No abstract

The gene for the extracellular alpha antigen of Mycobacterium bovis BCG was cloned by using a single probe restricted to G or C in the third position. This technique should have great potential for the isolation of mycobacterial antigen genes. The gene analysis revealed that the alpha antigen gene encoded 323 amino acid residues, including 40 amino acids for signal peptide followed by 283 amino acids for mature protein. This is the first report on the structure of the mycobacterial signal peptide. The promoter-like sequence and ribosome-binding site were observed upstream of the open reading frame. In the coding region, the third position of the codon showed high G + C content (86%). The gene was expressed as an unfused protein in Escherichia coli by using an E. coli expression vector. This protein, which reacted with polyclonal antibody raised against alpha antigen from Mycobacterium tuberculosis, would be applicable to the immunodiagnosis of tuberculosis.

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.

Resin composites with antibacterial activity may be useful to decrease the frequency of secondary caries around restorations. The purposes of this study were to investigate the antibacterial activity of light-activated resin composites incorporating one of three silver-containing materials and to evaluate their long-term inhibitory effect against Streptococcus mutans The three types of silver-containing materials, Novaron (N), Amenitop (AM), and AIS, were incorporated into TEGDMA-UDMA-based light-activated resin composites, and the antibacterial activities, mechanical properties and release of silver ions were examined. Minimum inhibitory concentrations in suspensions of N, AM, and AIS against S. mutans were 1.1, 1.2, and 23.0 mg/ml, respectively. Resin composites incorporating 5 wt% of Novaron (N-5) and 7 wt% of Amenitop (AM-7) inhibited the growth of S. mutans after immersion in water for 3 months, whereas the resin composite incorporating 10 wt% of AIS did not. No significant difference in either compressive or flexural strength was observed between the control and N-5 composites after 1 d and 3 months storage in water. However, for AM-5 composite, there was a significant difference in both strength parameters between the two immersion periods. There was no or extremely little release of silver ions from the N-5 and AM-5 composites after 1 d or 3 months immersion in water. These results indicated that a light-activated resin composite incorporating silver-containing materials such as Novaron may be clinically useful due to its long-term inhibitory effect against S. mutans and favorable mechanical properties.