bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel prohormone processing site in Aplysia californica: the Leu–Leu rule
Authors: Hummon Amanda B. , Huang He‐Qing , Kelley Wayne P. , Sweedler Jonathan V. 
Published: March 2003

AbstractNeuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non‐basic sites. Cleavage at Leu–Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non‐native synthetic peptides containing Leu–Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu–Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu–Leu residues. Products resulting from cleavage at Leu–Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity.
Nicotinic‐acetylcholine receptors are functionally coupled to the nitric oxide/cGMP‐pathway in insect neurons
Authors: Zayas Ricardo M. , Qazi Sanjive , Morton David B. , Trimmer Barry A. 
Published: March 2003

AbstractIn addition to their ionotropic role, neuronal nicotinic acetylcholine receptors (nAChRs) can influence second messenger levels, transmitter release and gene transcription. In this study, we show that nAChRs in an insect CNS control cGMP levels by coupling to NO production. In conditions that inhibit spiking, nicotine induced cGMP synthesis. This increase in cGMP was blocked by nicotinic antagonists, and by inhibitors of both nitric oxide synthase and soluble guanylyl cyclase. The nicotinic‐evoked increase in cGMP was localized to specific NO‐sensitive neurons in the CNS, several of which are identified motoneurons. Because NO production requires Ca2+, we investigated the effect of nicotinic stimulation on [Ca2+]i in cultured neurons. We found that activation of nAChRs increased [Ca2+]i, which was blocked by nAChR antagonists. Nicotinic stimulation of neurons in the isolated CNS in low‐Na+, also evoked increases in [Ca2+]i independent of fast changes in voltage. In addition, approximately 10% of the nicotinic‐evoked [Ca2+]i increase in cultured neurons persisted when voltage‐gated Ca2+ channels were blocked by Ni2+. Under the same conditions, nicotinic stimulation of cGMP in the CNS was unaffected. These combined results suggest that nicotinic stimulation is coupled to NOS potentially by directly gating Ca2+.
Caspase‐3 is not activated in seizure‐induced neuronal necrosis with internucleosomal DNA cleavage
Authors: Fujikawa Denson G. , Ke Xingrao , Trinidad Rosen B. , Shinmei Steve S. , Wu Aiguo 
Published: March 2003

AbstractA caspase‐3‐activated DNase produces internucleosomal DNA cleavage (DNA laddering). We determined whether caspase‐3 is activated by lithium–pilocarpine‐induced status epilepticus in six brain regions with necrosis‐induced DNA laddering. The thymuses of adult rats given methamphetamine or normal saline were used as controls for apoptosis. Some 6–8 h after methamphetamine treatment, thymocytes showed apoptosis by electron‐microscopic examination, positive terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL), DNA laddering, cleavage of caspase‐3 into its active p17 subunit, active caspase‐3 immunoreactivity, and a 25‐fold increase in caspase‐3‐like activity. Six hours after SE, necrotic neurons by electron‐microscopic examination in hippocampus, amygdala and piriform, entorhinal and frontal cortices showed no TUNEL and no DNA laddering. Twenty‐four hours after seizures, most necrotic neurons were negative for TUNEL, some were positive, but all regions showed DNA laddering. However, 6 and 24 h after seizures, active caspase‐3 immunoreactivity was negative, caspase‐3‐like activity did not increase, and western blot analysis failed to show the p17 subunit. In addition, 24 h after seizures,microdialytic perfusion of carbobenzoxy‐valyl‐alanyl‐aspartyl (O‐methylester) fluoromethylketone was not neuroprotective. Thus, caspase‐3 is not activated in brain regions with seizure‐induced neuronal necrosis with DNA laddering. Either caspase‐activated DNase is activated by another enzyme, or a caspase‐independent DNase is responsible for the DNA cleavage.
Activation of the rat dopamine D2 receptor promoter by mitogen‐activated protein kinase and Ca2+/calmodulin‐dependent protein kinase II pathways
Authors: Takeuchi Yusuke , Miyamoto Eishichi , Fukunaga Kohji 
Published: March 2003

AbstractTo investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin‐dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP‐dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5‐kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R‐expressing NB2A cells or nonexpressing NG108‐15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5‐ and 1.5‐fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)‐JNK1 or DN‐p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK‐induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK‐responsive region was Sp1 site B between nucleotides −56 and −47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK‐induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides −56 and −36. Increased activity by Zif268 was additive with CaMKII‐induced activation but not with activation induced by MEKK. Co‐transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII‐dependent pathway.
Expression of interleukin‐1 receptors and their role in interleukin‐1 actions in murine microglial cells
Authors: Pinteaux Emmanuel , Parker Lisa C. , Rothwell Nancy J. , Luheshi Giamal N. 
Published: March 2003

AbstractInterleukin (IL)‐1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL‐1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL‐1 receptors [IL‐1 type‐I receptor (IL‐1RI), IL‐1 type‐II receptor (IL‐1RII) and IL‐1 receptor accessory protein (IL‐1RAcP)] on microglia. In the present study we investigated whether microglia express IL‐1 receptors and whether they present target or modulatory properties for IL‐1 actions. RT–PCR analysis demonstrated lower expression of IL‐1RI and higher expression of IL‐1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL‐1RI, IL‐1RII and IL‐1RAcP mRNAs, induced the release of IL‐1β, IL‐6 and prostaglandin‐E2 (PGE2), and activated nuclear factor κB (NF‐κB) and the mitogen‐activated protein kinases (MAPKs) p38, and extracellular signal‐regulated protein kinase (ERK1/2), but not c‐Jun N‐terminal kinase (JNK) in microglial cultures. In comparison, IL‐1β induced the release of PGE2, IL‐6 and activated NF‐κB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL‐1β also failed to affect LPS‐primed microglial cells. Interestingly, a neutralizing antibody to IL‐1RII significantly increased the concentration of IL‐1β in the medium of LPS‐treated microglia and exacerbated the IL‐1β‐induced IL‐6 release in mixed glia, providing the first evidence that microglial IL‐1RII regulates IL‐1β actions by binding excess levels of this cytokine during brain inflammation.
Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress
Authors: Scheggi Simona , Leggio Benedetta , Masi Flavio , Grappi Silvia , Gambarana Carla , Nanni Giulio , Rauggi Riccardo , De Montis Maria Graziella 
Published: March 2003

AbstractStressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3‐week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate–putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393‐stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.
Control of the Na+/Ca2+exchanger 3 promoter by cyclic adenosine monophosphate and Ca2+in differentiating neurons
Authors: Gabellini Nadia , Bortoluzzi Stefania , Danieli Gian A. , Carafoli Ernesto 
Published: March 2003

AbstractThe human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1. This was confirmed by luciferase reporter assays of pGL3‐promoter constructs in transfected SH‐SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain‐derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+induced by the partial depolarization of the plasma membrane with KCl down‐regulated both the basal and the cAMP‐stimulated transcription. The down‐regulation of the latter may be mediated by the phosphorylation of the CRE‐binding protein by a calmodulin‐dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE‐binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF‐stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE‐binding protein.
Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways
Authors: Noda Mami , Yasuda Satsuki , Okada Mitsuko , Higashida Haruhiro , Shimada Aki , Iwata Nakao , Ozaki Norio , Nishikawa Kaori , Shirasawa Sakiko , Uchida Mayumi , Aoki Shunsuke , Wada Keiji 
Published: March 2003

AbstractHuman serotonin 5A (5‐HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5‐HT5A receptors‐expressing cells, accumulation of cAMP by forskolin was inhibited by 5‐HT as reported previously. Pertussis toxin‐sensitive inhibition of ADP‐ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine‐sensitive Ca2+ mobilization. On the other hand, 5‐HT‐induced outward currents were observed using the patch‐clamp technique in whole‐cell configuration. The 5‐HT‐induced outward current was observed in 84% of the patched 5‐HT5A receptor‐expressing cells and was concentration‐dependent. The 5‐HT‐induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5‐HT‐induced current was significantly attenuated by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5‐HT‐induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5‐HT‐induced current. IP3 showed a transient increase after application of 5‐HT in 5‐HT5A receptor‐expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP‐ribosyl cyclase activity, 5‐HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3‐sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.
Down‐regulation of Bcl‐2 and Bcl‐xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death
Authors: Jiang Zhihong , Zheng Xiao , Rich Keith M. 
Published: March 2003

AbstractThe functions of the antiapoptotic proteins Bcl‐2 and Bcl‐xL were examined in glioblastoma cells. Expression of both Bcl‐2 and Bcl‐xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non‐neoplastic glial cells. Down‐regulation of both bcl‐2 and bcl‐xL expression in glioblastoma cell lines U87 and NS008 with bcl‐2/bcl‐xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase‐dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl‐2/bcl‐xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl‐2 and Bcl‐xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase‐dependent cell death in gliomas. These findings support a potential role of bcl‐2/bcl‐xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase‐dependent cell death in patients with glioblastoma.
Functional analysis of connexin‐26 mutants associated with hereditary recessive deafness
Authors: Wang Hung‐Li , Chang Wen‐Teng , Li Allen H. , Yeh Tu‐Hsueh , Wu Ching‐Yi , Chen Mei‐Shin , Huang Pei‐Chen 
Published: March 2003

