Juan Bruses - Academia.edu (original) (raw)

Papers by Juan Bruses

Neuropharmacology, 2015

High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transm... more High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca(2+) channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca(2+) channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba(2+) currents through CaV2.1, CaV2.2 and CaV2.3 Ca(2+) channels independently of CaVβ subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca(2+) current amplitude. Altogether our data revealed that HVA Ca(2+) channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out b... more The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself.

Neuron, 1992

The pharmacological specificity of Ca2+ channel-secretion coupling in acetylcholine (ACh) and som... more The pharmacological specificity of Ca2+ channel-secretion coupling in acetylcholine (ACh) and somatostatin (SOM) release was studied in the chick eye choroid neuromuscular junctions and in dissociated ciliary ganglion (CG) neurons. ACh secretion changes in development from stage (St) 40, when release is dihydropyridine (DHP) and partially omega-conotoxin (omega-CgTX) sensitive, to posthatch, when release is insensitive to DHPs but sensitive to omega-CgTX. St 40 CG neurons cultured with striated muscle have release properties similar to those of St 40 iris and choroid but different from those of St 34 neurons, which are neither DHP nor omega-CgTX sensitive. SOM (also coreleased from posthatch choroid terminals) can inhibit ACh release in both posthatch and St 40 choroids, suggesting that the SOM receptor interacts with both DHP-sensitive and -insensitive channels.

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001

Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically... more Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta-cyclodextrin. This treatment caused the d...

Journal of neurophysiology, 1995

1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived ... more 1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons, to analyze the biological process of neuronal death caused by trophic factor withdrawal. Tissue culture conditions were refined by characterizing the additional medium components required to obtain 100% survival, for at least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris, and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly enriched neuronal cultures almost devoid of nonneuronal cells were used. 2. The time at which trophically deprived neurons cannot be rescued by the addition of trophic support, "commitment point," was established to be between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of protein translation, reduced 3H-leucine incorporation by 90-95%, at a concentration of 10-100 micrograms/ml. Th...

Frontiers in Synaptic Neuroscience, 2012

N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions... more N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChAT(BAC)EGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and β4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

Journal of Neuroscience, 2004

The juxtamembrane domain (JMD) of N-cadherin cytoplasmic tail is an important regulatory region o... more The juxtamembrane domain (JMD) of N-cadherin cytoplasmic tail is an important regulatory region of the clustering and adhesion activities of the protein. In addition, the JMD binds a diversity of proteins capable of modifying intracellular processes including cytoskeletal rearrangement mediated by Rho GTPases. These GTPases also function as regulators of voltage-activated calcium channels, which in turn modulate neuronal excitability. The present study was designed to determine whether there is a direct functional link, via Rho GTPase, between the N-cadherin JMD and these voltage-activated channels. It was found that the infusion of the soluble JMD into chick ciliary neurons causes a substantial decrease in the amplitude of the high-threshold voltage-activated (HVA) calcium current. The activation time is increased while the inactivation process is reduced, suggesting that the decreased current amplitude reflects a reduction in the number of channels available to open. This effect was reversed by inhibition of RhoA or its downstream effector, Rho-associated kinase (ROCK). Because ROCK determines the active state of myosin, these results suggest that the modulation of HVA by the JMD could be mediated by changes in the status of the actin-myosin cytoskeleton.

Journal of Neuroscience, 2005

Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions ... more Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions with a visible cytoplasm. One of these cytoplasmic compartments is the Schmidt-Lanterman incisure, which spirals through the compacted layers and is believed to help sustain the growth and function of compact myelin. Incisures contain adherens junctions (AJs), the key components of which are E-cadherin, its cytoplasmic partners called catenins, and F-actin. To explore in vivo the role of cadherin and catenins in incisures, E-cadherin mutant proteins that completely replace endogenous cadherin have been delivered to the cells using adenovirus. When the introduced cadherin lacked its extracellular domain, association of p120 catenin (p120ctn) with the cadherin did not occur, and incisures disappeared. Remarkably, the additional replacement of two phosphorylatable tyrosines by phenylalanine in the cytoplasmic tail of the mutant cadherin restored both p120ctn binding and incisure architecture, indicating that p120ctn recruitment is critical for incisures maintenance and might be regulated by phosphorylations. In addition, the ability of the p120ctn/cadherin complex to support incisures was blocked by mutation of the Rho GTPase regulatory region of the p120ctn, and downregulation of Rac1 activity at the junction reversed this inhibition. Because Rho GTPases regulate the state of the actin filaments, these findings suggest that one role of p120ctn in incisures is to organize the cytoskeleton at the AJ. Finally, developmental studies of Schwann cells demonstrated that p120ctn recruitment from the cytoplasm to the AJ occurs before the appearance of Rac1 GTPase and F-actin at the junction.

The Journal of Cell Biology, 1998

The up-and downregulation of polysialic

Proceedings of the National Academy of Sciences, 2004

Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their p... more Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

PLoS ONE, 2013

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central a... more Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central and peripheral nervous system and are localized at synaptic and extrasynaptic sites of the cell membrane. However, the mechanisms regulating the localization of nicotinic receptors in distinct domains of the cell membrane are not well understood. N-cadherin is a cell adhesion molecule that mediates homotypic binding between apposed cell membranes and regulates the actin cytoskeleton through protein interactions with the cytoplasmic domain. At synaptic contacts, N-cadherin is commonly localized adjacent to the active zone and the postsynaptic density, suggesting that N-cadherin contributes to the assembly of the synaptic complex. To examine whether N-cadherin homotypic binding regulates the cell surface localization of nicotinic receptors, this study used heterologous expression of N-cadherin and a3b4 nAChR subunits C-terminally fused to a myc-tag epitope in Chinese hamster ovary cells. Expression levels of a3b4 nAChRs at cell-cell contacts and at contact-free cell membrane were analyzed by confocal microscopy. a3b4 nAChRs were found distributed over the entire surface of contacting cells lacking N-cadherin. In contrast, N-cadherin-mediated cell-cell contacts were devoid of a3b4 nAChRs. Cellcell contacts mediated by N-cadherin-deleted proteins lacking the b-catenin binding region or the entire cytoplasmic domain showed control levels of a3b4 nAChRs expression. Inhibition of actin polymerization with latrunculin A and cytochalasin D did not affect a3b4 nAChRs localization within N-cadherin-mediated cell-cell contacts. However, treatment with the Rho associated kinase inhibitor Y27632 resulted in a significant increase in a3b4 nAChR levels within N-cadherinmediated cell-cell contacts. Analysis of a3b4 nAChRs localization in polarized Caco-2 cells showed specific expression on the apical cell membrane and colocalization with apical F-actin and the actin nucleator Arp3. These results indicate that actomyosin contractility downstream of N-cadherin homotypic binding regulates the cell surface localization of a3b4 nAChRs presumably through interactions with a particular pool of F-actin. Citation: Brusés JL (2013) Cell Surface Localization of a3b4 Nicotinic Acetylcholine Receptors Is Regulated by N-Cadherin Homotypic Binding and Actomyosin Contractility. PLoS ONE 8(4): e62435.

Pharmacology Biochemistry and Behavior, 1991

The properties of circling training (CT) for detecting behavioral teratologic drug-induced effect... more The properties of circling training (CT) for detecting behavioral teratologic drug-induced effects was evaluated by prenatal administration of two behavioral teratogenic drugs: vitamin A (80,000 IU/kg/day) and haloperidol (2.5 mg/kg/day). The circling training was started at 30 days of age and performed for 8 days in an automated apparatus. Statistically significant differences between drug-treated and control animals regarding the measured response (turns per minute) were found. Two components may affect the response measured by the CT: associative learning and motor performance. The incidence of these components was discriminated with behavioral and mathematical approaches. In the experimental conditions used the most affected parameter was motor performance. The results indicate that CT can be used as an instrumental conditioning test where the quantifiable endpoint is the on-going motor performance. Further applications of the CT for neurochemical evaluation of drug induced effects are also discussed.

