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PubMed1997

Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles.

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.

Animals↗

PubMed1997

Syndet is a novel SNAP-25 related protein expressed in many tissues.

SNAP-25 is a synaptosomal associated protein localized at the plasma membrane of nerve terminals. SNAP-25 associates with syntaxin 1 and vesicle-associated membrane protein-2 (VAMP-2) and is thought to form a complex essential for neurotransmitter release. We have identified syndet, a novel protein related to the family of SNAP-25 isoforms. Like SNAP-25, syndet has regions with high probability of forming coiled coils, a cysteine rich-domain, and lacks a signal sequence or transmembrane domains. Syndet is tightly bound to membranes, possibly by acylation within the cysteine-rich domain. Syndet is expressed in non-neuronal tissues. In adipocytes, syndet is found at the plasma membrane and in an intracellular compartment. The identification of syndet supports the hypothesis that multiple SNAP-25 related proteins ensure specificity of vesicle fusion at the cell surface.

3T3 Cells↗

PubMed2003

Regulation of presynaptic calcium channels by synaptic proteins.

Calcium entry into nerve termini via voltage gated calcium channels is an essential step in neurotransmission. Consequently, second messenger regulation of calcium channel activity modulates synaptic activity. It has been suggested that calcium channels must physically couple to the release machinery, and a physical interaction between a synaptic protein interaction (synprint) site contained within mammalian presynaptic calcium channels and synaptic proteins such as syntaxin 1, SNAP-25, and synaptotagmin has been demonstrated. Interestingly, synaptic calcium channels in invertebrates lack this region. In invertebrates, synaptic transmission is instead dependent on a presynaptic calcium channel splice variant that can physically associate with the adaptor proteins Mint-1 and CASK. We suggest that in the absence of a synprint region, these proteins may localize calcium channels to the synaptic release machinery. The interactions between synaptic proteins and mammalian N-type calcium channels serves to regulate calcium channel activity directly, as well as indirectly by altering second messenger regulation of the channels. This provides for a feedback mechanism that allows the fine-tuning of calcium channel activity during various steps in neurotransmitter release. This does not occur with invertebrate synaptic calcium channel homologs, suggesting that the regulation of calcium channel activity by synaptic proteins is a mechanism unique to vertebrates.

Animals↗

PubMed1999

Exocytotic protein components in rat pituitary gland after long-term estrogen administration.

Recently, a set of proteins involved in the docking and fusion machinery of secretory organelles has been identified in anterior pituitary cells. In this study we analyzed, by Western blotting and immunocytochemistry, the expression of several proteins involved in exocytosis after long-term administration of 17beta-estradiol (E2) in Fischer 344 rats. No differences were observed in the amount of synaptosomal-associated protein of 25 kDa, synaptobrevin 2, syntaxin 1, synaptotagmin I and Rab3a in total brain homogenates from treated rats after E2 administration. In striking contrast, the levels of all of these exocytotic proteins, including cellubrevin, were notably decreased in pituitary glands of E2-treated rats. In addition, no differences were observed in the in vitro basal and 8-Br-cAMP-induced prolactin (PRL) release between pituitary cells from control and E2-treated rats, whereas TRH-induced PRL release in anterior pituitary cells from E2-treated animals was higher than in control donors. In conclusion, this study shows that protein components of the exocytotic machinery are specifically down-regulated in the pituitary gland of E2-treated Fischer 344 rats.

Animals↗

PubMed2001

The detection of central nervous system tissue on beef carcasses and in comminuted beef.

We report the development and validation of a fluorescent enzyme-linked immunosorbent assay (ELISA) for glial fibrillary acidic protein (GFAP), which can be used as a rapid and sensitive method to detect CNS tissue in meat products. The fluorometric assay is sensitive to 0.2 ng GFAP and has an intra-assay coefficient of variation (CV) of 2.0% and an interassay CV of 14.1%. Bovine spinal cord and brain demonstrate dose-response curves that are parallel to GFAP standards, whereas peripheral sciatic nerve and cervical ganglia also cross-react at high tissue levels. The use of another central nervous system marker, syntaxin 1-B, was not effective for neural tissue detection. Less than 1.0 ng GFAP per mg tissue was found on most beef subprimals and advanced meat recovery (AMR) product. Occasional samples contained higher levels of GFAP, probably because of contamination by the carcass-splitting saw, incomplete removal of the spinal cord, or a chance sampling of a major nerve. Further reduction of CNS content was facilitated by removal of the cervical vertebrae and the spinal canal prior to processing beef chuck bones through AMR equipment. The presence of GFAP was very low (0.037 ng/mg) in beef patties collected from major processors throughout the USA. The presence of normal sausage ingredients or heating the product to 80 degrees C for 60 min did not affect the detection of GFAP. Heating the product to 115 degrees C for 100 min eliminated the detectability of GFAP.

Animals↗

PubMed2000

Direct interaction of Rab4 with syntaxin 4.

In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.

Adipocytes↗

PubMed1996

Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.

SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

3T3 Cells↗

PubMed1996

Characterization of SNARE protein expression in beta cell lines and pancreatic islets.

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.

Animals↗

PubMed1994

Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking.

Intracellular vesicles destined to fuse with the plasma membrane and secrete their contents must have a mechanism for specifically interacting with the appropriate target membrane. Such a mechanism is now suggested by the demonstration of specific interaction between vesicular proteins and plasma membrane proteins. The vesicle-associated membrane proteins (VAMPs) 1 and 2 specifically bind the acceptor membrane proteins syntaxin 1A and 4 but not syntaxin 2 or 3. The binding site is within amino acids 194 to 267 of syntaxin 1A, and the approximate equilibrium dissociation constants is 4.7 x 10(-6) molar. These data suggest a physical basis for the specificity of intracellular vesicular transport.

Amino Acid Sequence↗

PubMed2002

Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691-36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming endocytic material.

Amino Acid Sequence↗

PubMed1994

Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.

omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.

Amino Acid Sequence↗

PubMed2003

Acid-sensing ion channels in malignant gliomas.

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.

Acid Sensing Ion Channels↗

PubMed2001

Loss of heterozygosity and microsatellite instability at chromosomal sites 1Q and 10Q in morphologically distinct regions of late stage prostate lesions.

PURPOSE: We investigated the incidence of loss of heterozygosity (LOH) and microsatellite instability in sporadic prostate cancer and surrounding tissue at loci encompassing the HPC1 and PTEN genes. MATERIALS AND METHODS: Surgical specimens from 63 patients with sporadic stage T3 or T4 prostatic adenocarcinoma were analyzed for LOH and microsatellite instability. Microdissected tissue included morphologically normal foci, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma. LOH analysis was performed using 4 microsatellite markers that map in the region of the 1q24 to 25 locus of the putative prostate cancer susceptibility gene HPC1 and 4 that map in the region of the 10q23 locus of the PTEN gene. RESULTS: The incidence of LOH on 10q was consistent with that previously reported in prostatic tumors. LOH associated with the PTEN locus was recorded in morphologically normal foci, BPH and adenocarcinoma. Sequence analysis of PTEN in a limited number of lesions revealed mutations in nontumor and tumor tissue. Analysis of the DS10215 locus showed significant LOH in tumor but not in benign tissue, suggestive of a tumor suppressor gene in this region associated with prostatic neoplastic progression. In contrast, no significant LOH was observed in the same tissues at 4 loci on chromosome 1q. In this study we recorded elevated levels of microsatellite instability in benign prostatic tissue with an additional increase associated with prostatic adenocarcinoma. CONCLUSIONS: The low incidence of LOH in the region of the HPC1 locus in all prostate lesions studied suggests that this putative hereditary prostate cancer susceptibility locus does not appear to have a role in sporadic prostate cancer, at least not in the context of LOH. In contrast, analysis of the same tissues for LOH at chromosome 10q confirmed frequent alterations in this region linked to late stage prostate cancer. PTEN mutations in microdissected morphologically normal and BPH tissue showed alterations in nontumor tissue surrounding adenocarcinoma. Microsatellite instability was increased in adenocarcinomas over an elevated background recorded in surrounding tissues.

Adenocarcinoma↗

PubMed2001

African-American heredity prostate cancer study: a model for genetic research.

A genome-wide scan of high-risk prostate cancer families in North America has demonstrated linkage of a particular marker to Chromosome 1q (HPC1). An even greater proportion of African-American families have shown linkage to HPC1. Therefore, investigators at the National Human Genome Research Institute (NHGRI) in collaboration with Howard University and a predominantly African-American group of urologists established the African-American Hereditary Prostate Cancer (AAHPC) Study Network to confirm the suggested linkage of HPC in African Americans with a gene on Chromosome 1. Blood samples from recruited families were sent to Howard University for extraction of DNA. The DNA was sent to NHGRI at NIH where the genotyping and genetic sequence analysis was conducted. Genotype data are merged with pedigree information so that statistical analysis can be performed to establish potential linkage. From March 1, 1998, to June 1, 1999, a total of 40 African-American families have been recruited who met the study criteria. Preliminary results suggest that racial/ethnicity grouping may affect the incidence and extent of linkage of prostate cancer to specific loci. The importance of these findings lays in the future treatment of genetic-based diseases.

Aged↗

PubMed2003

False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions.

The techniques of co-immunoprecipitation and immunocytochemical co-labelling are classically used to identify protein-protein interactions. We have used an antibody to the rat small conductance calcium-activated potassium channel subtype 1 (rSK1) to immunoprecipitate proteins from rat brain. A 35 kDa protein was recognized by two monoclonal antibodies to syntaxin 1 and a polyclonal antibody to syntaxin 1A, but not by antibodies to syntaxins 2, 3 or 4. These data suggested that syntaxin 1A is specifically associated with rSK1 in rat brain. A GST construct of the carboxyl terminus of rSK1 was able to pull-down syntaxin 1A from rat brain. Immunocytochemistry showed somatic labelling for both rSK1 and syntaxin 1A in acutely dissociated hippocampal CA1 neurons, confirming that these proteins could interact in vivo. However, control immunoprecipitations showed that antibodies to eight potassium channels could also immunoprecipitate syntaxin, even though some of these channels would not be expected to reside in the same subcellular compartment. Mock immunoprecipitations and pull-down assays showed that syntaxin 1 could directly interact with sepharose and agarose resins. Hence immunoprecipitation and pull-down assays do not provide evidence that syntaxin is specifically associating with a protein, placing doubt on a number of reported interactions with syntaxin 1A.

Animals↗

PubMed1998

Mint 3: a ubiquitous mint isoform that does not bind to munc18-1 or -2.

Mint 1 and 2 are proteins that bind to munc18-1, an essential component of the synaptic vesicle fusion machinery, and are detectably expressed only in neurons [Okamoto and Südhof, J. Biol. Chem. 272, 31459-31464 (1997)]. Mint 1 and 2 are composed of a variable N-terminal region that includes a conserved munc18-1-binding site, and a constant C-terminal region that contains one PTB and two PDZ domains. We have now identified a third mint isoform, mint 3. Similar to mint 1 and 2, the C-terminal half of mint 3 is composed of one PTB domain and two PDZ domains. However, in contrast to mint 1 and 2, mint 3 lacks an N-terminal munc18-binding domain and does not interact with munc18-1 in yeast two-hybrid assays. Mint 3 is ubiquitously expressed in all tissues, with lowest levels in brain and testis whereas mint 1 and 2 appear to be brain-specific. Our data suggest that mints form a diverse family of proteins with specialized neuronal and ubiquitous isoforms.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Developing rat pineal cells manifest potential of neuronal differentiation in vitro.

The pineal gland in mammals is an endocrine organ and generally does not exhibit neuronal characteristics. However, it is known that under culture conditions, cells from newborn rat pineals express properties characteristic of photoreceptors. Here, we studied the potential of rat pineal cells to differentiate into neuronal cell types using different neural markers. Three phenotype markers characteristic of nerve cells, i.e., intense GABA, neuron-specific antigen (HPC-1) and microtubule-associated protein 2 (MAP2) immunoreactivities, were detected in the pineal culture of newborn rats. Expression of the respective neuronal phenotypes appears to be controlled by different mechanisms; in the normal culture medium containing 5.4 mM KCl, numerous cells were stained intensely with anti-GABA antiserum, whereas only a few were stained intensely either with HPC-1 or MAP2 antibody. In a culture medium with a high concentration of KCl (35 mM), which may induce depolarization of nerve cells, numerous cells became strongly positive for HPC-1 or MAP2; both the cell bodies and the neuritic fibers were stained positively. Since cells intensely immunoreactive to GABA, HPC-1 or MAP2 were not found in intact pineals of the rat, the present results indicate that the neuronal potency of the rat pineal cells is expressed only in vitro and is suppressed in vivo, and that the potency is lost during postnatal development. Norepinephrine at 1 microM, which suppresses differentiation of rhodopsin immunoreactive cells, was ineffective in inducing phenotypic expression of neuronal properties in the present system, indicating that the mechanism of suppression of neuronal properties in the intact pineal may be different from the one for photoreceptors.

Animals↗

PubMed2001

Characterization of a transformed rat retinal ganglion cell line.

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Animals↗