bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.
Authors: Deshmukh M , Vasilakos J , Deckwerth T L , Lampe P A , Shivers B D , Johnson E M
Published: May 2004

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.
Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.
Authors: Didenko V V , Hornsby P J
Published: May 2004

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.
Inhibition of pp125FAK in cultured fibroblasts results in apoptosis.
Authors: Hungerford J E , Compton M T , Matter M L , Hoffstrom B G , Otey C A
Published: May 2004

The tyrosine kinase called pp125FAK is believed to play an important role in integrin-mediated signal transduction. pp125FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125FAK is not known, two possibilities have been proposed: pp125FAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the beta 1 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125FAK to endogenous integrin, thus inhibiting activation of pp125FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125FAK was microinjected, as an alternative means of inhibiting pp125FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125FAK may play a critical role in suppressing apoptosis in fibroblasts.
Lamin proteolysis facilitates nuclear events during apoptosis.
Authors: Rao L , Perez D , White E
Published: May 2004

Expression of the adenovirus E1A oncogene stimulates both cell proliferation and p53-dependent apoptosis in rodent cells. p53 implements apoptosis in all or in part through transcriptional activation of bax, the product of which promotes cell death. The adenovirus E1B 19K product is homologous in sequence and in function to Bcl-2, both of which bind to and inhibit the activity of Bax and thereby suppress apoptosis. The E1B 19K protein also interacts with the nuclear lamins, but the role of this interaction in the regulation of apoptosis is not known. Lamins are, however, substrates for members of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases that are activated during apoptosis and function downstream of Bcl-2 in the cell death pathway. lamins are degraded during E1A-induced p53-dependent apoptosis. Lamin A and C are cleaved into 47- and 37-kD fragments, respectively, and the site of proteolysis is mapped to a conserved aspartic acid residue at position 230. The cleavage of lamins during apoptosis is consistent with the activation of an ICE-related cysteine protease down-stream of p53. No lamin protease activity was detected in cells expressing the E1B 19K protein, indicating that 19K functions upstream of protease activation in inhibiting apoptosis. Substitution of the aspartic acid at the cleavage site produced a mutant lamin protein that was resistant to proteolysis both in vitro and in vivo. Expression of uncleavable mutant lamin A or B attenuated apoptosis, delaying cell death and the associated DNA fragmentation by 12 h. Mutant lamin expressing cells failed to show the signs of chromatin condensation and nuclear shrinkage typical of cell death by apoptosis. Instead, the nuclear envelope collapsed and the nuclear lamina remained intact. However, the late stage of apoptosis was morphologically unaltered and formation of apoptotic bodies was evident. Thus, lamin breakdown by proteolytic degradation facilitates the nuclear events of apoptosis perhaps by facilitating nuclear breakdown.
Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.
Authors: Austin C D , Shields D
Published: May 2004

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.
Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.
Authors: Rodgers W , Rose J K
Published: May 2004

p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.
Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration.
Authors: Wang C , Pralong W F , Schulz M F , Rougon G , Aubry J M , Pagliusi S , Robert A , Kiss J Z
Published: May 2004

The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.
Native chick laminin-4 containing the beta 2 chain (s-laminin) promotes motor axon growth.
Authors: Brandenberger R , Kammerer R A , Engel J , Chiquet M
Published: May 2004

After denervation of muscle, motor axons reinnervate original synaptic sites. A recombinant fragment of the synapse specific laminin beta 2 chain (s-laminin) was reported to inhibit motor axon growth. Consequently, a specific sequence (leucine-arginine-glutamate, LRE) of the laminin beta 2 chain was proposed to act as a stop signal and to mediate specific reinnervation at the neuromuscular junction (Porter, B.E., J. Weis, and J.R. Sanes. 1995. Neuron. 14:549-559). We demonstrate here that native chick laminin-4, which contains the beta 2 chain and is present in the synaptic basement membrane, does not inhibit but rather promotes motor axon growth. In native heterotrimeric laminin, the LRE sequence of the beta 2 chain is found in a triple coiled-coil region that is formed by all three subunits. We show here that the effect of LRE depends on the structural context. Whereas a recombinant randomly coiled LRE peptide indeed inhibited outgrowth by chick motoneurons, a small recombinant triple coiled-coil protein containing this sequence did not.
Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.
Authors: Miyamoto S , Teramoto H , Gutkind J S , Yamada K M
Published: May 2004

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.
Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene.
Authors: Alexander C M , Howard E W , Bissell M J , Werb Z
Published: May 2004

