Associations between hepatocyte growth factor, c-Met, and basic fibroblast growth factor and survival in endometrial cancer patients.

BACKGROUND: Hepatocyte growth factor (HGF), c-Met, and basic fibroblast growth factor (bFGF) are molecular markers that contribute to angiogenesis and proliferation in numerous cancers. We assessed the prognostic significance of these factors in tumour and stroma of endometrial cancer (EC) patients (n=211). METHODS: Immunohistochemistry (IHC) was used to detect tumour and stromal protein expression of the biomarkers. Associations between expression and clinicopathological factors were assessed using Chi-square tests. Kaplan-Meier curves, log-rank tests, and Cox regression were used to summarise associations between biomarker expression and overall survival (OS) and recurrence-free survival (RFS). RESULTS: Tumour bFGF was significantly associated with high-grade endometrioid and clear cell histology (P<0.001), advanced stage (P=0.008), positive lymph-node involvement (P=0.002), poor OS (log-rank test, P=0.009), and poor RFS (P<0.001). In multivariable analyses, cases with HGF-positive, stromal bFGF-positive tumours had a lower risk of death compared with cases with HGF-positive, stromal bFGF-negative tumours (hazard ratio (HR): 0.14, 95% CI: 0.03, 0.60). Cases with HGF-positive, bFGF-positive tumours had a higher risk of recurrence compared with cases with negative expression of both markers (HR: 9.88, 95% CI: 2.63, 37.16). CONCLUSION: These IHC data show that tumour and stromal bFGF expression have opposite associations with survival outcomes in EC patients. If confirmed in larger studies, tumour-derived bFGF could be an attractive target in EC therapy.

Global alterations to the choroid plexus blood-CSF barrier in amyotrophic lateral sclerosis.

The choroid plexus (CP) is a highly vascularized structure located in the ventricles that forms the blood-CSF barrier (BCSFB) and separates the blood from the cerebrospinal fluid (CSF). In addition to its role as a physical barrier, the CP functions in CSF secretion, transport of nutrients into the central nervous system (CNS) and a gated point of entry of circulating immune cells into the CNS. Aging and neurodegeneration have been reported to affect CP morphology and function and increase protein leakage from blood to the CSF. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with both upper and lower motor neuron loss, as well as altered proteomic and metabolomic signatures in the CSF. The role of the BCSFB and the CP in ALS is unknown. Here we describe a transcriptomic and ultrastructural analysis of BCSFB and CP alterations in human postmortem tissues from ALS and non-neurologic disease controls. ALS-CP exhibited widespread disruptions in tight junctional components of the CP epithelial layer and vascular integrity. In addition, we detected loss of pericytes around ALS blood vessels, accompanied by activation of platelet aggregation markers vWF and Fibrinogen, reminiscent of vascular injury. To investigate the immune component of ALS-CP, we conducted a comprehensive analysis of cytokines and chemokine panels in CP lysates and found a significant down-regulation of M-CSF and V-CAM1 in ALS, as well as up-regulation of VEGF-A protein. This phenotype was accompanied by an infiltration of MERTK positive macrophages into the parenchyma of the ALS-CP when compared to controls. Taken together, we demonstrate widespread structural and functional disruptions of the BCSFB in human ALS increasing our understanding of the disease pathology and identifying potential new targets for ALS therapeutic development.

Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle.

SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast. Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane. The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl. Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane. Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p. However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes. Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable. During the course of these studies we have found that two activities associated with the yeast Golgi apparatus, Kex2 endopeptidase and GDPase, are in separable subcompartments.

Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis.

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.

The role of Myo2, a yeast class V myosin, in vesicular transport.

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

The septin CDCrel-1 binds syntaxin and inhibits exocytosis.

Septins are GTPases required for the completion of cytokinesis in diverse organisms, yet their roles in cytokinesis or other cellular processes remain unknown. Here we describe studies of a newly identified septin, CDCrel-1, which is predominantly expressed in the nervous system. This protein was associated with membrane fractions, and a significant fraction of the protein copurified and coprecipitated with synaptic vesicles. In detergent extracts, CDCrel-1 and another septin, Nedd5, immunoprecipitated with the SNARE protein syntaxin by directly binding to syntaxin via the SNARE interaction domain. Transfection of HIT-T15 cells with wild-type CDCrel-1 inhibited secretion, whereas GTPase dominant-negative mutants enhanced secretion. These data suggest that septins may regulate vesicle dynamics through interactions with syntaxin.

