Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane.

The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

Large recovery strain in Fe-Mn-Si-based shape memory steels obtained by engineering annealing twin boundaries.

Shape memory alloys are a unique class of materials that can recover their original shape upon heating after a large deformation. Ti-Ni alloys with a large recovery strain are expensive, while low-cost conventional processed Fe-Mn-Si-based steels suffer from a low recovery strain (<3%). Here we show that the low recovery strain results from interactions between stress-induced martensite and a high density of annealing twin boundaries. Reducing the density of twin boundaries is thus a critical factor for obtaining a large recovery strain in these steels. By significantly suppressing the formation of twin boundaries, we attain a tensile recovery strain of 7.6% in an annealed cast polycrystalline Fe-20.2Mn-5.6Si-8.9Cr-5.0Ni steel (weight%). Further attractiveness of this material lies in its low-cost alloying components and simple synthesis-processing cycle consisting only of casting plus annealing. This enables these steels to be used at a large scale as structural materials with advanced functional properties.

Ultrahigh frequency nanotube resonators.

We report carbon-nanotube-based electromechanical resonators with the fundamental mode frequency over 1.3 GHz, operated in air at room temperature. A new combination of drive and detection methods allows for unprecedented measurement of both oscillation amplitude and phase and elucidates the relative mobility of static charges near the nanotube. The resonator serves as an exceptionally sensitive mass detector capable of approximately 10(-18) g resolution.

Contact-dependent aggregation of functional Ca2+ channels, synaptic vesicles and postsynaptic receptors in active zones of a neuromuscular junction.

To examine whether Ca2+ channels aggregate in a contact-dependent manner, we characterized the distribution of synaptic vesicles and postsynaptic receptors, and compared it to the location of Ca2+ entry sites, in a Xenopus laevis nerve-muscle coculture preparation using a localized Ca2+ detection method. The majority (75%) of Ca2+ entry sites at spontaneously formed nerve-muscle contacts were associated with enhanced immunofluorescence to the synaptic vesicle protein, SV2. In contrast, only 11% of recorded sites without Ca2+ transients exhibited significant SV2 immunofluorescence. When comparing the spatial distribution of synaptic markers with that of Ca2+ entry sites, we found that the majority of Ca2+ entry sites (61%) were associated with both enhanced SV2 immunofluorescence and R-BTX fluorescence, thereby identifying putative neurotransmitter release sites where Ca2+ channels, synaptic vesicles and postsynaptic receptors are colocalized. Using polystyrene beads coated with a heparin binding protein known to mediate in vitro postsynaptic receptor clustering, we show that the location of Ca2+ domains was associated with enhanced SV2 immunofluorescence at neurite-to-bead contacts. We conclude that the localization of functional Ca2+ channels to putative active zones follows a contact-dependent signalling mechanism similar to that known to mediate vesicle aggregation and AChR clustering.

Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles.

We have used fluorescent protein tagging to study the localization and dynamics of the actin-binding protein cortactin in living NIH 3T3 fibroblast cells. Cortactin was localized to active lamellipodia and to small cytoplasmic spots. Time-lapse imaging revealed that these cortactin labeled structures were very dynamic. In the lamellipodia, cortactin labeled structures formed at the leading edge and then moved toward the cell center. Experiments with green fluorescent protein (GFP)-tagged actin showed that cortactin movement was coincident with the actin retrograde flow in the lamellipodia. Cytoplasmic cortactin spots also contained F-actin and were propelled by actin polymerization. Arp3, a component of the arp2/3 complex which is a key regulator of actin polymerization, co-localized with cortactin. Cytoplasmic cortactin-labeled spots were found to be associated with endosomal vesicles. Association was asymmetric and approximately half of the endosomes were associated with cortactin spots. Time-lapse imaging suggested that these cortactin and F-actin-containing spots propelled endosomes. Actin polymerization based propulsion may be a common mechanism for endomembrane trafficking in the same manner as used in the plasma membrane protrusions. As cortactin is known to interact with membrane-associated signaling proteins it could have a role in linking signaling complexes with dynamic actin on endosomes and in lamellipodia.

The actin-driven movement and formation of acetylcholine receptor clusters.

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

[Studies on shape selective isopropylation products of coal tar naphthalene by gas chromatography/mass spectrometry].

Shape selective isopropylation products of coal tar naphthalene over different zeolite catalysts have been studied by capillary gas chromatography/mass spectrometry (CGC/MS) combined with computerized library search and manual interpretations of mass spectra. The analytical results have shown that products mainly consisted of some monosubstituted, disubstituted and a few trisubstituted naphthalenes. Except the low activity of HZSM-5 catalyst, all other zeolite catalysts have higher activity. HY and H beta catalytic activities were the highest, and their naphthalene conversions attained to 79.12 and 55.79 percent respectively, but their selectivities for 2-isopropylnaphthalene (beta-IPN), 2, 6-diisopropylnaphthalene (2, 6-DIPN) and 2, 7-diisopropylnaphthalene (2, 7-DIPN) were too low. ZM catalysts had good activity and much better selectivity, especially for ZM4-3, of which its 2,6-DIPN/2,7-DIPN ratio was 2.97. Therefore, some zeolite catalysts are recommended for shape selective isopropylation of coal tar naphthalene.

VEGF increases retinal vascular ICAM-1 expression in vivo.

PURPOSE: Intraocular injections of vascular endothelial growth factor (VEGF), a peptide implicated in the pathogenesis of diabetic retinopathy, can induce retinal ischemia. Diabetic retinal ischemia may be caused, in part, by the adhesion of leukocytes to the retinal vasculature. In this study, the ability of VEGF to increase the expression of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in capillary endothelium and the retinal vasculature was examined. METHODS: The expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin on human brain capillary endothelial cell monolayers exposed to VEGF was quantitated by immunoassay. The effect of VEGF on retinal vascular ICAM-1 expression was determined in ICAM-1 immunofluorescence studies of retinal flat-mounts and in RNase protection assays. RESULTS: VEGF increased capillary endothelial cell ICAM-1 levels in a dose- and time-dependent manner (6-24 hours, plateau after 6 hours; EC50, 25 ng/ml). VEGF failed to alter E-selectin, P-selectin, or VCAM-1 levels under the conditions tested. Intravitreal injections of pathophysiologically relevant concentrations of VEGF increased ICAM-1 protein and mRNA levels in the retinal vasculature. CONCLUSIONS: VEGF increases retinal vascular ICAM-1 expression. VEGF-induced increases in ICAM-1 may promote retinal leukostasis in diabetic eyes.

A cysteine-rich form of Xenopus neuregulin induces the expression of acetylcholine receptors in cultured myotubes.

Neuregulin-1 (NRG-1) has diverse functions in neural development, and one of them is to up regulate the expression of acetylcholine receptors (AChRs) at muscle fibers during the formation of neuromuscular junctions. NRG-1 has two prominent alternative splicing sites at the N-terminus; it could be an immunoglobulin (Ig)-like domain named Ig-NRG-1 or an apolar cysteine-rich domain (CRD) named CRD-NRG-1. cDNAs encoding Xenopus CRD-NRG-1 were isolated by cross-hybridization with Xenopus Ig-NRG-1 cDNA fragment. The amino acid sequence of Xenopus CRD-NRG-1 is 45 to 70% identical to the human, rat, and chick homologs. Similar to Ig-NRG-1, two variation sites within CRD-NRG-1 were identified at the spacer domain with 0 or 43 amino acids inserted and at the C-terminus of the EGF-like domain to derive either alpha or beta isoform. Two transcripts encoding CRD-NRG-1, approximately 7.5 and approximately 9.0 kb, were revealed in adult brain and spinal cord, but the expression in muscle was below the detectable level. The recombinant Xenopus CRD-NRG-1 when applied onto cultured myotubes was able to induce the tyrosine phosphorylation of ErbB receptors and the expression of AChR. The AChR-inducing activity of CRD-NRG-1 was precipitated by anti-NRG-1 antibody but not by heparin. In situ hybridization showed a strong expression of CRD-NRG-1 mRNA in developing brain, spinal cord, and myotomal muscles of Xenopus embryo. Similar to the results in other species, both CRD-NRG-1 and Ig-NRG-1 may play a role in the developing Xenopus neuromuscular junctions.

Acetylcholinesterase clustering at the neuromuscular junction involves perlecan and dystroglycan.

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve- muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.

The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction.

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

Targeting acetylcholinesterase molecules to the neuromuscular synapse.

The functional integrity of the neuromuscular synapse requires that sufficient numbers of acetylcholinesterase (AChE) molecules be localized on the specialized extracellular matrix between the nerve terminal and the post-synaptic membrane. Multiple interrelated levels of regulation are necessary to accomplish this complex task including the spatial and temporal restriction of AChE mRNA expression within the muscle fiber, local translation and assembly of AChE polypeptides, and focused accumulation of AChE molecules on the extracellular matrix. This is accomplished in part through the organization of other extracellular matrix molecules into a complex which further associates with acetylcholine receptors and their accompanying molecules. Finally, the mature neuromuscular junction contains molecules which can act as receptors for the attachment of AChE which in turn may allow for the turnover of this enzyme at the synapse. This brief review will focus mainly on contributions from our laboratory towards understanding the mechanisms involved in organizing AChE molecules at the neuromuscular synapse.

Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development.

Neuregulin has diverse functions in neural development, and one of them is the up regulation of acetylcholine receptors (AChRs) at the muscle fiber during the formation of neuromuscular junctions. Although the primary source of neuregulin is derived from motor neuron, the expression in muscle has also been demonstrated. The precise role of neuron-derived and muscle-derived neuregulin during the early stages of development is not known. In order to study the role of neuregulin during early embryo development, we isolated the cDNAs encoding Xenopus neuregulin by cross-hybridization with its chick homologue. The amino acid sequence of Xenopus protein is 50 to 70% identical to members of the neuregulin family. The cDNAs encoding different isoforms of Xenopus neuregulin were identified, and these isoforms have two variation sites: (i) the spacer domain with either 0 or 43 amino acid insertion; and (ii) the C-terminus of EGF-like domain to derive either alpha or beta isoform. When the EGF-like domain of Xenopus neuregulin was expressed in mammalian cells, the recombinant protein was able to induce the expression of AChR and the tyrosine phosphorylation of erbB receptors in cultured myotubes. An approximately 6.5 kb transcript corresponding to neuregulin was detected in RNA isolated from brain and muscle. Various splicing variants were expressed in different Xenopus tissues. In situ hybridization showed a strong expression of neuregulin in developing brain and spinal cord of Xenopus embryo. In addition, it was also prominently expressed in the myotomal muscle. These data suggest that in addition to motor neurons, the postsynaptic muscle cells can also contribute neuregulin for synaptogenesis.

Inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation.

Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine-induced endothelial cell activation in a separated and mixed endothelial-RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-gamma. Inhibition of iNO synthase (iNOS) activity with N omega-monomethyl-L-arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-gamma and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on kappa B cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-kappa B revealed that iNO inhibited the activation of nuclear factor-kappa B. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation.

A role of tyrosine phosphatase in acetylcholine receptor cluster dispersal and formation.

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor-coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot-associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.

Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth.

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture.

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naïve neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article.

Association of cortactin with developing neuromuscular specializations.

During the development of the neuromuscular junction (NMJ), motoneurons grow to the muscle cell and the nerve-muscle contact triggers the development of both presynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of synaptic vesicles (SVs), and postsynaptic specialization, consisting of clusters of acetylcholine receptors (AChRs). Previous studies have shown that the activation of tyrosine kinases and the local assembly of an actin-based cytoskeletal specialization are involved in the development of both types of specializations. To understand the link between tyrosine phosphorylation and the assembly of the cytoskeleton, we examined the localization of cortactin in relationship to synaptic development. Cortactin is a 80/85 kD F-actin binding protein and is a substrate for tyrosine kinases. It contains a proline-rich motif and an SH3 domain and is localized at sites of active F-actin assembly. Using a monoclonal antibody against cortactin, its localization at developing NMJs in culture was observed. To understand the spatial and temporal relationship between cortactin and developing synaptic structures, cultured muscle cells and spinal neurons from Xenopus embryos were treated with beads coated with heparin-binding growth-associated molecule to induce the formation of AChR clusters and SV clusters and the localization of cortactin was followed by immunofluorescence. In untreated muscle cells, cortactin is often co-localized with spontaneously formed AChR clusters. After cells were treated with beads, cortactin became localized at bead-induced AChR clusters at their earliest appearance (1 h after the addition of beads). This association was most reliably detected at the early stage of the clustering process. On the presynaptic side, cortactin localization could be detected as early as 10 min after the bead-neurite contact was established. Cortactin-enriched contacts later showed concentration of F-actin (at 1 h) and clusters of SVs (at 24 h). These data suggest that cortactin mediates the local assembly of the cytoskeletal specialization triggered by the synaptogenic signal on both nerve and muscle.

Hydrogen peroxide-mediated transcriptional induction of macrophage colony-stimulating factor by TGF-beta1.

TGF-beta1 and macrophages are important regulators of tissue fibrosis and remodeling. Here we show that TGF-beta1 induces the expression of macrophage-CSF (M-CSF) in vascular endothelial cells via a signaling pathway(s) involving hydrogen peroxide (H2O2). In a time-dependent manner, TGF-beta1 produced a 10- and a 6-fold increase in M-CSF mRNA and protein levels after 12 h, respectively. This increase in M-CSF expression was attenuated by a nitric oxide donor, S-nitrosoglutathione (GSNO), and by a nonspecific oxidase inhibitor, diphenylene iodonium. Furthermore, the TGF-beta1-induced M-CSF mRNA expression was inhibited by catalase, but not by superoxide dismutase, suggesting that H2O2 rather than superoxide anion (O2.-) is the primary mediator of the effects of TGF-beta1. Transient transfection studies using deletional M-CSF promoter constructs demonstrated that TGF-beta1 produced a 13-fold induction in M-CSF promoter activity that was repressed by >85% with GSNO and catalase, in part through inhibitory effects on kappaB cis-acting elements. Electrophoretic mobility shift assays revealed that the activation of nuclear factor-kappaB by TGF-beta1 was also inhibited by GSNO and catalase, but not by superoxide dismutase. In a concentration-dependent manner, treatment with exogenous H2O2 produced 14- and 4.6-fold increases in M-CSF promoter activity and mRNA expression, respectively. These results indicate that the generation of H2O2 and activation of NF-kappaB by TGF-beta1 are required for the induction of M-CSF gene transcription.

HB-GAM (heparin-binding growth-associated molecule) and heparin-type glycans in the development and plasticity of neuron-target contacts.

HB-GAM is a secretory, extracellular matrix-associated protein that was isolated by screening for factors that enhance neurite outgrowth in rat brain neurons. The HB-GAM sequence clearly (about 50%) is homologous to that of MK (midkine) sequence, a protein discovered through screening for factors that mediate retinoic acid-induced cell differentiation. These lysine- and cysteine-rich sequences define a novel family of differentiation/growth factors, which are conserved in their structures from mammals to amphibians. HB-GAM is expressed strongly along axon pathways and target regions of axons during and prior to the stage of axonal growth in tissues. These findings, together with in vitro interactions with neurons, suggest that HB-GAM is a cell matrix-associated cue for growth cone migration. N-syndecan (syndecan-3) functions as a receptor/coreceptor in HB-GAM-induced neurite outgrowth in perinatal rat brain neurons. In addition to enhancing neurite growth in a developmentally regulated manner in early neurons, HB-GAM is accumulated at the growth cone-target interphase accompanying the onset of synaptogenesis, as evidenced by its presence at the neuromuscular junction of Xenopus and rat. In vitro studies suggest that HB-GAM functions as a local, synaptic matrix-associated factor that enhances both presynaptic and postsynaptic differentiation during development. In addition, a role in adult plasticity is suggested by studies on injury-induced and activity-dependent plasticity in rat hippocampus.