bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of multiwall Kraft paper or plastic bags on physico‐chemical changes in milk powder during storage at high temperature and humidity
Authors: Shakeel‐Ur‐Rehman  , Farkye Nana Y , Schaffner Andrew A
Published: March 2003

Plastic bags have recently been introduced as an alternative to multiwall Kraft paper bags in packaging milk powder in 25‐kg sizes as it is thought that plastic bags are more durable and would preserve the quality of milk powders during storage better than paper bags. However, there is little scientific information available on the performance of plastic bags for milk powder storage. The aim of this study was to compare the properties of skimmed milk powder (SMP) packaged in plastic or paper bags. Commercially produced and packaged SMP in 25‐kg plastic or paper bags was stored at 37°C and 90% relative humidity. Samples were analysed at intervals (0, 45, 90 and 135 days) for moisture, water activity, lactic acid content, pH, insolubility index, bulk density, flowability, dispersibility and particle size. The magnitude of change in the majority of SMP properties measured during storage suggests that plastic bags are similar to paper bags in maintaining milk powder quality during storage.
Regulation of the L‐Type Ca2+ Channel Current in Rat Pinealocytes
Authors: Chik Constance L. , Li Bing , Karpinski Edward , Ho Anthony K.
Published: March 2003

Abstract : In the present study, the role of phosphoprotein
phosphatase in the regulation of L‐type Ca2+ channel currents in
rat pinealocytes was investigated using the whole‐cell version of the
patch‐clamp technique. The effects of three phosphatase inhibitors, calyculin
A, tautomycin, and okadaic acid, were compared. Although all three inhibitors
were effective in inhibiting the L‐type Ca2+ channel current,
calyculin A was more potent than either tautomycin or okadaic acid, suggesting
the involvement of phosphoprotein phosphatase‐1. To determine the kinase
involved in the regulation of these channels, cells were pretreated with H7 (a
nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic
AMP‐dependent kinase), KT5823 (a specific inhibitor of cyclic GMP‐dependent
kinase), or calphostin C (a specific inhibitor of protein kinase C).
Pretreatment with either H7 or calphostin C decreased the inhibitory effect of
calyculin A on the L‐type Ca2+ channel current. In contrast,
pretreatment with H89 or KT5823 had no effect on the inhibition caused by
calyculin A. Based on these observations, we conclude that basal phosphatase
activity, probably phosphoprotein phosphatase‐1, plays an important role in
the regulation of L‐type Ca2+ channel currents in rat pinealocytes by counteracting protein kinase C‐mediated phosphorylation.
Glucose 6‐Phosphate and Fructose 1,6‐Bisphosphate Can Be Used as ATP‐Regenerating Systems by Cerebellum Ca2+‐Transport ATPase
Authors: Ramos Renata C. S. , De Meis Leopoldo
Published: March 2003

Abstract : In this work, it is shown that the
Ca2+‐transport ATPase found in the microsomal fraction of the
cerebellum can use both glucose 6‐phosphate/hexokinase and fructose
1,6‐bisphosphate/phosphofructokinase as ATP‐regenerating systems. The vesicles
derived from the cerebellum were able to accumulate Ca2+ in a
medium containing ADP when either glucose 6‐phosphate and hexokinase or
fructose 1,6‐bisphosphate and phosphofructokinase were added to the medium.
There was no Ca2+ uptake if one of these components was omitted
from the medium. The transport of Ca2+ was associated with the
cleavage of sugar phosphate. The maximal amount of Ca2+ accumulated
by the vesicles with the fructose 1,6‐bisphosphate system was larger than that
measured either with glucose 6‐phosphate or with a low ATP concentration and
phosphoenolpyruvate/pyruvate kinase. The Ca2+ uptake supported by
glucose 6‐phosphate was inhibited by glucose, but not by fructose 6‐phosphate.
In contrast, the Ca2+ uptake supported by fructose 1,6‐bisphosphate
was inhibited by fructose 6‐phosphate, but not by glucose. Thapsigargin, a
specific SERCA inhibitor, impaired the transport of Ca2+ sustained
by either glucose 6‐phosphate or fructose 1,6‐bisphosphate. It is proposed
that the use of glucose 6‐phosphate and fructose 1,6‐bisphosphate as an
ATP‐regenerating system by the cerebellum Ca2+‐ATPase may represent
a salvage route used at early stages of ischemia ; this could be used to
energize the Ca2+ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.
Geldanamycin Provides Posttreatment Protection Against Glutamate‐Induced Oxidative Toxicity in a Mouse Hippocampal Cell Line
Authors: Xiao Nianqing , Callaway Clifton W. , Lipinski Christopher A. , Hicks Shawn D. , DeFranco Donald B.
Published: March 2003

