Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum.

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.

Afuni, a novel transforming growth factor-beta gene is involved in arm regeneration by the brittle star Amphiura filiformis.

The bone morphogenetic proteins (BMPs) are a family of the transforming growth factor-beta (TGF-beta) superfamily that perform multiple roles during vertebrate and invertebrate development. Here, we report the molecular cloning of a novel BMP from regenerating arms of the ophiuroid Amphiura filiformis. The theoretically translated amino acid sequence of this novel BMP has high similarity to that of the sea urchin BMP univin. This novel BMP has been named afuni. Whole-mount in situ hybridisation implicates afuni in arm regeneration. Expression occurs in distinct proximal and distal regions of late regenerates (3- and 5-week postablation). These sites are at different stages of regeneration, suggesting multiple roles for this gene in adult arm development. Cellular expression of this gene occurs in migratory cells within the radial water canal (RWC) of regenerating and nonregenerating arms. These migrating coelomocytes suggest a key role for the coelomic RWC as a source of the cellular material for use in arm regeneration by A. filiformis.

Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats.

The effect of cerebral hypoxia-ischemia (HI) on levels and tyrosine phosphorylation of the NMDA receptor was examined in 7- (P7) and 21 (P21)-day-old rats. Unilateral HI was administered by ligation of the right common carotid artery and exposure to an atmosphere of 8% O2/92% N2 for 2 (P7) or 1.5 (P21) h. This duration of HI produces significant infarction in nearly all of the survivors with damage being largely restricted to the cortex, striatum, and hippocampus of the hemisphere ipsilateral to the carotid artery ligation. NR2A levels in the right hemisphere of P7 pups were markedly reduced after 24 h of recovery, while NR1 and NR2B remained unchanged. In contrast, NR2B, but not NR2A, was reduced after HI at P21. At both ages, HI resulted in a transient increase in tyrosine phosphorylation of a number of forebrain proteins that peaked between 1 and 6 h of recovery. At both P7 and P21, tyrosine phosphorylation of NR2B was enhanced 1 h after HI and had returned to basal levels by 24 h. HI induced an increase in tyrosine phosphorylation of NR2A in 21 day, but not in 7-day-old animals. The differential effects of HI on the NMDA receptor at different post-natal ages may contribute to changing sensitivity to hypoxia-ischemia.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor-beta (TGF-beta) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF-beta-like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF-beta-like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non-neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF-beta-like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Distribution of transcript and protein isoforms of the synaptic glycoprotein neuroplastin in rat retina.

PURPOSE: To examine the expression and localization of the neuroplastins (np), two synapse-enriched members of the immunoglobulin (Ig) superfamily of cell-adhesion molecules, in the developing and adult retina and optic nerve. METHODS: Expressions of the two isoforms np55 and np65 and carboxyl-terminal splice variants were investigated by immunocytochemistry, Western blot analysis, RT-PCR, and in situ hybridization. RESULTS: Immunoreactivity for both neuroplastins was confined to the two synaptic layers of the retina: the inner (IPL) and outer plexiform layer (OPL). Significant overlap was found in staining at synaptic structures with synaptophysin. A large proportion of immunoreactivity for both isoforms, however, was of perisynaptic origin. In situ hybridization studies were suggestive of a pre- and postsynaptic localization of np65 in the OPL. Transcripts for np55 were already present at birth in the inner retina, but the hybridization signals increased during postnatal development. Np65 transcripts and immunosignals appeared at later developmental ages, concomitant with synapse formation in the OPL. Several C-terminal neuroplastin cDNA clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in the intracellular domain of neuroplastins. Splice isoforms containing the insert exhibited a developmental expression pattern similar to that of np55; however, both neuroplastins could harbor the C-terminal insert. Neuroplastins were also detected in optic nerve homogenates. RT-PCR and blockade of axonal transport by nerve crush confirmed transcript and protein expression in optic nerve tissue. CONCLUSIONS: The findings suggest a role for neuroplastins in cell adhesion in the plexiform layers during histogenesis, as well as in maintenance of connections between specific cellular structures.