We have used transgenic mice overexpressing the human tissue inhibitor of metalloproteinases (TIMP)-1 gene under the control of the ubiquitous beta-actin promoter/enhancer to evaluate matrix metalloproteinase (MMP) function in vivo in mammary gland growth and development. By crossing the TIMP-1 transgenic animals with mice expressing an autoactivating stromelysin-1 transgene targeted to mammary epithelial cells, we obtained a range of mice with genetically engineered proteolytic levels. The alveolar epithelial cells of mice expressing autoactivating stromelysin-1 underwent unscheduled apoptosis during late pregnancy. When stromelysin-1 transgenic mice were crossed with mice overexpressing TIMP-1, apoptosis was extinguished. Entactin (nidogen) was a specific target for stromelysin-1 in the extracellular matrix. The enhanced cleavage of basement membrane entactin to above-normal levels was directly related to the apoptosis of overlying mammary epithelial cells and paralleled the extracellular MMP activity. These results provide direct evidence for cleavage of an extracellular matrix molecule by an MMP in vivo.
Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein.
Authors: Collas P , Courvalin J C , Poccia D
Published: May 2004

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.
Cell fusion during yeast mating requires high levels of a-factor mating pheromone.
Authors: Brizzio V , Gammie A E , Nijbroek G , Michaelis S , Rose M D
Published: May 2004

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.
Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation.
Authors: Chilcoat N D , Melia S M , Haddad A , Turkewitz A P
Published: May 2004

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.
The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.
Authors: Tan P K , Howard J P , Payne G S
Published: May 2004

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p. Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.
AP-2-containing clathrin coats assemble on mature lysosomes.
Authors: Traub L M , Bannykh S I , Rodel J E , Aridor M , Balch W E , Kornfeld S
Published: May 2004

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.
The initial fusion pore induced by baculovirus GP64 is large and forms quickly.
Authors: Plonsky I , Zimmerberg J
Published: May 2004

The formation of the fusion pore is the first detectable event in membrane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994. J. Cell Biol. 127:1885-1894). To date, fusion pores measured in exocytosis and viral fusion have shared features that include reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the triggering and the pore opening. We investigated baculovirus GP64-induced Sf9 cell-cell fusion, triggered by external acid solution, using two different electrophysiological techniques: double whole-cell recording (for high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave essentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was first detected by abrupt pore formation with a wide distribution of initial conductance, centered around 1 nS. Often the initial fusion pore conductance was stable for many seconds. Fluctuations in semistable conductances were much less than those of other fusion pores. The waiting time distribution, measured between pH onset and initial pore appearance, fits best to a model with many (approximately 19) independent elements. Thus, unlike previously measured fusion pores, GP64-mediated pores do not flicker, can have large, stable initial pore conductances lasting up to a minute, and have typical lag times of < 1 s. These findings are consistent with a barrel-shaped model of an initial fusion pore consisting of five to eight GP64 trimers that is lined with lipid.
Regulation of epidermal differentiation by a Distal-less homeodomain gene.
Authors: Morasso M I , Markova N G , Sargent T D
Published: May 2004

The Distal-less-related homeodomain gene Dlx3 is expressed in terminally differentiated murine epidermal cells. Ectopic expression of this gene in the basal cell layer of transgenic skin results in a severely abnormal epidermal phenotype and leads to perinatal lethality. The basal cells of affected mice ceased to proliferate, and expressed the profilaggrin and loricrin genes which are normally transcribed only in the latest stages of epidermal differentiation. All suprabasal cell types were diminished and the stratum corneum was reduced to a single layer. These data indicate that Dlx3 misexpression results in transformation of basal cells into more differentiated keratinocytes, suggesting that this homeoprotein is an important regulator of epidermal differentiation.
Treatment of melanoma cells with the synthetic retinoid CD437 induces apoptosis via activation of AP-1 in vitro, and causes growth inhibition in xenografts in vivo.
Authors: Schadendorf D , Kern M A , Artuc M , Pahl H L , Rosenbach T , Fichtner I , Nürnberg W , Stüting S , von Stebut E , Worm M , Makki A , Jurgovsky K , Kolde G , Henz B M
Published: May 2004

