Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the T1rA and T1rD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related (‘erm-type’) genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.     

Release of 12 Adipokines by Adipose Tissue,Nonfat Cells,and Fat Cells From Obese Women

The relative release in vitro of endothelin‐1, zinc‐α2‐glycoprotein (ZAG), lipocalin‐2, CD14, RANTES (regulated on activation, normal T cell expressed and secreted protein), lipoprotein lipase (LPL), osteoprotegerin (OPG), fatty acid–binding protein 4 (FABP‐4), visfatin/PBEF/Nampt, glutathione peroxidase‐3 (GPX‐3), intracellular cell adhesion molecule 1 (ICAM‐1), and amyloid A was examined using explants of human adipose tissue as well as the nonfat cell fractions and adipocytes from obese women. Over a 48‐h incubation the majority of the release of LPL was by fat cells whereas that of lipocalin‐2, RANTES, and ICAM‐1 was by the nonfat cells present in human adipose tissue. In contrast appreciable amounts of OPG, amyloid A, ZAG, FABP‐4, GPX‐3, CD14, and visfatin/PBEF/Nampt were released by both fat cells and nonfat cells. There was an excellent correlation (r = 0.75) between the ratios of adipokine release by fat cells to nonfat cells over 48 h and the ratio of their mRNAs in fat cells to nonfat cells at the start of the incubation. The total release of ZAG, OPG, RANTES, and amyloid A by incubated adipose tissue explants from women with a fat mass of 65 kg was not different from that by women with a fat mass of 29 kg. In contrast that of ICAM‐1, FABP‐4, GPX‐3, visfatin/PBEF/Nampt, CD14, lipocalin‐2, LP, and endothelin‐1 was significantly greater in tissue from women with a total fat mass of 65 kg.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).     

Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

Characterization of Cytomegalovirus Disease in Solid Organ Transplant Recipients by Markers of Inflammation in Plasma

Background

While several studies have examined the general inflammatory responses in relation to cytomegalovirus infection, the identification of the various inflammatory mediators as well as their relative importance is far from clear.

Patients and Methods

Solid organ recipients enrolled in an international multicenter trial of cytomegalovirus disease treatment (the VICTOR study) were analyzed (n = 289) (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00431353","term_id":"NCT00431353"}}NCT00431353). Plasma markers of inflammation and endothelial cell activation were assessed at baseline by enzyme immunoassays.

Results

The major findings were: (i) Plasma levels of the CXC-chemokine interferon-inducible protein-10 (P<0.001) and C-reactive protein (P = 0.046) were independently associated with the presence of cytomegalovirus DNAemia above lower level of quantification. (ii) High levels of CC-chemokine ligand 21 (P = 0.027) and pentraxin 3 (P = 0.033) were independently associated with tissue invasive cytomegalovirus disease as opposed to cytomegalovirus syndrome.

Conclusion

Our findings illustrate the complex interaction between cytomegalovirus and the immune system, involving a wide range of inflammatory mediators that could be associated to disease manifestations in cytomegalovirus related disease.