Pulmonary fibrosis in L-NAME-treated mice is dependent on an activated endothelin system

Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8 weeks were randomly allocated to the following 6 groups: WT (n = 11), wild-type animals without treatment; WT + l-NAME (n = 14), wild-type animals receiving l-NAME, an inhibitor of NO synthase; WT + l-NAME + LU (n = 13), wild-type animals receiving l-NAME and LU 302872, a dual ETA/ETB-receptor antagonist; ET1tg (n = 10), ET-1 transgenic mice; ET1tg + l-NAME (n = 13); and ET1tg + l-NAME + LU (n = 13). After 6 weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation, as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after l-NAME treatment. The combined ETA/ETB-receptor antagonist LU 302872 abolished inflammation and interstitial fibrosis in l-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study l-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU 302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.     

Human Alzheimer’s disease synaptic <Emphasis Type="Italic">O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Cloning,expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII

A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

     

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

     

TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue

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Quenching in Arabidopsis thaliana mutants lacking monomeric antenna proteins of photosystem II

The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.     

Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 degrees C) and high (18 degrees C) temperatures leads to differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5'-UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3'-UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH-A(alpha) and LDH-A(beta) subunits. It is expected that the beta-subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH-A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm-acclimated fish the relative expression between alpha and beta isoforms of the LDH-A mRNA is around 5 : 1, whereas in cold-acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH-A is more homogeneous in terms of alpha/beta-subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine-tuning of the properties of skeletal LDH-A, which is in line with previously observed kinetic and thermodynamic differences between 'cold' and 'warm' LDH-A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.     

Micro-CT based analysis of a new paradigm for vulnerable plaque rupture: cellular microcalcifications in fibrous caps

In this paper, we further investigate the new paradigm for the rupture of thin cap fibroatheroma (TCFA) proposed in Vengrenyuk et al. (2006 PNAS 103:14678) using a multilevel micro-CT based 3D numerical modeling. The new paradigm proposes that the rupture of TCFA is due to stress-induced interfacial debonding of cellular--level, 10-20 microm microcalcifications in the fibrous cap proper. Such microcalcifications, which lie below the visibility of current in vivo imaging techniques, were detected for the first time using confocal microscopy and high resolution microcomputed tomography (micro-CT) imaging in Vengrenyuk et al. (2006) In the present study, we use high resolution (7 microm) micro-CT imaging to construct accurate geometries of both these microcalcifications and larger mm size macrocalcifications at the cap shoulders to evaluate their biomechanical stability. The analysis shows that cellular-level calcifications by themselves are not dangerous unless they lie in a region of high background stress. This high level of background stress only occurs in caps whose thickness is < approximately 80 microm. Whereas a spherical microcalcification will increase peak circumferential stress (PCS) by a factor of two, in agreement with previous local analytical solutions, this can be increased several fold by elongated microcalcifications. The most dangerous situation is when a microinclusion appears in close proximity to a region where the PCS is already high. This stress will be substantially increased if the inclusion is elongated. In contrast, macrocalcifications at the cap shoulders are shown to actually increase plaque stability.