Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg. mL-1), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.     

Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of > or =100 nM for 18-44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.     

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer–dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.     

Galectins-3 and -7, but not galectin-1, play a role in re-epithelialization of wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study, using cornea as a model, we demonstrate for the first time the importance of carbohydrate-binding proteins galectins-3 and -7 in re-epithelialization of wounds. In two different models of corneal wound healing, re-epithelialization of wounds was significantly slower in galectin-3-deficient (gal3(-/-)) mice compared with wild-type (gal3(+/+)) mice. In contrast, there was no difference in corneal epithelial wound closure rates between galectin-1-deficient and wild-type mice. Quantitation of the bromodeoxyuridine-labeled cells in gal3(+/+) and gal3(-/-) corneas revealed that corneal epithelial cell proliferation rate is not perturbed in gal3(-/-) corneas. Exogenous galectin-3 accelerated re-epithelialization of wounds in gal3(+/+) mice but, surprisingly, not in the gal3(-/-) mice. Gene expression analysis using cDNA microarrays revealed that healing corneas of gal3(-/-) mice contain markedly reduced levels of galectin-7 compared with those of gal3(+/+) mice. More importantly, unlike galectin-3, galectin-7 accelerated re-epithelialization of wounds in both gal3(-/-) and gal3(+/+) mice. In corresponding experiments, recombinant galectin-1 did not stimulate the corneal epithelial wound closure rate. The extent of acceleration of re-epithelialization of wounds with both galectin-3 and galectin-7 was greater than that observed in most of the published studies using growth factors. These findings have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.     

Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1

To identify critical amino acid residues for carbohydrate binding of galectins (soluble -galactoside-binding lectins found in the animal kingdom), site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992)J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.Abbreviations EDTA-PBS 2mm EDTA, 20mm Na-phosphate, pH 7.2, 150mm NaCl - MEPBS EDTA-PBS containing 4mm -mercaptoethanol - IPTG isopropyl--(d)-thiogalactoside - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Diabetes is not a potent inducer of neuronal cell death in mouse sensory ganglia,but it enhances neurite regeneration in vitro

We examined the effects of diabetes on the morphological features and regenerative capabilities of adult mouse nodose ganglia (NG) and dorsal root ganglia (DRG). By light and electron microscopy, no apoptotic cell death was detected in the ganglia obtained from either streptozotocin (STZ)-induced diabetic or normal C57BL/6J mice in vivo. Neurite regeneration from transected nerve terminals of NG and DRG explants in culture at normal (10 mM) and high (30 mM) glucose concentrations was significantly enhanced in the diabetic mice. Chromatolytic changes (i.e. swelling and migration of the nucleus to an eccentric position in the neurons, and a loss of Nissl substance in the neuronal perikarya) and apoptotic cell death (less than one-fifth of the neurons) in the cultured ganglia were present, but neither hyperglycemia in vivo nor high glucose conditions in vitro altered the morphological features of the ganglia or the ratios of apoptotic cells at 3 days in culture. By semiquantitative RT-PCR analysis, the mRNA expressions of ciliary neurotrophic factor (CNTF) in DRG from both mice were down-regulated at 1 day in culture. The expression in diabetic DRG, but not in control DRG, was significantly up-regulated at later stages (3 and 7 days) in culture. In summary, hyperglycemia is unlikely to induce cell death in the sensory ganglia, but enhances the regenerative capability of vagal and spinal sensory nerves in vitro. The up-regulation of CNTF mRNA expression during the culture of diabetic DRG may play a role in the enhanced neurite regeneration.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.     

Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1

Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N-acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function.     

NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin

The NG2 proteoglycan is expressed by microvascular pericytes in newly formed blood vessels. We have used in vitro and in vivo models to investigate the role of NG2 in cross-talk between pericytes and endothelial cells (EC). Binding of soluble NG2 to the EC surface induces cell motility and multicellular network formation in vitro and stimulates corneal angiogenesis in vivo. Biochemical data demonstrate the involvement of both galectin-3 and alpha3beta1 integrin in the EC response to NG2 and show that NG2, galectin-3, and alpha3beta1 form a complex on the cell surface. Transmembrane signaling via alpha3beta1 is responsible for EC motility and morphogenesis in this system. Galectin-3-dependent oligomerization may potentiate NG2-mediated activation of alpha3beta1. In conjunction with recent studies demonstrating the early involvement of pericytes in angiogenesis, these data suggest that pericyte-derived NG2 is an important factor in promoting EC migration and morphogenesis during the early stages of neovascularization.     

Siramesine triggers cell death through destabilisation of mitochondria,but not lysosomes

A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 μM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 μM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5–40 μM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 μM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential.     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.     

Internal ribosome entry site-mediated translation of Apaf-1, but not XIAP, is regulated during UV-induced cell death

Components of the cellular translation machinery are targets of caspase-mediated cleavage during apoptosis that correlates with the inhibition of protein synthesis, which accompanies apoptosis. Paradoxically, protein synthesis is required for apoptosis to occur in many experimental settings. Previous studies showed that two proteins that regulate apoptosis by controlling caspase activity, XIAP and Apaf-1, are translated by a unique, cap-independent mechanism mediated by an internal ribosome entry site (IRES) that is used preferentially under conditions in which normal cap-dependent translation is repressed. We investigated the regulation of XIAP and Apaf-1 following UVC irradiation. We show that UVC irradiation leads to the inhibition of translation and cell death. Furthermore, IRES-mediated translation of Apaf-1, but not XIAP, is enhanced by UVC irradiation, and this increase in Apaf-1 translation correlated with cell death. The enhanced Apaf-1 IRES-mediated translation is caspase-independent but is negatively modulated by the eIF2alpha kinase protein kinase RNA-like endoplasmic reticulum kinase. These data suggest that progression of UV-induced apoptosis requires IRES-mediated translation of Apaf-1 to ensure continuous levels of Apaf-1 despite an overall suppression of protein synthesis.     

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.     

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

Bax κ, a novel Bax splice variant from ischemic rat brain lacking an ART domain, promotes neuronal cell death

Bax is a pro-apoptotic Bcl-2 family protein that regulates programmed cell death through homodimerization and through heterodimerization with Bcl-2. Bax alpha is encoded by six exons and undergoes alternative splicing. Bax kappa, a splice variant of Bax with conserved BH1, BH2 and BH3 binding domains and a C-terminal transmembrane domain (TM), but with an extra 446-bp insert between exons 1 and 2 leading to loss of an N-terminal ART domain, was identified from an ischemic rat brain cDNA library. Expression of Bax kappa mRNA and protein was up-regulated in hippocampus after cerebral ischemic injury. The increased Bax kappa mRNA was distributed mainly in selectively vulnerable hippocampal CA1 neurons that are destined to die after global ischemia. Overexpression of Bax kappa protein in HN33 mouse hippocampal neuronal cells induced cell death, which was partially abrogated by co-overexpression of Bcl-2. Moreover, co-overexpression of Bax kappa and Bax alpha increased HN33 cell death. The results suggest that the Bax kappa may have a role in ischemic neuronal death.     

Bcl-rambo beta,a special splicing variant with an insertion of an Alu-like cassette,promotes etoposide- and Taxol-induced cell death

The exonization of an Alu-like element into a coding sequence is unique to primates and this phenomenon distinguishes our genome from other mammals. Here, we report the presence of a special splicing variant of a proapoptotic protein Bcl-rambo in human lymph node, designated as Bcl-rambo beta. This variant contains a 98 bp Alu-like sequence which acts as an exon. There exists an in-frame stop codon within this inserted Alu-like cassette, resulting in generation of a premature protein of 104 amino acid residues. Unlike the Bcl-rambo, Bcl-rambo beta is lacking of the BH1, BH2 and BH3 motifs and becomes a BH4-only protein. Bcl-rambo beta is detected in several adult human tissues such as heart, lymph node and cervix but is absent in human brain tissue. In addition, Bcl-rambo beta is found not to be associated with mitochondria due to the absence of its C-terminal membrane anchor region. Nevertheless, this cytosol-localized protein is capable of promoting etoposide- and Taxol-induced cell death. Although the exact function of the Alu sequence is not fully characterized, the Alu element within the Bcl-rambo beta appeared to contribute to the proapoptotic capability, since removing of the Alu sequence from Bcl-rambo beta abrogates its ability to induce cell death. Our data support the speculation that the Alu element insertion during the splicing process may play an important role in the generation of protein diversity in primate cells by a yet uncharacterized mechanism.     

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate

Although neural c-Jun is essential for successful peripheral nerve regeneration, the cellular basis of this effect and the impact of c-Jun activation are incompletely understood. In the current study, we explored the effects of neuron-selective c-Jun deletion, substitution of serine 63 and 73 phosphoacceptor sites with non-phosphorylatable alanine, and deletion of Jun N-terminal kinases 1, 2 and 3 in mouse facial nerve regeneration. Removal of the floxed c-jun gene in facial motoneurons using cre recombinase under control of a neuron-specific synapsin promoter (junΔS) abolished basal and injury-induced neuronal c-Jun immunoreactivity, as well as most of the molecular responses following facial axotomy. Absence of neuronal Jun reduced the speed of axonal regeneration following crush, and prevented most cut axons from reconnecting to their target, significantly reducing functional recovery. Despite blocking cell death, this was associated with a large number of shrunken neurons. Finally, junΔS mutants also had diminished astrocyte and microglial activation and T-cell influx, suggesting that these non-neuronal responses depend on the release of Jun-dependent signals from neighboring injured motoneurons. The effects of substituting serine 63 and 73 phosphoacceptor sites (junAA), or of global deletion of individual kinases responsible for N-terminal c-Jun phosphorylation were mild. junAA mutants showed decrease in neuronal cell size, a moderate reduction in post-axotomy CD44 levels and slightly increased astrogliosis. Deletion of Jun N-terminal kinase (JNK)1 or JNK3 showed delayed functional recovery; deletion of JNK3 also interfered with T-cell influx, and reduced CD44 levels. Deletion of JNK2 had no effect. Thus, neuronal c-Jun is needed in regeneration, but JNK phosphorylation of the N-terminus mostly appears to not be required for its function.     

Anosmin-1, defective in the X-linked form of Kallmann syndrome,promotes axonal branch formation from olfactory bulb output neurons

The physiological role of anosmin-1, defective in the X chromosome-linked form of Kallmann syndrome, is not yet known. Here, we show that anti-anosmin-1 antibodies block the formation of the collateral branches of rat olfactory bulb output neurons (mitral and tufted cells) in organotypic cultures. Moreover, anosmin-1 greatly enhances axonal branching of these dissociated neurons in culture. In addition, coculture experiments with either piriform cortex or anosmin-1-producing CHO cells demonstrate that anosmin-1 is a chemoattractant for the axons of these neurons, suggesting that this protein, which is expressed in the piriform cortex, attracts their collateral branches in vivo. We conclude that anosmin-1 has a dual branch-promoting and guidance activity, which plays an essential role in the patterning of mitral and tufted cell axon collaterals to the olfactory cortex.     

A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative transposition, of Mu DNA

The phage-encoded proteins required for conservative integration of infecting bacteriophage Mu DNA were investigated. Our findings show that functional gpA, an essential component of the phage transposition system, is required for integration. The Mu B protein, which greatly enhances replicative transposition of Mu DNA, is also required. Furthermore, a truncated form of gpB lacking 18 amino acids from the carboxy terminus is blocked in replicative transposition, but not conservative integration. Our results point to a more prominent role for gpB than simply a replication enhancer in Mu DNA transposition. The ability of a truncated form of B to function in conservative integration, but not replicative transposition, also suggests a key role for the carboxy-terminal domain of the protein in the replicative reaction. The existence of a shortened form of gpB, which uncouples conservative integration from replicative transposition, should be invaluable for future dissection of Mu DNA transposition.