High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo-uterine interactions during the implantation process.

A number of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy are characterized by the formation and intraneuronal accumulation of fibrillar aggregates of alpha-synuclein (alpha-syn) protein in affected brain regions. These and other findings suggest that the accumulation of alpha-syn in the brain plays an important role in the pathogenesis of these diseases. However, more recently it has been reported that early amyloid aggregates or 'soluble oligomers' are the pathogenic species that lead to neurodegeneration and neuronal cell death rather than the later 'mature fibrils'. In this study, we investigated the presence of alpha-syn oligomers in brain lysates prepared from frozen post-mortem brains of normal, Alzheimer's disease and DLB patients. The brain extracts were subjected to high speed centrifugation, to remove insoluble alpha-syn aggregates, followed by specific detection of soluble oligomers in the supernatants by employing FILA-1, an antibody that specifically binds to alpha-syn aggregates, but not to alpha-syn monomers, or to tau or beta-amyloid aggregates. Using this novel enzyme-linked immunosorbent assay (ELISA) method to quantify the amounts of alpha-syn oligomers in the brain extracts, our data clearly show an increase in the levels of soluble oligomers of alpha-syn in the DLB brains compared to those with Alzheimer's disease and the controls (P < 0.0001). Our findings provide strong evidence to support the contention that elevated soluble oligomers of alpha-syn are involved in the pathogenesis of DLB. Furthermore, these findings establish FILA-1 as a very sensitive tool for the detection of oligomeric forms of alpha-syn in human brain lysates.

This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration.

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