Transforming growth factor-alpha induces neurogenesis and behavioral improvement in a chronic stroke model.

Transforming growth factor-alpha (TGFalpha) is a powerful endogenous mitogen and neurotrophic factor, which has previously been shown to induce a massive proliferative response in the brains of Parkinson's disease model rats injured by an acute neurotoxic lesion. We now show that TGFalpha can also produce a massive proliferative response in rat brains subjected to stroke caused by a middle cerebral artery occlusion (MCAO), even when the growth factor is administered as late as 4 weeks after injury. This combination of stimuli provokes DNA synthesis, shown by 5'-bromo-2-deoxyuridine incorporation, throughout the ependymal layer and subventricular zone (SVZ) of the forebrain during the 4 weeks of growth factor administration. The newly generated cells migrate preferentially along and ventral to the corpus callosum (CC) and external capsule to the site of the injury where many of them differentiate into several site-appropriate neuronal phenotypes in association with near complete (99%) behavioral recovery. We conclude that the injury response of endogenous neural stem cells as well as behavioral recovery can be significantly enhanced by application of TGFalpha, and that this approach represents a potential therapeutic strategy for chronic stroke and other neurological damage in human patients.

Ependymal stem cells divide asymmetrically and transfer progeny into the subventricular zone when activated by injury.

Evidence is presented to show that cells of the ependymal layer surrounding the ventricles of the mammalian (rat) forebrain act as neural stem cells (NSCs), and that these cells can be activated to divide by a combination of injury and growth factor stimulation. Several markers of asymmetric cell division (ACD), a characteristic of true stem cells, are expressed asymmetrically in the ependymal layer but not in the underlying subventricular zone (SVZ), and when the brain is treated with a combination of local 6-hydroxydopamine (6-OHDA) with systemic delivery of transforming growth factor-alpha (TGFalpha), ependymal cells divide asymmetrically and transfer progeny into the SVZ. The SVZ cells then divide as transit amplifying cells (TACs) and their progeny enter a differentiation pathway. The stem cells in the ependymal layer may have been missed in many previous studies because they are usually quiescent and divide only in response to strong stimuli.

Lignosulfonate adsorption on and stabilization of lead zirconate titanate in aqueous suspension.

The adsorption of lignosulfonate onto a commercial, modified lead zirconate titanate (PZT-PNN) powder in aqueous suspension and its effect on particle zeta potential and suspension rheology were investigated as functions of pH and lignosulfonate dosage. Langmuir analysis of the adsorption data demonstrated that a significant component of the overall driving force of adsorption at all pH values examined was specific (nonelectrostatic) bonding. Electrostatic bonding provided a significant contribution to adsorption at pH 6.0, but diminished at lower pH owing to decreased lignosulfonate ionization and at higher pH due to decreased positive surface site concentration on the PZT-PNN. The affinity of adsorption was highest at pH 6.0 because the electrostatic component was maximal at this pH. The zeta potential magnitude increased and the apparent viscosity decreased with increasing pH and increasing lignosulfonate dosage, up to approximately monolayer coverage. The lignosulfonate dosage required for monolayer coverage decreased with increasing pH owing to increasing lignosulfonate expansion and the decrease in concentration of positive surface sites on the PZT-PNN. Suspension stabilization was considered to occur by an electrosteric mechanism.

Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer.

INTRODUCTION: ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation. AIM: To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP). METHODOLOGY AND RESULTS: Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining. CONCLUSION: These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Growth factors controlling imaginal disc growth in Drosophila.

In the imaginal discs of Drosophila, contact-dependent cell interactions are important both for promoting cell proliferation and for limiting it at the end of the growth period. However, recent work indicates that diffusible growth factors are also important in regulating growth and proliferation. We have identified a family of five imaginal disc growth factors (IDGFs) by purifying mitogenic proteins that accumulate in conditioned medium during culture of imaginal disc cell lines. These proteins cooperate with insulin to stimulate not only proliferation, but also polarization and motility of imaginal disc cells. They are produced by the fat body and are probably active on a variety of peripheral tissues. The IDGFs are structurally related to chitinases, but they show an amino acid substitution that is known to abrogate catalytic activity and to transform chitinases into lectins. We suggest that these proteins act as endogenous mitogenic lectins and mediate nutritional effects on tissue growth, possibly by interacting with the insulin receptor pathway. Glycoproteins similar to the IDGFs are found in mammals and may constitute a novel class of growth factors and/or inflammatory cytokines.

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

Current role of beta-adrenergic blockers in the treatment of chronic congestive heart failure.

