Lysophosphatidic acid (LPA) effects on endometrial carcinoma in vitro proliferation, invasion, and matrix metalloproteinase activity.

OBJECTIVES: Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation. METHODS: All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 microM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay. RESULTS: LPA, at the concentrations of 0.1 and 1.0 microM, significantly induced the proliferation of HEC-1A cells (p<0.01). At 10 microM, LPA- induced HEC-1A proliferation to a less extent and showed no significant effect on HEC-1A invasion and migration (p>0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner. CONCLUSIONS: LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer.

Elevation in Cell Cycle and Protein Metabolism Gene Transcription in Inactive Colonic Tissue From Icelandic Patients With Ulcerative Colitis.

Background: A combination of genetic and environmental factors is thought to be involved in the pathogenesis of ulcerative colitis (UC). In Iceland, the incidence of UC is one of the highest in the world. The aim of this study was to characterize patients with UC and identify potential germline mutations and pathways that could be associated with UC in this population. Methods: Exome sequencing and genome-wide microarray analysis on macroscopically noninflamed colonic mucosa from patients and controls were performed. Exome sequence data were examined for very rare or novel mutations that were over-represented in the UC cohort. Combined matching of variant analysis and downstream influence on transcriptomic expression in the rectum were analyzed. Results: One thousand eight hundred thirty-eight genes were differentially expressed in rectal tissue from UC patients and identified an upregulation in genes associated with cell cycle control and protein processing in the endoplasmic reticulum (ER). Two missense mutations in thiopurine S-methyltransferase (TPMT) with a minor allele frequency of 0.22 in the UC patients compared with a reported 0.062 in the Icelandic population were identified. A predicted damaging mutation in the gene SLC26A3 is potentially associated with increased expression of DUOX2 and DUOXA2 in rectal tissue. Conclusions: Colonic mucosa of UC patients demonstrates evidence of an elevation in genes involving cell proliferation and processing of proteins within the ER. Exome sequencing identified a possible increased prevalence of 2 damaging TPMT variants within the UC population, suggesting screening the UC population before initiation of thiopurine analogue therapy to avoid toxicity associated with these mutations.

Lysophosphatidic acid (LPA) promotes E-cadherin ectodomain shedding and OVCA429 cell invasion in an uPA-dependent manner.

OBJECTIVES: To evaluate the role of LPA in regulating E-cadherin cell surface expression, adhesion, and invasion in epithelial ovarian carcinoma (EOC) cells. METHODS: E-cadherin mRNA expression in OVCA429 and IOSE-29 cells was evaluated by real-time RT-PCR. Immunofluorescence and Western blot analysis were performed to determine cell surface expression and shedding of E-cadherin 80-kDa soluble fragment by LPA. Kinetics of LPA-induced uPA activity was followed with a colorimetric enzymatic assay. Invasion assays were performed in a modified Boyden chamber where cells were allowed to migrate to the bottom compartment through a porous filter coated with collagen. Additionally we measured the 80-kDa form from the ascites of women with stage III/IV EOC. RESULTS: LPA induces E-cadherin shedding of a soluble 80-kDa fragment. We found that this process is mediated by the uPA proteolytic cascade. High levels of soluble E-cadherin were found in the ascites from women with advanced stage EOC. LPA and a soluble recombinant E-cadherin-Fc chimera promotes invasion of OVCA429 cells. CONCLUSIONS: LPA induces shedding of an 80-kDa E-cadherin-soluble fragment in an uPA-dependent manner and promotes in vitro invasion. High levels of soluble E-cadherin in malignant ascites may also affect ovarian metastasis.

7,8-Dihydroxyflavone reduces sleep during dark phase and suppresses orexin A but not orexin B in mice.

Brain-derived neurotrophic factor (BDNF) binds to Tropomyosin-receptor-kinase B (TrkB) receptors that regulate synaptic strength and plasticity in the mammalian nervous system. 7,8-Dihydroxyflavone (DHF) is a recently identified small molecule Trk B agonist that has been reported to ameliorate depression, attenuate the fear response, improve memory consolidation, and exert neuroprotective effects. Poor and disturbed sleep remains a symptom of major depressive disorder and most current antidepressants affect sleep. Therefore, we conducted sleep/wake recordings and concomitant measurement of brain orexins, endogenous peptides that suppress sleep, in mice for this study. Baseline polysomnograph recording was performed for 24 h followed by treatment with either 5 mg/kg of DHF or vehicle at the beginning of the dark phase. Animals were sacrificed the following day, one hour after the final treatment with DHF. Orexin A and B were quantified using ELISA and radioimmunoassay, respectively. Total sleep was significantly decreased in the DHF group, 4 h after drug administration in the dark phase, when compared with vehicle-treated animals. This difference was due to a significant decrease of non-rapid eye movement sleep, but not rapid eye movement sleep. DHF increased power of alpha and sigma bands but suppressed power of gamma band during sleep in dark phase. Interestingly, hypothalamic levels of orexin A were also significantly decreased in the DHF group (97 pg/mg) when compared with the vehicle-treated group (132 pg/mg). However, no significant differences of orexin B were observed between groups. Additionally, no change was found in immobility tests.

Significance of bacterial biomass in the nutrition of a freshwater isopod (Lirceus sp.).

The quantitative significance of bacterial biomass in the nutrition of detritivores remains equivocal. We have used tritiated thymidine to specifically label stable macromolecules in natural assemblages of sediment-associated and detritus-associated bacteria. This material was presented to the isopod (Lirceus sp.) and incorporation of bacterial biomass measured. The isopod incorporated roughly 1 ng bacterial carbon (mg wet wt.)-1 h-1 from leaf discs and about 6 ng mg-1 h-1 from sediment. Calculation of grazing rate from changes in cell counts yields grazing rates from 2.3-17.9 ng C mg-1 h-1. Even the maximum grazing rate, which is an overestimate of C assimilated, represents only 14.7% of C respired by the isopod.