The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2

In response to hydrogen peroxide (H2O2), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H2O2 induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H2O2, but alternative effectors

Nickel-Catalyzed Carbon–Carbon Bond-Forming Reactions of Unactivated Tertiary Alkyl Halides: Suzuki Arylations

The first Suzuki cross-couplings of unactivated tertiary alkyl electrophiles are described. The method employs a readily accessible catalyst (NiBr[subscript 2]·diglyme/4,4′-di-tert-butyl-2,2′-bipyridine, both commercially available) and represents the initial example of the use of a group 10 catalyst to cross-couple unactivated tertiary electrophiles to form C–C bonds. This approach to the synthesis of all-carbon quaternary carbon centers does not suffer from isomerization of the alkyl group, in contrast with the umpolung strategy for this bond construction (cross-coupling of a tertiary alkylmetal with an aryl electrophile). Preliminary mechanistic studies are consistent with the generation of a radical intermediate along the reaction pathway.National Institute of General Medical Sciences (U.S.) (R01-GM62871)Merck Research Laboratories (Summer Fellowship

Intramolecular additions of various π-nucleophiles to chemoselectively activated amides and application to the synthesis of (±)-tashiromine

Abstract: Vilsmeier-Haack type cyclizations proved to be particularly efficient for generating parts of the polycyclic cores of many alkaloids, although only monocyclizations have so far been reported. With the goal of rapidly and efficiently constructing polycyclic alkaloids, we decided to exploit the Vilsmeier-Haack reaction by utilizing iminium ions successively generated and trapped with tethered nucleophiles. To develop such a strategy, we had to set the first cyclization. This constitutes a great challenge in itself because amide activation conditions are usually not compatible with tethered nucleophiles, except for indoles and aromatic rings which have already been reported. This paper describes the comprehensive study of intramolecular addition of silyl enol ethers, allylsilanes, and enamines to chemoselectively activated formamides, aliphatic amides, and lactams. Good to excellent yields were obtained for the 5-exo, 6-exo, and 6-endo modes of cyclization. Moreover, we demonstrated that the species in solution after the cyclization are iminium ions. This is highly encouraging for the development of bis-cyclization strategies. An expeditious total synthesis of (()-tashiromine is also reported

Images of colonic real-time tissue sonoelastography correlate with those of colonoscopy and may predict response to therapy in patients with ulcerative colitis

<p>Abstract</p> <p>Background</p> <p>Real-time tissue sonoelastography (EG) is a new non-invasive technique that visualizes differences in tissue strain. We evaluated the usefulness of EG in patients with ulcerative colitis (UC) by investigating the association between EG and colonoscopic findings and disease activity.</p> <p>Methods</p> <p>Thirty-seven UC patients undergoing EG and colonoscopy were invited to enroll. EG findings were classified as normal, homogeneous, random, or hard, and colonoscopic findings as normal, mucosal edema and erosion, punched-out ulcer, and extensive mucosal abrasion. Clinical findings were evaluated using clinical activity index (CAI) scores for each patient at colonoscopy.</p> <p>Results</p> <p>On EG, 10 cases were classified as normal, 11 as homogeneous, 6 as random, and 10 as hard. EG findings showed a significant correlation those of colonoscopy (<it>p </it>< 0.001). Seven of 10 (70%) normal-type patients were in the remission phase, while all 6 random-type patients were in the active phase. Among active-phase patients, 4 of 7 (57%) homogeneous-type patients responded to steroid or leukocytapheresis therapy, while 3 of 6 (50%) random-type patients required treatment with cyclosporine. Three of 10 (30%) hard-type patients required colectomy.</p> <p>Conclusions</p> <p>In this small series, EG findings reflected colonoscopic findings and correlated with disease activity among patients with UC.</p

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter

The RNA Binding Protein Csx1 Promotes Sexual Differentiation in Schizosaccharomyces pombe

Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

Addition of calcium ions to the Ca2+-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (25000 cm-1) assigned as from the Ca2+-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t1/2 2 ps) in the case of the Ca2+-discharged obelin than aequorin (t1/2 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19400 cm-1, bound to Ca2+-discharged obelin, and 21300 cm-1, bound to Ca2+-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavit

Improved detectability of small-bowel lesions via capsule endoscopy with computed virtual chromoendoscopy: A pilot study

Objective. Real-time video capsule endoscopy (CE) with flexible spectral imaging color enhancement (FICE) improves visibility of small-bowel lesions. This article aims to clarify whether CE-FICE also improves detectability of small-bowel lesions. Patients and methods. A total of 55 patients who underwent CE at Hiroshima University Hospital during the period November 2009 through March 2010 were enrolled in the study. Five patients were excluded from the study because residues and transit delays prevented sufficient evaluation. Thus, 50 patients participated. Two experienced endoscopists (each having interpreted more than 50 capsule videos) analyzed the images. One interpreted conventional capsule videos; the other, blinded to interpretation of the conventional images, interpreted CE-FICE images obtained at settings 1-3 (setting 1: red 595 nm, green 540 nm, blue 535 nm; setting 2: red 420 nm, green 520 nm, blue 530 nm; setting 3: red 595 nm, green 570 nm, blue 415 nm). Lesions were classified as angioectasia, erosion, ulceration, or tumor. Detectability was compared between the two modalities. Time taken to interpret the capsule videos was also determined. Results. Seventeen angioectasias were identified by conventional CE; 48 were detected by CE-FICE at setting 1, 45 at setting 2, and 24 at setting 3, with significant differences at settings 1 and 2 (p = 0.0003, p < 0.0001, respectively). Detection of erosion, ulceration, and tumor did not differ statistically between conventional CE and CE-FICE, nor did interpretation time (conventional CE 36 ± 6.9 min; CE-FICE setting 1, 36 ± 6.4 min; setting 2, 38 ± 5.8 min; setting 3, 35 ± 6.7 min). Conclusions. CE-FICE is superior in the lesion detection in comparison with conventional CE and improves detection of angioectasia

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene