bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the N‐acetylaspartate biosynthetic enzyme from rat brain
Authors: Madhavarao C. N. , Chinopoulos C. , Chandrasekaran K. , Namboodiri M. A. A.
Published: July 2003

Aspartate N‐acetyltransferase (Asp‐NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp‐NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp‐NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size‐exclusion high performance liquid chromatography. Asp‐NAT activity and the optical density pattern of the eluted protein from size‐exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi‐subunit complex Asp‐NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3‐[(3‐cholamidopropyl)dimethylammonia]‐1‐propanesulfonate (CHAPS) treatment followed by size‐exclusion chromatography. Asp‐NAT showed an order of magnitude difference in Km between l‐aspartate (l‐Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp‐NAT showed high specificity towards l‐Asp with 3% or less activity towards l‐Glu, l‐Asn, l‐Gln and Asp‐Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.
Molecular modeling and mutagenesis of the ligand‐binding pocket of the mGlu3 subtype of metabotropic glutamate receptor
Authors: Yao Yi , Pattabiraman N. , Michne William F. , Huang Xi‐Ping , Hampson David R.
Published: July 2003

A homology model of the extracellular domain of the mGlu3 subtype of metabotropic glutamate (mGlu) receptor was generated and tested using site‐directed mutagenesis, a radioligand‐binding assay using the Group II selective agonist (2S,2′R,3′R)‐2‐(2′,3′‐[3H]dicarboxycyclopropyl) glycine ([3H]DCG‐IV), and in a fluorescence‐based functional assay in live transiently transfected human embryonic kidney cells. Ten of the 12 mGlu3 mutants (R64A, R68A, Y150A, S151A, T174A, D194A, Y222A, R277A, D301A and K389) showed either no binding or a 90% or greater loss of specific [3H]DCG‐IV binding. Several analogous mutations in mGlu2 supported the results obtained with mGlu3. These results demonstrate that the binding of [3H]DCG‐IV to mGlu3 is exceptionally sensitive to mutagenesis‐induced perturbations. In silico docking of DCG‐IV into the agonist binding pocket of mGlu3 facilitated the interpretation the mutagenesis results. Tyrosines 150 and 222, and arginine 277 show close contacts with the third carboxylic acid group in DCG‐IV, which is not present in glutamate or (2S,1′S,2′S)‐2‐(carboxycyclopropyl)glycine (L‐CCG‐I). Mutation of these three amino acids to alanine resulted in a near complete loss of receptor activation by DCG‐IV and retention of near wild‐type affinity for L‐CCG‐I. It is proposed that hydrogen bonding between this carboxylate and tyrosines 150 and 222 and arginine 277 provide a partial explanation for the high affinity and Group II selectivity of DCG‐IV. These findings define the essential features of the ligand‐binding pocket of mGlu3 and, together with other recent studies on mGlu receptors, provide new opportunities for structure‐based drug design.
Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARγ) in murine primary astrocytes and neurons
Authors: Klotz Luisa , Sastre Magdalena , Kreutz Anne , Gavrilyuk Vitaliy , Klockgether Thomas , Feinstein Douglas L. , Heneka Michael T.
Published: July 2003

Cerebral inflammatory events play an important part in the pathogenesis of Alzheimer's disease (AD). Agonists of the peroxisome proliferator‐activated receptor gamma (PPARγ), a nuclear hormone receptor that mediates anti‐inflammatory actions of non‐steroidal anti‐inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) – a pathological hallmark of AD – plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARγ‐mediated suppression of inflammation we investigated the influence of NA on PPARγ expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 µm NA resulted in an increase of PPARγ mRNA as well as PPARγ protein levels in both cell types. These effects were blocked by the β‐adrenergic antagonist propranolol but not by the α‐adrenergic antagonist phentolamine, suggesting that they might be mediated by β‐adrenergic receptors. Our results indicate for the first time that PPARγ expression can be modulated by the cAMP signalling pathway, and suggest that the anti‐inflammatory effects of NA on brain cells may be partly mediated by increasing PPARγ levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARγ expression and therefore potentiate neuroinflammatory processes.
Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis
Authors: Battaglioli Gino , Liu Hongcheng , Martin David L.
Published: July 2003

