Involvement of gicerin in the extension of microvilli.

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.

Amida predominantly expressed and developmentally regulated in rat testis.

Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Previous studies showed that Amida is a nuclear protein and overexpression of Amida induces cell apoptosis. In this study, we found that Amida mRNA was expressed predominantly in rat testis by Northern blot analysis. During the development of testis, Amida mRNA was barely detectable until postnatal days 24 to 29 during which it increased to levels found in adults. However, Amida protein was not detected until postnatal day 32. Amida mRNA was found to be enriched in spermatocytes and less in round spermatids, but was undetectable in elongated spermatids by in situ hybridization. In addition, Amida protein was observed in the nucleus of spermatocytes and even in the elongated spermatids by immunohistochemistry. The development and cellular localization differences of Amida mRNA and protein implicates that Amida mRNA may undergo posttranscriptional regulation. Furthermore, Amida mRNA decreased significantly in the 8-day experimental cryptorchid testis when spermatogenesis was disrupted. Taken together, these data suggest that Amida is involved in spermatogenesis and may play an important role in development of testicular germ cells.

The role of gicerin, a novel cell adhesion molecule, in development, regeneration and neoplasia.

Neurite outgrowth factor (NOF) is an extracellular matrix (ECM) protein in the laminin family and its ligand, gicerin, is a novel cell adhesion molecule in the immunoglobulin superfamily. Gicerin has a homophilic adhesive activity as well as a heterotypic manner to NOF. In the nervous systems, gicerin is expressed during developmental stage when neurons migrate or extend neurites to form a neural network. Gicerin promotes neurite extension and migration of embryonic neurons in vitro by its homophilic and heterophilic adhesion activities. Introduction of antigicerin antibody into early developing eyes perturbs the layer formation of neural retina. These data suggest that gicerin participates in the formation of neural tissues. Gicerin is also expressed in other non-neural tissues; in epithelia of trachea, kidney and oviduct, gicerin expression is restricted in the developmental period. In contrast, muscular tissues and endothelial cells express gicerin continuously even after maturation. Interestingly, gicerin re-appears strongly in the regenerating epithelia of trachea, kidney and oviduct, and also anti-gicerin antibody disrupts the healing process of trachea. Furthermore, gicerin and NOF are overexpressed in the chicken nephroblastomas (Wilm's tumor) and oviductal adenocarcinomas. In vitro analyses show that gicerin adhesive activities can promote binding among tumor cells and adhesion of tumor cells to NOF. A polyclonal antibody against gicerin also perturbs the re-attachment of cancer cells onto metastasizing sites. It is clear from these studies that gicerin is a potential effector for pathological tissue formation as well as for normal development.

Expression and adhesive ability of gicerin, a cell adhesion molecule, in the pock lesions of chorioallantoic membranes infected with an avian poxvirus.

The expression and adhesive activities of gicerin, a cell adhesion protein, in the pock lesions on chicken chorioallantoic membranes (CAM) infected with an avian poxvirus were studied. In normal CAMs, gicerin was found on the flattened epithelial cells, and neurite outgrowth factor (NOF) was in the basement membrane. However, in the pock lesions on infected CAMs, gicerin was overexpressed on the cell membranes of hyperplastic epithelial cells forming thick epithelial layers. Neurite outgrowth factor was also found mainly in the basement membrane, but occasionally showed aberrant expression among hyperplastic cells. In vitro analyses, using the dissociated cells from pock lesions, demonstrated that an anti-gicerin polyclonal antibody inhibit cell aggregation activity and cell adhesion to NOF. These results suggest that gicerin might promote the cell-cell and cell-extracellular matrix protein bindings of the hyperplastic epithelial cells by its homophilic and heterophilic adhesive activities, and contribute to pock formation on the infected CAMs.

Kheper, a novel ZFH/deltaEF1 family member, regulates the development of the neuroectoderm of zebrafish (Danio rerio).

Kheper is a novel member of the ZFH (zinc-finger and homeodomain protein)/deltaEF1 family in zebrafish. kheper transcripts are first detected in the epiblast of the dorsal blastoderm margin at the early gastrula stage and kheper is expressed in nearly all the neuroectoderm at later stages. kheper expression was expanded in noggin RNA-injected embryos and also in swirl mutant embryos and was reduced in bmp4 RNA-injected embryos and chordino mutant embryos, suggesting that kheper acts downstream of the neural inducers Noggin and Chordino. Overexpression of Kheper elicited ectopic expansion of the neuroectoderm-specific genes fkd3, hoxa-1, and eng3, and the ectopic expression of hoxa-1 was not inhibited by BMP4 overexpression. Kheper interacted with the transcriptional corepressors CtBP1 and CtBP2. Overexpression of a Kheper mutant lacking the homeodomain or of a VP16-Kheper fusion protein disturbed the development of the neuroectoderm and head structures. These data underscore the role of Kheper in the development of the neuroectoderm and indicate that Kheper acts as a transcriptional repressor.

Somatodendritic localization of Translin, a component of the Translin/Trax RNA binding complex.

Recent studies implicating dendritic protein synthesis in synaptic plasticity have focused attention on identifying components of the molecular machinery involved in processing dendritic RNA. Although Translin was originally identified as a protein capable of binding single-stranded DNA, subsequent studies have demonstrated that it also binds RNA in vitro. Because previous studies indicated that Translin-containing RNA/single-stranded DNA binding complexes are highly enriched in brain, we and others have proposed that it may be involved in dendritic RNA processing. To assess this possibility, we have conducted studies aimed at defining the localization of Translin and its partner protein, Trax, in brain. In situ hybridization studies demonstrated that both Translin and Trax are expressed in neurons with prominent staining apparent in cerebellar Purkinje cells and neuronal layers of the hippocampus. Subcellular fractionation studies demonstrated that both Translin and Trax are highly enriched in the cytoplasmic fraction compared with nuclear extracts. Furthermore, immunohistochemical studies with Translin antibodies revealed prominent staining in Purkinje neuron cell bodies that extends into proximal and distal dendrites. A similar pattern of somatodendritic localization was observed in hippocampal and neocortical pyramidal neurons. These findings demonstrate that Translin is expressed in neuronal dendrites and therefore support the hypothesis that the Translin/Trax complex may be involved in dendritic RNA processing.

Inhibition by naloxone of promoter activity of the neurofilament gene in SK-N-SH cells.

Chronic administration of morphine is known to decrease the levels of neurofilaments (NFs) in the ventral tegmental area. We ligated a promoter region of the mouse 68-KDa neurofilament (NF-68) gene to the pGL3-enhancer vector containing a luciferase gene, transfected it into SK-N-SH cells and then analyzed transcriptional activity in the cells treated with agonists or antagonists of opiate receptors. The activity of the NF-68 promoter was suppressed by naloxone about 55% at 10(-5) M and 30% at 10(-7) M at 48 h, but suppressed not by morphine. Naltrexone at 10(-5) M suppressed the promoter activity about 20%, but levallorphan, DAMGO, DPDPE and U50488 did not. The inhibition by naloxone was dose-dependent and not reversed by morphine. The inhibitory effect of naloxone was not observed in N18TG-2 cells and PC12 cells. Experiments with various deletion mutants revealed that a region responsible for naloxone suppression spans from -328 to -101 in the gene. These results suggest that naloxone has the ability to suppress transcriptional activity in some neurons.

Molecular cloning and characterization of Amida, a novel protein which interacts with a neuron-specific immediate early gene product arc, contains novel nuclear localization signals, and causes cell death in cultured cells.

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule.

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.

Expression of gicerin, a cell adhesion molecule, in the abnormal retina in silver plumage color mutation of Japanese quail (Coturnix japonica).

Silver plumage color mutant (B/B) quail has an abnormal retina characterizing the transdifferentiation of retinal pigment epithelium (RPE) following the retinal separation in the early developmental stage. In the present study; (i) the expression of gicerin, an immunoglobulin-superfamily cell adhesion molecule, was examined in the retina of B/B quail. In the wild-type quail, gicerin protein was enriched in the apical membrane (facing the neural retina, NR) of RPE cells on embryonic day (E) 4 and then appeared also in NR cells from E5. However, in the B/B retina, no gicerin expression was found in the transdifferentiation area of RPE prior to the retinal separation. (ii) In addition to this, microinjection of anti-gicerin polyclonal antibody into the eyeball of wild-type quail on E3 caused the retinal separation and induced the transdifferentiation of RPE into new NR. These observations suggest that the decrease of gicerin expression might participate in the retinal separation and RPE-transdifferentiation in B/B quail.

[Functional analysis of a novel cell adhesion molecule, gicerin].

Recent studies identified a novel cell adhesion molecule, gicerin, that exists on the surface of developing neurons. Determination of the amino acid sequence revealed that this molecule has five immunoglobulin-like structures in its extracellular domain and a short cytoplasmic tail. Gicerin binds in a homophilic manner and also displays heterophilic binding activity to NOF (Neurite Outgrowth Factor), which belongs to the laminin family extracellular matrix molecule. We clarified that there are two subtypes of gicerin that differ only in the cytoplasmic domain. S-gicerin, which has smaller tail, has stronger activity in cell aggregation or NOF binding. This suggests a physiological difference in the activity of each subtype. In the nervous system, gicerin is expressed during its developmental stage when neurons migrate or extend neurites to form a neural network. Gicerin promotes neurite extension from embryonic neurons by both homophilic adhesion and heterophilic adhesion to NOF. These data suggest that gicerin participates in the formation of neural tissues. Gicerin is also expressed in other tissues such as the kidney and trachea. In these tissues, gicerin expression is also observed during the regeneration process and in tumors in addition to being present during the developmental stage. We believe that gicerin plays an important role in the histogenesis of the nervous system as well as other tissues.

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken.

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.

Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process.

The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.

Identification of translin and trax as components of the GS1 strand-specific DNA binding complex enriched in brain.

In previous gel-shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence-specific manner to single-stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1-DNA complex was subjected to UV irradiation to induce cross-linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel-shift assays, we were able to achieve approximately 500,000-fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV cross-linking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined.

Adhesive activity of gicerin, a cell-adhesion molecule, in kidneys and nephroblastomas of chickens.

Gicerin, a cell-adhesion molecule belonging to the immunoglobulin superfamily, has both homophilic and heterophilic binding activities to neurite outgrowth factor, an extracellular matrix molecule in the laminin family. Gicerin is thought to play a role in the normal development of chicken kidney, because it is expressed abundantly in the embryonic organ and only slightly in the mature organ. In this study, we have examined the adhesive activity of gicerin in the kidney to characterize its function in organogenesis. We have also examined the function of gicerin in chicken nephroblastomas ("embryonic nephromas"), which show various structures resembling those in embryonic kidneys. Immunohistochemically, the expression patterns of gicerin and neurite outgrowth factor in nephroblastomas are similar to those of embryonic kidneys. Cell-aggregation assays have shown that primary culture cells from both embryonic kidneys and nephroblastomas have strong aggregation activities, and that each aggregation is partially inhibited by gicerin antibody. In contrast, cells from adult kidney exhibit weak aggregation activity that is not inhibited by the antibody. In addition, ligand blot analysis has revealed that gicerins in embryonic kidney and nephroblastoma bind to purified neurite outgrowth factor, whereas extracts from adult kidney show no positive reaction. These findings suggest that the homophilic and heterophilic adhesive activities of gicerin are involved in the formation of both normal kidney and nephroblastoma.

Neurite promotion from ciliary ganglion neurons by gicerin.

Gicerin is a cell adhesion molecule of an immunoglobulin superfamily member and transiently expressed on the surface of neurons such as retinal ganglion cells during synaptogenesis. Gicerin is a receptor for NOF (neurite outgrowth factor) that belongs to the laminin family, and mediates neurite extension induced by NOF. As we have reported, gicerin also exhibits homophilic cell adhesion activity, we compared the patterns of extending neurites induced by homophilic and heterophilic cell adhesion activities of gicerin using ciliary ganglion (CG) neurons. CG neurons expressed gicerin and extended neurites on a feeder layer of gicerin-transfected cells, suggesting a neurite extension by gicerin-gicerin (homophilic) interaction. We found that CG neurons cultured on gicerin-transfected cells extended slightly branched neurites, while those cultured on NOF-coated substratum extended many long branched neurites. It is suggested that neurites induced by homophilic or heterophilic cell adhesion activities of gicerin differ in the length and branching.

Gicerin, a cell adhesion molecule, participates in the histogenesis of retina.

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis.

Expression of neurite outgrowth factor and gicerin during inner ear development and hair cell regeneration in the chick.

Several cell adhesion molecules are expressed in the developing inner ear. The present study focused on gicerin, a novel member of the immunoglobulin superfamily, in an attempt to improve our understanding of the development and regeneration of chick inner ear. Gicerin is known to homophilically interact with itself and to bind to neurite outgrowth factor (NOF). The data collected herein show that gicerin is highly expressed in auditory epithelium and acoustic ganglion during early embryogenesis. The immunoreactivity of gicerin in the auditory epithelium decreases more rapidly than that in the acoustic ganglion as the mature hair cells become distinguishable. At the post-hatch stage, the expression of gicerin is not observed. In contrast, NOF was expressed on the basement membranes around the auditory epithelium, and in the acoustic ganglion during development and after birth, but not in the auditory epithelium. Following noise damage, gicerin is transiently re-expressed on the damage receptor epithelium when active cell proliferation is observed in the epithelium. This positive reaction immediately disappears as immature short hair cells appear. These results suggest that gicerin may be associated with cell proliferation in the auditory epithelium, and play a role in neurite extension of the acoustic ganglion cells in conjunction with NOF.

Identification of a strand-specific Egr response element binding complex enriched in rat brain.

Multiple members of the Egr family of transcription regulatory factors are rapidly induced in response to neuronal stimulation and share a common double-stranded DNA binding consensus sequence, referred to as the Egr response element. Recent studies have identified transcription regulatory factors that bind preferentially to short segments of single-stranded DNA, rather than the conventional double-stranded versions of regulatory elements. Accordingly, in the present study, we have investigated whether the Egr response element may also be regulated by trans factors that bind to single-stranded versions of this cis element. Using gel-shift studies, we have identified a protein complex that binds selectively to the G-rich strand of the Egr response element. In competition studies, an RNA oligonucleotide containing the corresponding G-rich sequence is approximately 25-fold less potent than its DNA counterpart. This DNA binding complex, referred to as GS1, is present in several regions of the rat brain with highest levels in cerebellum; negligible binding activity was detected in multiple peripheral tissues surveyed. UV cross-linking studies revealed two major protein bands with estimated molecular masses of 36 and 30 kDa. The highly restricted tissue distribution of this complex and its sequence-specific binding properties indicate that GS1 may be involved in regulating transcription directed by the Egr response element in brain.