Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4‐excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction‐modification (RM) protein and turned on by a phage‐encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double‐stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild‐type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.     

BMI may overestimate the prevalence of obesity among women of lower socioeconomic status

Objective: Our objective was to examine gender differences in height and weight associated with socioeconomic status (SES) and the consequent effect on body mass index in a multiethnic society. Research Methods and Procedures: A cross‐sectional study, the First Israeli National Health and Nutrition Survey, was performed on a representative population sample of 3246 adults 25 to 64 years of age, between the years 1999 to 2001. Height and weight were measured, and BMI and other weight‐height indices were calculated. SES was assessed by income and education. Results: Age‐adjusted height was significantly lower at lower levels of SES among both women and men (p < 0.001). As opposed to men, women of lower SES were heavier than those of higher SES, and the mean age‐adjusted weight was 4.6 kg higher among those of lower SES (p < 0.001). Thus, using the standard index of BMI, the prevalence of obesity was significantly higher among shorter women. Discussion: In this group of Israeli adults, the unfavorable effect of low SES on BMI was evident among women, partly due to their decreased height combined with increased weight common in this socioeconomic sector. Since BMI is only partly independent of height, it may overestimate the prevalence of obesity among women of lower SES. Alternative measures for classifying obesity in the lower SES groups that put less emphasis on height may be considered and studied.     

Identification of RNA editing sites in the SNP database

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications.     

Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.     

Tuning DO:DM Ratios Modulates MHC Class II Immunopeptidomes

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM sensitivity, peptide abundance, binding affinity and motif, peptide length, and choice of binding register along the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.     

Cellular localization of cytochrome bd in cyanobacteria using genetic code expansion

Photosynthesis is one of the most fundamental and complex mechanisms in nature. It is a well-studied process, however, some photosynthetic mechanisms are yet to be deciphered. One of the many proteins that take part in photosynthesis, cytochrome bd, is a terminal oxidase protein that plays a role both in photosynthesis and in respiration in various organisms, specifically, in cyanobacteria. To clarify the role of cytochrome bd in cyanobacteria, a system for the incorporation of an unnatural amino acid into a genomic membrane protein cytochrome bd was constructed in Synechococcus sp. PCC7942. N-propargyl- l -lysine (PrK) was incorporated into mutants of cytochrome bd. Incorporation was verified and the functionality of the mutant cytochrome bd was tested, revealing that both electrochemical and biochemical activities were relatively similar to those of the wild-type protein. The incorporation of PrK was followed by a highly specific labeling and localization of the protein. PrK that was incorporated into the protein enabled a “click” reaction in a bio-orthogonal manner through its alkyne group in a highly specific manner. Cytochrome bd was found to be localized mostly in thylakoid membranes, as was confirmed by an enzyme-linked immunosorbent assay, indicating that our developed localization method is reliable and can be further used to label endogenous proteins in cyanobacteria.     

Potentiation of Regulatory Volume Decrease by a P2-Like Receptor and Arachidonic Acid in American Alligator Erythrocytes

This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy. Gadolinium (50 μM), hexokinase (2.5 U/ml), and suramin (100 μM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca²⁺ that normally occurs during cell swelling. The P2X antagonists PPADS (50 μM) and TNP-ATP (10 μM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10 μM), α,β-methylene-ATP (50 μM) and Bz-ATP (50 μM) had the opposite effect, whereas 2-methylthio-ATP (50 μM) and UTP (10 μM) had no effect. In addition, the phospholipase A₂ (PLA₂) inhibitors ONO-RS-082 (10 μM), chlorpromazine (10 μM), and isotetrandrine (10 μM) increased osmotic fragility and blocked volume recovery, whereas AA (10 μM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10 μM) had the reverse effect. Further, AA (10 μM), but not ATPγS (10 μM), prevented the inhibitory effect of a low Ca²⁺-EGTA Ringer on RVD, whereas both AA (10 μM) and ATPγS (10 μM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.