Molecular Neurobiology, 2006

Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuronto- ... more Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuronto- neuron synapses. N-cadherin contains an extracellular domain that binds to other cadherins on juxtaposed cell membranes, a single-pass transmembrane region, and a cytoplasmic tail that interacts with various proteins, including catenins, kinases, phosphatases, and presenilin 1. N-cadherin contributes to the structural and functional organization of the synaptic complex by ensuring the adhesion between synaptic membranes and organizing the underlying actin cytoskeleton. Additionally, recent findings have shown that N-cadherin may participate in synaptic physiology by regulating calcium influx through voltage-activated calcium currents. The diverse activities of N-cadherin stem from its ability to operate as both an adhesion molecule that links cytoskeletons across cell membranes and a ligand-activated homophilic receptor capable of initiating intracellular signaling. An important mechanism of cadherin signaling is the regulation of small Rho guanosine triphosphatase activity that affects cytoskeleton dynamics and calcium influx. Because both the regulation of cadherin adhesive activity and cadherin-mediated signaling are affected by the binding of molecules to the intracellular domain, changes in the composition of the N-cadherin complex are central to the regulation of cadherin-mediated functions. This article focuses on the roles that N-cadherin might play at the level of the synapse through its effect on adhesion and signaling in the proximity of the synaptic junction.

Molecular and Cellular Neuroscience, 2005

N-cadherin is an adhesion receptor that participates in both interaction between immature pre-and... more N-cadherin is an adhesion receptor that participates in both interaction between immature pre-and postsynaptic neurons and in the stabilization and function of matured neuron -neuron synapses. To better understand how the N-cadherin complex contributes to synapse formation, we examined its distribution and composition during synapse formation in the chick ciliary neurons. It was found that at early phases of synaptogenesis, N-cadherin is distributed in small clusters on the cell surface and primarily associates with p120-catenin and B-catenin. In contrast, as synaptic contacts matured, larger Ncadherin clusters were found localized adjacent to the active zone and associated with PS1 and ;-catenin, while p120-and B-catenin were dispersed among other cell regions, including axons. As it is known that PS1 binds ;-catenin and that uncoupled p120-catenin can alter the cytoskeleton via its effect on Rho GTPases, these changes in the molecular composition of the N-cadherin complex (represented by the uncoupling of p120-catenin and association with PS1) may correspond to distinct functional states of the complex involved in synaptic maturation. D

Molecular and Cellular Neuroscience, 2009

N-cadherin is a transmembrane adhesion receptor that contributes to neuronal development and syna... more N-cadherin is a transmembrane adhesion receptor that contributes to neuronal development and synapse formation through homophilic interactions that provide structural-adhesive support to contacts between cell membranes. In addition, N-cadherin homotypic binding may initiate cell signaling that regulates neuronal physiology. In this study, we investigated signaling capabilities of N-cadherin that control voltage activated calcium influx. Using whole-cell voltage clamp recording of isolated inward calcium currents in freshly isolated chick ciliary ganglion neurons we show that the juxtamembrane region of N-cadherin cytoplasmic domain regulates high-threshold voltage activated calcium currents by interacting with p120-catenin and activating RhoA. This regulatory mechanism requires myosin interaction with actin. Furthermore, N-cadherin homophilic binding enhanced voltage activated calcium current amplitude in dissociated neurons that have already developed mature synaptic contacts in vivo. The increase in calcium current amplitude was not affected by brefeldin A suggesting that the effect is caused via direct channel modulation and not by increasing channel expression. In contrast, homotypic N-cadherin interaction failed to regulate calcium influx in freshly isolated immature neurons. However, RhoA inhibitors enhanced calcium current amplitude in these immature neurons, suggesting that the inhibitory effect of RhoA on calcium entry is regulated during neuronal development and synapse maturation. These results indicate that N-cadherin modulates voltage activated calcium entry by a mechanism that involves RhoA activity and its downstream effects on the cytoskeleton, and suggest that N-cadherin provides support for synaptic maturation and sustained synaptic activity by facilitating voltage activated calcium influx.

