Induced expression of pattern recognition receptors in cumulus oocyte complexes: novel evidence for innate immune-like functions during ovulation

Ovulation is the complex, inflammatory-like process by which the cumulus oocyte complex (COC) is released from a mature, preovulatory follicle through a rupture site at the ovarian surface and requires expression of genes that generate and stabilize the expanded extracellular COC matrix. Gene profiling analyses of COCs at selected time intervals during ovulation revealed that many genes associated with immune related surveillance functions were also induced in cumulus cells. Specifically, cell surface signaling molecules known as pattern recognition receptors that act as sensors of the external environment important for the innate immune system to detect self from nonself or altered self are induced and/or expressed in cumulus cells as well as granulosa cells. These include the complement factor q1, CD14, and the Toll-like receptors (TLRs) 4, 8, and 9 as well as mediators of TLR activation, myeloid differentiation primary response gene 88 and interferon regulatory factor 3. COCs exposed to bacterial lipopolysaccharide exhibit enhanced phosphorylation of p38MAPK, ERK1/2 and nuclear factor-kappaB and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and scavenger receptor type B1 and exhibited phagocytic uptake of fluorescently tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process.     

Chymase Mediates Injury and Mitochondrial Damage in Cardiomyocytes during Acute Ischemia/Reperfusion in the Dog

Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.     

Distribution and Outcomes of a Phenotype-Based Approach to Guide COPD Management: Results from the CHAIN Cohort

RationaleThe Spanish guideline for COPD (GesEPOC) recommends COPD treatment according to four clinical phenotypes: non-exacerbator phenotype with either chronic bronchitis or emphysema (NE), asthma-COPD overlap syndrome (ACOS), frequent exacerbator phenotype with emphysema (FEE) or frequent exacerbator phenotype with chronic bronchitis (FECB). However, little is known on the distribution and outcomes of the four suggested phenotypes.ObjectiveWe aimed to determine the distribution of these COPD phenotypes, and their relation with one-year clinical outcomes.MethodsWe followed a cohort of well-characterized patients with COPD up to one-year. Baseline characteristics, health status (CAT), BODE index, rate of exacerbations and mortality up to one year of follow-up were compared between the four phenotypes.ResultsOverall, 831 stable COPD patients were evaluated. They were distributed as NE, 550 (66.2%); ACOS, 125 (15.0%); FEE, 38 (4.6%); and FECB, 99 (11.9%); additionally 19 (2.3%) COPD patients with frequent exacerbations did not fulfill the criteria for neither FEE nor FECB. At baseline, there were significant differences in symptoms, FEV₁ and BODE index (all p<0.05). The FECB phenotype had the highest CAT score (17.1±8.2, p<0.05 compared to the other phenotypes). Frequent exacerbator groups (FEE and FECB) were receiving more pharmacological treatment at baseline, and also experienced more exacerbations the year after (all p<0.05) with no differences in one-year mortality. Most of NE (93%) and half of exacerbators were stable after one year.ConclusionsThere is an uneven distribution of COPD phenotypes in stable COPD patients, with significant differences in demographics, patient-centered outcomes and health care resources use.     

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.     

Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor

The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Herein we document that epidermal growth factor (EGF)-like factors amphiregulin (Areg), epiregulin (Ereg), and betacellulin (Btc) are induced in cumulus oocyte complexes (COCs) by autocrine and paracrine mechanisms that involve the actions of prostaglandins (PGs) and progesterone receptor (PGR). Areg and Ereg mRNA and protein levels were reduced significantly in COCs and ovaries collected from prostaglandin synthase 2 (Ptgs2) null mice and Pgr null (PRKO) mice at 4 h and 8 h after human chorionic gonadotropin, respectively. In cultured COCs, FSH/forskolin induced Areg mRNA within 0.5 h that peaked at 4 h, a process blocked by inhibitors of p38MAPK (SB203580), MAPK kinase (MEK) 1 (PD98059), and PTGS2 (NS398) but not protein kinase A (PKA) (KT5720). Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. PGE2 reversed the inhibitory effects of AG on AREG-induced expression of Areg but not Ptgs2, placing Ptgs2 downstream of EGF-R signaling. Phorbol 12-myristate 13-acetate (PMA) and adenovirally expressed PGRA synergistically induced Areg mRNA in granulosa cells. In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). Collectively, FSH-mediated induction of Areg mRNA via p38MAPK precedes AREG induction of Ptgs2 mRNA via ERK1/2. PGs acting via PTGER2 in cumulus cells provide a secondary, autocrine pathway to regulate expression of Areg in COCs showing critical functional links between G protein-coupled receptor and growth factor receptor pathways in ovulating follicles.     

N',2-diphenylquinoline-4-carbohydrazide based NK3 receptor antagonists II

Introduction of selected amine containing side chains into the 3-position of N',2-diphenylquinoline-4-carbohydrazide based NK3 antagonists abolishes unwanted hPXR activation. Introduction of a fluorine at the 8-position is necessary to minimize unwanted hI(Kr) affinity and a piperazine N-tert-butyl group is necessary for metabolic stability. The lead compound (8m) occupies receptors within the CNS following oral dosing (Occ(90) 7 mg/kg po; plasma Occ(90) 0.4 microM) and has good selectivity and excellent PK properties.     

Overexpression, Purification, and Biochemical Characterization of the Esterase Est0796 from Lactobacillus plantarum WCFS1

Lactobacillus plantarum is a lactic acid bacterium that is encountered in an extensive range of foods such as fermented dairy products, meat, vegetables, and bakery products. Given the little molecular information available on the lipolytic activity of L. plantarum, the aim of this study was to clone, purify, and biochemically characterize the esterase coded by gene lp_0796 (Est0796). The esterase was cloned in pET28a and purified in two steps, using solid ammonium sulfate and His tag affinity chromatography. The molecular mass of the purified Est0796 was 28.7 kDa (by SDS-PAGE) and 26.6 kDa (by gel filtration chromatography), pointing to a monomeric structure. Est0796 showed maximum activity at pH 8.0 and 35 °C and toward shorter acyl chain lengths (C₂–C₄). The activity was resistant to organic solvents and cations, suggesting that this esterase may play a role in the fermentation of food products.     

Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress

A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and-II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants.