bluuebunny/crossref_metadata_2025 huggingface dataset:
Messenger RNA and protein expression analysis of voltage‐gated potassium channels in the brain of Aβ25–35‐treated rats
Authors: Pan Yaping , Xu Xianghua , Tong Xiaoyong , Wang Xiaoliang
Published: May 2004

Potassium channel dysfunction has been implicated in Alzheimer's disease. In the present study, the expression of voltage‐gated potassium channel (Kv) subunits in rat brain was measured after a single intracerebroventricular injection of β‐amyloid peptide 25–35 (Aβ25–35). After injection of Aβ, the spatial memory of rats was significantly impaired in the Morris water maze. Expression of five main Kv channel subunits (Kv1.5, Kv2.1, Kv1.4, Kv4.2, and Kv4.3) in mRNA level was assessed by using reverse transcription‐polymerase chain reaction (RT‐PCR). The mRNA levels of Kv2.1 and Kv1.4 were increased by 72% and 67%, respectively, in hippocampus, and Kv4.2 mRNA was increased by 58% in cortex. No other significant mRNA expression changes were found in Aβ‐treated rats. The protein expression of Kv2.1, Kv1.4, and Kv4.2 was detected by using Western blotting. Kv2.1 and Kv1.4 protein levels were increased by 48% and 50%, respectively, in hippocampus of Aβ‐treated rats, and Kv4.2 protein was increased by 42% in cerebral cortex. This study indicates that the expression up‐regulation of Kv1.4, Kv2.1, and Kv4.2 in Aβ‐induced cognitive impairment might play an important role in the pathogenesis of Alzheimer's disease. © 2004 Wiley‐Liss, Inc.
Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5‐triphosphate receptors in the neurotoxic effects induced by the amyloid‐β peptide
Authors: Ferreiro Elisabete , Oliveira Catarina R. , Pereira Cláudia
Published: May 2004

Studies with in‐vitro‐cultured neurons treated with amyloid‐β (Aβ) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca2+ homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)‐specific apoptotic pathway mediated by caspase‐12, which is activated upon the perturbation of ER Ca2+ homeostasis, may contribute to Aβ toxicity. To elucidate the involvement of deregulation of ER Ca2+ homeostasis in neuronal death induced by Aβ peptides, we have performed a comparative study using the synthetic peptides Aβ25–35 or Aβ1–40 and thapsigargin, a selective inhibitor of Ca2+ uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 μM) increased the intracellular Ca2+ levels and activated caspase‐3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the Aβ peptides (Aβ25–35, 25 μM; Aβ1–40, 0.5 μM), we observed a significant increase in [Ca2+]i, in caspase‐3‐like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca2+ release through ryanodine receptors (RyR) or inositol 1,4,5‐trisphosphate receptors (IP3R) in Aβ neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca2+ release through RyR or IP3R, were able to prevent the increase in [Ca2+]i and the activation of caspase‐3 and to protect partially against apoptosis induced by treatment with Aβ25–35 or Aβ1–40. In conclusion, our results demonstrate that the release of Ca2+ from the ER, mediated by both RyR and IP3R, is involved in Aβ toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to Aβ‐induced neuronal death. This study suggests that Aβ accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca2+ homeostasis. © 2004 Wiley‐Liss, Inc.
On the neurotoxicity of glutaric, 3‐hydroxyglutaric, and trans‐glutaconic acids in glutaric acidemia type 1
Authors: Lund T.M. , Christensen E. , Kristensen A.S. , Schousboe A. , Lund A.M.
Published: May 2004

Glutaric acidemia type 1 (GA1) is an autosomal recessively inherited deficiency of glutaryl‐CoA dehydrogenase. Accumulating metabolites, 3‐hydroxyglutaric (3‐OH‐GA), glutaric (GA), and trans‐glutaconic (TG) acids, have been proposed to be involved in the development of the striatal degeneration seen in children with GA1 via an excitotoxic mechanism. We have studied the extent to which 3‐OH‐GA, GA, and TG are neurotoxic and whether neurotoxicity is caused by an excitotoxic mechanism in which 3‐OH‐GA, GA, or TG overactivates N‐methyl‐D‐aspartate (NMDA) receptors. In cultured mouse neocortical neurons, all three compounds were weakly neurotoxic, possibly through activation of NMDA receptors. However, further studies in the rat cortical wedge preparation and with NMDA receptors expressed in Xenopus oocytes could not confirm an interaction of the compounds with NMDA receptors. It is concluded that the metabolites 3‐OH‐GA, GA, and TG are only weak neurotoxins and that the neurodegenerative cascade destroying the striatum in patients with GA1 involves mainly mechanisms other than excitoxicity. © 2004 Wiley‐Liss, Inc.
Molecular mimicry: Cross‐reactive antibodies from patients with immune‐mediated neurologic disease inhibit neuronal firing
Authors: Kalume Franck , Lee Sang Min , Morcos Yvette , Callaway Joseph C. , Levin Michael C.
Published: May 2004