Human malignant melanoma is notoriously resistant to pharmacological modulation. We describe here for the first time that the synthetic retinoid CD437 has a strong dose-dependent antiproliferative effect on human melanoma cells (IC50: 5 x 10(-6) M) via the induction of programmed cell death, as judged by analysis of cell morphology, electron microscopical features, and DNA fragmentation. Programmed cell death was preceded by a strong activation of the AP-1 complex in CD437-treated cells as demonstrated by gel retardation and chloramphenicol transferase (CAT) assays. Northern blot analysis showed a time-dependent increase in the expression of c-fos and c-jun encoding components of AP-1, whereas bcl-2 and p53 mRNA levels remained constant. CD437 also exhibited a strong growth inhibitory effect on MeWo melanoma cells in a xenograft model. In tissue sections of CD437-treated MeWo tumors from these animals, apoptotic melanoma cells and c-fos overexpressing cells were colocalized by TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) staining and in situ hybridization. Taken together, this report identifies CD437 as a retinoid that activates and upregulates the transcription factor AP-1, leading eventually to programmed cell death of exposed human melanoma cells in vitro and in vivo. Further studies are needed to evaluate whether synthetic retinoids such as CD437 represent a new class of retinoids, which may open up new ways to a more effective therapy of malignant melanoma.
Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.
Authors: Adams C L , Nelson W J , Smith S J
Published: May 2004

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.
Calreticulin modulates cell adhesiveness via regulation of vinculin expression.
Authors: Opas M , Szewczenko-Pawlikowski M , Jass G K , Mesaeli N , Michalak M
Published: May 2004

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.
Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway
Authors: van Bakel Harm , Huynen Martijn , Wijmenga Cisca
Published: May 2004

Motivation: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional context as members of the same pathway in the prokaryotic domain is less clear.

Results: The co-occurrence of gene families involved in Atx1p-mediated copper transport in the genomes and operon structures of 80 non-redundant prokaryotes was investigated. For this purpose, we developed a Web tool (SHOPS) to display the operon context for a given set of proteins. In total, a set of 43 putative operons was identified. These were found to be involved in a variety of pathways and indicate a large diversity in the functional context of the individual gene family members.

Availability: The SHOPS tool can be found at http://www.bioinformatics.med.uu.nl/shops. Supplemental data are available at http://humgen.med.uu.nl/publications/vanbakel/pathway/

Supplementary information: For additional information on our datasets and for the supplemental figures referred to in the text, please see http://humgen.med.uu.nl/publications/vanbakel/pathway/. The Web-based tool, SHOPS, can be found at http://www.bioinformatics.med.uu.nl/shops/
GeneRecords: a relational database for GenBank flat file parsing and data manipulation in personal computers
Authors: D'Addabbo P. , Lenzi L. , Facchin F. , Casadei R. , Canaider S. , Vitale L. , Frabetti F. , Carinci P. , Zannotti M. , Strippoli P.
Published: May 2004

Summary: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer.

Availability: the current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/
Late Eocene and early Oligocene teleost and associated ichthyofauna of the Jebel Qatrani Formation, Fayum, Egypt
Authors: Murray Alison M.
Published: May 2004

Fishes from the late Eocene and Oligocene Jebel Qatrani Formation of the Fayum, Egypt, have been collected for many years, but have not been extensively studied. Collections from various sites in the formation, predominantly representing riverine and shallow lake deposits, include remains of several fishes not known previously. The teleost fishes from these collections [representing Characiformes, Siluriformes, Cichlidae, Latidae (= Centropomidae), and Channidae] include species that are similar to those found in the older, underlying, Qasr el Sagha Formation (catfishes), as well as species of fishes previously unrecorded from the Fayum (cichlids and latids), or even from the Tertiary of Africa (channids). It has been suggested that the Jebel Qatrani Formation represents an area of swampy rivers with overgrown banks and floating vegetation and at least one small lake. The fish remains support this reconstruction of the palaeoenvironment, and further indicate that open riverine habitat was also probably available.
Strategic approaches to osteoporosis in transplantation
Authors: Mattano Leonard A.
Published: May 2004

Abstract:  Recipients of stem cell transplantation are at risk for osteopenia and osteoporosis. Longitudinal studies performed in adults have shown that significant bone demineralization occurs following myeloablative therapy and subsequent immune suppression. Among children and adolescents, cross‐sectional analyses indicate that younger patients are also at risk for long‐term bone toxicity. Strategies to detect and manage this disorder in pediatric SCT recipients are presented.
Aboriginal perspective on middle ear disease in the arid zone of Western Australia
Authors: Jeffries‐Stokes C , Lehmann D , Johnston J , Mason A , Evans J , Elsbury D , Wood K
Published: May 2004

Objectives: To explore perceptions, knowledge and experience of otitis media (OM) and barriers to compliance with treatment among Aboriginal people of the Kalgoorlie‐Boulder area, Western Australia.

Methods: This qualitative applied research study is based on a holistic design. We conducted structured interviews with three community focus groups, 56 key informants, and 22 mothers of babies known to have suffered from OM. Written records of interviews were checked with participants. The three sources of data enabled comparison and verification of results.

