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Biomedical subjects

N R Smalheiser

Publications and source records attributed to N R Smalheiser.

At least 37 records · Page 2Linked to original sources
PubMed1994

Acute neurite retraction triggered by lysophosphatidic acid: timing of the inhibitory effects of genistein.

Acute neurite retraction, elicited by diverse agents in several neuronal cell types, has been reported to be inhibited by genistein, a kinase antagonist that is relatively (though not absolutely) selective for tyrosine kinases. It was hypothesized that genistein acts upon some final common pathway that integrates multiple extrinsic and intrinsic signals to regulate whether neurites will execute a retraction response (J. Neurochem., 61 (1993) 340-343). To define this pathway in more detail, a quantitative study of NG108-15 cell rapid-onset neurites was carried out as they retract in response to lysophosphatidic acid (LPA, 10 microM). Following the application of LPA, most neurites exhibited early morphologic changes between 0.5 and 1.5 min, followed by progressive shortening and eventual retraction, with 50% of neurites completely retracted by 5 min and 80% gone by 10 min. Genistein did not inhibit the formation of subcortical F-actin, nor its functional competence in several assays. Genistein protected neurites when added at any time prior to the onset of the earliest morphologic changes, but failed to block progression when added to neurites that were already undergoing retraction. These findings imply that the final common pathway (i.e. the critical target(s) for genistein) must be activated late, after the increase in F-actin levels has peaked and just before retraction is initiated.

Actins↗
PubMed1993

Cranin interacts specifically with the sulfatide-binding domain of laminin.

Cranin is a 120 kDa integral membrane glycoprotein which binds laminin under conditions of physiologic ionic strength in a calcium-dependent manner. Here, binding of cranin to laminin has been characterized using both ligand-blotting assays and laminin affinity bead assays. Binding was specifically inhibited by anti-laminin antibodies against the A chain terminal domain G, but not by several other region-specific antibodies. Dextran sulfate, fucoidin, and sulfatide were potent inhibitors of binding (50% inhibition at 0.03, 0.5, and 1.7 micrograms/ml, respectively); heparin was a weaker inhibitor (50% inhibition approximately 5 micrograms/ml), and mannan and chondroitin sulfate were not inhibitory at 100 micrograms/ml. Binding was not inhibited by lactose or the A chain peptide PA22-2. The mobility of the broad, fuzzy cranin band was shifted after digestion with neuraminidase, N-glycanase, and O-glycanase, though none of these treatments decreased band heterogeneity nor destroyed the ability to bind laminin. Cranin bound to Jacalin lectin, which recognizes the Gal beta 1-3GalNAc linkage expressed in certain classes of mucins. These findings indicate that cranin binds at or near the high affinity sulfatide-binding site previously mapped to the E3 domain of laminin, which is known to exhibit bioactivity for neural cells. In view of the extremely low abundance of cranin in brain membranes (approximately 0.005%), its avid laminin-binding activity is remarkable, and strongly suggests that cranin may play a physiologic role in regulating specific neural cell interactions.

Animals↗
PubMed1993

Monensin-sensitive cellular events modulate neurite extension on laminin: an example of higher-order regulation of cell motility.

NG108-15 cells extend "rapid-onset" neurites vigorously within the first hour after plating in minimal serum-free medium on Petri dishes coated with polylysine and laminin (1 ng/mm2). We recently reported that the initial rates of neurite formation and cell translocation are further accelerated in this system when non-specific substratum attachment sites are partially blocked by polyglutamate, bovine serum albumin, or polyethylene glycol polymers [Smalheiser, N. R. (1991): Dev. Brain Res. 62:81-89]. When cells were plated in the presence of the monovalent cation ionophore monensin (1-5 microM) or hypertonic sucrose (50-100 mM), the initial rate of outgrowth on laminin/polylysine-treated Petri dishes was not affected, yet the acceleration produced by polyglutamate was strongly inhibited. These data indicate that monensin-sensitive intracellular events can regulate neurite extension on laminin indirectly, through modulating the effects exerted on cells by nonspecific substratum sites. Although the critical events affected by monensin remain to be identified, movements of laminin receptors (their clustering, internalization, and recycling) are likely targets for further study.

Anti-Bacterial Agents↗
PubMed1993

Expression of Egr-1 in the brain of sleep deprived rats.

In previous research, rats subjected to prolonged sleep deprivation have shown disturbances of thermoregulation, hormonal and metabolic changes in apparent response to the thermoregulatory problems, lesions on the tail and paws, and eventual death. To search for alterations of functional activity in brain, the expression of the immediate early gene Egr-1 was examined by immunocytochemistry and Northern blotting in rats subjected to total sleep deprivation (TSD) for 10 days. Controls included yoked stimulus-control (TSC) rats, surgically implanted but otherwise undisturbed control rats, and unoperated control rats. Photographs of immunoreacted coronal sections from four sets of rats were ranked blindly for 25 brain regions. TSD rats showed tendencies for regionally specific increases in Egr-1-like immunoreactivity in dorsal raphe, lateral habenula, superior colliculus, and ventral periaqueductal grey. However, most regions showed no differences in Egr-1-like immunoreactivity between TSD and control rats. Neither was there a difference in whole brain Egr-1 mRNA by Northern blot in two additional sets of rats. Thus, this study, like previous studies of brain histology, amines, adrenoceptors, and glucose utilization, does not provide positive support for the hypothesis that sleep protects the central nervous system against massive global damage, fatigue, or dysfunction.

Animals↗
PubMed1993

Acute neurite retraction elicited by diverse agents is prevented by genistein, a tyrosine kinase inhibitor.

The retraction of axonal branches is a prominent feature of nervous system development and function. Although various biological and pathological signals can elicit retraction, little is known regarding their underlying mode of action. An in vitro assay using NG108-15 cells was used to demonstrate that rapid-onset neurites exposed acutely to trypsin, serum, lysophosphatidic acid, extracellular ATP, the phorbol ester phorbol 12-myristate 13-acetate, and nocodazole were all protected from retraction by the tyrosine kinase inhibitor genistein. This finding indicates that a common (genistein-sensitive) cellular event is involved in integrating the influence of multiple extrinsic and intrinsic signals and in regulating whether or not neurites will execute a retraction response.

Cells, Cultured↗
PubMed1992

Characterization of a novel set of membrane antigens associated with axonal growth. I. Biochemical and functional studies.

Cranin, a prominent 120 kDa laminin-binding protein of cell membranes, was originally identified and characterized by virtue of its ability to bind laminin directly in ligand-blotting assays. We have now raised polyclonal ('3070') and monoclonal antibodies (MAbs 4 and 199) against a partially purified preparation of cranin, and have characterized the properties and expression of the corresponding antigens in further detail. In immunoblots of E14 chick brain membranes, these antibodies all recognized a single major band migrating at approximately 125 kDa, with minor bands at 115 kDa and 170/180 kDa. The major 125 kDa antigen bound to laminin affinity beads in a divalent cation- and conformation-dependent manner. The 125 and 115 kDa bands were also the most prominent HNK-1 positive proteins detected overall within E14 chick brain membranes. MAbs 4 and 199 specifically inhibited the attachment of NG108-15 cells to low, but not high amounts of substratum-bound laminin. While the relation of 3070/MAb 4 antigens to cranin requires further elucidation, these data, together with evidence presented in the companion papers and elsewhere, suggest that the antigens are important in neural development by mediating at least some of the responses of neural cells to laminin--for example, by acting as a laminin receptor guiding axonal outgrowth and neuronal migration, or by involvement in the transport and binding of laminin to the surface of neurons and reactive glial cells that synthesize laminin.

Animals↗
PubMed1992

Characterization of a novel set of membrane antigens associated with axonal growth. II. Expression in the chick central nervous system.

In the preceding paper (Dev. Brain Res., 69 (1992) 215-223), a polyclonal antiserum ('3070') and several monoclonal antibodies (MAbs 4 and 199) were used to define a novel set of laminin-binding membrane antigens in chick brain. These MAbs specifically inhibited attachment of NG108-15 cells to low, but not high amounts of laminin. Here, the immunocytochemical localization of MAb 4 and 3070 antigens was examined in frozen sections of E3-P21 chick brain, with particular reference to retina, optic tectum, cerebellum and spinal cord. Growing axons and migrating nascent neurons were stained transiently, while projection neurons were intensely labelled at all stages. Some interneurons and glial cells were stained faintly; others, apparently not at all. Finally, 3070 antiserum stained neuropil regions in mature brain. Cells in the CNS expressing high levels of MAb 4 antigens corresponded to those which, in rodents, are known to migrate in response to laminin or to synthesize forms of laminin themselves. These data, together with evidence presented elsewhere (J. Comp. Neurol., 313 (1991) 625-642) and in the next paper in this series (Dev. Brain Res., 69 (1992) 277-282), suggest that MAb 4 and 3070 antigens are important in neural development by mediating at least some of the responses of neural cells to laminin--for example, by acting as a laminin receptor guiding axonal outgrowth and neuronal migration, or by involvement in the transport and binding of laminin to the surface of neurons and reactive glial cells.