Recent data indicate that molecular mimicry may play a role in the pathogenesis of human‐T‐lymphotropic virus type‐1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune‐mediated disease of the central nervous system (CNS). Specifically, HAM/TSP patients developed antibodies that cross‐react with heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an antigen highly expressed in neurons. Antibodies to HTLV‐1‐tax cross‐reacted with hnRNP A1, suggesting molecular mimicry between the two proteins. In support of this hypothesis, HAM/TSP IgG and antibodies to hnRNP A1 and HTLV‐1‐tax inhibited neuronal firing, suggesting that these antibodies can be pathogenic. We extended these observations by carrying out studies on over 20 different neurons. We also tested IgG isolated from six different HAM/TSP patients and two HTLV‐1 seronegative controls and added experiments that control for antibody isotype, antibody target, and neuron viability. In these studies, IgG was infused into the extracellular space during whole‐cell current clamp recordings of neurons. Our results confirm that in contrast to normal IgG, IgG from all HAM/TSP patients completely inhibited neuronal firing. Affinity‐purified antibodies specific for hnRNP A1 and a monoclonal antibody to HTLV‐1‐tax (which reacted with hnRNP A1 and whose epitope overlaps the human immunodominant epitope of tax) also inhibited neuronal firing. Monoclonal antibodies to neurofilament did not change neuronal firing. These data indicate that antibodies to neurons can be pathogenic, that biologic activity can be affected by a cross‐reactive epitope between HTLV‐1‐tax and hnRNP A1, and that molecular mimicry may play a role in the pathogenesis of HAM/TSP. © 2004 Wiley‐Liss, Inc.
Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre‐ and postsynaptic sites in the rat striatum
Authors: Tebano Maria Teresa , Pintor Annita , Frank Claudio , Domenici Maria Rosaria , Martire Alberto , Pepponi Rita , Potenza Rosa Luisa , Grieco Rosa , Popoli Patrizia
Published: May 2004

Adenosine A2A receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A2A receptor antagonists in models of pre‐ and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A2A receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose‐dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30–100 nM) significantly reduced 4‐aminopyridine (4‐AP)‐induced paired‐pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N‐methyl‐D‐aspartate (NMDA; 12.5 and 50 μM). The A2A antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A2A agonist CGS 21680 (100 nM) showed a protective influence toward 50 μM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca2+]i or the release of lactate dehydrogenase (LDH) induced by 100 and 300 μM NMDA, respectively. The ability of ZM 241385 to prevent QA‐induced glutamate outflow and 4‐AP‐induced effects confirms that A2A receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA‐induced effects suggest that A2A receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A2A antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role. © 2004 Wiley‐Liss, Inc.
Characterization of V642I‐AβPP‐induced cytotoxicity in primary neurons
Authors: Niikura Takako , Yamada Marina , Chiba Tomohiro , Aiso Sadakazu , Matsuoka Masaaki , Nishimoto Ikuo
Published: May 2004

Amyloid precursor protein (AβPP), a precursor of amyloid β (Aβ) peptide, is one of the molecules involved in the pathogenesis of Alzheimer's disease (AD). Specific mutations in AβPP have been found in patients inheriting familial AD (FAD). These mutant AβPP proteins cause cell death in neuronal cell lines in vitro, but the molecular mechanism of cytotoxicity has not yet been clarified completely. We analyzed the cytotoxic mechanisms of the London‐type AβPP mutant, V642I‐AβPP, in primary cortical neurons utilizing an adenovirus‐mediated gene transfer system. Expression of V642I‐AβPP protein induced degeneration of the primary neurons. This cytotoxicity was blocked by pertussis toxin, a specific inhibitor for heterotrimeric G proteins, Go/i, and was suppressed by an inhibitor of caspase‐3/7 and an antioxidant, glutathione ethyl ester. A specific inhibitor for NADPH oxidase, apocynin, but not a xanthine oxidase inhibitor or a nitric oxide inhibitor, blocked V642I‐AβPP‐induced cytotoxicity. Among mitogen‐activated protein kinase (MAPK) family proteins, c‐Jun N‐terminal kinase (JNK) and p38MAPK, but not extracellular regulated kinase (ERK), were involved in this cytotoxic pathway. The V642I‐AβPP‐induced cytotoxicity was not suppressed by two secretase inhibitors, suggesting that Aβ does not play a major role in this cytotoxicity. Two neuroprotective factors, insulin‐like growth factor I (IGF‐I) and Humanin, protected these primary neurons from V642I‐AβPP‐induced cytotoxicity. Furthermore, interleukin‐6 and ‐11 also attenuated this cytotoxicity. This study demonstrated that the signaling pathway activated by mutated AβPP in the primary neurons is the same as that by the other artificial insults such as antibody binding to AβPP and the artificial dimerization of cytoplasmic domain of AβPP. The potential of neurotrophic factors and cytokines in AD therapy is also indicated. © 2004 Wiley‐Liss, Inc.
Does phosphorylation of eukaryotic elongation factor eEF2 regulate protein synthesis in ischemic preconditioning?
Authors: García L. , O'Loghlen A. , Martín M.E. , Burda J. , Salinas M.
Published: May 2004

Ischemia/reperfusion‐associated translation inhibition in the hippocampus is attenuated significantly at reinitiation and elongation steps by ischemic preconditioning (Burda et al. [2003] Neurochem. Res. 28:1237–1243). To address potential regulation of the elongation step by changes in eukaryotic elongation factor 2 (eEF2) phosphorylation with and without acquired ischemic tolerance (IT), Wistar rats were preconditioned by 5‐min sublethal ischemia and 2 days later, 30‐min lethal ischemia was induced. Given the important role that oxidative stress plays in the ischemic process, eEF2 phosphorylation was also studied in a model of oxidative stress in vitro. Three blocks of our results support a lack of correlation between eEF2 phosphorylation status and protein synthesis rate. First, eEF2 was dephosphorylated significantly (activated) after transient cerebral ischemia in rats with and without IT or H2O2‐treated cells; however, protein synthesis was significantly inhibited under these three conditions. Second, after 30‐min reperfusion, the protein synthesis rate was maintained below control levels in cortex and hippocampus of rats without IT. Eukaryotic EF2 phosphorylated levels were notably low only in the cortex, whereas levels in the hippocampus were close to that of sham controls. In rats with IT, protein synthesis was virtually restored in both brain regions, but phosphorylated eEF2 levels were even higher than in rats without IT. Third, after 4‐hr reperfusion, the protein synthesis rate in cortex and hippocampus was observed to be below sham control values in rats with and without IT. Conversely, phosphorylated eEF2 levels were below sham control in rats with IT and reached sham control values in rats without IT. © 2004 Wiley‐Liss, Inc.
Insulin‐like growth factor type 1 prevents hyperglycemia‐induced uncoupling protein 3 down‐regulation and oxidative stress
Authors: Gustafsson Helena , Söderdahl Therése , Jönsson Gunn , Bratteng Jan‐Ove , Forsby Anna
Published: May 2004

Uncoupling proteins (UCPs) have been reported to decrease the mitochondrial production of reactive oxygen species (ROS) by lowering the mitochondrial inner membrane potential (MMP). We have previously shown that UCP3 expression is positively regulated by insulin‐like growth factor‐1 (IGF‐1). The aim of this study was to investigate the role of UCPs in IGF‐1‐mediated protection from hyperglycemia‐induced oxidative stress and neurodegeneration. Human neuroblastoma SH‐SY5Y cells were differentiated with retinoic acid for 6 days, after which exposure to 8, 30, or 60 mM glucose with or without 10 nM IGF‐1 was started. After 48–72 hr, the number of neurites per cell, UCP3 protein expression, MMP, and intracellular levels of ROS and total glutathione were examined. These studies showed that glucose concentration‐dependently reduced the number of neurites per cell, with a 50% reduction at 60 mM. In parallel, the UCP3 protein expression was down‐regulated, and the MMP was raised 3.5‐fold, compared with those in cells incubated with 8 mM glucose. Also, the ROS levels were increased, showing a twofold maximum at 60 mM glucose. This was accompanied by a twofold elevation of total glutathione levels, confirming an altered cellular redox state. IGF‐1 treatment prevented the glucose‐induced neurite degeneration and UCP3 down‐regulation. Furthermore, the MMP and the intracellular levels of ROS and glutathione were normalized to those of control cells. These data indicate that IGF‐1 may protect from hyperglycemia‐induced oxidative stress and neuronal injuries by regulating MMP, possibly by the involvement of UCP3. © 2004 Wiley‐Liss, Inc.
Recurrent paraplegia after remyelination of the spinal cord
Authors: Jasmin Luc , Ohara Peter T.
Published: May 2004

We have conducted a long‐term study of spinal cord morphology and motor function recovery in rats that have undergone lumbar spinal demyelination induced by the B‐fragment of cholera toxin (CTB)‐saporin. We found that, after the initial demyelination and paraplegia, motor function recovered and was stable for up to 9 months, after which there occurred a slow deterioration of motor function accompanied by loss of motoneurons and loss of spinal white matter. A striking morphological feature was the appearance of large spheroids of calcium in the ventral and dorsal horns and occasionally in the white matter. Motor performance deterioration occurred earlier and was more severe in rats that had been exercised on a treadmill, but the same morphological changes occurred in both exercise‐ and nonexercise‐treated animals. Rats given treadmill exercise starting 3 weeks after toxin injection had a mean motor deficit score of 3.0 (i.e., paraplegia) at perfusion, whereas the nontreadmill‐treated rats had a mean score of 1.8 (SD 0.38; n = 6; P < .05). These findings suggest that, in addition to the acute effects of the toxin‐induced demyelination from which there is recovery of motor function, there are chronic irreversible effects of the toxin, or the initial demyelination, that cause a slow progressive degeneration of the spinal cord. This model might therefore be useful in studying the long‐term effects of spinal insult of the type associated with conditions such as postpolio syndrome. © 2004 Wiley‐Liss, Inc.
Caloric restriction augments brain glutamic acid decarboxylase‐65 and ‐67 expression
Authors: Cheng Clara M. , Hicks Kristin , Wang Jie , Eagles Douglas A. , Bondy Carolyn A.
Published: May 2004

The ketogenic diet is a very low‐carbohydrate, high‐fat diet used to treat refractory epilepsy. We hypothesized that this diet may act by increasing expression of glutamic acid decarboxylase (GAD), the rate‐limiting enzyme in γ‐aminobutyric acid (GABA) synthesis. Thus, we evaluated brain GAD levels in a well‐established, seizure‐suppressing, rodent model of the ketogenic diet. Because the diet is most effective when administered with a modest (∼10%) calorie restriction, we studied three groups of animals: rats fed ad libitum standard rat chow (Ad lib‐Std); calorie‐restricted standard chow (CR‐Std); and an isocaloric, calorie‐restricted ketogenic diet (CR‐Ket). We found that GAD67 mRNA was significantly increased in the inferior and superior colliculi and cerebellar cortex in both CR diet groups compared with control (e.g., by 45% in the superior colliculus and by 71% in the cerebellar cortex;P< .001). GAD65 mRNA was selectively increased in the superior colliculus and temporal cortex in both CR‐Std and CR‐Ket diet groups compared with ad lib controls. The only apparent CR‐Ket‐specific effect was a 30% increase in GAD67 mRNA in the striatum (P= .03). Enhanced GAD immunoreactivity was detected in parallel with the mRNA changes. These data clearly show that calorie restriction increases brain GAD65 and ‐67 expression in several brain regions, independent of ketogenic effects. These observations may explain why caloric restriction improves the efficacy of the ketogenic diet in treating epilepsy and suggest that diet modification might be useful in treatment of a number of brain disorders characterized by impaired GAD or GABA activity. © 2004 Wiley‐Liss, Inc.
Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis
Authors: Matejuk Agata , Bakke Antony C. , Hopke Corwyn , Dwyer Jami , Vandenbark Arthur A. , Offner Halina
Published: May 2004

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogen's action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45dim) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA‐4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45dimVLA‐4+ cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45dimVLA‐4+ cells conferred protection from spontaneous EAE (Sp‐EAE). In contrast, injection of CD45highVLA‐4+ cells exacerbated the disease course. CD45dimVLA‐4+ cells also suppressed antigen‐specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45dimVLA‐4+ cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases. © 2004 Wiley‐Liss, Inc.
Beta‐secretase BACE1 is differentially controlled through muscarinic acetylcholine receptor signaling
Authors: Züchner Thole , Perez‐Polo J. Regino , Schliebs Reinhard
Published: May 2004

The β‐amyloid peptides derived by proteolytic cleavage from the amyloid precursor protein (APP) play a major role in the pathogenesis of Alzheimer's disease (AD) by forming aggregated, fibrillary complexes that have been shown to be neurotoxic. The β‐site APP‐cleaving enzyme (BACE1) has been identified as the key enzyme leading to β‐amyloid formation, and cholinergic mechanisms have been shown to control APP processing. The present study sought to determine whether BACE1 expression is controlled by muscarinic acetylcholine receptor (mAChR) subtypes in the neuroblastoma cell line SK‐SH‐SY5Y. Stimulation of cells with the M1/M3‐selective mAChR agonist talsaclidine for 1 hr resulted in a dose‐dependent increase in BACE1 expression up to twofold over basal levels. Similar effects of BACE1 up‐regulation were observed when protein kinase C was directly activated by phorbol esters. However, when the MAP kinases MEK/ERK were inhibited, BACE1 expression was no longer up‐regulated by the activation of M1‐mAChR. In contrast, BACE1 expression was suppressed by stimulation of M2‐mediated pathways via selective M2‐agonist binding or direct activation of adenylate cyclase with forskolin, an effect that was prevented by inhibiting protein kinase A. These results may explain the observed deterioration of AD patients after initial improvements with AChE inhibitor or M1‐mAChR agonist treatment. © 2004 Wiley‐Liss, Inc.
Metallothionein prevents neurodegeneration and central nervous system cell death after treatment with gliotoxin 6‐aminonicotinamide
Authors: Penkowa Milena , Quintana Albert , Carrasco Javier , Giralt Mercedes , Molinero Amalia , Hidalgo Juan
Published: May 2004

Transgenic expression of interleukin‐6 (IL‐6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP‐IL6 mice) induces significant inflammation and neurodegeneration but also affords neuroprotection against acute traumatic brain injury. This neuroprotection is likely mediated by the IL‐6‐induced protective factors metallothioneins‐I and ‐II (MT‐I+II). Here we evaluate the neuroprotective roles of IL‐6 vs. MT‐I+II during 6‐aminonicotinamide (6‐AN)‐induced neurotoxicity, by using GFAP‐IL6 mice and transgenic mice overexpressing MT‐I (TgMT) as well as GFAP‐IL6 mice crossed with TgMT mice (GFAP‐IL6 × TgMT). 6‐AN caused acute damage of brainstem gray matter areas identified by necrosis of astrocytes, followed by inflammatory responses. After 6‐AN‐induced toxicity, secondary damage was observed, consisting of oxidative stress, neurodegeneration, and apoptotic cell death. We hereby show that the primary injury caused by 6‐AN was comparable in wild‐type and GFAP‐IL6 mice, but MT‐I overexpression could significantly protect the brain tissue. As expected, GFAP‐IL6 mice showed increased CNS inflammation with more gliosis, macrophages, and lymphocytes, including increased cytokine expression, relative to the other mice. However, GFAP‐IL6 mice showed reduced oxidative stress (judged from nitrotyrosine, malondialdehyde, and 8‐oxoguanine stainings), neurodegeneration (accumulation of neurofibrillary tangles), and apoptosis (determined from TUNEL and caspase‐3). MT‐I+II expression was significantly higher in GFAP‐IL6 mice than in wild types, which may contribute to the IL‐6‐induced neuroprotection. In support of this, overexpression of MT‐I in GFAP‐IL6 × TgMT as well as TgMT mice protected the brainstem tissue significantly from 6‐AN‐induced toxicity and secondary brain tissue damage. Overall, the results demonstrate that brain MT‐I+II proteins are fundamental neuroprotective factors, which in the future may become therapeutic agents. © 2004 Wiley‐Liss, Inc.
Dysmyelination and reduced myelin basic protein gene expression by oligodendrocytes of SHP‐1‐deficient mice
Authors: Massa Paul T. , Wu Charlene , Fecenko‐Tacka Karen
Published: May 2004

We have shown previously that myelin‐forming oligodendrocytes express the protein tyrosine phosphatase SHP‐1 and that myelin formation was decreased in SHP‐1‐deficient motheaten mice compared to that in normal littermates. These studies suggested a potential importance for SHP‐1 in oligodendrocyte and myelin development. To address further this possibility, we analyzed myelin formation by microscopy and myelin basic protein (MBP) gene expression in motheaten mice at ages when myelination occurs in the developing central nervous system (CNS). Furthermore, we correlate these findings with MBP gene expression in oligodendrocytes grown in vitro. We have found that CNS myelination was significantly reduced in SHP‐1‐deficient mice relative to their normal littermates at multiple times during the active period of myelination. Under electron microscopy, greater numbers of axons in spinal cords of motheaten mice were either unmyelinated or had thinner myelin sheathes compared to those in matched areas of normal littermates. Accordingly, MBP protein and mRNA levels were reduced in SHP‐1‐deficient mice compared to that in the CNS of normal littermates. In vitro, O1+ oligodendrocytes from motheaten mice expressed much less MBP than O1+ oligodendrocytes of normal littermates indicating an alteration in oligodendrocyte differentiation. The latter correlated with reduced MBP mRNA relative to cerebroside galactosyl transferase (CGT) gene mRNA in SHP‐1‐deficient oligodendrocytes in purified cultures. We propose that SHP‐1 is a critical regulator of developmental signals leading to terminal differentiation and myelin sheath formation by oligodendrocytes. © 2004 Wiley‐Liss, Inc.
Vibrational spectra of some pharmacologically interesting pyroglutamato complexes
Authors: Wagner Claudia C. , Baran Enrique J.
Published: May 2004

The infrared and Raman spectra of lithium, zinc, calcium and magnesium complexes of L‐pyroglutamic acid were recorded and are briefly discussed. For the first three compounds the spectral discussion is based on their known structural characteristics. Comparing the spectroscopic behaviour of the complexes and the free acid, some comments on the structural peculiarities of the magnesium compound could be advanced. The pharmacological interest of these complexes is also briefly discussed. Copyright © 2004 John Wiley & Sons, Ltd.
A theoretical and spectroscopic study of carbamazepine polymorphs
Authors: Strachan Clare J. , Howell Sarah L. , Rades Thomas , Gordon Keith C.
Published: May 2004

The drug carbamazepine has been modeled, using ab initio density functional theory calculations [B3LYP/6–31G(d)], both as a single molecule and as a dimer. The predicted geometry of the single molecule calculation is compared with the crystallographic data on each of the polymorphs (carbamazepine forms I and III). From the predicted geometry it is possible to calculate the IR and Raman spectra; these predictions compare favorably with the observed spectra of both polymorphs of carbamazepine for most of the bands. The spectral differences between the polymorphs are more striking in the IR than the Raman spectra, with strong IR bands at 1688 and 1396 cm−1 in form I shifting to 1678 and 1388 cm−1 in form III. Analysis of the potential energy distributions for the calculated normal modes reveals that the vibrations are localized across different ring systems of the carbamazepine structure. Most notably, the polymorph‐sensitive modes in the IR spectra are localized to the pendant CONH2 group; it is these modes that show the greatest disparity from the calculated spectra, and it is this group that is perturbed in the polymorph crystal structures. The calculated dimer structure is similar to that of the single molecule, but the polymorph‐sensitive IR modes are significantly better predicted by the dimer calculation. Copyright © 2004 John Wiley & Sons, Ltd.
Surface‐enhanced Raman scattering of D‐penicillamine on silver colloids
Authors: López‐Ramírez M. R. , Arenas J. F. , Otero J. C. , Castro J. L.
Published: May 2004

The Raman spectra of D‐penicillamine in H2O and D2O were recorded under different acidity conditions, together with the respective SERS spectra on silver colloids. Vibrational assignments of the spectra obtained are proposed. The analysis of the SERS spectra shows that two of the conformers of D‐penicillamine are adsorbed at least in their dianionic forms, and a stable chemical bond is established between the sulfur atom and silver. Additionally, carboxylate and amino groups also interact with the metal. The pK and pK apparent values are decreased upon adsorption. Copyright © 2004 John Wiley & Sons, Ltd.
Polymorphism and devitrification of nifedipine under controlled humidity: a combined FT‐Raman, IR and Raman microscopic investigation
Authors: Chan K. L. A. , Fleming O. S. , Kazarian S. G. , Vassou D. , Chryssikos G. D. , Gionis V.
Published: May 2004

FT‐Raman and FT‐IR spectroscopy and Raman microscopy were employed to identify the structural characteristics of one amorphous and three crystalline phases of nifedipine, a common antihypertension drug of the 1,4‐dihydropyridine family. A significant fraction of the nifedipine molecules in the glass are found to be frozen into metastable rotational conformations as a result of melt quenching. Moreover, the effect of relative humidity (RH = 20–80%) on the onset and rate of crystallization was studied at 40 °C by means of in situ IR and Raman microscopy employing a controlled humidity cell. The nature and the relative abundance of the various crystallization products were analysed on the basis of the corresponding spectra of the neat compounds. Glassy nifedipine is found to crystallize to the thermodynamically stable α‐polymorph via a transient metastable β‐nifedipine phase. Increasing RH in the range 20–60% is found to favour the surface crystallization of the glass to β‐nifedipine. At high RH (80%), β‐nifedipine is rapidly converted to α‐nifedipine. Copyright © 2004 John Wiley & Sons, Ltd.
Quantitative analysis of polymorphic mixtures of carbamazepine by Raman spectroscopy and principal components analysis
Authors: Strachan Clare J. , Pratiwi Destari , Gordon Keith C. , Rades Thomas
Published: May 2004