AbstractThe physiological importance of connexin‐26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication‐deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell–cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild‐type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.
Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms
Authors: Mattson Mark P. , Duan Wenzhen , Guo Zhihong 
Published: March 2003

AbstractAlthough all cells in the body require energy to survive and function properly, excessive calorie intake over long time periods can compromise cell function and promote disorders such as cardiovascular disease, type‐2 diabetes and cancers. Accordingly, dietary restriction (DR; either caloric restriction or intermittent fasting, with maintained vitamin and mineral intake) can extend lifespan and can increase disease resistance. Recent studies have shown that DR can have profound effects on brain function and vulnerability to injury and disease. DR can protect neurons against degeneration in animal models of Alzheimer's, Parkinson's and Huntington's diseases and stroke. Moreover, DR can stimulate the production of new neurons from stem cells (neurogenesis) and can enhance synaptic plasticity, which may increase the ability of the brain to resist aging and restore function following injury. Interestingly, increasing the time interval between meals can have beneficial effects on the brain and overall health of mice that are independent of cummulative calorie intake. The beneficial effects of DR, particularly those of intermittent fasting, appear to be the result of a cellular stress response that stimulates the production of proteins that enhance neuronal plasticity and resistance to oxidative and metabolic insults; they include neurotrophic factors such as brain‐derived neurotrophic factor (BDNF), protein chaperones such as heat‐shock proteins, and mitochondrial uncoupling proteins. Some beneficial effects of DR can be achieved by administering hormones that suppress appetite (leptin and ciliary neurotrophic factor) or by supplementing the diet with 2‐deoxy‐d‐glucose, which may act as a calorie restriction mimetic. The profound influences of the quantity and timing of food intake on neuronal function and vulnerability to disease have revealed novel molecular and cellular mechanisms whereby diet affects the nervous system, and are leading to novel preventative and therapeutic approaches for neurodegenerative disorders.
Employing a superior BACE1 cleavage sequence to probe cellular APP processing
Authors: Tomasselli Alfredo G. , Qahwash Isam , Emmons Thomas L. , Lu Yifeng , Leone Joseph W. , Lull June M. , Fok Kam F. , Bannow Carol A. , Smith Clark W. , Bienkowski Michael J. , Heinrikson Robert L. , Yan Riqiang 
Published: March 2003

AbstractThe involvement of β‐secretase (BACE1; β‐site APP‐cleaving enzyme) in producing the β‐amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3‐P2′ sequence, …VKM‐↓‐DA…, was replaced by …ISY‐↓‐EV… (‐↓‐ = β site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL‐↓‐DAEFR. The expanded peptide, GLTNIKTEEISEISY‐↓‐EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these β‐site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the β‐site. Insertion of the optimized ISY‐↓‐EV motif at either the β‐site (Asp1) or β′‐site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.
Brain region‐specific alterations of 5‐HT2A and 5‐HT2C receptors in serotonin transporter knockout mice
Authors: Li Qian , Wichems Christine H. , Ma Li , Van de Kar Louis D. , Garcia Francisca , Murphy Dennis L. 
Published: March 2003

AbstractThe aim of the present studies was to determine the effects of reduced or absent serotonin (5‐HT) transporters (5‐HTTs) on 5‐HT2A and 5‐HT2C receptors. The density of 5‐HT2C receptors was significantly increased in the amygdala and choroid plexus of 5‐HTT knockout mice. On the other hand, the density of 5‐HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5‐HTT knockout mice. However, 5‐HT2A mRNA was not changed in any brain region measured. 5‐HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5‐HT2A receptors was evaluated by hormonal responses to (+/–)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5‐HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5‐HTTs changes the density of 5‐HT2A and 5‐HT2C receptors in a brain region‐specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5‐HTT knockout mice.
Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells
Authors: Ohsawa Ikuroh , Nishimaki Kiyomi , Yasuda Chie , Kamino Kouzin , Ohta Shigeo 
Published: March 2003

AbstractMitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late‐onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2‐deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2‐deficient transfectants were highly vulnerable to exogenous 4‐hydroxy‐2‐nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2‐deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4‐hydroxy‐2‐nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.