Effect of lower chlorinated hydroxylated-polychlorobiphenyls on development of PC12 cells.

Hydroxylated polychlorobiphenyls (OH-PCBs) are major metabolites of PCBs that are widely distributed in the environment. While the effects of penta- to hepta-chlorinated OH-PCBs on neuronal differentiation have been widely reported, those of lower chlorinated OH-PCBs have not been extensively studied. To investigate the effects of lower chlorinated OH-PCBs on neuronal development, we studied the effects of mono- to hexa-chlorinated OH-PCBs on PC12 cells. Morphological changes were examined using an automatic system IN Cell Analyzer. Seventeen of the 20 OH-PCBs investigated promoted neuronal elongation in an OH-PCB concentration-dependent manner, while three OH-PCB congeners suppressed neuronal elongation based on Dunnett's analysis. In particular, the top five OH-PCBs (4OH-PCB2, 4'OH-PCB3, 4'OH-PCB25, 4'OH-PCB68, and 4'OH-PCB159), which have hydroxyl groups at the para-position and chlorine substitutions at the 2, 4, or 3' positions, significantly promoted neuronal elongation. Moreover, these neuronal elongations were suppressed by U0126, and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was observed in PC12 cells treated with 4OH-PCB2, 4'OH-PCB25, and 4'OH-PCB159. Taken together, our results indicate that the effect of OH-PCB on neuronal development is not dependent on the number of chlorine groups but on the chemical structure, and the mitogen-activated kinase kinase (MEK)-ERK1/2 signaling pathway is involved in this process.

Re-evaluation of Culture Condition of PC12 and SH-SY5Y Cells Based on Growth Rate and Amino Acid Consumption.

BACKGROUND/AIM: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively). MATERIALS AND METHODS: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization. RESULTS: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aß1-42 and Aß25-35 suppressed their growth nearly by 50%. CONCLUSION: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity.

Probing the biocompatibility of MoS2 nanosheets by cytotoxicity assay and electrical impedance spectroscopy.

Transition metal dichalgogenides such as MoS2 have recently emerged as hot two-dimensional (2D) materials due to their superior electronic and catalytic properties. Recently, we have reported the usefulness of MoS2 nanosheets toward the electrochemical detection of neurotransmitters and glucose (Narayanan et al 2014 Nanotechnology 25 335702). Furthermore, there are reports available in the literature that demonstrate the usefulness of MoS2 nanosheets for biosensing and energy storage applications (Zhu et al 2013 J. Am. Chem. Soc. 135 5998-6001; Pumera and Loo 2014 Trends Anal. Chem. 61 49-53; Lee et al 2014 Sci. Rep. 4 7352; Stephenson et al 2014 Energy Environ. Sci. 7 209-31). Understanding the cytotoxic effect of any material is very important prior to employing them for any in vivo biological applications such as implantable sensors, chips, or carriers for drug delivery and cell imaging purposes. Herein, we report the cytotoxicity of the MoS2 nanosheets based on the cytotoxic assay results and electrical impedance analysis using rat pheochromocytoma cells (PC12) and rat adrenal medulla endothelial cells (RAMEC). Our results indicated that the MoS2 nanosheets synthesized in our work are safe 2D nanosheets for futuristic biomedical applications.

Systematic Asymmetric Synthesis of All Diastereomers of (-)-Talaumidin and Their Neurotrophic Activity.

(-)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan isolated from Aristolochia arcuata Masters, shows significant neurite-outgrowth promotion and neuroprotection in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells. The four stereogenic centers on the tetrahydrofuran moiety in 1 result in the presence of seven diastereomers except for their enantiomers. In order to investigate the stereochemistry-activity relationships of the stereoisomers, the systematic synthesis of all stereoisomers of 1 was accomplished by employing Evans aldol, diastereoselective hydroboration, reductive deoxygenation, and Mitsunobu reactions as key steps. The ability of all of the synthesized stereoisomers to promote neurite-outgrowth in PC12 and neuronal cells was evaluated. All stereoisomers exhibited moderate to potent neurotrophic activities in NGF-differentiated PC12 cells at 30 µM and in primary cultured rat cortical neuronal cells at 0.01 µM. In particular, 1e bearing all cis substituents resulted in the most potent neurite-outgrowth promotion.

