The synthesis of methylated, phosphorylated, and phosphonated 3'-aminoacyl-tRNA(Sec) mimics.

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA(Sec)) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl-tRNA(Sec) intermediate is converted into selenocysteinyl-tRNA(Sec). The SepSecS architecture and the mode of tRNA(Sec) recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNA(Sec) mimics that contain a hydrolysis-resistant ribose 3'-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid-oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNA(Sec) derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNA(Sec) mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes.

Synthesis and decoding of selenocysteine and human health.

Selenocysteine, the 21st amino acid, has been found in 25 human selenoproteins and selenoenzymes important for fundamental cellular processes ranging from selenium homeostasis maintenance to the regulation of the overall metabolic rate. In all organisms that contain selenocysteine, both the synthesis of selenocysteine and its incorporation into a selenoprotein requires an elaborate synthetic and translational apparatus, which does not resemble the canonical enzymatic system employed for the 20 standard amino acids. In humans, three synthetic enzymes, a specialized elongation factor, an accessory protein factor, two catabolic enzymes, a tRNA, and a stem-loop structure in the selenoprotein mRNA are critical for ensuring that only selenocysteine is attached to selenocysteine tRNA and that only selenocysteine is inserted into the nascent polypeptide in response to a context-dependent UGA codon. The abnormal selenium homeostasis and mutations in selenoprotein genes have been causatively linked to a variety of human diseases, which, in turn, sparked a renewed interest in utilizing selenium as the dietary supplement to either prevent or remedy pathologic conditions. In contrast, the importance of the components of the selenocysteine-synthetic machinery for human health is less clear. Emerging evidence suggests that enzymes responsible for selenocysteine formation and decoding the selenocysteine UGA codon, which by extension are critical for synthesis of the entire selenoproteome, are essential for the development and health of the human organism.

Nonadenomatous dysplasia in barrett esophagus: a clinical, pathologic, and DNA content flow cytometric study.

Rarely, dysplasia in Barrett's esophagus (BE) is composed of crypts lined by cuboidal-shaped cells that contain a centrally located nucleus, markedly increased nuclear/cytoplasmic ratio, but without nuclear stratification characteristic of conventional "adenomatous" dysplasia. The aim of this study was to evaluate the clinical and pathologic features, natural history, and DNA content flow cytometric abnormalities of BE patients with non-adenomatous dysplasia (NAD) in a cohort of BE patients enrolled in a prospective surveillance program. Eighteen patients with NAD identified over a 6 year period, in a cohort of 270 consecutive patients with BE and without esophageal adenocarcinoma (EA) at baseline, were evaluated for clinical and pathologic features, including association with conventional adenomatous dysplasia and EA, DNA content flow cytometric abnormalities (tetraploidy and aneuploidy) and outcome, over a mean follow-up period of 4.1 years. The findings in the 18 study patients were compared to those in the 252 remaining (control) patients without NAD. Control patients included 228 with metaplasia/indefinite for dysplasia, and 24 with conventional adenomatous dysplasia (13 low-grade, 11 high-grade). The prevalence rate of NAD in our BE cohort was 6.7% Of the 18 study patients, there were 17 were males and 1 female of mean age 66.7 years. The mean length of BE was 3.9 cm NAD foci were associated with goblet or non-goblet epithelium in 62% and 38% of cases, respectively. Ninety-four percent of patients with NAD (17/18) also had conventional adenomatous dysplasia (four with low-grade, 13 with high-grade) elsewhere in the esophagus at the same endoscopic procedure as the one that detected NAD. Patients with NAD had a significantly shorter length of BE compared to control patients with conventional adenomatous dysplasia (N=24) (p=0.03). Patients with NAD also showed a significantly higher rate of DNA content flow cytometric abnormalities compared to the entire cohort of control patients (38% vs. 11%, p=0.05). However, no significant differences regarding either flow cytometric abnormalities or progression to EA were found when the NAD patients were compared only to the 24 controls with conventional adenomatous dysplasia. NAD is a high grade histologic variant of intraepithelial neoplasia that is episodic in nature, and shows a high association with conventional adenomatous high-grade dysplasia.