-release devices that can be implanted directly into the vitreous of the eye, at the site of the inflammatory disease. Preliminary studies of such a device, the fluocinolone acetonide (Retisert) implant, have shown significant reductions in the number of inflammatory episodes and decreased reliance on systemic corticosteroids or other IMT. This review explores the current research evaluating the fluocinolone sustained-release intravitreal implant in the treatment of posterior uveitis and the implications for its future use on a wider scale.     

Immunological roles of Pasteurella multocida toxin (PMT) using a PMT mutant strain

The immunological role of the Pasteurella multocida toxin (PMT) in mice was examined using a PMT mutant strain. After a nasal inoculation, the mutant strain failed to induce interstitial pneumonia. Moreover, PMT had no significant effect on the populations of CD4+, CD8+, CD3+, and CD19+ immunocytes in blood or on the populations of CD4+ and CD8+ splenocytes (P<0.01). However, there was a significant increase in the total number of cells in the BAL samples obtained from the wild-type P. multocida-inoculated mice. On the other hand, the level of IL-1 expression decreased when the macrophages from the bronchio-alveolar lavage were stimulated with PMT. Overall, PMT appears to play some role (stimulating and/or inhibiting) in the immunological responses but further studies will be required to confirm this.     

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.     

A novel cold-inducible expression system for Bacillus subtilis

Production of recombinant proteins at low temperatures is one strategy to prevent formation of protein aggregates and the use of an expensive inducer such as IPTG. We report on the construction of two expression vectors both containing the cold-inducible des promoter of Bacillus subtilis, where one allows intra- and the other extracellular synthesis of recombinant proteins. Production of recombinant proteins started within the first 30min after temperature downshock to 25 degrees C and continued for about 5h.     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Association of the FADS2 gene with omega-6 and omega-3 PUFA Concentration in the egg yolk of Japanese quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low omega-6:omega-3 PUFA ratio (h2 = 0.36-0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the omega-6 and omega-3 PUFAs (P < 0.05). No effects of the other SNPs were found - indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to omega-6 and omega-3 PUFA concentration in the egg yolk.     

Combined mitochondrial and nuclear sequences support the monophyly of forcipulatacean sea stars

Previous molecular phylogenetic analyses of forcipulatacean sea stars (Echinodermata: Asteroidea) have reconstructed a non-monophyletic order Forcipulatida, provided that two or more forcipulate families are included. This result could mean that one or more assumptions of the reconstruction method was violated, or else the traditional classification could be erroneous. The present molecular phylogenetic analysis included 12 non-forcipulatacean and 39 forcipulatacean sea stars, with multiple representatives of all but one of the forcipulate families and/or subfamilies. Bayesian analysis of approximately 4.2kb of sequence data representing seven partitions (nuclear 18S rRNA and 28S rRNA, mitochondrial 12S rRNA, 16S rRNA, 5 tRNAs and cytochrome oxidase I with first and second codon positions analyzed separately from third codon positions) recovered a consensus tree with three well-supported clades (78%-100% bootstrap support) that corresponded at least approximately to traditional taxonomic ranks: the superorder Forcipulatacea (Forcipulatida + Brisingida) + Pteraster, the Brisingida/Brisingidae and Asteriidae + Rathbunaster + Pycnopodia. When a molecular clock was enforced, the partitioned Bayesian analysis recovered the traditional Forcipulatacea. Five of six genera represented by two or more species were monophyletic with 100% bootstrap support. Most of the traditional subfamilial and familial groupings within the Forcipulatida were either unresolved or non-monophyletic. The separate partitions differed considerably in estimates of model parameters, mainly between nuclear sequences (with high GC content, low rates of sequence substitution and high transition/transversion rate ratios) and mitochondrial sequences.     

Endothelial cell and macrophage regulation of vascular smooth muscle cell calcification modulated by cholestane-3beta, 5alpha, 6beta-triol

The cellular and molecular mechanisms that mediate vascular calcification remain poorly understood. In our previous study, oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) was shown to promote vascular smooth muscle cells (VSMCs) calcification. In this study, by using direct coculture, non-contact transwell coculture, and culture with conditioned media, we investigated the roles of endothelial cells (ECs) and macrophages in the regulation of VSMCs calcification in the absence or presence of Triol. In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. The results showed that ECs inhibited VSMCs calcification, as manifested by the reduction of calcium deposition in extracellular matrix. This effect of ECs on calcification was via the secreted soluble factors. Furthermore, the stimulation of ECs by Triol had no influence on ECs inhibition of calcification. On the other hand, macrophages promoted VSMCs calcification via the secreted soluble factors such as reactive oxygen species, which was further enhanced by Triol. Our results supported the roles for ECs and macrophages in vascular calcification, modulated by oxysterols in atherosclerotic plaque.     

Phylogeographic structure and late Quaternary population history of the Japanese oak Quercus mongolica var. crispula and related species revealed by chloroplast DNA variation

Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnH-psbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (HT) and haplotype diversity within populations (HS) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.