Saffloflavonesides A and B, two rearranged derivatives of flavonoid C-glycosides with a furan-tetrahydrofuran ring from Carthamus tinctorius.

Two new rearranged derivatives of flavonoid C-glycosides, saffloflavonesides A (1) and B (2), were isolated from the florets of Carthamus tinctorius. Their structures were determined using UV, IR, HRESIMS, and 1D and 2D NMR data and by comparing experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1 and 2 were unprecedented chalcone and flavanone derivatives possessing a furan conjoining tetrahydrofuran ring. A potential biosynthetic pathway was proposed. At 10 µM, 1 and 2 both showed strong inhibitory activity against PC12 cell damage induced by rotenone.

Is the PentaBDE replacement, tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a developmental neurotoxicant? Studies in PC12 cells.

Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (2,3-dibromopropyl) phosphate (TDBPP), and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants.

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells.

AIMS: We investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells. MAIN METHODS: PC12 cells were treated with 40mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of ß-amyloid (Aß), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay. KEY FINDINGS: DG (40mM) completely retarded cell growth after incubation for 72h, and this effect was not due to osmotic changes, as 40mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4h and increased Aß and AGEs at 24h. DG treatment for 24h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5-10µM). SIGNIFICANCE: The present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.

Inhibition of medulloblastoma tumorigenesis by the antiproliferative and pro-differentiative gene PC3.

Medulloblastoma, the most common brain tumor in childhood, appears to originate from cerebellar granule cell precursors (GCPs), located in the external granular layer (EGL) of the cerebellum. The antiproliferative gene PC3 (Tis21/BTG2) promotes cerebellar neurogenesis by inducing GCPs to shift from proliferation to differentiation. To assess whether PC3 can prevent the neoplastic transformation of GCPs and medulloblastoma development, we crossed transgenic mice conditionally expressing PC3 (TgPC3) in GCPs with Patched1 heterozygous mice (Ptc(+/-)), a model of medulloblastoma pathogenesis characterized by hyperactivation of the Sonic Hedgehog pathway. Perinatal up-regulation of PC3 in Ptc(+/-)/TgPC3 mice results in a decrease of medulloblastoma incidence of approximately 40% and in a marked reduction of preneoplastic abnormalities, such as hyperplastic EGL areas and lesions. Moreover, overexpression of cyclin D1, hyperproliferation, and defective differentiation--observed in Ptc(+/-) GCPs--are restored to normality in Ptc(+/-)/TgPC3 mice. The PC3-mediated inhibition of cyclin D1 expression correlates with recruitment of PC3 to the cyclin D1 promoter, which is accompanied by histone deacetylation. Remarkably, down-regulation of PC3 is observed in preneoplastic lesions, as well as in human and murine medulloblastomas. As a whole, this indicates that PC3 may prevent medulloblastoma development by controlling cell cycle and promoting differentiation of GCPs.

Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein-protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated 'signalling particles' with an estimated size of 60-75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein.

OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface. METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal. Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1.

Retinoic acid-receptor-related orphan receptor (ROR) alpha is a nuclear receptor involved in many pathophysiological processes such as cerebellar ataxia, inflammation, atherosclerosis and angiogenesis. In the present study we first demonstrate that hypoxia increases the amount of Rora transcripts in a wide panel of cell lines derived from diverse tissues. In addition, we identified a functional promoter sequence upstream of the first exon of the human Rora gene, spanning -487 and -45 from the translation initiation site of RORalpha1. When cloned in a luciferase reporter vector, this sequence allowed the efficient transcription of the luciferase gene in several cell lines. Interestingly, the activity of the Rora promoter was enhanced by hypoxia in HepG2 human hepatoma cells, and this effect was dependent on an HRE (hypoxia response element) spanning from -229 to -225. Using electrophoretic-mobility-shift assays, we showed that HIF-1 (hypoxia-inducible factor 1), which plays a key role in the transcriptional response to hypoxia, bound to this HRE. Overexpression of HIF-1alpha increased the activity of the Rora promoter through the HRE. Overexpression of a dominant-negative form of HIF-1alpha producing transcriptionally inactive HIF-1alpha/HIF-1beta dimers abolished hypoxic activation of the Rora promoter. This indicated that HIF-1 is involved in the response of RORalpha to hypoxia. Taken together, our data reveal Rora as a new HIF-1 target gene. This illustrates, at the molecular level, the existence of cross-talk between signalling pathways mediated by HIF-1 and those mediated by nuclear receptors.

Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B).

Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41-49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108-170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B-EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)-Rattus norvegicus IP3K (aa 108-170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.

RNA interference-mediated silencing of synaptotagmin IX, but not synaptotagmin I, inhibits dense-core vesicle exocytosis in PC12 cells.

Although PC12 cells express three synaptotagmin isoforms (Syts I, IV and IX), all of which have been proposed to regulate dense-core vesicle exocytosis, it remains unknown which of the Sytisoforms acts as the major Ca2+ sensor for dense-core vesicle exocytosis. In the present study, it has been shown by immunoaffinity purification and immunocytochemistry that Syts I and IX, but not Syt IV, are present on the same secretory vesicles in PC12 cells. Silencing of Syt IX with specific small interfering RNA significantly reduced high KCl-dependent neuropeptide Y secretion from PC12 cells, whereas silencing of Syt I with specific small interfering RNA had no significant effect. The results indicate that Syts I and IX are not functionally equivalent and that Syt IX, and not Syt I, is indispensable for the regulation of Ca2+-dependent dense-core vesicle exocytosis in PC12 cells.

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase.

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and brain ischemia, and hydrogen peroxide (H(2)O(2)) plays a central role in the stress. In this study, we have examined the kinetics of H(2)O(2) elimination by PC12 cells as a neuronal model in connection with the enzyme activities supporting the reaction. Similarly to other cell lines previously studied, H(2)O(2) removal kinetics could be divided into two reactions: one apparently following the Michaelis-Menten kinetics (GSH-dependent reaction) and the other following the first-order kinetics (mainly catalyzed by catalase). Based on the enzyme activities in the cell homogenate, it was inferred that glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the GSH- and NADPH-dependent H(2)O(2) elimination by PC12 cells. This is in contrast with fibroblasts and endothelial cells previously examined, in which glutathione reductase (GR) is rate-limiting in the reaction sequence. Treatment of PC12 cells with nerve growth factor increased G6PD activity in the cell homogenate and H(2)O(2) removal activity of the whole cells, with a concomitant increase in the resistance against H(2)O(2) toxicity. These results suggest the importance of G6PD in the antioxidant function of brain and pathogenesis of the oxidative stress-related diseases.

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence.

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.

Synaptotagmin I-DeltaC2B. A novel synaptotagmin isoform with a single C2 domain in the bovine adrenal medulla.

Synaptotagmin I is a 65 kDa type 1 membrane glycoprotein found in secretory organelles that plays a key role in regulated exocytosis. We have characterised two forms (long and short) of synaptotagmin I that are present in the bovine adrenal medulla. The long form is a type I integral membrane protein which has two cytoplasmic C2 domains and corresponds to the previously characterised full-length synaptotagmin I isoform. The short-form synaptotagmin I-DeltaC2B has the same structure in the lumenal and transmembrane sequences, but synaptotagmin I-DeltaC2B is truncated such that it only has a single cytoplasmic C2 domain. Analysis of synaptotagmin I-DeltaC2B expression indicates that synaptotagmin I-DeltaC2B is preferentially expressed in the bovine adrenal medulla. However, it is absent from the dense core chromaffin granules. Furthermore, when expressed in the rat pheochromocytoma cell line PC12 bovine synaptotagmin I-DeltaC2B is largely absent from dense core granules and synaptic-like microvesicles. Instead, indirect immunofluorescence microscopy reveals the intracellular location of synaptotagmin I-DeltaC2B to be the plasma membrane.

Plasma membrane localization of palmitoylated tubulin.

PC12 pheochromocytoma cells incorporate [(3)H]palmitic acid into tubulin in a time- and cell-density-dependent manner. The plasma membrane-enriched fraction contains most of the radioactivity of the membrane pellet. While palmitoylated tubulin is found in both the cytoplasm and particulate fraction, the bulk of [(3)H]palmitic acid bound to tubulin is present in the crude membrane pellet and the tubulin extracted from the plasma membrane is more heavily palmitoylated than that extracted from endoplasmic reticulum. Detergent-extracted tubulin from plasma membrane is, to a large extent, polymerization competent; a substantial fraction, increasing as a function of labeling time, is not hydroxylamine-labile. The requirement for detergent extraction, the accompanying changes in tubulin properties and the present findings of preferential incorporation of labeled tubulin into plasma membranes, make it clear that direct incorporation of tubulin into the plasma membrane can occur.