Results: People were concerned about serious consequences of OM, especially deafness and learning difficulties. Since early disease may have no localizing symptoms, not surprisingly, people had limited understanding of the aetiology of OM and were often only aware of disease once ear discharge was visible. Nevertheless, they usually sought treatment for non‐specific symptoms. Competing demands in people's daily lives and the unpleasant, intensive nature of treatment result in families becoming resigned to a child's chronic ear discharge. Someone other than the biological mother within the extended family may be responsible for administering treatments. Half the carers thought passive smoking may predispose children to OM and 70% suggested clearing the nasal passages to prevent OM. Results of surgery were viewed positively but specialist services were not always readily accessible.

Conclusions: Since responsibility for treatment may not lie with the biological mother, awareness campaigns must target the entire community. As early OM may be asymptomatic, health personnel should be encouraged to do otoscopy on all children with non‐specific symptoms.
Newly developed blockers of the M‐current do not reduce spike frequency adaptation in cultured mouse sympathetic neurons
Authors: Romero M. , Reboreda A. , Sánchez E. , Lamas J. A.
Published: May 2004

The M‐current (IK(M)) is believed to modulate neuronal excitability by producing spike frequency adaptation (SFA). Inhibitors of M‐channels, such as linopirdine and 10,10‐bis(4‐pyridinylmethyl)‐9(10H)‐anthracenone (XE991), enhance depolarization‐induced transmitter release and improve learning performance in animal models. As such, they are currently being tested for their therapeutic potential for treating Alzheimer's disease. The activity of these blockers has been associated with the reduction of SFA and the depolarization of the membrane observed when IK(M) is inhibited. To test whether this is the case, the perforated patch technique was used to investigate the capacity of IK(M) inhibitors to alter the resting membrane potential and to reduce SFA in mouse superior cervical ganglion neurons in culture. Linopirdine and XE991 both proved to be potent blockers of IK(M) when the membrane potential was held at −30 mV (IC50 2.56 and 0.26 µm, respectively). However, their potency gradually declined upon membrane hyperpolarization and was almost null when the membrane potential was kept at −70 mV, indicating that their blocking activity was voltage dependent. Nevertheless, IK(M) could be inhibited at these hyperpolarized voltages by other inhibitors such as oxotremorine‐methiodide and barium. Under current‐clamp conditions, neither linopirdine (10 µm) nor XE991 (3 µm) was effective in reducing the SFA and both provoked only a small slowly developed depolarization of the membrane (2.27 and 3.0 mV, respectively). In contrast, both barium (1 mm) and oxotremorine‐methiodide (10 µm) depolarized mouse superior cervical ganglion neurons by about 10 mV and reduced the SFA. In contrast to classical IK(M) inhibitors, the activity of linopirdine and XE991 on the IK(M) is voltage dependent and, thus, these newly developed IK(M) blockers do not reduce the SFA. These results may shed light on the mode of action of these putative cognition enhancers in vivo.
Soluble Arctic amyloid β protein inhibits hippocampal long‐term potentiation in vivo
Authors: Klyubin Igor , Walsh Dominic M. , Cullen William K. , Fadeeva Julia V. , Anwyl Roger , Selkoe Dennis J. , Rowan Michael J.
Published: May 2004

Mutations in the amyloid precursor protein that result in substitutions of glutamic acid at residue 22 of the amyloid β protein (Aβ) with glutamine (Q22, Dutch) or glycine (G22, Arctic) cause aggressive familial neurological diseases characterized by cerebrovascular haemorrhages or Alzheimer's‐type dementia, respectively. The present study compared the ability of these peptides to block long‐term potentiation (LTP) of glutamatergic transmission in the hippocampus in vivo. The effects of intracerebroventricular injection of wild‐type, Q22 and G22 Aβ(1–40) peptides were examined in the CA1 area of urethane‐anaesthetized rats. Both mutant peptides were ≈ 100‐fold more potent than wild‐type Aβ at inhibiting LTP induced by high‐frequency stimulation when solutions of Aβ were freshly prepared. Fibrillar material, as determined by electron microscopy, was obvious in all these peptide solutions and exhibited appreciable Congo Red binding, particularly for Aβ(1–40)G22 and Aβ(1–40)Q22. A soluble fraction of Aβ(1–40)G22, obtained following high‐speed centrifugation, retained full activity of the peptide solution to inhibit LTP, providing strong evidence that in the case of the Arctic disease a soluble nonfibrillar form of Aβ may represent the primary mediator of Aβ‐related cognitive deficits, particularly early in the disease. In contrast, nonfibrillar soluble Aβ(1–40)Q22 supernatant solution was ≈ 10‐fold less potent at inhibiting LTP than Aβ(1–40)G22, a finding consistent with fibrillar Aβ contributing to the inhibition of LTP by the Dutch peptide.
This new science of ours: a more or less systematic history of consciousness and transcendence  Part II
Authors: Tresan David I.
Published: May 2004