The polymorphic behaviour of drugs is a major concern of the pharmaceutical industry as it may have considerable formulation, therapeutic, legal and commercial implications. It is therefore crucial to be able adequately to identify and quantify different polymorphic forms of drugs as early as possible in the drug discovery and development process. The aim of the present study was to investigate the application of Raman spectroscopy and principal components analysis (PCA) to the quantitative analysis of polymorphic mixtures of carbamazepine forms III and I, based on both individual peak areas and heights in the Raman spectra and whole wavenumber ranges. Various linear regressions indicated that in general using every fourth wavenumber value of a larger region of the Raman spectrum gives better quantitative results than choosing defined peak areas and peak heights, as the spectral differences between the polymorphs were only subtle. Baseline correction of the spectral data did not appear to improve the quantitative analysis, with detection and quantification limits being the lowest (0.33 and 1.09%, respectively) and R2 being the highest (0.96) when using every fourth wavenumber value from 2950 to 3100 cm−1 as input data for the PCA analysis without baseline correction. This study demonstrated that PCA of Raman spectroscopic data provides a sensitive method for the quantitative analysis of polymorphic forms, with a quantitation limit of <2%, if the spectral input data are selected carefully. In the absence of clear and distinct spectral differences at certain wavenumbers between the polymorphic forms, selection of larger spectral regions that contain most of the detectable differences between the two forms appears to be the best data selection technique. Copyright © 2004 John Wiley & Sons, Ltd.
Structural compatibility of novel nucleotide modifications with shortened linkages designed for antigene/antisense therapy
Authors: Hanuš J. , Němeček D. , Štěpánek J. , Turpin P.‐Y. , Králíková Š. , Bok J. , Rosenberg I.
Published: May 2004

The impact of isopolar shortened internucleotide linkage modification on the hybridization properties of potential antisense or antigene oligonucleotides was studied by using a model molecular system consisting of polyuridylic acid (PolyU) and analogs of diadenosine monophosphate with the modified linkage. Raman spectra of mixed aqueous solutions were measured at various temperatures and compositions of the mixtures for four types of modified linkage and natural ApA (3′–5′) as a reference. Analysis of the spectral sets provided amounts of formed complexes and their Raman spectra. It has been found that as in the case of ApA (3′–5′), the studied ApA analogs form with PolyU triplex‐like complexes containing a central pseudo‐chain of closely arranged adenosine dimers. In the case of S‐ and R‐configured 2′–5′ and 3′–5′ ApCOH A analogs, respectively, the amounts of complexes formed even exceed the ApA. This hybridization effectiveness is reached, however, by a more feasible prolongation of the pseudo‐chain, while the stability constant for the initiation step is significantly lower. Raman spectra of the complexes showed that for both of the above analogs the structural compatibility with the natural nucleic acid chain is decreased, because of the distorted position of one adenine residue. This distortion influences the Watson–Crick hydrogen bonds. The two types of linkages may be suitable just for construction of longer antigene or antisense oligonucleotides with alternating lengthened and shortened linkages. There is, however, a warning of possible decreased binding specificity. Copyright © 2004 John Wiley & Sons, Ltd.
Joint application of micro‐Raman and surface‐enhanced Raman spectroscopy to the interaction study of the antitumoral anthraquinone drugs danthron and quinizarin with albumins
Authors: Fabriciova G. , Sanchez‐Cortes S. , Garcia‐Ramos J. V. , Miskovsky P.
Published: May 2004

We report on the joint application of surface‐enhanced Raman scattering (SERS) and micro‐Raman techniques to the study of danthron (DT) and quinizarin (QZ) and their complexes with human and bovine albumins. We propose a novel method based on the combination of SERS and micro‐Raman spectroscopy by using immobilized Ag nanoparticles, which we have named micro‐SERS, to improve the sensitivity of the SERS technique and that could be applied in recognition processes of drugs with large biomolecules. The micro‐SERS technique demonstrated clear advantages over direct measurements in air: reproducibility, rapidity and high sensitivity due to a large coverage of the metal surface and good organization of the adsorbate on the surface. Furthermore, the chemical damage of the analyte induced by laser irradiation is substantially reduced. By means of this method, we were able to deduce important information concerning the binding properties of DT and QZ when interacting with human and bovine albumins. For instance, the interaction mechanism can be drastically modified when fatty acids are present in albumins, there are large differences between human and bovine albumins in the binding of the studied drugs related to changes in the amino‐acid sequence of the binding pockets of these proteins and the interaction mechanism of the analysed drugs can be different depending on their structure. Copyright © 2004 John Wiley & Sons, Ltd.
Identification and characterization of pharmaceuticals using Raman and surface‐enhanced Raman scattering
Authors: Cîntǎ Pînzaru S. , Pavel I. , Leopold N. , Kiefer W.
Published: May 2004