Identification and analysis of the complete cDNA sequence for the human FAC1 gene.

The complete cDNA sequence for the FAC1 gene has been isolated from human fetal brain. The FAC1 cDNA encodes for a protein of 810 amino acids that contains a putative Zn(2+)-finger binding domain, nuclear transport signals and a charged acidic domain that may function in protein-protein interactions. Identification of the complete cDNA and protein sequence of FAC1 permits further insight into potential functions for this developmentally regulated gene product.

Immunolocalization and redistribution of the FAC1 protein in Alzheimer's disease.

The presence of senile plaques and neurofibrillary tangles are hallmark neuropathologic features of Alzheimer's disease (AD). Many proteins have previously been immunolocalized to amyloid-containing plaques in AD brain. Using a monoclonal antibody to a recently described developmentally regulated gene product, we demonstrate the presence of FAC1 protein in a subset of diffuse and neuritic plaques in AD brain. FAC1 is not observed in neurofibrillary tangles common in the hippocampus or entorhinal cortex, nor is it localized in diffuse plaques of nondemented elderly control subjects. FAC1 protein is also immunolocalized in swollen dendrites of hippocampal pyramidal cells observed in some cases of early stage AD. Therefore, FAC1 is a novel protein localized in early pathologic features of AD and in a subset of plaques.

A confocal microscopic analysis of galaninergic hyperinnervation of cholinergic basal forebrain neurons in Alzheimer's disease.

The galanin (GAL) containing peptide fiber system innervates the basal forebrain and has been shown to hyperinnervate remaining cholinergic neurons in Alzheimer's disease (AD). GAL modulates the release of acetylcholine and, therefore, may depress this neurotransmitter in surviving cholinergic basal forebrain (CBF) neurons in AD. The aim of this study was to identify putative synaptic contacts between GAL immunoreactive processes and CBF neurons and evaluate whether these processes hypertrophy in AD patients. We observed by confocal laser microscopy a hyperinnervation of GAL-containing fibers in both AD and Parkinson's disease patients with concurrent AD (PD/AD). Galaninergic fibers were often seen in direct apposition to remaining CBF neurons and enwrapped cholinergic cell soma and dendrites. Our results demonstrate that GAL-containing fibers are in direct apposition to CBF neurons in normal-aged humans and that this phenotype is enhanced in AD and PD/AD, suggesting that direct synaptic contacts occur between GAL-containing fibers and CBF neurons. Because GAL can modulate acetylcholine release from cholinergic neurons, hyperinnervation of GAL fibers in AD and PD/AD patients may further decrease release of acetylcholine from remaining CBF neurons. We propose that therapies based solely on acetylcholinesterase inhibitors may be insufficient to effectively increase cortical levels of acetylcholine.

Expression of fetal ALZ-50 reactive clone 1 (FAC1) in dentate gyrus following entorhinal cortex lesion.

The Fetal ALZ-50 Reactive Clone 1 (FAC1) gene is expressed at high levels during brain development and is re-expressed in some neurodegenerative diseases. It is hypothesized that FAC1 functions during neuronal differentiation and may play an active role in neuritic re-organization following brain injury. We have previously employed the entorhinal cortex lesion model to examine reactive synaptogenesis and plasticity in the hippocampal dentate molecular layer following denervating lesion. We now report re-expression of FAC1 in the molecular layer (ML) of the dentate gyrus following entorhinal cortex (ERC) lesion. Denervated hippocampi (2,6,15, and 30 days post ERC lesion) were stained with anti-FAC1 antibody and processed for both light and electron microscopy. FAC1 was rapidly re-expressed (by 2 days) following ERC lesion, paralleling our previous observations with embryonic neural cell adhesion molecule (eN-CAM). Like eN-CAM, FAC1 expression was restricted to the denervated outer ML (OML) at 2 days post lesion. Analysis of later time points revealed an elimination of FAC1 immunostaining at the inner ML (IML)/(OML) interface as IML sprouts into the denervated zone. Image analysis confirmed the diminution of FAC1 staining in the OML as the IML sprouted into the denervated zone and revealed that FAC1 expression paralleled the temporal and spatial expression of eN-CAM following ERC lesion. Ultrastructural analysis of FAC1 staining at 6 and 30 days post lesion revealed immunoreactive profiles with the morphological characteristics of dendrites and cytoplasmic staining of granule cell perikarya. Dendritic staining was localized to the denervated OML and was not associated with any other neuropil profiles within this zone; IML staining was rare and restricted to large apical dendrites proximal to granule cell perikarya. These findings suggest that re-expression of FAC1 in the denervated OML is a rapid response to brain injury and may be important in synaptic plasticity and sprouting.