Glutamate decarboxylase (GAD) exists as two isoforms, GAD65 and GAD67. GAD activity is regulated by a cycle of activation and inactivation determined by the binding and release of its co‐factor, pyridoxal 5′‐phosphate. Holoenzyme (GAD with bound co‐factor) decarboxylates glutamate to form GABA, but it also catalyzes a slower transamination reaction that produces inactive apoGAD (without bound co‐factor). Apoenzyme can reassociate with pyridoxal phosphate to form holoGAD, thus completing the cycle. Within cells, GAD65 is largely apoenzyme (∼93%) while GAD67 is mainly holoenzyme (∼72%). We found striking kinetic differences between the GAD isoforms that appear to account for this difference in co‐factor saturation. The glutamate dependent conversion of holoGAD65 to apoGAD was about 15 times faster than that of holoGAD67 at saturating glutamate. Aspartate and GABA also converted holoGAD65 to apoGAD at higher rates than they did holoGAD67. Nucleoside triphosphates (such as ATP) are known to affect the activation reactions of the cycle. ATP slowed the activation of GAD65 and markedly reduced its steady‐state activity, but had little affect on the activation of GAD67 or its steady‐state activity. Inorganic phosphate opposed the effect of ATP; it increased the rate of apoGAD65 activation but had little effect on apoGAD67 activation. We conclude that the apo‐/holoenzyme cycle of inactivation and reactivation is more important in regulating the activity of GAD65 than of GAD67.
Regulation of expression of sulfoglucuronyl carbohydrate (HNK‐1), Amphoterin and RAGE in retinoic acid‐differentiated P19 embryonal carcinoma cells
Authors: Chou Denise K. H. , Henion Timothy R. , Jungalwala Firoze B.
Published: July 2003

HNK‐1 antibody reactive sulfoglucuronyl carbohydrate (SGC) and SSEA‐1 antibody reactive Lewis X (Lex) epitope are expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and have been implicated in cell–cell recognition, neurite outgrowth, and/or neuronal migration during development. Interaction of SGC with its binding protein Amphoterin and interaction of Amphoterin with a cell‐signaling molecule, receptor for advance glycation end product (RAGE) have been suggested to regulate neurite outgrowth and neuronal migration. The regulation of expression of SGC, Lex, Amphoterin, and RAGE was studied in embryonal carcinoma P19 cells after treatment with retinoic acid (RA). The untreated proliferating P19 cells strongly expressed the Lex epitope, which was mostly due to Lex‐glycoproteins. P19 cells, when differentiated into neuron‐like cells by RA, did not express the Lex epitope, but expressed increasing levels of SGC, with time in culture. Quantitative biochemical analyses showed that in the P19 cells after RA treatment, the amount of SGC‐glycoproteins increased at a significantly higher level than sulfoglucuronyl glycolipid‐1 (SGGL‐1). The increase in the levels of SGGL‐1 was due to 16‐fold upregulation in the activity of lactosylceramide: N‐acetylglucosaminyl‐transferase (Lc3 synthase), which synthesizes the key intermediate lactotriosylceramide (Lc3Cer), for lacto‐ and neolacto‐glycolipids. The large increase in the activity of Lc3 synthase appeared to regulate the levels of other neolacto glycolipids, such as Lc3Cer, nLc4Cer, nLc6Cer, disialosyl‐nLc4Cer (LD1), and Lex‐glycolipids. Strong upregulation of glucuronyl‐transferase and modest twofold enhancement in the activity of the glucuronyl‐sulfotransferase, which catalyze the final steps in the SGC synthesis, also would account for the large increase in the synthesis SGC‐glycoproteins. RA also upregulated the synthesis of Amphoterin and RAGE in P19 cells. SGC, RAGE, and Amphoterin were co‐localized in the RA‐differentiated neurons. The initiation of neurite outgrowth along with co‐ordinated upregulation of Amphoterin, RAGE, SGC‐glycoproteins, and SGGLs in RA‐treated P19 cells support the hypothesis that these molecules are involved in the neuronal process formation.
Diabetic neuropathy: inhibitory G protein dysfunction involves PKC‐dependent phosphorylation of Goα
Authors: Shangguan Yu , Hall Karen E. , Neubig Richard R. , Wiley John W.
Published: July 2003

We examined the hypothesis that decreased inhibitory G protein function in diabetic neuropathy is associated with increased protein kinase C (PKC)‐dependent phosphorylation of the Goα subunit. Streptozotocin‐induced diabetic rats were studied between 4 and 8 weeks after onset of diabetes and compared with aged‐matched healthy animals as controls. Opioid‐mediated inhibition of forskolin‐stimulated cyclic AMP was significantly less in dorsal root ganglia (DRGs) from diabetic rats compared with controls. Activation of PKC in DRGs from control rats was associated with a significant decrease in opioid‐mediated inhibition of forskolin‐stimulated cyclic AMP that was similar to the decrease in inhibition observed in DRGs from diabetic rats. Both basal and PKC‐mediated labeling of Goα with 32Pi was significantly less in DRGs from diabetic rats, supporting increased endogenous PKC‐dependent phosphorylation of Goα. Probing of immunoprecipitated Goα with an anti‐phospho‐serine/threonine specific antibody revealed a significant increase in baseline phosphorylation in diabetic DRGs. Activation of PKC produced a significant increase in phosphorylation in control DRGs but no significant increase in Goα in diabetic DRGs. Phosphorylation of PKC‐α was increased, PKC‐βII was unchanged and PKC‐δ decreased in diabetic DRGs. These results suggest that diminished inhibitory G protein function observed in DRGs neurons from diabetic rats involves an isoform‐specific PKC‐dependent pathway.
Involvement of mitochondrial K+ release and cellular efflux in ischemic and apoptotic neuronal death
Authors: Liu Dong , Slevin John R. , Lu Chengbiao , Chan Sic L. , Hansson Magnus , Elmér Eskil , Mattson Mark P.
Published: July 2003

