Roflumilast N-Oxide Prevents Cytokine Secretion Induced by Cigarette Smoke Combined with LPS through JAK/STAT and ERK1/2 Inhibition in Airway Epithelial Cells

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative in vitro model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 µg/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5–120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E₂ alone or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-α/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of LPS 0.1 µg/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE₂ reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is reduced by RNO.     

Bioreduction of α-chloroacetophenone by whole cells of marine fungi

The asymmetric reduction of 2-chloro-1-phenylethanone (1) by seven strains of marine fungi was evaluated and afforded (S)-(-)-2-chloro-1-phenylethanol with, in the best case, an enantiomeric excess of 50% and an isolated yield of 60%. The ability of marine fungi to catalyse the reduction was directly dependent on growth in artificial sea water-based medium containing a high concentration of Cl⁻ (1.2 M). When fungi were grown in the absence of artificial sea water, no reduction of 1 by whole cells was observed. The biocatalytic reduction of 1 was more efficient at neutral rather than acidic pH values and in the absence of glucose as co-substrate.     

Local interactions in protein folding determined through an inverse folding model

We adopt a model of inverse folding in which folding stability results from the combination of the hydrophobic effect with local interactions responsible for secondary structure preferences. Site-specific amino acid distributions can be calculated analytically for this model. We determine optimal parameters for the local interactions by fitting the complete inverse folding model to the site-specific amino acid distributions found in the Protein Data Bank. This procedure reduces drastically the influence on the derived parameters of the preference of different secondary structures for buriedness, which affects local interaction parameters determined through the standard approach based on amino acid propensities. The quality of the fit is evaluated through the likelihood of the observed amino acid distributions given the model and the Bayesian Information Criterion, which indicate that the model with optimal local interaction parameters is strongly preferable to the model where local interaction parameters are determined through propensities. The optimal model yields a mean correlation coefficient r = 0.96 between observed and predicted amino acid distributions. The local interaction parameters are then tested in threading experiments, in combination with contact interactions, for their capacity to recognize the native structure and structures similar to the native against unrelated ones. In a challenging test, proteins structurally aligned with the Mammoth algorithm are scored with the effective free energy function. The native structure gets the highest stability score in 100% of the cases, a high recognition rate comparable to that achieved against easier decoys generated by gapless threading. We then examine proteins for which at least one highly similar template exists. In 61% of the cases, the structure with the highest stability score excluding the native belongs to the native fold, compared to 60% if we use local interaction parameters derived from the usual amino acid propensities and 52% if we use only contact interactions. A highly similar structure is present within the five best stability scores in 82%, 81%, and 76% of the cases, for local interactions determined through inverse folding, through propensity, and set to zero, respectively. These results indicate that local interactions improve substantially the performances of contact free energy functions in fold recognition, and that similar structures tend to get high stability scores, although they are often not high enough to discriminate them from unrelated structures. This work highlights the importance to apply more challenging tests, as the recognition of homologous structures, for testing stability scores for protein folding.     

黑龙江和图们江流域菱属(Trapa L.)植物分布格局及形态多样性

菱属(Trapa L.)的系统分类一直存在较大分歧,至今还没有一个比较公认的分类系统。黑龙江和图们江流域是菱属物种多样性的重要分布区之一,为了揭示该流域菱属植物的地理分布格局和形态多样性,我们进行了大量实地调查和研究。结果显示,从该地区28个湖中共采集到菱属11个种及8个种内变异类型,表明它们具有丰富的形态多样性;结合查阅菱属354份标本资料,共获得92个分布地点数据;采集到的11个物种的地理分布格局呈不均衡性,其中细果野菱(Trapa maximowiczii Korsch.)分布最广,野菱(Trapa incisa Siebold et Zucc.)、兴凯菱(Trapa khankensis Pshennikova)和科热夫尼科夫菱(Trapa kozhevnikovirum Pshennikova)为狭域分布种;东部乌苏里江和图们江流域是菱属物种多样性的分布中心,可能是第四纪冰期避难所;菱属植物多数种间形态特性相对稳定,东北菱(Trapa manshurica Fler.)、耳菱(Trapa potaninii V.Vassil)、丘角菱(Trapa japonica Fler.)、西伯利亚菱(Trapa sibirica Fler.)和细果野菱共有8个种内形态变异类型;种群内多数分类性状稳定,种群间形态变异较明显;菱属植物分布格局不均衡和种内形态变异的形成可能与基因流的扩散限制有关。本研究结果为进一步澄清菱属分类混乱问题奠定了基础,进一步结合分子标记技术研究系统演化关系将对揭示菱属的进化历史具有重要意义。     