Altered subcellular distribution of transcriptional regulators in response to Abeta peptide and during Alzheimer's disease.

Recent studies have shown that various cell cycle proteins are expressed in post-mitotic neurons within affected brain regions during Alzheimer's disease (AD). Cell cycle proteins have been proposed to function in mechanisms of neuronal cell death during AD. To further explore the role of cell cycle proteins in neurodegeneration associated with AD, we utilized PC12 cells to examine the subcellular distribution of cell cycle transcriptional regulators, including the retinoblastoma gene product (pRb), E2F1 and FAC1, during beta-amyloid (Abeta)-induced neurodegeneration. Moreover, we examined the immunolocalization of pRb and E2F1 in non-demented control and AD brain tissue. We found that pRb exhibited increased levels of Ser795 phosphorylation in response to Abeta in the nucleus of PC12 cells and also in the nucleus of a subset of neurons during AD. E2F1 was distributed throughout the cytoplasm and neurites of PC12 cells in response to Abeta and in the cytoplasm of cells in AD brain. FAC1 exhibited a rapid redistribution from the cytoplasm to the perinuclear region in PC12 cells treated with Abeta. These data indicate that altered phosphorylation and subcellular distribution of transcriptional regulators occur in response to Abeta-induced neurotoxicity and during AD.

Alzheimer's disease and brain development: common molecular pathways.

Research on the causes and treatments of Alzheimer's disease (AD) has led investigators down numerous avenues. Although many models have been proposed, no single model of AD satisfactorily accounts for all neuropathologic findings as well as the requirement of aging for disease onset. The mechanisms of disease progression are equally unclear. We hypothesize that alternative gene expression during AD plays a critical role in disease progression. Numerous developmentally regulated genes and cell cycle proteins have been shown to be re-expressed or activated during AD. These proteins include transcription factors, members of the cell cycle regulatory machinery, and programmed cell death genes. Such proteins play an important role during brain development and would likely exert powerful effects if re-expressed in the adult brain. We propose that the re-expression or activation of developmentally regulated genes define molecular mechanisms active both during brain development and in AD.

SEPT5_v2 is a parkin-binding protein.

Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.

Galanin expression within the basal forebrain in Alzheimer's disease. Comments on therapeutic potential.

The inhibitory neuropeptide galanin has widespread distribution throughout the central nervous system. Studies indicate that galanin modulates cognition by regulating cholinergic basal forebrain (CBF) neuron function. The chemoanatomic organization of galanin within the mammalian CBF differs across species. In monkeys, all CBF neurons coexpress galanin, whereas in apes and humans galanin is found within a separate population of interneurons that are in close apposition to the CBF perikarya. Pharmacologic investigations revealed a low and high affinity galanin receptor within the basal forebrain in humans. In vitro autoradiographic investigations of the primate brain indicate that galanin receptors are concentrated within the anterior subfields of the CBF as well as bed nucleus of the stria terminalis, amygdala, and entorhinal cortex. Galaninergic fibers hyperinnervate remaining CBF neurons in Alzheimer's disease. Because galanin inhibits the release of acetylcholine in the hippocampus, it has been suggested that the overexpression of galanin in Alzheimer's disease may downregulate the production of acetylcholine within CBF perikarya, further exacerbating cholinergic cellular dysfunction in this disorder. These observations suggest that the development of a potent galanin antagonist would be a useful step towards the successful pharmacologic treatment of Alzheimer's disease.