Animals↗
PubMed1992

Characterization of novel set of membrane antigens associated with axonal growth. III: Expression in the regenerating goldfish optic nerve and tectum.

In the preceding two papers in this series, a polyclonal antiserum (3070) and several monoclonal antibodies (MAbs 4 and 199) were used to define a novel set of laminin-binding membrane antigens in chick brain. In chick brain, 3070 and MAb 4 antigens stained growing axons and migrating nascent neurons transiently; though projection neurons were intensely labelled at all stages, normal glial cells were stained faintly or not at all. To learn if these antigens are associated more generally with active states of nerve growth, the present paper examined the expression of 3070 and MAb 4 antigens during regeneration of the goldfish optic nerve. Only a few optic fibers or glial cells were stained by 3070 antiserum in the resting (uninjured) optic nerve, but 10 days following a unilateral nerve crush, when the growth response is maximal in these fish, distal regenerating neuritic sprouts and glial cell bodies and processes were profusely labelled. The intensity of labelling was diminished by 135 days post-crush, when most active growth was completed. Since 3070/MAb 4 antigens exhibit properties expected for functional laminin receptors, these findings suggest several possible roles that they could play to aid nerve regeneration.

Animals↗
PubMed1991

Expression of a neurally related laminin binding protein by neural crest-derived cells that colonize the gut: relationship to the formation of enteric ganglia.

In order to give rise to the enteric nervous system (ENS), cells migrating from the neural crest must find the bowel and cease migrating at appropriate locations within the gut. Previous studies of the development of the ENS in a mutant mouse have led to the hypothesis that laminin in the enteric mesenchyme may act as a signal to crest-derived cells to cease migrating and extend neurites (or glial processes). Implied in this hypothesis is the idea that crest-derived cells, as a prelude to their participation in ganglion formation, acquire a neurally related laminin receptor, which they do not express at pre-enteric stages of migration. As a partial test of this hypothesis, single and double label immunocytochemistry at light and electron microscopic (EM) levels were used to study the expression of cell surface laminin binding proteins by crest-derived cells in the process of migrating to or within the developing chick gut. Two antibodies (called 3070 and alpha-110) raised against neuronal cell surface laminin binding proteins were employed for this purpose. Laminin binding protein immunoreactivity was found to be expressed within the bowel and ganglion of Remak by a subset of crest-derived cells (identified immunocytochemically with NC-1/HNK-1 antibodies) and by all of those developing as neurons (identified immunocytochemically with antibodies to neurofilament-associated proteins). Laminin binding protein immunoreactivity was also found to be expressed in fixed neural structures elsewhere in the embryos, including cranial and spinal roots, nerves, and ganglia. In contrast, laminin binding protein immunoreactivity was not expressed by migrating crest-derived cells in the vicinity of the vagal or sacral regions of the neuraxis (from which the precursors of the ENS take origin); nor was it expressed by juxta-pharyngeal vagal crest-derived cells migrating to the foregut through the caudal branchial arches or by the caudal stream of sacral crest-derived cells approaching the hindgut. EM immunocytochemistry confirmed that laminin binding protein immunoreactivity in the bowel was located on the surfaces of crest-derived cells, and was exhibited both by those cells that could only be distinguished from their neighbors by their NC-1/HNK-1 immunoreactivity and by cells developing as neurons or glia. EM immunocytochemistry also revealed that the surfaces of crest-derived cells migrating through the enteric mesenchyme were contacted by many small osmiophilic "puffs" of laminin-immunoreactive extracellular material. These puffs coincided in location with membrane sites that expressed the immunoreactivity of the laminin binding protein. These observations are consistent with the hypothesis that laminin plays a role in the formation of enteric ganglia.

Animals↗
PubMed1991

Role of laminin in stimulating rapid-onset neurites in NG108-15 cells: relative contribution of attachment and motility responses.