Many recent papers reflect ongoing research and development concerning pharmaceutical applications of Raman techniques. This short review highlights different Raman techniques (dispersive, Fourier transform, resonance Raman, SERS, SERRS, FT‐SERS) employed in pharmaceutical investigations. Several Raman applications such as fundamental structural investigations, quantitative analysis, drug–excipient interaction, formulation, limit of detection, pH‐dependent pharmaceutical species, adsorption geometry at a given surface and functional groups involved in adsorption for several widely used pharmaceutical compounds are presented. Copyright © 2004 John Wiley & Sons, Ltd.
Near‐infrared Fourier transform surface‐enhanced Raman scattering spectroscopy of 1,4‐benzodiazepine drugs employing gold films over nanospheres
Authors: Trachta Gerd , Schwarze Bernd , Brehm Georg , Schneider Siegfried , Hennemann Matthias , Clark Timothy
Published: May 2004

SERS spectra of eight benzodiazepine drugs were recorded with an FT‐NIR Raman spectrometer employing AuFONs (gold films over nanospheres) as SERS‐active substrates. A tentative assignment of the vibrational bands was made by comparison with the Raman spectra of crystalline reference samples and the results of density functional theory (DFT) calculations. It is concluded that the bands observed in the SERS spectra are almost exclusively due to vibrations located on rings a and b of the benzodiazepine moiety. The latter should be adsorbed with the molecular plane essentially orthogonal to the metal surface. However, different adsorption geometries are postulated for derivatives containing NH or NCH3 groups. It is demonstrated that a few hundred nanograms of analyte per well are sufficient to record spectra that allow identification and discrimination of the drugs. Such amounts can be collected at the output of an HPLC instrument employed for separating these drugs from blood serum. Copyright © 2004 John Wiley & Sons, Ltd.
Polyethylene oxides: protection potential against polymorphic transitions of drugs?
Authors: Schmidt Andrea G. , Wartewig Siegfried , Picker Katharina M.
Published: May 2004

Polyethylene oxides (PEOs) were analysed in mixtures with polymorphic drugs to test their potential in order to prevent polymorphic or pseudopolymorphic transitions, which are induced by the tableting process. Five PEOs with different molecular weights were tested in comparison with the well‐known tableting excipients microcrystalline cellulose (MCC), hydroxypropylmethylcellulose (HPMC) and dicalcium phosphate dihydrate (DCPD). Amorphous indomethacin was chosen as the model drug. Its recrystallization behaviour is well known and can be mechanically stimulated. Theophylline monohydrate was also used; its dehydration is induced by tableting. Pure materials and mixtures containing 20% (w/w) of drug were compressed at different maximum relative densities. The tablets were broken in order to determine the degree of transformation inside by FT‐Raman spectroscopy. For quantitative interpretation, the intensities of representative Raman bands were used. Differential scanning calorimetry (DSC) was applied additionally. In tablets and mixtures of amorphous indomethacin and PEOs, recrystallization was dramatically lowered in comparison with the other excipients. The molecular weights of the various PEOs seem to have no influence. The Raman spectra indicate chemical interactions as the reason. Thus, PEO can stabilize amorphous indomethacin. DSC investigations verified these results. For theophylline monohydrate, no interactions could be detected and DCPD showed the best protection properties. Hence the results concerning indomethacin cannot be generalized, but blending with PEO could be a simple method to stabilize amorphous indomethacin. Copyright © 2004 John Wiley & Sons, Ltd.
Development of sampling methods for Raman analysis of solid dosage forms of therapeutic and illicit drugs
Authors: Bell Steven E. J. , Beattie J. Renwick , McGarvey John J. , Peters K. Laota , Sirimuthu N. M. S. , Speers S. James
Published: May 2004

The results of a study aimed at determining the most important experimental parameters for automated, quantitative analysis of solid dosage form pharmaceuticals (seized and model ‘ecstasy’ tablets) are reported. Data obtained with a macro‐Raman spectrometer were complemented by micro‐Raman measurements, which gave information on particle size and provided excellent data for developing statistical models of the sampling errors associated with collecting data as a series of grid points on the tablets' surface. Spectra recorded at single points on the surface of seized MDMA–caffeine–lactose tablets with a Raman microscope (λex = 785 nm, 3 µm diameter spot) were typically dominated by one or other of the three components, consistent with Raman mapping data which showed the drug and caffeine microcrystals were ca 40 µm in diameter. Spectra collected with a microscope from eight points on a 200 µm grid were combined and in the resultant spectra the average value of the Raman band intensity ratio used to quantify the MDMA: caffeine ratio, µr, was 1.19 with an unacceptably high standard deviation, σr, of 1.20. In contrast, with a conventional macro‐Raman system (150 µm spot diameter), combined eight grid point data gave µr = 1.47 with σr = 0.16. A simple statistical model which could be used to predict σr under the various conditions used was developed. The model showed that the decrease in σr on moving to a 150 µm spot was too large to be due entirely to the increased spot diameter but was consistent with the increased sampling volume that arose from a combination of the larger spot size and depth of focus in the macroscopic system. With the macro‐Raman system, combining 64 grid points (0.5 mm spacing and 1–2 s accumulation per point) to give a single averaged spectrum for a tablet was found to be a practical balance between minimizing sampling errors and keeping overhead times at an acceptable level. The effectiveness of this sampling strategy was also tested by quantitative analysis of a set of model ecstasy tablets prepared from MDEA–sorbitol (0–30% by mass MDEA). A simple univariate calibration model of averaged 64 point data had R2 = 0.998 and an r.m.s. standard error of prediction of 1.1% whereas data obtained by sampling just four points on the same tablet showed deviations from the calibration of up to 5%. Copyright © 2004 John Wiley & Sons, Ltd.
Applications of Raman spectroscopy to pharmacy
Authors: Fini Giancarlo
Published: May 2004