We measured and manipulated intracellular potassium (K+) fluxes in cultured hippocampal neurons in an effort to understand the involvement of K+ in neuronal death under conditions of ischemia and exposure to apoptotic stimuli. Measurements of the intracellular K+ concentration using the fluorescent probe 1,3‐benzenedicarboxylic acid, 4,4′‐[1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecane‐7,16‐diylbis(5‐methoxy‐6,2‐benzofurandiyl)]bis‐, tetrakis [(acetyloxy) methyl] ester (PBFI) revealed that exposure of neurons to cyanide (chemical hypoxia), glutamate (excitotoxic insult) or staurosporine (apoptotic stimulus) results in efflux of K+ and cell death. Treatment of neurons with 5‐hydroxydecanoate (5HD), an inhibitor of mitochondrial K+ channels, reduced K+ fluxes in neurons exposed to each insult and increased the resistance of the cells to death. K+ efflux was attenuated, levels of oxyradicals were decreased, mitochondrial membrane potential was stabilized and release of cytochrome c from mitochondria was attenuated in neurons treated with 5HD. K+ was rapidly released into the cytosol from mitochondria when neurons were exposed to the K+ channel opener, diazoxide, or to the mitochondrial uncoupler, carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), demonstrating that the intramitochondrial K+ concentration is greater than the cytosolic K+ concentration. The release of K+ from mitochondria was followed by efflux through plasma membrane K+ channels. In vivo studies showed that 5HD reduces ischemic brain damage without affecting cerebral blood flow in a mouse model of focal ischemic stroke. These findings suggest that intracellular K+ fluxes play a key role in modulating neuronal oxyradical production and cell survival under ischemic conditions, and that agents that modify K+ fluxes may have therapeutic benefit in stroke and related neurodegenerative conditions.
Central catecholamine depletion inhibits peripheral lymphocyte responsiveness in spleen and blood
Authors: Pacheco‐López Gustavo , Niemi Maj‐Britt , Kou Wei , Bildhäuser Andre , Gross Claus M. , Goebel Marion U. , Del Rey Adriana , Besedovsky Hugo O. , Schedlowski Manfred
Published: July 2003

Experimental and clinical evidence has demonstrated extensive communication between the CNS and the immune system. To analyse the role of central catecholamines in modulating peripheral immune functions, we injected the neurotoxin 6‐hydroxydopamine (6‐OHDA) i.c.v. in rats. This treatment significantly reduced brain catecholamine content 2, 4 and 7 days after injection, and in the periphery splenic catecholamine levels were reduced 4 days after treatment. Central catecholamine depletion induced an inhibition of splenic and blood lymphocyte proliferation and splenic cytokine production and expression (interleukin‐2 and interferon‐γ) 7 days after injection. In addition, central treatment with 6‐OHDA reduced the percentage of spleen and peripheral blood natural killer (CD161 +) cells, and T‐cytotoxic (CD8 +) cells in peripheral blood. The reduction in splenocyte proliferation was not associated with a glucocorticoid alteration but was completely abolished by prior peripheral sympathectomy. These data demonstrate a crucial role of central and peripheral catecholamines in modulating immune function.
Oxidative phosphorylation by in situ synaptosomal mitochondria from whole brain of young and old rats
Authors: Joyce O. J. P. , Farmer M. K. , Tipton K. F. , Porter R. K.
Published: July 2003

Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 ± 0.42, n = 11) when compared with those from the young rats (7.87 ± 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age‐dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.
The structure of neuromelanin as studied by chemical degradative methods
Authors: Wakamatsu Kazumasa , Fujikawa Kenichi , Zucca Fabio A. , Zecca Luigi , Ito Shosuke
Published: July 2003

The biosynthesis, structure and function of neuromelanin (NM), the dark brown melanin‐like pigment present in the substantia nigra (SN), are not well characterized, in spite of the possible involvement of NM in the etiology and pathogenesis of Parkinson's disease. NM was isolated from the SN of five non‐Parkinsonian human brains. NM and synthetic melanins, employed as models, were characterized by chemical analysis. Alkaline hydrogen peroxide (H2O2) oxidation of NM generated four degradation products, pyrrole‐2,3‐dicarboxylic acid (PDCA), pyrrole‐2,3,5‐tricarboxylic acid (PTCA), thiazole‐4,5‐dicarboxylic acid (TDCA) and thiazole‐2,3,5‐tricarboxylic acid (TTCA), whose ratios, especially the TTCA to PDCA ratio, indicate that NM is derived mostly from dopamine (DA) with 25% incorporation of cysteine (Cys) in the form of a benzothiazine structure. Hydriodic acid (HI) reductive hydrolysis of NM yielded 4‐amino‐3‐hydroxyphenylethylamine (4‐AHPEA) as a marker of cysteinyldopamine (CysDA)‐derived units. The 4‐AHPEA to PDCA ratio indicates a 21% incorporation of CysDA‐derived units into NM. These degradative experiments also suggest that DOPA is not incorporated into NM to a significant extent (approximately 6% the level of DA). It is concluded that the TTCA to PDCA ratio is a useful indicator of CysDA‐derived units in NM, and NM consists mainly of DA‐melanin with some contribution from CysDA‐melanin. The involvement of DA and CysDA as building blocks of NM demonstrates the detoxifying role of NM synthesis, since it prevents the intraneuronal accumulation of DA and CysDA, which would cause toxic effects.
Mitochondrial peroxiredoxin‐3 protects hippocampal neurons from excitotoxic injury in vivo
Authors: Hattori Fumiyuki , Murayama Norihito , Noshita Takafumi , Oikawa Shinzo
Published: July 2003

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium‐buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin‐3 (Prx‐3), to investigate the mechanism by which toxic properties of ROS were up‐regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx‐3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx‐3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx‐3 completely inhibited protein nitration and markedly reduced gliosis, a post‐neuronal cell death event. Since mitochondrial Prx‐3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx‐3 up‐regulation might be a useful novel approach for the management of neurodegenerative diseases.
Evidence for functional adenosine A3 receptors in microglia cells
Authors: Hammarberg Christian , Schulte Gunnar , Fredholm Bertil B.
Published: July 2003

Adenosine exerts its effects through four subtypes of G‐protein‐coupled receptors (GPCRs): adenosine A1 and A3 receptors (A3R), which generally couple to Gi proteins and adenosine A2A and A2B receptors that activate Gs proteins. Though there is evidence for the expression of mRNA for the A3R in the central nervous system, evidence for functional receptors has depended on drugs with uncertain specificity. Here, we show that A3Rs mediating functional responses are present in microglia cells. By selectively stimulating the A3R in both primary mouse microglia cells and the N13 microglia cell line with the agonist Cl‐IB‐MECA, we have found a biphasic, partly Gi protein‐dependent influence on the phosphorylation of the extracellular signal‐regulated protein kinase 1/2 (ERK1/2). ERK1/2 activation was assessed by immunoblotting with phospho‐specific antibodies. The involvement of the A3R in Cl‐IB‐MECA‐induced ERK1/2 phosphorylation was confirmed by demonstrating that those effects are absent in primary mouse microglia cells isolated from mice lacking the gene for the A3R.
Collapsin response mediator protein‐2 accelerates axon regeneration of nerve‐injured motor neurons of rat
Authors: Suzuki Yasuhiro , Nakagomi Saya , Namikawa Kazuhiko , Kiryu‐Seo Sumiko , Inagaki Naoyuki , Kaibuchi Kozo , Aizawa Hitoshi , Kikuchi Kenjiro , Kiyama Hiroshi
Published: July 2003

The rat collapsin response mediator protein‐2 (CRMP‐2) is a member of CRMP family (CRMP‐1–5). The functional consequence of CRMP‐2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP‐2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP‐1, CRMP‐2, CRMP‐5 mRNA expressions increased after nerve injury, whereas CRMP‐3 and CRMP‐4 mRNA did not show any significant change. In the N1E‐115 cells, CRMP‐2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP‐2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP‐2 (ΔC381), which acts as a dominant negative form of CRMP‐2, inhibited neurite formation. Collectively, it would be plausible that CRMP‐2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP‐2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde‐tracer, Fluoro‐Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP‐2‐overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP‐2 has potent neurite elongation activity in nerve regeneration in vivo.
A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases
Authors: Kurrasch‐Orbaugh Deborah M. , Parrish Jason C. , Watts Val J. , Nichols David E.
Published: July 2003

Previous studies in our laboratory have shown that in NIH3T3–5HT2A cells, 5‐HT‐induced AA release is PLA2‐coupled and independent of 5‐HT2A receptor‐mediated PLC activation. Although 5‐HT2A receptor‐mediated PLC activation is known to be Gαq‐coupled, much less is understood about 5‐HT2A receptor‐mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5‐HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5‐HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin‐sensitive G proteins, namely Gαi/o, as well as pertussis toxin‐insensitive G proteins, namely Gα12/13, in 5‐HT‐induced AA release. Furthermore, inactivation of both Gβγ heterodimers and Rho proteins resulted in decreased agonist‐induced AA release, without having any effect on PLC‐IP accumulation. We also demonstrated 5‐HT2A receptor‐mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5‐HT‐induced AA release. Taken together, these results suggest that the 5‐HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Gαi/o‐associated Gβγ‐mediated ERK1,2 activation and Gα12/13‐coupled, Rho‐mediated p38 activation.
The role of the RE1 element in activation of the NR1 promoter during neuronal differentiation
Authors: Bai Guang , Zhuang Zhiye , Liu Anguo , Chai Yanfeng , Hoffman Peter W.
Published: July 2003

To understand the genetic mechanism controlling the expression of the NMDA subtype of glutamate receptors during neuronal differentiation, we studied activation of the N‐methyl‐D‐aspartate receptor subunit 1 (NR1) gene and the role of the repressor element‐1 (RE1) element in NR1 promoter activation. Following neuronal differentiation of P19 embryonic carcinoma cells, the NR1 transcription rate and mRNA level were significantly increased, while the nuclear level of the repressor RE1 silencing transcription factor (REST)/neuron‐restriction silencer factor (NRSF) was reduced. Nuclear REST/NRSF from undifferentiated cells formed a large complex with the NR1 RE1 element. While this complex was significantly reduced after the differentiation, REST/NRSF from differentiated cells formed a new, faster migrating complex. In transient transfections, deletion of the RE1 element increased activity of the 5.4‐kb NR1 promoter sixfold in undifferentiated cells, but only induced approximately 1.4‐fold increase in differentiated cells. Forced expression of REST/NRSF in differentiated cells suppressed the promoter, while forced expression of a dominant‐negative REST/NRSF induced promoter activity as well as the mRNA of the NR1 gene in undifferentiated cells. In stable transfectants, the wild‐type promoter showed a robust increase in activity following differentiation in a pattern similar to the NR1 mRNA increase. Conversely, the promoter lacking the RE1 element showed only a moderate increase. Our data suggest that the NR1 gene up‐regulation during neuronal differentiation is controlled by its promoter activation, which is largely determined by the interaction between the RE1 element and the repressor REST/NRSF.
Neurotoxins in the Neurobiology of Pain
Authors: Silberstein Stephen D.
Published: July 2003

Migraine is a common, chronic, incapacitating, neurovascular disorder that affects an estimated 12% of the population. Understanding the basic mechanisms of pain is important when treating patients with chronic pain disorders.

Pain, an unpleasant sensory and emotional experience, is usually triggered by stimulation of peripheral nerves and often associated with actual or potential tissue damage. Peripheral nerve fibers transmit pain signals from the periphery toward the spinal cord or brain stem. The different diameter pain fibers (A and C) vary in the speed of conduction and the type of pain transmitted (eg, sharp versus dull). When stimulated, peripheral pain fibers carrying sensory input from the body enter at different layers of the dorsal horn, which is then propagated toward the thalamus via the spinothalamic tract within the spinal cord. Conversely, sensory input from the face does not enter the spinal cord but enters the brain stem via the trigeminal nerve.

This review describes in detail the neurobiological mechanisms and pathways for pain sensation, with a focus on migraine pain.
Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain Management
Authors: Aoki K. Roger
Published: July 2003

The neurotoxin, botulinum toxin type A, has been used successfully, in some patients, as an analgesic for myofascial pain syndromes, migraine, and other headache types. The toxin inhibits the release of the neurotransmitter, acetylcholine, at the neuromuscular junction thereby inhibiting striated muscle contractions. In the majority of pain syndromes where botulinum toxin type A is effective, inhibiting muscle spasms is an important component of its activity. Even so, the reduction of pain often occurs before the decrease in muscle contractions suggesting that botulinum toxin type A has a more complex mechanism of action than initially hypothesized. Current data points to an antinociceptive effect of botulinum toxin type A that is separate from its neuromuscular activity. The common biochemical mechanism, however, remains the same between botulinum toxin type A's effect on the motor nerve or the sensory nerve: enzymatic blockade of neurotransmitter release. The antinociceptive effect of the toxin was reported to block substance P release using in vitro culture systems.1

The current investigation evaluated the in vivo mechanism of action for the antinociceptive action of botulinum toxin type A. In these studies, botulinum toxin type A was found to block the release of glutamate. Furthermore, Fos, a product of the immediate early gene, c‐fos, expressed with neuronal stimuli was prevented upon peripheral exposure to the toxin.

These findings suggest that botulinum toxin type A blocks peripheral sensitization and, indirectly, reduces central sensitization. The recent hypothesis that migraine involves both peripheral and central sensitization may help explain how botulinum toxin type A inhibits migraine pain by acting on these two pathways. Further research is needed to determine whether the antinociceptive mechanism mediated by botulinum toxin type A affects the neuronal signaling pathways that are activated during migraine.
Synaptic Transmission: Inhibition of Neurotransmitter Release by Botulinum Toxins
Authors: Dolly Oliver
Published: July 2003

Botulinum toxin type A, a protein long used in the successful treatment of various dystonias, has a complex mechanism of action that results in muscle relaxation. At the neuromuscular junction, the presynaptic nerve ending is packed with synaptic vesicles filled with acetylcholine, and clustered at the tip of the folds of the postsynaptic muscle membrane are the acetylcholine receptors. Synaptic vesicles fuse with the membrane in response to an elevation of intraneuronal calcium concentration and undergo release of their transmitter by exocytosis. Intracellular proteins that contribute to the fusion of the vesicles with the plasma membrane during exocytosis include synaptosomal protein with a molecular weight of 25 kDa (SNAP‐25); vesicle‐associated membrane protein (VAMP), also known as synaptobrevin; and syntaxin.

Through their proteolytic action on these proteins, botulinum toxins prevent exocytosis, thereby inhibiting the release of acetylcholine. There are 7 serotypes of this toxin—A, B, C1, D, E, F, and G—and each cleaves a different intracellular protein or the same target at distinct bonds. The separate cleavage sites in SNAP‐25 for botulinum toxin types A and E contribute to their dissimilar durations of muscle relaxation.

This report describes the molecular basis for the inhibition by botulinum toxins of neuroexocytosis and subsequent functional recovery at the neuromuscular junction.
Botulinum Neurotoxin for the Treatment of Migraine and Other Primary Headache Disorders: From Bench to Bedside
Authors: Dodick David W.
Published: July 2003

Botulinum toxin type A, a neurotoxin, is effective for treating a variety of disorders of involuntary muscle contraction including cervical dystonia, blepharospasm, and hemifacial spasm. It inhibits neuromuscular signaling by blocking the release of acetylcholine at the neuromuscular junction. The biological effects of the toxin are transient, with normal neuronal signaling returning within approximately 3 to 6 months postinjection.

Recent clinical findings suggest that botulinum toxin type A may inhibit pain associated with migraine and other types of headache. However, the mechanism by which this toxin inhibits pain is not fully understood and is under investigation. Research findings suggest that botulinum toxin type A inhibits the release of neurotransmitters from nociceptive nerve terminals and, in this way, may possess an analgesic effect. A number of retrospective open‐label chart reviews and 3 double‐blind, placebo‐controlled trials have demonstrated that localized injections of botulinum toxin type A significantly reduce the frequency, severity, and disability associated with migraine headaches. Although the majority of patients in these studies experienced no botulinum toxin type A‐mediated side effects, a small percentage of patients did report transient minor side effects including blepharoptosis, diplopia, and injection‐site weakness. Currently, 4 randomized, placebo‐controlled, clinical trials are being conducted to evaluate the efficacy, optimal dosing, and side‐effect profile of botulinum toxin type A as a novel treatment for migraine and other types of headache. These studies may provide further evidence that botulinum toxin type A is an effective option for the preventive treatment of migraine.
A STUDY OF THE PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA/CUSPIDATA COMPLEX (BACILLARIOPHYCEAE): WHAT IS P. PSEUDODELICATISSIMA? 1
Authors: Lundholm Nina , Moestrup Øjvind , Hasle Grethe Rytter , Hoef‐Emden Kerstin
Published: July 2003

Based on morphological variation found in specimens ascribed to Pseudo‐nitzschia pseudodelicatissima and uncertainty regarding the delineation of P. pseudodelicatissima and P. cuspidata, cultures and field material of diatoms in the P. pseudodelicatissima/cuspidata complex were studied in morphological detail. Four different species were identified. The descriptions of the species P. pseudodelicatissima and P. cuspidata were emended on the basis of studies of type material. In addition, P. calliantha sp. nov. and P. caciantha sp. nov. were described as new species based on morphological and molecular data. The morphological differences among the species were found in characters such as width and shape of the valve, density of fibulae and striae, structural pattern of the poroid hymen, and structure of the girdle bands. The morphological studies were supported by phylogenetic analyses of the nuclear‐encoded internal transcribed spacer 1, 5.8S, and internal transcribed spacer 2 rDNA of 24 strains representing 16 different Pseudo‐nitzschia species. The description of the four species helps to explain the variation observed in mating experiments on cultures originally designated as P. pseudodelicatissima. At least two previous reports of toxin production in species identified as P. pseudodelicatissima have been identified as being caused by P. calliantha, and one additional report of toxin production has been identified as either P. pseudodelicatissima or P. cuspidata.
MORPHOLOGY OF THE DIATOM GENUS CAMPYLONEIS (BACILLARIOPHYCEAE, BACILLARIOPHYTA), WITH A DESCRIPTION OF CAMPYLONEIS JULIAE SP. NOV. AND AN EVALUATION OF THE FUNCTION OF THE VALVOCOPULAE1
Authors: De Stefano Mario , Kooistra Wiebe H. C. F. , Marino Donato
Published: July 2003

