Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Persistent cloaca with fetal ascites: clinical features and perinatal management.

PURPOSE: Fetuses with persistent cloaca are known to develop urine or meconium backflow into the abdominal cavity caused by obstruction of the common channel, thus leading to fetal peritonitis with fetal ascites. We analyzed the impact of prenatal fetal ascites on postnatal clinical features and management. METHODS: This retrospective single-center cohort study was conducted to compare the perinatal parameters of patients with isolated persistent cloaca who were born and treated at our hospital between 1991 and 2021. The clinical features and management of those with and without fetal ascites were compared. RESULTS: Among the 17 eligible patients, fetal ascites were recognized in seven. The occurrence of fetal ascites was significantly related to preterm birth, higher birth weight z-score, birth via emergency cesarean delivery, low Apgar scores at 1 min and 5 min, higher C-reactive protein levels at birth, longer duration of oxygen administration, the need for a urinary drainage catheter at initial discharge, and shorter neonatal hospital stays. CONCLUSIONS: The postnatal management of patients with persistent cloaca with fetal ascites differed significantly from that of patients without fetal ascites. For patients with unexplained fetal ascites, magnetic resonance imaging may be helpful for determining the definite diagnosis of persistent cloaca.

Factors associated with neonatal surgical site infection after abdominal surgery.

PURPOSE: To assess the risk factors for surgical site infections (SSIs) post-abdominal surgery in neonates. METHODS: A retrospective, single-center cohort study was conducted using patient data from 2009 to 2018. Patient characteristics and several variables were analyzed to identify independent risk factors for SSI. RESULTS: SSI occurred in 39/406 procedures (9.6%). Univariate analysis showed that the incidence of SSI was significantly higher in patients who had undergone multiple surgical procedures (P = 0.032), prolonged operations (P = 0.016), long-term hospitalization (P < 0.001), long-term antibiotic administration (P < 0.001), with methicillin-resistant Staphylococcus aureus (MRSA) colonization (P = 0.044), contaminated/dirty wounds (P < 0.001), and American Society of Anesthesiologists physical status of 3 or 4 (P = 0.021). Multivariate analysis identified prolonged operations [odds ratio (OR): 2.91 (1.21-8.01)] and contaminated/dirty wounds [OR: 5.42 (2.41-12.1)] as independent risk factors. Patients with SSI had a higher incidence of MRSA colonization (27.8% vs. 14.8%, P = 0.044), longer antibiotic administration (24 days vs. 8 days, P = 0.049), and longer hospitalization times (98 days vs. 43 days, P = 0.007) than those without SSIs. CONCLUSIONS: Long operations exceeding 100 min and surgical procedures with contaminated/dirty wounds are independent risk factors for neonatal SSIs after abdominal surgery. SSIs were related to MRSA colonization during hospitalization, long-term antibiotic administration, and long-term hospitalization.

The effects of phospholipids and fatty acids on the oligomer formation of NAP-22.

Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM fraction of the neuronal cell membrane. Previous studies showed tight binding activity of NAP-22 to acidic membrane lipids and the self-interaction of NAP-22, i.e., oligomerization. In this study, the effect of various phospholipids, lysophospholipids and fatty acids on the oligomerization of NAP-22 was studied through SDS-PAGE after chemical cross-linking and electron microscopic observation. High molecular mass oligomers were detected by SDS-PAGE after incubation in solutions containing over 20 mM NaCl at pH 6.5-8.5, even in the absence of lipid addition, and the addition of Ca2+/calmodulin abolished oligomerization. Higher molecular mass oligomer formation after incubation with acidic phospholipids was detected with gradient SDS-PAGE. Much higher mass oligomers were detected in the presence of polyunsaturated fatty acids. Electron microscopic analysis of the samples after SDS treatment showed tangled rope-like structures. Liposome-bound NAP-22 showed small oval or annular structures after cross-linking and SDS treatment. These oligomers were suggested to make the tangled rope-like structures, for annular structures of the same size were observed in the structure. These results suggest the participation of NAP-22 to liquid-liquid phase separation through oligomerization.

Raftophilic rhodopsin-clusters offer stochastic platforms for G protein signalling in retinal discs.

Rhodopsin is a G protein-coupled receptor (GPCR) that initiates the phototransduction cascade in retinal disc membrane. Recent studies have suggested that rhodopsin forms highly ordered rows of dimers responsible for single-photon detection by rod photoreceptors. Dimerization is also known to confer to rhodopsin a high affinity for ordered lipids (raftophilicity). However, the role of rhodopsin organization and its raftophilicity in phototransduction remains obscure, owing to the lack of direct observation of rhodopsin dynamics and distribution in native discs. Here, we explore the single-molecule and semi-multimolecule behaviour of rhodopsin in native discs. Rhodopsin forms transient meso-scale clusters, even in darkness, which are loosely confined to the disc centre. Cognate G protein transducin co-distributes with rhodopsin, and exhibits lateral translocation to the disc periphery upon activation. We demonstrate that rhodopsin offers inherently distributed and stochastic platforms for G protein signalling by self-organizing raftophilic clusters, which continually repeat generation/extinction in the disc membrane.

Interaction of dynamin I with NAP-22, a neuronal protein enriched in the presynaptic region.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant low-density membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM and localizes in the presynaptic region. In order to know the role of NAP-22 in the synaptic transmission, NAP-22 binding proteins in the cytosol were searched with an affinity screening with NAP-22 as a bait and several protein bands were detected. Using mass-analysis and western blotting, one of the main band of ∼90 kDa was identified as dynamin I. The GTPase activity of dynamin I was partly inhibited by NAP-22 expressed in bacteria and this inhibition was recovered by the addition of calmodulin, a NAP-22 binding protein. The GTPase activity of dynamin was known to be activated with acidic membrane lipids such as phosphatidylserine and the addition of NAP-22, a phosphatidylserine binding protein, inhibited the activation of the GTPase by this lipid. Since NAP-22 localizes on the presynaptic plasma membrane and on synaptic vesicles, these results suggest the participation of NAP-22 in the membrane cycling through binding to dynamin and acidic membrane lipids at the presynaptic region.

Inhibitory effect of several sphingolipid metabolites on calcineurin.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM. In a previous study, we showed that DRM-derived NAP-22 binds ganglioside and the inhibitory effect of ganglioside to calcineurin (CaN), a neuron-enriched calmodulin-regulated phosphoprotein phosphatase. Considering the important roles of CaN in neurons, identification of other cellular regulators of CaN could be a good clue to understand the molecular background of neuronal function. In this study, we screened the effect of several membrane lipid-derived molecules on the CaN activity and found sphingosine and some sphingosine-derived metabolites such as sphingosylphosphorylcholine, galactosylsphingosine (psychosine), and glucosylsphingosine, have inhibitory effect on CaN through the interaction with calmodulin.

Astrocyte calcium waves propagate proximally by gap junction and distally by extracellular diffusion of ATP released from volume-regulated anion channels.

Wave-like propagation of [Ca2+]i increases is a remarkable intercellular communication characteristic in astrocyte networks, intercalating neural circuits and vasculature. Mechanically-induced [Ca2+]i increases and their subsequent propagation to neighboring astrocytes in culture is a classical model of astrocyte calcium wave and is known to be mediated by gap junction and extracellular ATP, but the role of each pathway remains unclear. Pharmacologic analysis of time-dependent distribution of [Ca2+]i revealed three distinct [Ca2+]i increases, the largest being in stimulated cells independent of extracellular Ca2+ and inositol 1,4,5-trisphosphate-induced Ca2+ release. In addition, persistent [Ca2+]i increases were found to propagate rapidly via gap junctions in the proximal region, and transient [Ca2+]i increases were found to propagate slowly via extracellular ATP in the distal region. Simultaneous imaging of astrocyte [Ca2+]i and extracellular ATP, the latter of which was measured by an ATP sniffing cell, revealed that ATP was released within the proximal region by volume-regulated anion channel in a [Ca2+]i independent manner. This detailed analysis of a classical model is the first to address the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP.

Multiple pathways for elevating extracellular adenosine in the rat hippocampal CA1 region characterized by adenosine sensor cells.

Extracellular adenosine in the brain, which modulates various physiological and pathological processes, fluctuates in a complicated manner that reflects the circadian cycle, neuronal activity, metabolism, and disease states. The dynamics of extracellular adenosine in the brain are not fully understood, largely because of the lack of simple and reliable methods of measuring time-dependent changes in tissue adenosine distribution. This study describes the development of a biosensor, designated an adenosine sensor cell, expressing adenosine A1 receptor, and a genetically modified G protein. This biosensor was used to characterize extracellular adenosine elevation in brain tissue by measuring intracellular calcium elevation in response to adenosine. Placement of adenosine sensor cells below hippocampal slices successfully detected adenosine releases from these slices in response to neuronal activity and astrocyte swelling by conventional calcium imaging. Pharmacological analyses indicated that high-frequency electrical stimulation-induced post-synaptic adenosine release in a manner dependent on L-type calcium channels and calcium-induced calcium release. Adenosine release following treatments that cause astrocyte swelling is independent of calcium channels, but dependent on aquaporin 4, an astrocyte-specific water channel subtype. The ability of ectonucleotidase inhibitors to inhibit adenosine release following astrocyte swelling, but not electrical stimulation, suggests that the former reflects astrocytic ATP release and subsequent enzymatic breakdown, whereas the latter reflects direct adenosine release from neurons. These results suggest that distinct mechanisms are responsible for extracellular adenosine elevations by neurons and astrocytes, allowing exquisite regulation of extracellular adenosine in the brain.

Pharmacological characterization of the involvement of protein kinase C in oscillatory and non-oscillatory calcium increases in astrocytes.

Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors.

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity.

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 104/cm2 showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 104/cm2 largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.

Tight binding of NAP-22 with acidic membrane lipids.

Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. Insolubility of the outer leaflet of DRM to the non-ionic detergent is ascribed to the tight association of cholesterol and sphingolipid. Since, poor localization of sphingolipid is observed in the inner leaflet, the physicochemical background of the insolubility of the inner leaflet is hence still an enigma. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM of the neuronal cell membrane. A previous study showed the presence of several lipids in a NAP-22 fraction after the process of extraction and column chromatography. In this study, the effect of lipid extraction on NAP-22 was studied through native-gel electrophoresis, ultracentrifugation, and electron microscopic observation. The mobility of NAP-22 in native-PAGE was shifted from low to high after delipidation. Delipidated NAP-22 bound phosphatidylserine (PS), phosphatidylinosotol, and ganglioside. Some part of the mixture of PS and NAP-22 was recovered in the insoluble fraction after Triton X-100 treatment and the addition of cholesterol enhanced the amount of NAP-22 in the insoluble fraction.

Ganglioside contained in the neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) fraction prepared from the detergent-resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast.

Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD).

NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched protein localized mainly in the synaptic vesicles and the synaptic plasma membrane. Biochemically, it is recovered in the lipid raft fraction. In order to understand the physiological function of the neuronal lipid raft, NAP-22 binding proteins were screened with a pull-down assay. Glutamic acid decarboxylase (GAD) was detected through LC-MS/MS, and Western blotting using a specific antibody confirmed the result. Two isoforms of GAD, GAD65 and GAD67, were expressed in bacteria as GST-fusion forms and the interaction with NAP-22 was confirmed in vitro. Partial co-localization of NAP-22 with GAD65 and GAD67 was also observed in cultured neurons. The binding showed no effect on the enzymatic activity of GAD65 and GAD67. These results hence suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.

Identification of dynamin as a septin-binding protein.

Lipid rafts (detergent-resistant low-density membrane microdomain: DRM) are signal-transducing membrane platforms. In a previous study, we showed maturation-dependent localization of septin in the DRM fraction of rat brain. Mammalian septin is composed with 13-14 isoforms and these isoforms assemble to form rod-shaped hetero-oligomeric complexes. End-to-end polymerization of these complexes results in the formation of higher order structures such as filamentous sheets or bundles of filaments that restrict the fluid-like diffusion of the membrane proteins and lipids. Considering the function of septin as the membrane scaffold, elucidation of the molecular interaction of septin in DRM could be a breakthrough to understand another role of lipid rafts. In order to identify septin-binding proteins in DRM, solubilization and fractionation of septin from DRM was attempted. Several proteins were co-fractionated with septin and LC-MS/MS analysis identified one of these proteins as dynamin and Western blotting using anti-dynamin confirmed this result. Immunoprecipitation of septin11 in a crude supernatant showed co-precipitation of dynamin and dynamin fraction prepared from brain contained several septin isoforms. Within bacterially expressed septin isoforms, septin5 and septin11 bound dynamin but septin9 did not. These results suggest that some septin isoforms participate in the dynamin-related membrane dynamics.

Structures of septin filaments prepared from rat brain and expressed in bacteria.

Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl(2) solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ~0.4µm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ~0.6µm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft.

Inhibitory effect of NAP-22 on the phosphatase activity of synaptojanin-1.

Endocytosis of the synaptic vesicle is a complicated process, in which many proteins and lipids participate. Phosphatidylinositol 4,5-bisphosphate (PIP(2) ) plays important roles in the process, and the dynamic regulation of this lipid is one of the key events. Synaptojanin is a PIP(2) phosphatase, and dephosphorylation of PIP(2) of the clathrin coated-vesicle results in the uncoating of the vesicle. NAP-22 is one of the major proteins of the neuronal detergent-resistant membrane microdomain and localizes in both the presynaptic plasma membrane and the synaptic vesicle. To elucidate the role of NAP-22 in synaptic function, a screening of the NAP-22 binding proteins through pull-down assay was performed. In addition to CapZ protein, synaptojanin-1 was detected by LC-MS/MS, and Western blotting using antisynaptojanin-1 confirmed this result. The interaction seems to be important in the course of synaptic vesicle endocytosis, because NAP-22 inhibited the phosphatase activity of synaptojanin in a dose-dependent manner. The inhibitory region for 5-phosphatase and the binding region for PIP(2) overlapped in the amino acid sequence of NAP-22, so elucidation of the regulatory mechanism of the PIP(2) binding ability of NAP-22 could be important in understanding the membrane dynamics at the presynaptic region.

Calcium fluxes cause nuclear shrinkage and the translocation of phospholipase C-delta1 into the nucleus.

Phospholipase C-delta1 (PLCdelta1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCdelta1 shuttles between the cytoplasm and nucleus, and an influx of Ca2+ triggers the nuclear import of PLCdelta1 via Ca2+-dependent interaction with importin beta1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCdelta1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCdelta1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCdelta1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca2+ caused similar results. Furthermore, overexpression of GFP-PLCdelta1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCdelta1 gene-knockout mice (PLCdelta1KO-MEF). By contrast, an E341A mutant that cannot bind with importin beta1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCdelta1KO-MEF. Nuclear translocation and the PLC activity of PLCdelta1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca2+.

OBCAM, an immunoglobulin superfamily cell adhesion molecule, regulates morphology and proliferation of cerebral astrocytes.

Opioid-binding cell adhesion molecule (OBCAM) is a member of the immunoglobulin superfamily. In the present study, we investigated the expression and functional significance of OBCAM in cerebral astrocytes in vitro and in vivo. The confocal microscopic study revealed that the expression of OBCAM was prominent at filopodia and cellular processes of type-1 and -2 astrocytes. Moreover, the expression of OBCAM was shown to be stronger at the growth phase than the stationary one in type-1 ones. Ki-67-positive proliferating type-1 astrocytes showed higher frequency of OBCAM expression as compared with Ki-67-negative cells. The application of the anti-OBCAM IgG facilitated the proliferation of type-1 astrocytes at relatively low concentration (1 and 5 microg/mL) and had no effects at relatively high concentration (25 and 50 microg/mL). The cellular proliferation was not promoted when the anti-OBCAM Fab fragment was used, indicating that the antibody-induced clustering of OBCAM promotes the proliferation. The over-expression of OBCAM increased the cell size of type-1 astrocytes. Moreover, intense expression of OBCAM was observed on reactive astrocytes in the injured cerebrum in vivo. These results suggest that OBCAM is responsible for controlling the cell proliferation and growth in cortical astrocytes.