Journal of Neurochemistry

Beckmann, Alison M.; Davidson, Mark S.; Goodenough, Sharon; Wilce, Peter A.

doi: 10.1046/j.1471-4159.1997.69062227.xpmid: 9375653

Abstract: Egr‐1 and related proteins are inducible transcription factors within the brain recognizing the same consensus DNA sequence. Three Egr DNA‐binding activities were observed in regions of the naive rat brain. Egr‐1 was present in all brain regions examined. Bands composed, at least in part, of Egr‐2 and Egr‐3 were present in different relative amounts in the cerebral cortex, striatum, hippocampus, thalamus, and midbrain. All had similar affinity and specificity for the Egr consensus DNA recognition sequence. Administration of the convulsants NMDA, kainate, and pentylenetetrazole differentially induced Egr‐1 and Egr‐2/3 DNA‐binding activities in the cerebral cortex, hippocampus, and cerebellum. All convulsants induced Egr‐1 and Egr‐2 immunoreactivity in the cerebral cortex and hippocampus. These data indicate that the members of the Egr family are regulated at different levels and may interact at promoters containing the Egr consensus sequence to fine tune a program of gene expression resulting from excitatory stimuli.

Vinnakota, Shyamala; Qian, Xiaojun; Egal, Hussein; Sarthy, Vijay; Sarkar, Hemanta K.

doi: 10.1046/j.1471-4159.1997.69062238.xpmid: 9375654

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high‐affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+‐ and Cl−‐dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl− was varied, a single saturation process with an average apparent equilibrium constant (KCl−) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, KNa+∼54.8 mM). Analyses of the Na+‐ and Cl−‐concentration dependence data suggest that at least two Na+ and one Cl− are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β‐alanine > l‐diaminopropionic acid > guanidinoethane sulfonate > β‐guanidinopropionic acid > chloroquine > γ‐aminobutyric acid > 3‐amino‐1‐propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α‐phorbol 12,13‐didecanoate but was inhibited significantly by the active phorbol ester phorbol 12‐myristate 13‐acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.

Barnes, T. M.; Hekimi, S.

doi: 10.1046/j.1471-4159.1997.69062251.xpmid: 9375655

Abstract: Mutations in the unc‐9 gene of the nematode Caenorhabditis elegans cause abnormal forward locomotion and an egg‐retention phenotype. unc‐9 mutations also reduce the worms' sensitivity to avermectin and block a form of hypersensitivity to volatile anesthetics. We report here the cloning and molecular characterization of unc‐9 and show that it encodes a member of the OPUS family of proteins that is 56% identical to another OPUS protein, UNC‐7. It is significant that unc‐9 mutants share all phenotypes with unc‐7 mutants. Mutants in another gene, unc‐124, also share all tested phenotypes with unc‐9 mutants, including identical locomotory and egg‐laying defects, suggesting that multiple genes are required for the same biochemical function. OPUS proteins are implicated in the function of invertebrate gap junctions, and, based on a new alignment including 24 members from C. elegans, we present a refined model for the structure of OPUS proteins suggesting that oligomers could form a hydrophilic pore. We also show that alteration of highly conserved proline residues in UNC‐9 leads to a cold sensitivity that likely affects a step in protein expression rather than function. Finally, we speculate on the basis of the avermectin resistance and anesthetic response phenotypes.

Wang, Li‐Hsien; Strittmatter, Stephen M.

doi: 10.1046/j.1471-4159.1997.69062261.xpmid: 9375656

Abstract: The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin‐1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two‐hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two‐hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two‐hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8–134 and 281–435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.

Bzdega, Tomasz; Turi, Thomas; Wroblewska, Barbara; She, Dewei; Chung, Hye Sun; Kim, Hyun; Neale, Joseph H.

doi: 10.1046/j.1471-4159.1997.69062270.xpmid: 9375657

Abstract: N‐Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate‐specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752‐amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by β‐NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5′ end, the central region, and the 3′ end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5′ end of the hippocampal cDNA.