The present paper analyzed how attachment and motility responses contribute to the initial formation of rapid-onset neurites in undifferentiated NG108-15 cells. Attachment responses played a positive role in stabilizing neurites against slippage, but exerted a negative effect upon active motility--which appears to be the major rate-determining event in initial outgrowth. The role of laminin in this paradigm was elucidated by analyzing the effects of polyglutamate and other agents (bovine serum albumin and polyethylene glycol chain derivatives) which reduced the number of passive substratum attachment sites, but which did not affect cells directly nor affect the binding of laminin to the substratum. Polyglutamate decreased attachment and spreading of cells, yet greatly accelerated the initial laminin-dependent formation of rapid-onset neurites, and greatly enhanced active neurite remodelling and cell translocation as well. Together with data presented in previous studies in this series, these findings indicate that laminin stimulates rapid-onset neurites primarily by stimulating active motility responses. Insofar as rapid-onset neurites appear to provide a valid model of how neuronal growth cones are regulated, it is likely that these findings will be relevant to developing primary neurons as well.

Cell Adhesion↗
PubMed1991

Cell attachment and neurite stability in NG108-15 cells: what is the role of microtubules?

Undifferentiated NG108-15 cells forming rapid-onset neurites were acutely exposed to nocodazole or trypsin. Resorption, cell rounding and detachment were delayed or prevented by 5'-deoxy,5'-methylthioadenosine (MTA), which selectively enhanced the strength of attachment responses. However, taxol, which stabilized microtubules, did not protect cell shape appreciably when trypsin or mechanical stimuli were used to decrease the strength of attachment. Together with numerous control experiments, this evidence suggests that the mechanical properties of microtubules do not contribute acutely to maintaining cell shape, though microtubules may play an indirect regulatory role (e.g. through their interactions with actin and substratum attachment sites). Patterns of trypsin-induced resorption resembled those seen 'spontaneously' in NG108-15 cells growing on laminin, and in fibroblastic CHO cells, suggesting that these results may be both physiologically relevant and applicable widely to many cell types.

Adenosine↗
PubMed1990

Cell attachment and neurite stability in NG108-15 cells: effects of 5'-deoxy, 5'-methyl thioadenosine (MTA) compared with laminin, kinase inhibitor H-7, and Mn2+ ions.

Methylation inhibitors such as 5'-deoxy, 5'-methyl thioadenosine (MTA) have been shown to exert an intriguing spectrum of effects upon neural cells: inhibiting responses to nerve growth factor in PC12 cells; stimulating outgrowth of rapid-onset neurites in NG108-15 cells; inhibiting the resorption and remodelling of these rapid-onset neurites; and inducing fibroblasts to extend long, multipolar, branched processes. To learn whether the apparently diverse effects of these agents might reflect some common underlying cellular sites of action, we examined simpler, short-term effects of MTA upon cell attachment and stability of rapid-onset neurites in NG108-15 cells, and compared the effects with those produced by the kinase inhibitor H-7, substratum-bound laminin, or Mn2+ ions. MTA was shown to selectively enhance the response of cells and their neurites in attaching to their substrata, without inducing other 'adhesive' responses such as cell spreading or motility. The data suggest that MTA activates or increases the effectiveness of 'attachment receptors', and that this at least partially explains its neurite-promoting effects. While it is still premature to propose a common mechanism of action for MTA and related agents, all of their known effects thus appear to involve modulating responses generated at cell-surface receptors. A further clue is that kinase C activity appears to be critical, since cells pretreated with phorbol esters for 24 h (which down-regulated levels of kinase C) selectively failed to extend neurites in response to MTA, but responded normally to laminin and Mn2+ ions.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Morphologic plasticity of rapid-onset neurites in NG108-15 cells stimulated by substratum-bound laminin.

Undifferentiated NG108-15 cells, when replated onto laminin-coated substrata, extend multipolar, highly branched neurite-like extensions up to 200 microns in length within 4 h; morphologic and pharmacologic properties of these 'rapid-onset neurites' have been described recently. The present study has extended these observations, using time lapse video recordings of their dynamic behavior and additional pharmacologic studies. Rapid-onset neurites and neuronal growth cones were shown to be regulated in an identical manner in all respects examined, including inhibition of outgrowth by cytochalasin B. Of particular interest was the observation that rapid-onset neurites in contact with laminin exhibited an extremely high rate of turnover, which was inhibited by 5'-deoxy-5'-methylthioadenosine (MTA). This system provides a uniquely favorable in vitro preparation in which neuritic plasticity can be elicited, directly observed and experimentally modulated under controlled conditions.