Abstract:  Part I of this paper having postulated the locus of consciousness as residing in a field between primitive mind in participation mystique with the world and a transcendent ground of being, Part II takes up the nature of mind itself in this field in terms of in vivo experiences. Operations and manifestations of consciousness are inexhaustively explored in four venues: 1) Jung's life experiences, 2) Joseph Henderson's theory of psychic development, 3) Eric Voegelin's theory of consciousness and 4) the experiences of analytic patients in far reaches of very long analyses.
Mechanosensory S‐neurons rather than AH‐neurons appear to generate a rhythmic motor pattern in guinea‐pig distal colon
Authors: Spencer Nick J. , Smith Terence K.
Published: May 2004

Simultaneous intracellular recordings were made from myenteric neurons and circular muscle (CM) cells in isolated, stretched segments of guinea‐pig distal colon. We have shown previously that maintained stretch generates a repetitive and coordinated discharge of ascending excitatory and descending inhibitory neuronal reflex pathways in the distal colon. In the presence of nifedipine (1–2 μm) to paralyse the muscle, simultaneous recordings were made from 25 pairs of AH (after‐hyperpolarization)‐neurons and CM cells separated by 100–500 μm. In all 25 AH‐neurons, proximal process potentials (PPPs) were never recorded, even though at the same time, all recordings from neighbouring CM cells showed an ongoing discharge of inhibitory junction potentials (IJPs) anally, or excitatory junction potentials (EJPs) orally. In fact, 24 of 25 AH‐neurons were totally silent, while in one AH‐cell, some spontaneous fast excitatory postsynaptic potentials (FEPSPs) were recorded. All 10 electrically silent AH‐cells that were injected with neurobiotin were found to be multipolar Dogiel type II neurons. In contrast, when recordings were made from myenteric S‐neurons, two distinct electrical patterns of electrical activity were recorded. Recordings from 25 of 48 S‐neurons showed spontaneous FEPSPs, the majority of which (22 of 25) showed periods when discrete clusters of  FEPSPs (mean duration 88 ms) could be temporally correlated with the onset of EJPs or anal IJPs in the CM. Nine S‐neurons were electrically quiescent. The second distinct electrical pattern in 14 S‐neurons consisted of bursts, or prolonged trains of action potentials, which could be reduced to proximal process potentials (PPPs) in six of these 14 neurons during membrane hyperpolarization. Unlike FEPSPs, PPPs were resistant to a low Ca2+–high Mg2+ solution and did not change in amplitude during hyperpolarizing pulses. Mechanosensory  S‐neurons were found to be uniaxonal or pseudounipolar filamentous neurons, with morphologies consistent with interneurons. No slow EPSPs were ever recorded from AH‐ or S‐type neurons when IJPs or EJPs occurred in the CM. In summary, we have identified a population of mechanosensory S‐neurons in the myenteric plexus of the distal colon which appear to be largely stretch sensitive, rather than muscle‐tension sensitive, since they generate ongoing trains of action potentials in the presence of nifedipine. No evidence was found to suggest that in paralysed preparations, the repetitive firing in ascending excitatory or descending inhibitory nerve pathways was initiated by myenteric AH‐neurons, or slow synaptic transmission.
Ionized magnetron sputter deposition of hard nanocomposite TiN/amorphous-silicon nitride films
Authors: Phinichka N. , Chandra R. , Barber Z. H.
Published: May 2004

Thin films of Ti–Si–N have been deposited by ionized magnetron sputter deposition using separate Ti and Si targets in an Ar/N2 gas mixture. A built-in rf coil generated an inductively coupled plasma close to the substrate. With this setup, the ion flux and energy can be controlled by adjusting the rf power to the coil and the dc substrate bias. Deposition was carried out under various conditions to investigate the effects of different operational parameters on film structure and mechanical properties. Crystallography, grain size, and film texture have been characterized by x-ray diffraction, showing that the addition of Si to TiN films transforms the (111)-oriented structure to a preferred (200)-oriented structure. The nanocrystallite domain size of the TiN, calculated from peak broadening, was found to be less than 10 nm. No signals from crystalline Si3N4 or other phases of titanium silicide were observed. The hardness of the films, measured using nanoindentation, increased to as much as twice that of pure TiN films.