Growth factors, heat-shock proteins and regeneration in echinoderms.

The study of regeneration in armed echinoderm species, including crinoids, ophiuroids and asteroids, is attracting increasing attention. Recent interest has focused on the presence and potential role of growth factors, including members of the nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) families, in the regenerative process and their possible relationship to the normal developmental (ontogenetic) regulatory cascade. In addition, the expression patterns of the heat-shock family of stress proteins (Hsps) during regeneration are also important. Their role forms part of a normal stress response to the trauma of autotomy in combination with a putative function in tissue remodelling and associated protein turnover during regeneration. The temporal dynamics of the stress response may also be strongly indicative of environmentally adaptive pressures operating on these systems.

Molecular approach to echinoderm regeneration.

Until very recently echinoderm regeneration research and indeed echinoderm research in general has suffered because of the lack of critical mass. In terms of molecular studies of regeneration, echinoderms in particular have lagged behind other groups in this respect. This is in sharp contrast to the major advances achieved with molecular and genetic techniques in the study of embryonic development in echinoderms. The aim of our studies has been to identify genes involved in the process of regeneration and in particular neural regeneration in different echinoderm species. Our survey included the asteroid Asterias rubens and provided evidence for the expression of Hox gene homologues in regenerating radial nerve cords. Present evidence suggests: 1) ArHox1 expression is maintained in intact radial nerve cord and may be upregulated during regeneration. 2) ArHox1 expression may contribute to the dedifferentiation and/or cell proliferation process during epimorphic regeneration. From the crinoid Antedon bifida, we have been successful in cloning a fragment of a BMP2/4 homologue (AnBMP2/4) and analysing its expression during arm regeneration. Here, we discuss the importance of this family of growth factors in several regulatory spheres, including maintaining the identity of pluripotent blastemal cells or as a classic skeletal morphogenic regulator. There is clearly substantial scope for future echinoderm research in the area of molecular biology and certain aspects are discussed in this review.

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

Different dystrophin-like complexes are expressed in neurons and glia.

Duchenne muscular dystrophy is a fatal muscle disease that is often associated with cognitive impairment. Accordingly, dystrophin is found at the muscle sarcolemma and at postsynaptic sites in neurons. In muscle, dystrophin forms part of a membrane-spanning complex, the dystrophin-associated protein complex (DPC). Whereas the composition of the DPC in muscle is well documented, the existence of a similar complex in brain remains largely unknown. To determine the composition of DPC-like complexes in brain, we have examined the molecular associations and distribution of the dystrobrevins, a widely expressed family of dystrophin-associated proteins, some of which are components of the muscle DPC. beta-Dystrobrevin is found in neurons and is highly enriched in postsynaptic densities (PSDs). Furthermore, beta-dystrobrevin forms a specific complex with dystrophin and syntrophin. By contrast, alpha-dystrobrevin-1 is found in perivascular astrocytes and Bergmann glia, and is not PSD-enriched. alpha-Dystrobrevin-1 is associated with Dp71, utrophin, and syntrophin. In the brains of mice that lack dystrophin and Dp71, the dystrobrevin-syntrophin complexes are still formed, whereas in dystrophin-deficient muscle, the assembly of the DPC is disrupted. Thus, despite the similarity in primary sequence, alpha- and beta-dystrobrevin are differentially distributed in the brain where they form separate DPC-like complexes.

Hypoxia-ischemia in perinatal rat brain induces the formation of a low molecular weight isoform of striatal enriched tyrosine phosphatase (STEP).

