Prostaglandin-like inhibitors of prostaglandin-H-synthase

The effect of none prostaglandin-like cyclopentanone derivatives on the prostaglandin H synthase activity was studied. Seven substances proved to be inhibitors of the enzyme, some of the being similar to the well-known nonsteroid antiinflammatory drugs with respect to their inhibitory activity.     

The artiodactyl <Emphasis Type="Italic">APOBEC3</Emphasis> innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals

Background  

APOBEC3 (A3) proteins deaminate DNA cytosines and block the replication of retroviruses and retrotransposons. Each A3 gene encodes a protein with one or two conserved zinc-coordinating motifs (Z1, Z2 or Z3). The presence of one A3 gene in mice (Z2–Z3) and seven in humans, A3A-H (Z1a, Z2a-Z1b, Z2b, Z2c-Z2d, Z2e-Z2f, Z2g-Z1c, Z3), suggests extraordinary evolutionary flexibility. To gain insights into the mechanism and timing of A3 gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cDNAs and activities of the full A3 repertoire of three artiodactyl lineages: sheep, cattle and pigs.     

Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.     

土曲霉金色变种AT8951菊粉酶的纯化和性质的研究

土典霉金色变种AT8951菊粉酶粗酶液经硫酸铵分段沉淀、DEAE Cellulose DE32离子交换、超滤、Sephadex G-150凝胶过滤和FPLC,获得两个菊粉酶组分EⅠ和EⅡ,经分析型FPLG和PAGE鉴定为单一纯和分析纯。EⅠ分子量为66KD,最适作用温度和pH分别55℃和5.8;EⅡ分子量为56KD,最适作用温度为57℃,最适pH为6.0。EⅠ和EⅡ皆为糖蛋白,多糖含量分别为24.7％和22％,都属于内切酶。本文还对EⅠ和EⅡ的Km值和I/s值,温度、pH、离子对酶活作用的影响等进行了研究。     

Relationships in the Drosophila obscura species group, inferred from mitochondrial cytochrome oxidase II sequences

We compare the sequences for the mitochondrial cytochrome oxidase II gene of 13 species of the Drosophila obscura group. The survey includes six members of the D. affinis subgroup, four of the D. pseudoobscura subgroup, and three of the D. obscura subgroup. In all species, the gene is 688 nucleotides in length, encoding a protein of 229 amino acids plus the first position T of the stop codon. The sequences show the typical high-transition bias for closely related species, but that bias is essentially eliminated for species pairs of > 5% sequence divergence. The phylogenetic relationships in the species group are inferred using both neighbor-joining and maximum parsimony. The two procedures give comparable results, showing that the D. affinis and D. pseudoobscura subgroups are monophyletic groupings that appear to have closer affinities to one another than either has to the D. obscura subgroup. We use transversion distances to estimate times of divergence, on the basis of three different estimates of the time of separation of the D. obscura species group from the D. melanogaster group. If that event occurred 35 Mya, then we can estimate the origin of the nearctic forms at approximately 22 Mya and the separation of the D. affinis and D. pseudoobscura subgroups at approximately 17 Mya.      

The nature of functional groups regulating the catalytic activity of prostaglandin endoperoxide synthetase

The influence of some reagents modifying NH2-, SH-groups or imidazole moiety, on the prostaglandin endoperoxide synthetase activity was studied. Acetaldehyde, pyridoxal phosphate, dithiobis (nitrobenzoic) acid and iodoacetamide were found not to affect the enzyme activity. The activity was abolished as a result of the interaction with p-chloromercuribenzoic acid and diethyl pyrocarbonate. The hemin completely protected the apo-enzyme against the inactivation with diethyl pyrocarbonate. The assumption about the presence of imidazole moiety in the active site of the enzyme was made.     

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model

Background

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Aim

We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.

Methods

BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.

Results

Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.

Conclusion

Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.     

SHEAR: sample heterogeneity estimation and assembly by reference

Background

Personal genome assembly is a critical process when studying tumor genomes and other highly divergent sequences. The accuracy of downstream analyses, such as RNA-seq and ChIP-seq, can be greatly enhanced by using personal genomic sequences rather than standard references. Unfortunately, reads sequenced from these types of samples often have a heterogeneous mix of various subpopulations with different variants, making assembly extremely difficult using existing assembly tools. To address these challenges, we developed SHEAR (Sample Heterogeneity Estimation and Assembly by Reference; http://vk.cs.umn.edu/SHEAR), a tool that predicts SVs, accounts for heterogeneous variants by estimating their representative percentages, and generates personal genomic sequences to be used for downstream analysis.

Results

By making use of structural variant detection algorithms, SHEAR offers improved performance in the form of a stronger ability to handle difficult structural variant types and better computational efficiency. We compare against the lead competing approach using a variety of simulated scenarios as well as real tumor cell line data with known heterogeneous variants. SHEAR is shown to successfully estimate heterogeneity percentages in both cases, and demonstrates an improved efficiency and better ability to handle tandem duplications.

Conclusion

SHEAR allows for accurate and efficient SV detection and personal genomic sequence generation. It is also able to account for heterogeneous sequencing samples, such as from tumor tissue, by estimating the subpopulation percentage for each heterogeneous variant.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-84) contains supplementary material, which is available to authorized users.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.