Journal of Neuroinflammation, 2012

Background: Maternal viral infection during pregnancy is associated with an increase in the incid... more Background: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro-and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment.

Journal of Neurochemistry, 1993

Some snake venoms contain toxins that are reported to be selective for damaging muscle. This spec... more Some snake venoms contain toxins that are reported to be selective for damaging muscle. This specificity can be used to design experiments intended to eliminate muscle. We studied the small myotoxins and fractions IV and V of Bothrops nunmfer venom to evaluate their direct effect on cultured muscle cells, neurons, macrophages, and a fibroblast cell line. The small myotoxins, at 100 pg/ml for 2 h, had no effect in vitro, contrary to the in vivo applications. Fractions IV and V were both myotoxic and, at 100 pg/ml, destroyed all cell types. However, at 10 pg/ml the effects of fraction IV were more selective for muscle. Vacuolation of the sarcoplasmic reticulum and T-tubules was first seen in the poisoned muscles, without initial lesions in the nuclei, sarcolemma, mitochondria, and rough endoplasmic reticulum. Fractions IV and V have different toxic activity in cells other than muscles and are a mixture of two basic proteins (i and ii). Protein ii is predominant in fraction IV and protein i is predominant in fraction V. The toxic effects may be mediated by the formation of nonspecific ionic pores in the sarcolemma and/or T-tubule muscle membrane. Key Words: Myotoxins-Snake venom-Tissue culture-Bolhrops nummifer. Brusks J. L. et al. Specific in vitro biological activity of snake venom myotoxins.

The Journal of Comparative Neurology, 2002

The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) participates in a va... more The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) participates in a variety of developmental processes, including axonal guidance and cell migration. PSA&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s function in these contexts stems from its ability to reduce cell interactions. The present study examines the regulation of PSA expression during formation of the calyciform synapse by the oculomotor axons on chick ciliary neurons. Prior to synaptogenesis, PSA is abundantly and uniformly expressed on the surface of the ciliary neuron body. However, at the time synaptic bonds start to form, as reflected in the localized accumulation of synaptic vesicles, PSA is lost from the point of synaptic contact. Thereafter, PSA is progressively lost from the ciliary neuron surface as the calyx grows. The dense mats of pseudodendritic-like somatic spines, which extend from the postsynaptic cell body, form an exception. These spines, which are known to undergo morphological remodeling, retain PSA expression until the end of embryogenesis. The experimental removal of PSA did not affect synaptogenesis itself, in that no significant changes were observed in the surface covered by the calyx, the number of spine aggregates, the size of acetylcholine receptor clusters, the cell surface area covered by these receptors, or the ultrastructure of the calyx, spine mats, and active zones. Together, these observations suggest that the synapse eliminates PSA as a part of its normal development and that the loss of PSA from the site of axon-target interaction may serve to stabilize structures formed during synaptogenesis.

The Journal of Comparative Neurology, 2011

N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by... more N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection.

Journal of Biological Chemistry, 1995

The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme ... more The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme activity responsible for extension of alpha 2,8-linked sialic acid homopolymers (PSA) associated with the fifth Ig domain of the neural cell adhesion molecule (NCAM). have been studied. The assay for PST used exogenous NCAM as a substrate, with a PSA-specific endoneuraminidase as a control for specificity. Optimal conditions for PST activity at 37 degrees C were found to be pH 6.0 in the presence of divalent cations (Mn2+, 20mM). The enzyme Km was found to increase with increasing polymer length, ranging from 0.7 to 0.07 microns. The developmental regulation both of PST activity and of the addition of PSA to NCAM were studied in chick whole brain, tectum, and cerebellum and found to be precisely coordinated. In each tissue PSA and PST were highest during early stages of morphogenesis, followed by a decrease as development reached completion. The insertion of the VASE exon in the fourth Ig domain of NCAM was also found to parallel closely the developmental down-regulation of PSA, and on this basis could be considered a potential determinant in the specific polysialylation of NCAM. However in direct tests of this hypothesis in transfected cells the presence of VASE did not markedly alter the level of NCAM polysialylation or alter the affinity of PST for the NCAM substrate.