Protein tyrosine phosphatases play a critical role in controlling tyrosine phosphorylation levels of proteins. Ischemia induces changes in tyrosine phosphorylation. As part of our investigations of the mechanisms responsible for these changes, we studied the effects of cerebral hypoxia-ischemia in 7-day-old (P7) and P21 rat brains on expression of the STEP (striatal enriched phosphatase) family of protein tyrosine phosphatases. P7 and P21 rats were subjected to unilateral hypoxia-ischemia, and brains were analyzed at various intervals of recovery for the presence of STEP. Hypoxia-ischemia induced the formation of a low Mr isoform of STEP, STEP33, in the ipsilateral (damaged) hemisphere but not in the contralateral (undamaged) side. STEP33 produced as a result of ischemia was located exclusively in the cell soluble fraction. In P21 rats, the ischemia-induced elevation in STEP33 was delayed relative to P7 rats. STEP33 was produced by digestion of postsynaptic densities with calpain I and by exposure of NT2/D1 cells expressing STEP to the calcium ionophore A23187. The results suggest that ischemia-induced calcium influx results in the calcium-dependent proteolysis of membrane-associated STEP61 and the concomitant release of STEP33 into the cytoplasm.

Age-associated differential expression of Na(+)-K(+)-ATPase subunit isoforms in skeletal muscles of F-344/BN rats.

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.

Preliminary observations on ascidian and echinoderm neurons and neural explants in vitro.

As part of a study on echinoderm and ascidian neural regeneration, attempts were made to develop a system for the maintenance of their neurons in vitro. It was found that neurons and neural tissue explants from the starfish, Asterias rubens, and the brittlestar, Ophiura ophiura, and explants from the brain of the ascidian, Ciona intestinalis, could be cultured for up to 6 weeks in a modified L15-based medium. Some cells extended axonal projections and produced growth cones under certain conditions. Attempts were made to stimulate neuron survival and outgrowth of echinoderm cultures with conditioned media containing growth factors or tissue extracts and with various substrates including extracellular matrix extracts from native tissue. Ascidian brain explants from both normal and regenerating animals were cultured in the standard conditions established for echinoderm tissue, with outgrowth being observed in 25% of explants. In these cultures labelling with bromodeoxyuridine suggested that regeneration continues in vitro, although results using substance P immunocytochemistry indicate neuronal differentiation may be impeded. These preliminary studies suggest it is possible to maintain adult echinoderm and ascidian neurons in vitro.

Immunoglobulin superfamily members gp65 and gp55: tissue distribution of glycoforms.

Gp65 and gp55 are immunoglobulin superfamily members produced by alternative splicing of the same gene transcript, and originally identified as components of synaptic membranes. A monoclonal antibody specific for gp65 and gp55 has been used to detect immunoreactive species in a wide range of tissues. All immunoreactive species bind to concanavalin A and deglycosylation studies show that in all tissues tested other than brain the immunoreactive species are derived from gp55. HEK cells transfected with gp65 or gp55 express different glycoforms from brain showing that the pattern of glycosylation of these molecules is dependent upon the cell type in which they are expressed.

Hypoxia-ischemia induces a rapid elevation of ubiquitin conjugate levels and ubiquitin immunoreactivity in the immature rat brain.

Postnatal rats at 7 and 21 days of age were subjected to unilateral hypoxia-ischemia (H/I) by right carotid artery ligation followed by 1.5 to 2 hours of hypoxia (8% oxygen). Brains were frozen at specific intervals of recovery from 0 to 24 hours. Western blots of samples of right and left forebrain were immunodeveloped with a monoclonal antibody specific for ubiquitin, RHUb1. An elevation of ubiquitin conjugate levels in the right compared with the left forebrain of 7-day-old animals was detectable immediately following H/I and increased by close to 60% of control level within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but had declined to control levels by 24 hours of recovery. No such increase was observed in response to hypoxia alone. Similar changes were observed in samples from the 21-day-old rat brain. However, the elevation of ubiquitin conjugate levels was of slower onset and persisted longer than observed for the 7-day-old animals. Immunocytochemical studies of brain fixed by immersion in formaldehyde/acetone/methanol showed that ubiquitin-like immunoreactivity was increased in the right, but not left, cerebral cortex and hippocampus of animals subjected to H/I. The data suggest that elevated ubiquitination may represent a neuroprotective response to H/I.

Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

Synaptic membrane glycoproteins gp65 and gp55 are new members of the immunoglobulin superfamily.