Identification of endogenous ouabain in culture supernatant of PC12 cells.

OBJECTIVE: Ouabain-like factor (OLF), assayed as ouabain-like immunoreactivity (OLI), is thought to represent an endogenous digitalis-like factor. We found increased plasma OLI during the surgical removal of a pheochromocytoma. The elution volume of the OLI extracted from plasma and the pheochromocytoma tissue was the same as that for authentic ouabain, using reverse phase high-performance liquid chromatography. The present study was performed to characterize OLF from the culture supernatant of a rat pheochromocytoma cell line, PC12 cells. DESIGN: OLI from culture supernatant and chromatographic fractions were assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. PC12 cells, subcultured in RPMI 1640 with 10% horse serum and 5% fetal bovine serum, were washed, and then cultured in Iscove's modified Dulbecco's medium (Life Technologies, Rockville, Maryland, USA) with 0.4% bovine serum albumin (without serum). Progesterone was added to augment the production or secretion of OLI. The conditioned medium was acidified to dissociate the binding protein, and OLI was purified by five steps of octadecylsilane (ODS) column chromatography. The structural identity of this OLI was determined by liquid chromatography and mass spectrometry (LC/MS). RESULTS: OLI in the culture medium increased after addition of progesterone in a dose-dependent manner. The concentration in the culture medium was approximately double of that in homogenized PC12 cells. After five rounds of ODS column chromatography, approximately 100 ng of OLI was purified from 21 of culture supernatant, without fetal calf serum, in the presence of progesterone. The molecular size of purified OLI was found to be identical to authentic ouabain, based on analysis by LC/ MS. CONCLUSION: Mammalian cells originating from a rat pheochromocytoma cell line were found to produce and/or secrete OLF by the addition of progesterone.

Differential expression of the G protein beta(5) gene: analysis of mouse brain, peripheral tissues, and cultured cell lines.

A neurally expressed heterotrimeric G protein beta subunit, Gbeta(5), has been found to exhibit functional specialization with respect to its interactions with effector targets and Galpha subunits. A splice variant of Gbeta(5) that contains an N-terminal 42-residue extension, Gbeta(5)-long, has been described in the retina. To define better the potential range of its specialized interactions, analysis of Gbeta(5) gene transcript and protein expression in mouse brain and other tissues and cell lines was performed. Quantification by ribonuclease protection assay of Gbeta(5) transcript expression in the developing brain demonstrates a fivefold increase that occurs postnatally. Analysis of transcript expression by in situ hybridization and ribonuclease protection assay indicates that the Gbeta(5) gene is differentially expressed among multiple adult mouse brain regions, including the motor and occipital cortex, the olfactory bulb and associated rhinencephalic structures, hypothalamus, pontine cochlear nuclei, and Purkinje cells in the cerebellum. Gbeta(5) is also expressed in several cultured cell lines of neuroendocrine origin, including murine alphaT3-1 pituitary gonadotrophs and GT1-7 hypothalamic cells, and rat PC12 pheochromocytoma cells. Immunoblotting of tissue homogenates with antibodies to two peptides common to Gbeta(5) and Gbeta(5)-long confirmed expression of Gbeta(5) in multiple brain regions and in spinal cord and expression of Gbeta(5)-long in retina. Taken together, these results suggest that the specialized molecular properties of Gbeta(5) have been adapted to diverse neural functions in the adult brain.

Identification of D-aspartate in rat pheochromocytoma PC12 cells.

D-Aspartate is now known to be present in mammalian neuronal and endocrine cells in vivo, and may play some role(s) in neurocrine and endocrine functions. However, origin of D-aspartate is unknown. Here, we report that free D-aspartate (108 pmoles/3 x 10(7) cells) is present in the cultured PC12 cells, a rat pheochromocytoma cell line, as determined with immunohistochemical techniques as well as high performance liquid chromatography (HPLC) on a Pirkle-type chiral column. The amount of D-aspartate does not change with the passage. The culture medium does not contain D-aspartate. These results strongly suggest the presence of a de novo biosynthetic pathway for D-aspartate in the endocrine cells.