Osteopontin (OPN) is a phosphorylated glycoprotein that binds to a v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

It is well established that inflammation and oxidative stress are key components of the pathology of Alzheimer's disease (AD), but how early in the pathological cascade these processes are involved or which specific molecular components are key, has not been fully elucidated. This paper describes the pharmacological approach to understand the molecular components of inflammation and oxidative stress on the activation of microglial cells and neuronal cell viability. We have shown that activation of microglia with the 42-amino-acid form of the ϐ-amyloid peptide (Aϐ42) activates the production of cyclooxygenase-2, the inducible form of nitric oxide synthase and tumour necrosis factor-α and there appears to be little interactive feedback between these three mediators. Moreover, we explore the effects of a series of salen-manganese complexes, EUK-8, -134 and -189, which are known to possess both superoxide and catalase activity. These compounds are able to protect cells from insults produced by hydrogen peroxide or peroxynitrite. Moreover, EUK-134 was also able to limit the output of prostaglandin E2 from activated microglial cells. The mechanisms underlying these effects are discussed. Together, these data support a pivotal role for oxidative stress and inflammation as key mediators of the pathological cascade in AD and provide some ideas about possible therapeutic targets.

The insect enzyme ecdysteroid phosphate phosphatase (EPP) mobilizes active ecdysteroids from an inactive phosphorylated pool. Previously assigned to a novel class, it is shown here that it resides in the large histidine phosphatase superfamily related to cofactor-dependent phosphoglycerate mutase, a superfamily housing notably diverse catalytic activities. Molecular modeling reveals a plausible substrate-binding mode for EPP. Analysis of genomic and transcript data for a number of insect species shows that EPP may exist in both the single domain form previously characterized and in a longer, multidomain form. This latter form bears a quite unexpected relationship in sequence and domain architecture to vertebrate proteins, including Sts-1, characterized as a key regulator of T-cell activity. Long form Drosophila melanogaster EPP, human Sts-1, and a related protein from Caenorhabditis elegans have all been cloned, assayed, and shown to catalyse the hydrolysis of ecdysteroid and steroid phosphates. The surprising relationship described and explored here between EPP and Sts-1 has implications for our understanding of the function(s) of both.

Muscarhic acetylcholine receptors (mAChRs) are involved in synaptic plasticity. In a screen designed to identify genes involved in mAChR signaling. w e found I 1 genes that were readily activated by mAChR These included the transcription factors Egr-I. Egr-2, Egr-3, c-jun, jun-D, and GOS3, the growth regulator hCyr61, the signaling factors NGFi-B, ETRIOl, and Gig-2 as well as the acetylcholine cstcrasc gene (AChE). hCyr61 encodes a secretory protein with groarrh-promoting functions mediated by interactions with inregrins and heparin-containing components of the extracellular matrix. Gig-2 is a novel gene with similarities to serine-threonine kinases, and with potential targeting to the secretory pathway. Both mAChR over-expressing 293 lines and primary cortical neurons responded to receptor stimulation with increased expression of both genes within 15 minutes, attained a maximum after 1 hour with sustained high expression for 4 hours. Receptor stimulation also triggered gene expression in the presence of cycloheximide, indicating that hCyr61 and gig-2 arc immediate urly genes. In vivo experiments with pilocarpine strongly induced gig-2 expression in neurons of several layers of the brain cortex, the hippoumpal CAI region, and the puumcn. Increased expression of both hCyr61 and gig-2 was coupled to mAChRs by PKC. whereas CAMP failed to affect expression. We also observed that mAChR rudily increased both Egr-l transcription and nuclear concentrations of Egr-l protein; with subsequent strong activation of transcription from the human AChE promoter, and expression of the AChE gene.Our data show that multiple immediate-urly genes arc under the control of mAChRs, and they suggest that both hCyr61 and gig-2 play important roles in coupling receptor stimulation to long-term neuronal responses. The results also suggest a feedback mechanism by which increases in cholinergic transmission are limited by upregulated AChE expression, and accelerated breakdown of ACh at the cholincrgic synapses.Evidence that aberrations in oxidative signalling and glial inflammatory pathways are features of the pathology of Alzheimer's disease (AD) remains undisputed. Data generated over the past 15 yCars have consistently reported the presence of a variety of inflammatory proteins in the brain of A D patients taken post-mortem (see McGccr & McGecr, 1995). There is also considerable evidence for damage caused by free radicals that arc produced by alterations in the homeostatic mechanisms controlling oxygen utilisation (see Smith et al, 1999). However. one of the key hurdles for research continues to be able to address whether oxidative stress andlor inflammation are early aetiological factors that contribute to the devclopment of the progressive pathology of the disease and thus provide specific targets for drug intervention. This talk will describe the utility of pharmacological tools to probe mechanisms of oxidative stress and inflammation, both in vitro and in vivo. The data to be presented support the premise that specific mediators of infl...