Recent findings on the use of beta-adrenergic blockers in patients with congestive heart failure (CHF) are reviewed. CHF is a progressive, debilitating disease that afflicts 4.6 million patients in the United States. Treatment has traditionally consisted of a diuretic, an angiotensin-converting-enzyme (ACE) inhibitor, and digoxin. Despite advances in ACE-inhibitor therapy, the five-year mortality rate remains nearly 50%. Overstimulation of the sympathetic nervous system is believed to contribute to mortality. Beta-blockers have recently been added to the standard of care for patients with New York Heart Association functional class II or III heart failure. Four randomized, double-blind, placebo-controlled clinical trials were recently completed that addressed the benefits of beta-blockers in CHF. The overall mortality rate was reduced 65% by carvedilol, 34% by metoprolol, and 33% by bisoprolol; all these reductions were significant compared with placebo, and the trials were ended early. Bucindolol, however, did not have a significant effect on mortality. These drugs are hepatically metabolized and may require dosage adjustment in hepatically impaired patients. Decompensation of heart failure is another consideration; a beta-blocker should be added only for patients with stable CHF. Dosages must be slowly adjusted to targeted levels. Adverse effects do not differ significantly among beta-blockers. In addition to their effect on mortality, beta-blockers reduce CHF-related morbidity, such as all-cause hospitalization. Carvedilol, metoprolol, and bisoprolol decrease the mortality and morbidity associated with CHF and can be used with limited adverse effects. The choice among these agents does not affect clinical outcomes; bucindolol, however, has proven ineffective.

Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster.

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5-8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.

LAP proteins: what's up with epithelia?

How cells maintain their overall shape and size, and the related question of how proteins and other molecules reach and stay at their specific subcellular locations, are among the most difficult and exciting problems in cell biology. Three recent studies have made a significant contribution to this area by identifying new proteins, called LAP proteins, that have critical functions in maintaining the shape and apical-basal polarity of epithelial cells.

The genetics of the protein 4.1 family: organizers of the membrane and cytoskeleton.

Protein 4.1 (also called band 4.1 or simply 4.1) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. The protein and its relatives have since been found in many cell types of metazoan organisms and they are often concentrated in the nucleus, as well as in cell-cell junctions. They form multimolecular complexes with transmembrane and membrane-associated proteins, and these complexes may be important for both structural stability and signal transduction at sites of cell contact.

Genomic organization of human DLG4, the gene encoding postsynaptic density 95.

We have determined the exon-intron organization and characterized the 5'-flanking promoter region of DLG4. Encompassing approximately 30 kb, the DLG4 locus is composed of 22 exons that range in size from 28 to 1,218 nucleotides. All splice sites conform to the GT-AG rule, except for the splice acceptor site of intron 5, which is TG instead of AG. Three different exons of DLG4 were found to be alternatively spliced in a subset of tissues. Two of these variants result in altered postsynaptic density 95 (PSD95) isoforms that dramatically truncate the protein. The third splicing variant represents an extension of exon 4 that encodes an additional 33-amino acid segment. Analysis of the core promoter region for DLG4 suggests that the expression of this gene is controlled by a TATA-less promoter using a single transcriptional start site embedded within a CpG island. DLG4 maps to a region on chromosome 17p13.1 known to contain a locus for autosomal dominant cone dystrophy 5. Scanning for mutations in the DLG4 coding region and splice sites was performed in 15 cone dystrophy patients, including probands from five families showing linkage to the DLG4 region. No disease-causing mutations were identified in any patients, suggesting that DLG4 is not the causative gene for this genetic eye disorder.

double-time is identical to discs overgrown, which is required for cell survival, proliferation and growth arrest in Drosophila imaginal discs.

We have isolated the discs overgrown gene of Drosophila and shown that it encodes a homolog of the Casein kinase I(delta)/(epsilon) subfamily and is identical to the double-time gene. However, in contrast to the weak double-time alleles, which appear to affect only the circadian rhythm, discs overgrown alleles, including bona fide null alleles, show strong effects on cell survival and growth control in imaginal discs. Analysis of their phenotypes and molecular lesions suggests that the Discs overgrown protein is a crucial component in the mechanism that links cell survival during proliferation to growth arrest in imaginal discs. This work provides the first analysis in a multicellular organism of Casein kinase I(delta)/(epsilon) functions necessary for survival. Since the amino acid sequences and three-dimensional structures of Casein kinase I(delta)/(epsilon) enzymes are highly conserved, the results suggest that these proteins may also function in controlling cell growth and survival in other organisms.

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.

Filopodia: fickle fingers of cell fate?

Epithelial cells often produce extensions, known variously as filopodia, cell feet or cytonemes, which can extend across many cell diameters to directly contact non-adjacent cells. Do they function in morphogenesis, cell-cell signaling or both?.

Progress of the Proton-Ion Medical Machine Study (PIMMS).

