Engineered,highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side‐chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site‐specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100‐fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5‐cd]indolizine‐type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α‐helical, β‐sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q‐GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG‐reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.     

Design, synthesis, and biological evaluation of 8-biarylquinolines: a novel class of PDE4 inhibitors

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC(50)<10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC(50)<0.5 microM) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis.     

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.     

Shrub cover in northern Nunavik: can herbivores limit shrub expansion?

Recent climate changes have increased the primary productivity of many Arctic and subarctic regions. Erected shrub has been shown to increase in abundance over the last decades in northern regions in response to warmer climate. At the same time, caribou herds are declining throughout the circumboreal regions. Based on observation of heavy browsing on shrubs at Deception Bay (Nunavik, Canada), we hypothesized that the densification of shrubs observed in nearby locations did not occur at our study site despite of observed warming because of a recent peak of the Rivière-aux-Feuilles caribou herd. To assess shrub cover changes, we compared a 1972 mosaic of aerial photos to a 2010 satellite image over a 5 km² area, divided into 56 grids of 100 30 m × 30 m cells. Most cells (n = 4,502) did not show any changes in the cover of shrubs but those who did were as likely to increase as to decrease. The relative cover of shrubs in cells who changed was not higher in 2010 (6.1 ± 0.2 %) than in 1972 (7.3 ± 0.4 %). More than 70 % of birch and willow had more than 50 % of their shoot browsed, suggesting that caribou may limit shrub expansion at this site. We cannot rule out that abiotic factors also contribute to the inertia in shrub cover. Increases in shrub abundance reported in Nunavik and elsewhere were located closer to the tree line or in discontinuous permafrost, whereas our site is characterized by herbaceous arctic tundra, continuous permafrost and relatively low annual precipitation.     

Resistance to Erythropoiesis Stimulating Agents in Patients Treated with Online Hemodiafiltration and Ultrapure Low-Flux Hemodialysis: Results from a Randomized Controlled Trial (CONTRAST)

Resistance to erythropoiesis stimulating agents (ESA) is common in patients undergoing chronic hemodialysis (HD) treatment. ESA responsiveness might be improved by enhanced clearance of uremic toxins of middle molecular weight, as can be obtained by hemodiafiltration (HDF). In this analysis of the randomized controlled CONvective TRAnsport STudy (CONTRAST; {"type":"clinical-trial","attrs":{"text":"NCT00205556","term_id":"NCT00205556"}}NCT00205556), the effect of online HDF on ESA resistance and iron parameters was studied. This was a pre-specified secondary endpoint of the main trial. A 12 months'' analysis of 714 patients randomized to either treatment with online post-dilution HDF or continuation of low-flux HD was performed. Both groups were treated with ultrapure dialysis fluids. ESA resistance, measured every three months, was expressed as the ESA index (weight adjusted weekly ESA dose in daily defined doses [DDD]/hematocrit). The mean ESA index during 12 months was not different between patients treated with HDF or HD (mean difference HDF versus HD over time 0.029 DDD/kg/Hct/week [−0.024 to 0.081]; P = 0.29). Mean transferrin saturation ratio and ferritin levels during the study tended to be lower in patients treated with HDF (−2.52% [−4.72 to −0.31]; P = 0.02 and −49 ng/mL [−103 to 4]; P = 0.06 respectively), although there was a trend for those patients to receive slightly more iron supplementation (7.1 mg/week [−0.4 to 14.5]; P = 0.06).In conclusion, compared to low-flux HD with ultrapure dialysis fluid, treatment with online HDF did not result in a decrease in ESA resistance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00205556","term_id":"NCT00205556"}}NCT00205556     

Sensitive high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine

A method to determine total and free mycophenolic acid (MPA) and its metabolites, the phenolic (MPAG) and acyl (AcMPAG) glucuronides, using HPLC and mass spectrometry was developed. Mean recoveries in plasma and urine samples were >85%, and the lower limits of quantification for MPA, MPAG and AcMPAG were 0.05, 0.05 and 0.01 mg/L, respectively. For plasma, the assay was linear over 0.05-50 mg/L for MPA and MPAG, and from 0.01 to 10mg/L for AcMPAG. A validation study demonstrated good inter- and intra-day precision (CV相似文献   

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.