Localization of a novel septin protein, hCDCrel-1, in neurons of human brain.

Synaptic function is critical for cell-cell communication and the characterization of proteins that function during vesicle formation, transport and fusion events will yield further insight into the mechanisms of synaptic transmission. We have cloned and characterized a gene product expressed in human brain called hCDCrel-1. This protein is a new member of the septin family of gene products that functions during cytokinesis in lower eukaryotes. In this study we characterize the expression of the hCDCrel-1 gene and localize the hCDCrel-1 protein to neurons in adult human brain. hCDCrel-1 co-purifies with SNAP-25 and synaptophysin marked synaptosomes, suggesting a novel function for this gene family in the brain. Our data indicate that members of the septin family of proteins may function in synaptic vesicle transport, fusion or recycling events in the human brain.

Binding of Alz 50 depends on Phe8 in tau synthetic peptides and varies between native and denatured tau proteins.

Alz 50 is a monoclonal antibody that in Western blotting analysis recognizes both normal tau as well as hyperphosphorylated tau proteins associated with paired helical filaments (PHF-tau) in Alzheimer disease (AD). Within tissue sections of AD brain, however, Alz 50 immunolabels only PHF, which suggests that the antibody recognizes a conformational epitope. Using competitive enzyme-linked immunosorbent assay, we demonstrate that Alz 50 binds to tau synthetic peptides with low affinity (KD between 0.27 to 2.7 x 10(-5) M) and that the binding is specific for the RQEF sequence corresponding to N-terminal residues 5-8 of tau. The Alz 50 epitope appears to be largely dependent on Phe8, a strongly hydrophobic amino acid residue, since the substitution of Phe8 with Ala8 in the synthetic peptide abolishes Alz 50 binding. The effects of tau conformation on Alz 50 binding were studied with various normal tau proteins with either low or high phosphate content (adult vs. fetal) and PHF-tau proteins. The normal tau fractions were isolated from both adult and fetal human brains using affinity chromatography (native form) and heat/perchloric acid treatments (denatured form). PHF-tau was isolated as Sarcosyl-insoluble fraction. With competitive ELISA, the denatured form of normal tau (fetal and adult) bound Alz 50 with the same high affinity as did PHF-tau (KD between 1.3 to 1.8 x 10(-7) M). In contrast, the native form of tau from either brain was unable to fully compete for Alz 50 and at most only 50% of the Alz 50 binding sites in native tau were occupied. These results suggest that native tau may exist either in complexes with other proteins or in a form of dimers/oligomers, in which only some N-termini are available for binding (e.g. head-to-tail assembly). The results also suggest that denaturation rather than phosphorylation of tau has more significant effect on interactions of tau with Alz 50.

Expression of FAC1 in activated microglia during Alzheimer's disease.

The presence of reactive microglia and astrocytes is a common observation in Alzheimer's disease brain. Microglia are present within the numerous beta-amyloid containing neuritic plaques, whereas reactive astrocytes usually surround the plaque perimeter. These glial cells express and secrete numerous neurotrophic and neurotoxic factors that contribute to the etiology of the disease. The molecular mechanisms that dictate glial cell activation and subsequent alternative gene expression are currently unknown. In the present study we determine that activated microglia in AD brain express the FAC1 protein, a developmentally regulated gene product, while astrocytes fail to express significant levels of FAC1 protein. Since FAC1 is a putative DNA binding protein, expression in microglia during AD suggests that FAC1 participates in the regulation of alternative gene expression.

Ironic effects of sleep urgency.

Normal sleepers were instructed either to fall asleep as quickly as they could or to fall asleep whenever they desired, under a high mental load (listening to John Philip Sousa marches) or a low mental load (listening to sleep-conducive new age music). Under low load, participants trying to fall asleep quickly did so faster than those attempting only to fall asleep whenever they desired. Under high load, however, and consistent with the ironic process theory of mental control (Wegner, D. M., 1994, Psychological Review, 101, 34-52), sleep onset latency was greater for participants attempting to fall asleep quickly than for those not attempting to do so.

Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: early introduction of lysosomal enzymes into the endocytic pathway.

The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3-10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R. F., J. Cell. Physiol., 131:200-209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20 degrees C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.