The Proton-Ion Medical Machine Study (PIMMS) was set up following an agreement between Professor M. Regler of the Med-AUSTRON (Austria) and Professor U. Amaldi of the TERA Foundation (Italy) to join their efforts in the design of a medical synchrotron that could later be adapted to individual national needs. CERN agreed to host this study inside its PS Division and to contribute one full-time member to the study team. The study group has worked in collaboration with GSI (Germany) and was more recently joined by Onkologie 2000 (Czech Republic). Work started in January 1996 and is expected to finish during 1998. The agreed aim of the study was to investigate and design a generic facility that would allow the direct clinical comparison of protons and carbon ions for cancer treatment. The accelerator was to be designed primarily for high-precision active beam scanning with both protons and ions, but was also to be capable of delivering proton beams with passive spreading.

A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells.

By fractionating conditioned medium (CM) from Drosophila imaginal disc cell cultures, we have identified a family of Imaginal Disc Growth Factors (IDGFs), which are the first polypeptide growth factors to be reported from invertebrates. The active fraction from CM, as well as recombinant IDGFs, cooperate with insulin to stimulate the proliferation, polarization and motility of imaginal disc cells. The IDGF family in Drosophila includes at least five members, three of which are encoded by three genes in a tight cluster. The proteins are structurally related to chitinases, but they show an amino acid substitution that is known to abrogate catalytic activity. It therefore seems likely that they have evolved from chitinases but acquired a new growth-promoting function. The IDGF genes are expressed most strongly in the embryonic yolk cells and in the fat body of the embryo and larva. The predicted molecular structure, expression patterns, and mitogenic activity of these proteins suggest that they are secreted and transported to target tissues via the hemolymph. However, the genes are also expressed in embryonic epithelia in association with invagination movements, so the proteins may have local as well as systemic functions. Similar proteins are found in mammals and may constitute a novel class of growth factors.

What is Drosophila telling us about cancer?

In Drosophila, genetic loss of the tumour suppressor protein Dlg (in dlg mutants) or p127 (in lgl mutants) leads to loss of epithelial structure and excess proliferation in the imaginal discs and brain of the developing larva. These phenotypes show most of the characteristic features of human neoplasia, so study of the gene products may contribute to our understanding of cancer. Both proteins occur in high molecular-mass complexes in the membrane-associated cytoskeleton, and they both appear to play dual roles as structural proteins and active partners in signal transduction. Dlg is a membrane-associated guanylate kinase homolog (MAGUK) found at septate junctions between epithelial cells, as well as at neuromuscular junctions. Specific domains of the protein are required for membrane targeting and for localisation injunctions, and for epithelial cell proliferation control; all of these functions are probably mediated through binding to other proteins. Loss of Dlg results in the absence of septate junctions, delocalisation of several proteins including Fasciclin III, Coracle, actin and tubulin, and loss of cell polarity. p127, although mostly associated with the plasma membrane, is in most cell types also present in the cytoplasm. It shows a dynamic subcellular distribution, and its cytosolic and membrane-associated forms play distinctive roles by interacting with different binding partners, in particular the non-muscle myosin II heavy chain. Defects associated with the lgl temperature-sensitive allele include loss of the columnar organisation of epithelial cells, indicating that p127 contributes to cell structure, presumably by stabilising the plasma membrane. In addition to their organising functions, both Dlg and p127 appear to be involved in signal transduction pathways. Study of these genes shows that some proteins play both structural and functional roles, and that cancer can involve changes in the organisation of signalling pathways in addition to changes in individual pathway components.

Loss of the tight junction MAGUK ZO-1 in breast cancer: relationship to glandular differentiation and loss of heterozygosity.

Membrane-associated guanylate kinase homologs (MAGUKs) may play a role in cellular functions preventing tumorigenesis as indicated by the neoplastic phenotype caused by genetic loss of the MAGUK Dlg in Drosophila. To test this possibility, we examined the expression and subcellular localization of the tight junction MAGUK ZO-1, as well as the cell adhesion molecule E-cadherin, in paraffin-embedded breast cancer samples, using immunohistochemistry and confocal microscopy. As expected, normal tissue showed intense staining for ZO-1 at the position of the epithelial tight junctions, but this staining was reduced or lost in 69% of breast cancers analyzed (n = 48). In infiltrating ductal carcinomas (n = 38) there was a reduction in staining in 42% of well differentiated, in 83% of moderately differentiated and 93% of poorly differentiated tumors. ZO-1 staining was positively correlated with tumor differentiation (P = .011) and more specifically with the glandular differentiation of tumors (P = .0019). Reduction in ZO-1 staining was strongly correlated with reduced E-cadherin staining (P = 4.9 x 10(-5)). The results suggest that down-regulation of ZO-1 expression and its failure to accumulate at cell junctions may be causally related to cancer progression. To detect loss of heterozygosity, the ZO-1 gene tjp-1 was mapped relative to other markers in 15q13 and polymorphic markers flanking tjp-1 were identified. The marker D15S1019 showed loss of heterozygosity in 23% of informative tumors (n = 13). Loss of a tjp-1-linked marker suggests that genetic loss may, in some cases, be responsible for the reduction in ZO-1 expression in breast cancer.