Glycoproteins gp65 and gp55 are major components of synaptic membranes prepared from rat forebrain. Both are recognized by the monoclonal antibody SMgp65. We have used SMgp65 to screen a rat brain cDNA expression library. Two sets of overlapping cDNAs that contain open reading frames of 397 and 281 amino acids were isolated. The deduced proteins are members of the immunoglobulin (Ig) superfamily containing three and two Ig domains, respectively. The common part has approximately 40% sequence identity with neurothelin/basigin. The identity of the proteins as gp65 and gp55 was confirmed by production of new antisera against a common recombinant protein fragment. These antisera immunoprecipitate gp65 and gp55. Furthermore, expression of gp65 and gp55 cDNAs in human 293 cells treated with tunicamycin results in the production of unglycosylated core proteins of identical size to deglycosylated gp65 and gp55. Northern analysis revealed that gp65 transcripts are brain-specific, whereas gp55 is expressed in most tissues and cell lines examined. The tissue distribution was confirmed at the protein level though the pattern of glycosylation of gp55 varies between tissues. In situ hybridization experiments with a common and a gp65-specific probe suggest differential expression of gp65 and gp55 transcripts in the rat brain.

JSJ-1, an anti-spermidine monoclonal antibody with potential clinical applications.

Polyamines have been implicated in a wide variety of functions including nucleic acid synthesis and protein synthesis. Their levels have been shown to increase in response to cell growth and differentiation. Use of polyamines as prognostic indicators of proliferative disease conditions has been hindered by the lack of suitable rapid and sensitive assays. We report the characterization of an anti-spermidine antibody, JSJ-1, with novel putrescine cross reactivity. JSJ-1 cross-reacts more strongly with putrescine (11%) than with spermine (6%). This suggests that the aminobutyl group common to both putrescine and spermidine is an important element in the antibody-antigen interaction. We have demonstrated that antibody-spermidine binding is effected by increased ionic strength. This finding is consistent with the antibody-antigen interaction being ionic. The JSJ-1 antibody has been successfully used to detect increased polyamine levels in clinical serum samples and identify those with increased polyamine levels.

Beta-dystroglycan: subcellular localisation in rat brain and detection of a novel immunologically related, postsynaptic density-enriched protein.

The distribution of a glycoprotein component of the muscle dystrophin complex, beta-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that beta-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle beta-dystroglycan) abolished reactivity against both beta-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of beta-dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species.

N-cadherin is a major glycoprotein component of isolated rat forebrain postsynaptic densities.

We have previously described a monoclonal antibody, PAC 1, that recognises two postsynaptic density (PSD)-enriched glycoproteins (pgps) of apparent M(r) 130,000 (pgp130) and 117,000 (pgp117). Immunodevelopment of western blots of rat forebrain homogenate, synaptic membrane (SM), and PSD samples with PAC 1 and an N-cadherin antiserum shows that pgp130 and N-cadherin are of identical apparent M(r) and show identical patterns of enrichment in these fractions. The apparent molecular masses of pgp130 and N-cadherin are both lowered by 11 kDa following removal of N-linked carbohydrate with endoglycosidase-F containing N-glycopeptidase. The two molecules show an identical pattern of migration when separated by two-dimensional electrophoresis. A single 130-kDa band immunoprecipitated from solubilised PSD preparations by the N-cadherin antiserum is recognised by PAC 1 on western blots. We conclude that pgp130 is N-cadherin. Development of western blots of two-dimensional gel separations of SM and PSD glycoproteins shows that N-cadherin is a major glycoprotein component of PSDs. The immunoprecipitation experiments show that the M(r) of N-cadherin is greater than that of the major pgp, PSD gp116. The PAC 1 antibody recognises two concanavalin A-binding glycoproteins with apparent molecular masses of 136 and 127 kDa in liver samples. The 136-kDa band is also recognised by the N-cadherin antiserum. These observations, together with data showing that the PAC 1 epitope is intracellular, suggest that PAC 1 is a pan-cadherin antibody and recognises an epitope on the conserved cadherin intracellular carboxyl-terminal domain.