Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina

Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death.

Synthesis of bis(3-{[2-(allyloxy)ethoxy]methyl}-2,4,6-trimethylbenzoyl)(phenyl)phosphine oxide - a tailor-made photoinitiator for dental adhesives

Because of the poor solubility of the commercially available bisacylphosphine oxides in dental acidic aqueous primer formulations, bis(3-{[2-(allyloxy)ethoxy]methyl}-2,4,6-trimethylbenzoyl)(phenyl)phosphine oxide (WBAPO) was synthesized starting from 3-(chloromethyl)-2,4,6-trimethylbenzoic acid by the dichlorophosphine route. The substituent was introduced by etherification with 2-(allyloxy)ethanol. In the second step, 3-{[2-(allyloxy)ethoxy]methyl}-2,4,6-trimethylbenzoic acid was chlorinated. The formed acid chloride showed an unexpected low thermal stability. Its thermal rearrangement at 180 ° C resulted in a fast formation of 3-(chloromethyl)-2,4,6-trimethylbenzoic acid 2-(allyloxy)ethyl ester. In the third step, the acid chloride was reacted with phenylphosphine dilithium with the formation of bis(3-{[2-(allyloxy)ethoxy]methyl}-2,4,6-trimethylbenzoyl)(phenyl)phosphine, which was oxidized to WBAPO. The structure of WBAPO was confirmed by ¹H NMR, ¹³C NMR, ³¹P NMR, and IR spectroscopy, as well as elemental analysis. WBAPO, a yellow liquid, possesses improved solubility in polar solvents and shows UV-vis absorption, and a high photoreactivity comparable with the commercially available bisacylphosphine oxides. A sufficient storage stability was found in dental acidic aqueous primer formulations.

The S-1/S-2 exciton interaction in 2-pyridone center dot 6-methyl-2-pyridone: Davydov splitting, vibronic coupling, and vibronic quenching

The excitonic splitting between the S-1 and S-2 electronic states of the doubly hydrogen-bonded dimer 2-pyridone center dot 6-methyl-2-pyridone (2PY center dot 6M2PY) is studied in a supersonic jet, applying two-color resonant two-photon ionization (2C-R2PI), UV-UV depletion, and dispersed fluorescence spectroscopies. In contrast to the C-2h symmetric (2-pyridone) 2 homodimer, in which the S-1 <- S-0 transition is symmetry-forbidden but the S-2 <- S-0 transition is allowed, the symmetry-breaking by the additional methyl group in 2PY center dot 6M2PY leads to the appearance of both the S-1 and S-2 origins, which are separated by Delta(exp) = 154 cm(-1). When combined with the separation of the S-1 <- S-0 excitations of 6M2PY and 2PY, which is delta = 102 cm(-1), one obtains an S-1/S-2 exciton coupling matrix element of V-AB, el = 57 cm(-1) in a Frenkel-Davydov exciton model. The vibronic couplings in the S-1/S-2 <- S-0 spectrum of 2PY center dot 6M2PY are treated by the Fulton-Gouterman single-mode model. We consider independent couplings to the intramolecular 6a' vibration and to the intermolecular sigma' stretch, and obtain a semi-quantitative fit to the observed spectrum. The dimensionless excitonic couplings are C(6a') = 0.15 and C(sigma') = 0.05, which places this dimer in the weak-coupling limit. However, the S-1/S-2 state exciton splittings Delta(calc) calculated by the configuration interaction singles method (CIS), time-dependent Hartree-Fock (TD-HF), and approximate second-order coupled-cluster method (CC2) are between 1100 and 1450 cm(-1), or seven to nine times larger than observed. These huge errors result from the neglect of the coupling to the optically active intra-and intermolecular vibrations of the dimer, which lead to vibronic quenching of the purely electronic excitonic splitting. For 2PY center dot 6M2PY the electronic splitting is quenched by a factor of similar to 30 (i.e., the vibronic quenching factor is Gamma(exp) = 0.035), which brings the calculated splittings into close agreement with the experimentally observed value. The 2C-R2PI and fluorescence spectra of the tautomeric species 2-hydroxypyridine center dot 6-methyl-2-pyridone (2HP center dot 6M2PY) are also observed and assigned. (C) 2011 American Institute of Physics.