Estrogen receptors mediate rapid activation of phospholipase C pathway in the rat endometrium

The aim of the present study was to investigate the activation of rapid signaling events by 17β-estradiol in the rat uterus. 17β-Estradiol induced a rapid increase of total [³H]-inositol phosphate accumulation in the whole uterus and endometrium, but not in the myometrium. The effect of 17β-estradiol in the endometrium was blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [³H]-inositol phosphate accumulation in the endometrium. The G-1 effects were blocked by GPER antagonist (G-15). 17β-Estradiol and G-1 promoted an additive effect on total [³H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17β-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormone stimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.     

IFN-gamma and IL-12B polymorphisms in women with cervical intraepithellial neoplasia caused by human papillomavirus

The Human Papillomavirus (HPV) is a sexually transmitted organism associated with Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, the second main cause of malignancy in women worldwide. The virus itself, however, is not enough to cause lesions on the cervix. Several studies suggest that some polymorphic sites changes the cytokines levels and influence the cancer development in HPV infected patients. In this study, we evaluated the presence of functional polymorphisms at +874 (T/A) IFNG and +1188 (A/C) IL-12B genes in cervical smears samples from 76 healthy women and 162 women, HPV positive, with CIN lesion--CIN I (45), CIN II (55), CIN III (53) and cervical cancer (9)--in Brazilian population. There was no significant differences in genotype (p = 0.4192) and allele (p = 0.370; OR = 1.20) distributions between CIN patients and control groups on IFNG allelic polymorphism. Moreover, for IL-12B gene, there was a significant difference in genotype (p = 0.015) and allele distribution (p = 0.014; OR = 0.5754) between the groups. When samples were stratified according to grade of cervical lesion, the AA genotype and A allele were less frequent in the group with low-grade cervical lesions than in group with high-grade cervical lesions (p = 0.0036 and p = 0.0010; OR = 0.3819, respectively), suggesting that the C allele (mutant) may protect against the emergence of CIN lesions and its progression.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.     

Some Mechanistic Aspects on Fmoc Solid Phase Peptide Synthesis

Peptides are biomolecules that may have several biological activities which makes them important to the environment in which they operate. Sometimes it is necessary for larger amounts of peptides to carry out some studies, like biological tests, NMR structural research or even interaction studies between peptides with other molecules. Expression can be an alternative for that. However, synthesis is specially useful when unnatural modifications or introduction of site specific tags are required. Synthetic peptides have been used for different studies such as cell signaling, development of epitope-specific antibodies, in cell-biology, biomarkers for diseases etc. Many different methodologies for peptide synthesis can be found in the literature. Solid phase peptide synthesis (SPPS) has been largely used and can be an excellent alternative to achieve larger quantities of these biomolecules. In this mini review, we aim to describe the SPPS and explain some of the mechanistic aspects and reagents involved in all phases of the synthesis: the use of resin, the ninhydrin test, some of the protecting groups, coupling reagents for peptide bond formation and the cleavage process.     

CD137 differentially regulates innate and adaptive immunity against Mycobacterium tuberculosis

Protective immunity against Mycobacterium tuberculosis is primarily mediated by the interaction of antigen-specific T cells and antigen presenting cells, which often depends on the interplay of cytokines produced by these cells. Costimulatory signals represent a complex network of receptor-ligand interactions that qualitatively and quantitatively influence immune responses. Thus, here we investigated the function of CD137 and CD137L, molecules known to have a central role in immune regulation, during human tuberculosis (TB). We demonstrated that M. tuberculosis antigen stimulation increased both CD137 and CD137L expression on monocytes and NK cells from TB patients and healthy donors, but only up-regulated CD137 on T lymphocytes. Blockage of the CD137 pathway enhanced the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by monocytes and NK against M. tuberculosis. In contrast, CD137 blockage significantly decreased the specific degranulation of CD8(+) T cells and the percentage of specific IFN-γ and TNF-α producing lymphocytes against the pathogen. Furthermore, inhibition of the CD137 pathway markedly increased T-cell apoptosis. Taken together, our results demonstrate that CD137:CD137L interactions regulate the innate and adaptive immune response of the host against M. tuberculosis.