A revision of the monoraphid pennate diatom genus Campyloneis Grunow was carried out based on LM and EM observations. The material examined originated from various herbarium collections and from extant epiphytic diatom communities on leaves of Posidonia spp. We also examined the generitype C. grevillei (Smith) Grunow and the fossil material of C. gheyselinchi Reinhold from which the author extracted the type. Our results clarified the fine structure of C. grevillei and C. gheyselinchi. Of the various varieties of C. grevillei, only the variety argus (Grunow) Cleve was retained. This differs from the nominate variety in the arrangement and shape of the areolae adjacent to the sternum of the araphid valve. The newly described taxon Campyloneis juliae De Stefano differs from all Campyloneis species in areolae ultrastructure and morphology of the valvocopulae. As for the fossil species C. gheyselinchi, the sternum valve areolae are similar to those of C. grevillei, but scarcity of frustules in the type material prohibited evaluation of its variability. For this reason we provisionally maintained its rank of species. The elaborate linking systems among the valvocopulae and valves in Campyloneis species appear to provide structural reinforcement against pressure from neighboring epiphytic diatoms and scouring of seagrass leaves.
IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1
Authors: Litaker R. Wayne , Vandersea Mark W. , Kibler Steven R. , Reece Kimberly S. , Stokes Nancy A. , Steidinger Karen A. , Millie Dave F. , Bendis Brian J. , Pigg Ryan J. , Tester Patricia A.
Published: July 2003

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL−1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.
LIGHT ACTION SPECTRA OF N2 FIXATION BY HETEROCYSTOUS CYANOBACTERIA FROM THE BALTIC SEA1
Authors: Staal Marc , Stal Lucas J. , Te Lintel Hekkert Sacco , Harren Frans J. M.
Published: July 2003

An on‐line, laser photo‐acoustic, trace gas detection system in combination with a stepper motor‐controlled monochromator was used to record semicontinuous light action spectra of nitrogenase activity in heterocystous cyanobacteria. Action spectra were made of cultures of Nodularia spumigena Mertens ex Bornet & Flahault, Aphanizomenon flos‐aquae Ralfs ex Bornet & Flahault, and Anabaena sp. and from field samples of a cyanobacterial bloom in the Baltic Sea. Nitrogenase activity was stimulated by monochromatic light coinciding the red and blue peaks of chl a, the phycobiliproteins phycocyanin (allophycocyanin) and phycoerythrin, and several carotenoids. Because nitrogenase is confined to the heterocyst, it was concluded that all photopigments must have been present in these cells, were involved in light harvesting and photosynthesis, and supplied the energy for N2 fixation. The species investigated showed marked differences in their nitrogenase action spectra, which might be related to their specific niches and to their success in cyanobacterial blooms. Moreover, light action spectra of nitrogenase activity shifted during the day, probably as the result of changes in the phycobiliprotein content of the heterocyst relative to chl a. Action spectra of nitrogenase and changes in pigment composition are essential for the understanding of the competitive abilities of species and for the estimation of N2 fixation by a bloom of heterocystous cyanobacteria.
SOME PRYMNESIACEAE (HAPTOPHYTA, PRYMNESIOPHYCEAE) FROM THE MEDITERRANEAN SEA, WITH THE DESCRIPTION OF TWO NEW SPECIES:CHRYSOCHROMULINA LANCEOLATASP. NOV. ANDC. PSEUDOLANCEOLATASP. NOV.1
Authors: Puigserver Margarita , Chrétiennot‐Dinet Marie‐Josèphe , Nezan Elisabeth
Published: July 2003

Five species belonging to the family Prymnesiaceae (onePrymnesiumand fourChrysochromulina) have been identified in cultures obtained from water collected in the Bay of Banyuls‐sur‐Mer (Mediterranean Sea, France) using LM, SEM, and TEM. Two are described as new species,Chrysochromulina lanceolatasp. nov. andC. pseudolanceolatasp. nov. Both species are large and lanceolate with an acute posterior and two anterior arms. They are easily detectable with LM but difficult to distinguish to species level with live cells, without experience. EM reveals two completely different scale patterns in the two species. Cells ofC. lanceolataare 21–38 μm long, 7–12 μm wide, and 3–7 μm thick. They possess two subequal flagella (30–51 and 29–44 μm), and the haptonema is shorter than the flagella (23–37 μm). The cell body is covered by plate and spine scales. Cells ofC. pseudolanceolatasp. nov. are slightly smaller (15–18 × 6–8 μm) with more rounded extremities, two subequal flagella (19–26 and 17–24 μm), and the haptonema is longer than the flagella (about 35 μm). Three types of plate scales are observed in this species. Other findings areC. aliferaParke et Manton andC. throndseniiEikrem (a new record for the Mediterranean Sea).Prymnesium faveolatumFresnel, a new toxic species recently described, is illustrated with both LM and SEM.
PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1
Authors: Negri Andrew , Llewellyn Lyndon , Doyle Jason , Webster Nicole , Frampton Dion , Blackburn Susan
Published: July 2003

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.
ESTUARINE HETEROTROPHIC CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE): LIFE CYCLE AND CULTURE STUDIES1
Authors: Parrow Matthew W. , Burkholder JoAnn M.
Published: July 2003

Cryptoperidiniopsoids are an unclassified group of delicately thecate heterotrophic dinoflagellates known to be common in eastern U.S. estuarine waters. Over the past 10 years cryptoperidiniopsoids were isolated from different geographical regions and cultured with cryptophyte algal prey. In the seven clonal isolates examined, reproduction was strongly linked to the availability of prey cells. The dinoflagellates phagocytized the contents of prey cells through a tube‐like peduncle, similarly as close relatives of Pfiesteria spp. and several other heterotrophic species. Cell division occurred while encysted, most commonly yielding two biflagellated offspring. Abundant fusing gametes, phagotrophic planozygotes, and cysts with a pronounced nuclear cyclosis characterized persistent sexuality. Cysts with nuclear cyclosis produced two flagellated offspring cells. The resistance of reproductive cysts to antimicrobial treatments was examined, and a simple high‐yield technique was developed for population synchronization while ridding the dinoflagellates of most contaminating vacuolar prey DNA and external contaminants. The DNA content and population DNA profiles of synchronously excysted cryptoperidiniopsoids from different isolates were measured using flow cytometry and were related to the life history of these and other dinoflagellates. Cryptophyte‐fed cultures with versus without extracellular bacteria were compared, and bacteria apparently promoted cryptoperidiniopsoid feeding and growth. Externally bacteria‐free dinoflagellates were cultured in media enriched with dissolved organic nutrients, and nutritional benefit may have occurred in some treatments. The potential for mixotrophic nutrition from maintenance of cryptophyte chloroplasts was examined using flow cytometrically sorted cells, but evidence of kleptoplastidy was not found in these isolates under the conditions imposed.
VARIABILITY IN THE ECOPHYSIOLOGY OF HALIMEDA SPP. (CHLOROPHYTA, BRYOPSIDALES) ON CONCH REEF, FLORIDA KEYS, USA1
Authors: Beach Kevin , Walters Linda , Vroom Peter , Smith Celia , Coyer James , Hunter Cynthia
Published: July 2003

The photosynthetic performance, pigmentation, and growth of a Halimeda community were studied over a depth gradient on Conch Reef, Florida Keys, USA during summer–fall periods of 5 consecutive years. The physiology and growth of H. tuna (Ellis & Solander) Lamouroux and H. opuntia (L.) Lamouroux on this algal dominated reef were highly variable. Maximum rate of net photosynthesis (Pmax), respiration rate, and quantum efficiency (α) did not differ between populations of either species at 7 versus 21 m, even though the 21‐m site received a 66% lower photon flux density (PFD). Physiological parameters, as well as levels of photosynthetic pigments, varied temporally. Pmax, saturation irradiance, compensation irradiance, and growth were greatest in summer months, whereas α, chl a, chl b, and carotenoid concentrations were elevated each fall. Halimeda tuna growth rates were higher at 7 m compared with 21 m for only two of five growth trials. This may have arisen from variability in light and nutrient availability. Individuals growing at 7 m received a 29% greater PFD in August 2001 than in 1999. In August 1999 and 2001 seawater temperatures were uniform over the 14‐m gradient, whereas in August 2000 cold water regularly intruded upon the 21‐m but not the 7‐m site. These results illustrate the potentially dynamic relationship between nutrients, irradiance, and algal productivity. This suggests the necessity of long‐term monitoring over spatial and temporal gradients to accurately characterize factors that impact productivity.
MIXOTROPHY IN THE ANTARCTIC PHYTOFLAGELLATE, PYRAMIMONAS GELIDICOLA (CHLOROPHYTA: PRASINOPHYCEAE)1
Authors: Bell Elanor M. , Laybourn‐Parry Johanna
Published: July 2003

Grazing by the planktonic phytoflagellate, Pyramimonas gelidicola McFadden (Chlorophyta: Prasinophyta), and heterotrophic nanoflagellates (HNAN) in meromictic saline Ace Lake in the Vestfold Hills, eastern Antarctica was investigated in the austral summers of 1997 and 1999. Up to 47% of the P. gelidicola population ingested fluorescently labeled prey (FLP). Ingestion rates varied with depth. In January 1997 and November 1999, maximum P. gelidicola ingestion rates of 6.95 and 0.79 FLP·cell−1·h−1, respectively, were measured at the chemocline (6–8 m) where a deep chl maximum composed of phototrophic nanoflagellates (PNAN DCM), predominantly P. gelidicola, persisted all year. During the summers of 1997 and 1999, the grazing P. gelidicola community removed between 0.4% and approximately 16% of in situ bacterial biomass, equivalent to between 4% and>100% of in situ bacterial production. Because of their higher abundance, the community clearance rates of HNAN in Ace Lake generally exceeded those of P. gelidicola, but HNAN removed approximately only 3%–4% of bacterial biomass, equivalent to between 28% and 32% of bacterial production. Pyramimonas gelidicola growth rates were highest at the PNAN DCM concomitant with the highest ingestion rates. It is estimated that during the summer P. gelidicola can derive up to 30% of their daily carbon requirements from bacterivory at the PNAN DCM. This study confirms mixotrophy as an important strategy by which planktonic organisms can survive in extreme, polar, lacustrine ecosystems.