S-0 and S-1 State Structure, Methyl Torsional Barrier Heights, and Fast Intersystem Crossing Dynamics of 5-Methyl-2-hydroxypyrimidine

We report the analysis of the SI So rotational band contours of jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine. Unlike thymine, which exhibits a structureless spectrum, the vibronic spectrum of 5M2HP is well structured, allowing us to determine the rotational constants and the methyl group torsional barriers in the S-0 and S-1 states. The 0(0)(0), 6a(0)(1), 6b(0)(1), and 14(0)(1) band contours were measured at 900 MHz (0.03 cm(-1)) resolution using mass-specific two-color resonant two-photon ionization (2C-R2PI) spectroscopy. All four bands are polarized perpendicular to the pyrimidine plane (>90% c type), identifying the S-1 <- S-0 excitation of 5M2HP as a 1n pi* transition. All contours exhibit two methyl rotor subbands that arise from the lowest 5-methyl torsional states 0A '' and 1E ''. The S-0 and S-1 state torsional barriers were extracted from fits to the torsional subbands. The 3-fold barriers are V-3 '' = 13 cm(-1) and V3' = SI cm(-1); the 6-fold barrier contributions V-6 '' and V-6' are in the range of 2-3 cm(-1) and are positive in both states. The changes of A, B, and C rotational constants upon S-1 <- S-0 excitation were extracted from the contours and reflect an "anti-quinoidal" distortion. The 0(0)(0) contour can only be simulated if a 3 GHz Lorentzian line shape is included, which implies that the S-1(1n pi*) lifetime is similar to 55 ps. For the 6a(0)(1) and 6b(0)(1) bands, the Lorentzian component increases to 5.5 GHz, reflecting a lifetime decrease to similar to 30 ps. The short lifetimes are consistent with the absence of fluorescence from the 1n pi* state. Combining these measurements with the previous observation of efficient intersystem crossing (ISC) from the Si state to a long-lived T-1((3)n pi*) state that lies similar to 2200 cm(-1) below [S. Lobsiger, S. et al. Phys. Chem. Chem. Phys. 2010, 12, 5032] implies that the broadening arises from fast intersystem crossing with k(ISC) approximate to 2 x 10(10) s(-1). In comparison to 5-methylpyrimidine, the ISC rate is enhanced by at least 10 000 by the additional hydroxy group in position 2.

Comparative stability studies of poly(2-methyl-2-oxazoline) and poly(ethylene glycol) brush coatings

Non-fouling surfaces that resist non-specific adsorption of proteins, bacteria, and higher organisms are of particular interest in diverse applications ranging from marine coatings to diagnostic devices and biomedical implants. Poly(ethylene glycol) (PEG) is the most frequently used polymer to impart surfaces with such non-fouling properties. Nevertheless, limitations in PEG stability have stimulated research on alternative polymers that are potentially more stable than PEG. Among them, we previously investigated poly(2-methyl-2-oxazoline) (PMOXA), a peptidomimetic polymer, and found that PMOXA shows excellent anti-fouling properties. Here, we compare the stability of films self-assembled from graft copolymers exposing a dense brush layer of PEG and PMOXA side chains, respectively, in physiological and oxidative media. Before media exposure both film types prevented the adsorption of full serum proteins to below the detection limit of optical waveguide in situ measurements. Before and after media exposure for up to 2 weeks, the total film thickness, chemical composition, and total adsorbed mass of the films were quantified using variable angle spectroscopic ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and optical waveguide lightmode spectroscopy (OWLS), respectively. We found (i) that PMOXA graft copolymer films were significantly more stable than PEG graft copolymer films and kept their protein-repellent properties under all investigated conditions and (ii) that film degradation was due to side chain degradation rather than due to copolymer desorption.

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

PhIP carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but < 1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P=0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered.

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.