Cheng, Jr‐Gang; Pennica, Diane; Patterson, Paul H.

doi: 10.1046/j.1471-4159.1997.69062278.xpmid: 9375658

Abstract: Cardiotrophin‐1 (CT‐1) was cloned from mouse embryoid body for its ability to induce growth of heart cells. Predictions of its secondary structure indicate that CT‐1 belongs to a family of cytokines with a four‐helical bundle structure, and sequence comparisons reveal a weak homology to leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). Using a reverse transcriptase‐polymerase chain reaction assay with rat sympathetic neuron cultures, we find that CT‐1 induces and suppresses the expression of the same set of neuropeptide and neurotransmitter synthetic enzyme mRNAs as do LIF and CNTF. In addition, the effects of CT‐1 and LIF are not additive, and CT‐1 does not require a GPI‐linked component to mediate its actions. Our functional data confirm that CT‐1 is a member of the neuropoietic cytokine family and suggest that the CT‐1 receptor complex contains the gp130 signal transducing component.

Liu, Yuanbin; Schubert, David

doi: 10.1046/j.1471-4159.1997.69062285.xpmid: 9375659

Abstract: Amyloid β peptide (Aβ) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of Aβ toxicity is the inhibition of cellular 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that Aβ and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide‐enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.

Hu, Jingru; Ferreira, Adriana; Van Eldik, Linda J.

doi: 10.1046/j.1471-4159.1997.69062294.xpmid: 9375660

Abstract: The glial‐derived neurotrophic protein S100β has been implicated in the development and maintenance of the nervous system. S100β has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100β overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100β results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100β‐induced NO release from astrocytes might influence neurons, we studied S100β effects on neuroblastoma B104 cells or primary hippocampal neurons co‐cultured with astrocytes. We found that S100β treatment of astrocyte‐neuron co‐cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100β required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100β may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.

Sciamanna, Michele A.; Griesmann, Guy E.; Williams, Carol L.; Lennon, Vanda A.

doi: 10.1046/j.1471-4159.1997.69062302.xpmid: 9375661

Abstract: SCC‐37 is a small cell lung carcinoma line that aberrantly expresses muscle‐type nicotinic acetylcholine receptors (nAChRs). It was established from a patient with a paraneoplastic autoimmune neuromuscular disorder, myasthenia gravis. When grown as a xenograft tumor, SCC‐37 cells express plasma membrane receptors that bind 125I‐labeled α‐bungarotoxin (125I‐α‐BTx), cosediment with 9S nAChR pentamers, and bind to a monoclonal antibody (MAb 35) specific for muscle‐type (α1 subunit) α‐BTx receptors. The agonist carbamylcholine (carbachol) stimulates influx of 22Na+ in SCC‐37 cells; this is inhibited by α‐BTx and by d‐tubocurarine. Long‐term cultured SCC‐37 cells have functional and ligand‐binding evidence for surface coexpression of both α1 and neuronal‐type (α7 subunit) α‐BTx receptors. A subclone of SCC‐37, designated SCC‐A9, expresses only the neuronal‐type (α7 subunit) α‐BTx receptors on its surface. Carbachol does not stimulate 22Na+ influx in SCC‐A9 cells, but cytisine initiates a sustained influx of Ca2+. Activation of this response is inhibited by α‐BTx and by the α7‐selective antagonist methyllycaconitine. Addition of Co2+ abrogates the sustained elevation of intracellular free Ca2+ concentration, implying that the cytisine‐stimulated influx of Ca2+ is sustained by secondary opening of voltage‐sensitive channels in the plasma membrane. Surface receptors for 125I‐α‐BTx are blocked by methyllycaconitine and d‐tubocurarine. Solubilized α‐BTx receptors from plasma membranes of SCC‐A9 cells cosediment with 10S neuronal nAChR pentamers and bind to an α7‐specific monoclonal antibody (MAb P27) but not to the muscle nAChR‐reactive MAb 35. However, MAb P27 and MAb 35 both bind to α‐BTx receptors solubilized from the cytoplasmic compartments of SCC‐A9 and the parental SCC‐37 line. Reverse transcription‐PCR analysis revealed RNA transcripts for α7 and α1 subunits in both SCC‐A9 and SCC‐37 cells. The nAChRs that are expressed in these novel human cell lines can regulate cation fluxes directly as well as indirectly by synergizing with the activity of voltage‐sensitive Ca2+ channels. These activities may influence the secretion of autocrine growth factors and the transcription of growth regulatory genes and thus be pertinent to the growth and metastasis of malignant neuroendocrine neoplasms.

Gamberino, William C.; Berkich, Deborah A.; Lynch, Christopher J.; Xu, Baiyang; LaNoue, Kathryn F.

doi: 10.1046/j.1471-4159.1997.69062312.xpmid: 9375662

Abstract: CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C‐labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α‐ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C‐labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α‐[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α‐ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α‐ketoglutarate to glutamate may frequently be the rate‐limiting step in this process of glutamate synthesis.