Adenosine↗
PubMed1989

Analysis of slow-onset neurite formation in NG108-15 cells: implications for a unified model of neurite elongation.

When undifferentiated NG108-15 cells are plated onto polylysine coated Petri dishes in serum-free medium, they form neurites within 1-4 h if plated in the presence of laminin or 5'-deoxy-5'-methylthioadenosine (rapid-onset neurites). In the absence of such agents, serum-deprived NG108-15 cells extend axon-like neurites onto polylysine over several days; here we characterize the dynamic behavior of this slow-onset outgrowth pattern in detail. Individual cells plated on laminin expressed a gradual multipolar-to-unipolar transition due to rapid-onset neurites becoming remodelled into the appearance of slow-onset neurites. This phenomenon reflected the selective stabilization of certain rapid-onset neurites, along with the restriction of motility to their distal tips. Based upon the properties and interactions of both rapid- and slow-onset neurites in NG108-15 cells, a unified model for neurite formation is presented.

Animals↗
PubMed1989

Altered cell shapes in fibroblasts treated with 5'-deoxy-5'-methylthioadenosine: relation to morphogenesis of neural cells.

In the present study, mouse 3T3 fibroblasts and primary human foreskin fibroblasts exposed to MTA (5'-deoxy-5'-methylthioadenosine) were found to achieve neural-like cell shapes and to extend long, multipolar processes rapidly and reversibly. Time lapse recordings and pharmacologic studies revealed that process formation in MTA-treated fibroblasts was mechanistically related to the rapid-onset mode of neurite formation previously characterized in neural hybrid NG108-15 cells. These data, together with evidence presented elsewhere, indicate that MTA selectively reorganizes the mode of expression of a specific cytoplasmic machinery that is active in many types of cells, and which is involved in regulating cell shape and neurite formation in developing neurons.

Adenosine↗
PubMed1987

Kinetic analysis of 'rapid onset' neurite formation in NG108-15 cells reveals a dual role for substratum-bound laminin.

Undifferentiated neural hybrid NG108-15 cells plated on laminin-coated, polylysine-treated plastic Petri dishes in minimal serum-free media formed long neurites within 1-4 h post-plating. Morphologic features and pharmacologic responses of these 'rapid onset' neurites were strikingly similar to those of neuronal growth cones. Cycloheximide (1-10 micrograms/ml) and forskolin (10(-7) to 10(-6) M) accelerated the initial formation of laminin-stimulated neurites, but did not cause rapid onset neurites to emerge upon Petri dishes coated with polylysine alone. Quantitative study of the substratum-dependent effect of cycloheximide showed that it was additive even with maximally effective amounts of laminin, independent of the magnitude of the laminin-stimulated baseline rate and not limited by an inherent ceiling in the rate at which neurites could form. The methylation inhibitor 5'-deoxy-5'-methyl thioadenosine (MTA) (3 X 10(-4) to 3 X 10(-3) M) did stimulate neurites to form on polylysine. MTA- and laminin-stimulated neurites were similar in their susceptibility to calmodulin antagonists and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, formation of MTA-stimulated neurites was not accelerated by cycloheximide. A simple two-compartment kinetic model of 'rapid onset' neurite formation is proposed: compartment A is common to both laminin- and MTA-stimulated neurites. Compartment B is affected by cycloheximide, and its access to the neurite formation machinery contained in compartment A is gated according to the nature of the substratum. In addition to its direct effects, laminin controls the relative dominance of the two kinetic compartments via modulating the effectiveness of other signals acting upon an endogenously active compartment B.

Adenosine↗
PubMed1987

Cranin: a laminin-binding protein of cell membranes.

We report that a 120-kDa glycoprotein is the predominant laminin-binding protein detected within plasma membranes of rodent NG108-15 neural hybrid cells, embryonic chicken brain, and mouse 3T3 fibroblasts. This protein was detected when membrane extracts were separated by PAGE, transferred to nitrocellulose, and incubated with laminin at concentrations as low as 2.8 X 10(-11) M, under conditions of physiological ionic strength and pH and in the presence of calcium ions. It behaves as an integral membrane component, and its laminin-binding moiety is accessible to the external face of the cell surface. Moreover, it appears to bind to a site on laminin that is very sensitive to proteolysis. The properties of this protein, which we have termed "cranin," distinguish it from other known laminin receptors and make it a candidate to mediate some of the effects of laminin upon cells.

Animals↗