This paper gives a summary of this special issue on ‘Pharmaceutical Applications of Raman Spectroscopy’. It summarizes the papers collected and introduces the possible applications of classical Raman spectroscopy (macro and micro) and surface‐enhanced Raman spectroscopy to the identification of pharmacologically active substances, their qualitative and quantitative analysis, characterization of crystalline forms and structure determination. Copyright © 2004 John Wiley & Sons, Ltd.
Rural tourism in Australia — has the gaze altered? Tracking rural images through film and tourism promotion
Authors: Beeton Sue
Published: May 2004

Australians' image of themselves and how others view them is embedded in the images presented by contemporary popular media, both domestic and international. Many of these images are rural; however, they are not based on a Romantic rural idyll but, rather, stem from notions of the Australian ‘bush’. The meaning behind these images not only reflects the attitudes and political environment of the day, but also can influence potential tourists' desire to visit the country. By looking at three film versions (spanning almost a century) of the popular Australian bushranging legend, Ned Kelly, and their relationship with tourism images, we can start to consider this relationship. While the Kelly legend is influential in terms of Australian self‐image, the images presented in the movies do not correlate with the tourism images of the time. Such dissonance creates confusion in visitors' perceptions and represents lost promotional opportunities possibly even having a negative effect on rural tourism. Copyright © 2004 John Wiley & Sons, Ltd.
Kinetic investigation of gas‐phase reactions between the OH‐radical and o‐, m‐, p‐ethyltoluene and n‐nonane in air
Authors: Colomb A. , Jacob V. , Kaluzny P. , Baussand P.
Published: May 2004

We have carried out relative rate experiments (T = 294 ± 2 K, atmospheric pressure) to investigate the OH‐oxidation of o‐, m‐, and p‐ethyltoluene and n‐nonane (k1, k2, k3, and k4 respectively). The experiments were performed in a 2‐m3 smog chamber with Teflon coated walls. The rate constants obtained are (in cm3 molecule−1 s−1 with two sigma uncertainties): k1 = (1.36 ± 0.07) × 10−11; k2 = (2.12 ± 0.26) × 10−11; k3 = (1.47 ± 0.04) × 10−11, and k4 = (0.95 ± 0.02) × 10−11. The measured rate constants are in accordance with previously published data, so that a coherent group of values for the compounds studied can be established. Atmospheric implications, ozone, and particle production are discussed. In addition, we have determined the amount of o‐, m‐, and p‐ethyltoluenes in different types of gasoline. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 367–378 2004
Rate coefficients for the gas‐phase reactions of OH radicals with methylbutenols at 298 K
Authors: Imamura Takashi , Iida Yumi , Obi Kinichi , Nagatani Ikue , Nakagawa Kazumichi , Patroescu‐Klotz Iulia , Hatakeyama Shiro
Published: May 2004

The relative‐rate method has been used to determine the rate coefficients for the reactions of OH radicals with three C5 biogenic alcohols, 2‐methyl‐3‐buten‐2‐ol (k1), 3‐methyl‐3‐buten‐1‐ol (k2), and 3‐methyl‐2‐buten‐1‐ol (k3), in the gas phase. OH radicals were produced by the photolysis of CH3ONO in the presence of NO. Di‐n‐butyl ether and propene were used as the reference compounds. The absolute rate coefficients obtained with the two reference compounds agreed well with each other. The O3 and O‐atom reactions with the target alcohols were confirmed to have a negligible contribution to their total losses by using two kinds of light sources with different relative rates of CH3ONO and NO2 photolysis. The absolute rate coefficients were obtained as the weighted mean values for the two reference compound systems and were k1 = (6.6 ± 0.5) × 10−11, k2 = (9.7 ± 0.7) × 10−11, and k3 = (1.5 ± 0.1) × 10−10 cm3 molecule−1 s−1 at 298 ± 2 K and 760 torr of air. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 379–385 2004
Thermal decomposition of tert‐butyl peroxide in a gas chromatographic reactor: A comparison of kinetic approaches
Authors: Skrdla Peter J.
Published: May 2004

The thermal decomposition of tert‐butyl peroxide is investigated utilizing both the column and the injection port of a commercial gas chromatograph (GC) as chemical reactors. Using the injector liner as the reactor, the chromatographic peak areas of the reactant, measured at various injector temperatures, are used in the determination of the activation energy of the decomposition (Ea). With the column serving as the reactor, both the reactant peak areas and the product peak shapes are similarly utilized for this purpose. Values of Ea obtained using different mathematical treatments for each of the three approaches are found to range from 115 to 164 kJ/mol. Of these methods, the column reactor approach utilizing peak area measurements (referred to as PACR, for “peak area, column reactor”) is found to be far superior in terms of its speed, robustness, and its accuracy in determining Ea. The PACR method's effectiveness can be largely attributed to the mathematical treatment that is described in the approach. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 386–393, 2004