Abstract : The benzoquinoid ansamycin geldanamycin interferes with many cell signaling pathways and is currently being evaluated as an anticancer agent. The main intracellular target of geldanamycin is the 90‐kDa heat shock protein, hsp90. In this report we demonstrate that geldanamycin is effective at preventing glutamate‐induced oxidative toxicity in the HT22 mouse hippocampal cell line, even if given 4 h after glutamate treatment. Geldanamycin prevents glutamate‐induced internucleosomal DNA cleavage in the HT22 cells but does not reverse the depletion of glutathione levels brought about by glutamate treatment. Both anabolic and catabolic effects are generated by geldanamycin treatment of HT22 cells, as evidenced by the induction of hsp70 expression and degradation of c‐Raf‐1 protein, respectively. Thus, geldanamycin may provide an effective strategy for manipulating signaling pathways in neuronal cells that use hsp90 as they proceed through a programmed cell death pathway in response to oxidative stress.
Oxidative Inactivation of Brain Alkaline Phosphatase Responsible for Hydrolysis of Phosphocholine
Authors: Sok Dai‐Eun
Published: March 2003

Abstract : Alkaline phosphatase, one of the enzymes responsible for
the conversion of phosphocholine into choline, was purified from bovine brain
membrane, where the phosphatase is bound as glycosylphosphatidylinositollinked
protein, and subjected to oxidative inactivation. The phosphatase activity,
based on the hydrolysis of p‐nitrophenyl phosphate and
phosphocholine, decreased slightly after the exposure to
H2O2. Inclusion of Cu2+ in the incubation
with 1 mM H2O2 led to a rapid decrease of
activity in a time‐ and concentration‐dependent manner. In comparison, the
H2O2/Cu2+ system was much more effective than
the H2O2/Fe2+ system in inactivating brain
phosphatase. In a further study, it was observed that the hydroxy radical
scavengers mannitol, ethanol, or benzoate failed to prevent against
H2O2/Cu2+‐induced inactivation of the
phosphatase, excluding the involvement of extraneous hydroxy radicals in
metalcatalyzed oxidation. In addition, it was found that both substrates,
p‐nitrophenyl phosphate and phosphocholine, and an inhibitor,
phosphate ion, at their saturating concentrations exhibited a remarkable,
although incomplete, protection against the inactivating action of
H2O2/Cu2+. A similar protection was also
expressed by divalent metal ions such as Mg2+ or Mn2+.
Separately, it was found that
H2O2/Fe2+‐induced inactivation was prevented
by p‐nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine‐hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal‐catalyzed oxidation.
Role of Protein Kinase C (PKC) in Agonist‐Induced μ‐Opioid Receptor Down‐Regulation
Authors: Kramer Kenneth H. , Simon Eric J. , Simon Eric J.
Published: March 2003

Abstract : Agonist‐induced down‐regulation of opioid receptors
appears to require the phosphorylation of the receptor protein. However, the
identities of the specific protein kinases that perform this task remain
uncertain. Protein kinase C (PKC) has been shown to catalyze the
phosphorylation of several G protein‐coupled receptors and potentiate their
desensitization toward agonists. However, it is unknown whether opioid
receptor agonists induce PKC activation under physiological conditions. Using
cultured SH‐SY5Y neuroblastoma cells, which naturally express μ‐ and
δ‐opioid receptors, we investigated whether μ‐opioid receptor
agonists can activate PKC by measuring enzyme translocation to the membrane
fraction. PKC translocation and opioid receptor densities were simultaneously
measured by 3H‐phorbol ester and [3H]diprenorphine
binding, respectively, to correlate alterations in PKC localization with
changes in receptor binding sites. We observed that μ‐opioid agonists have
a dual effect on membrane PKC density depending on the period of drug
exposure. Exposure for 2‐6 h to [d‐Ala2,
N‐Me‐Phe4, Gly‐ol]enkephalin or morphine promotes the
translocation of PKC from the cytosol to the plasma membrane. Longer periods
of opioid exposure (>12 h) produce a decrease in membrane‐bound PKC density
to a level well below basal. A significant decrease in
[3H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid‐mediated PKC translocation and receptor down‐regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.
Differential Regional Effects of Methamphetamine on the Activities of Tryptophan and Tyrosine Hydroxylase
Authors: Haughey Heather M. , Fleckenstein Annette E. , Hanson Glen R.
Published: March 2003

Abstract : Administration of high doses of methamphetamine (METH) produces both short‐ and long‐term enzymatic deficits in central monoaminergic systems. To determine whether a correlative relationship exists between these acute and long‐term consequences of METH treatment, in the present study we examined the regional effects of METH on tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) activities in various regions of the caudate nucleus, nucleus accumbens, and globus pallidus. A single METH administration decreased TPH activity 1 h after treatment in the globus pallidus, in the nucleus accumbens, and throughout the caudate ; in the anterior caudate, the ventral‐medial was more affected than the dorsal‐lateral region. In contrast, TH activity was not decreased in either the caudate or the globus pallidus after a single METH administration ; however, it was altered in the nucleus accumbens. Seven days after multiple METH administrations, TH and TPH activities were decreased in most caudate regions but not in the nucleus accumbens or globus pallidus. These data demonstrate that (1) the effects of METH on TPH and TH vary regionally ; and (2) the short‐term and long‐term regional responses of TPH to METH in the caudate and globus pallidus correlated. In contrast, METH‐induced acute TH responses did not predict the long‐term changes in TH activity.
Effects of MDL 72527, a Specific Inhibitor of Polyamine Oxidase, on Brain Edema, Ischemic Injury Volume, and Tissue Polyamine Levels in Rats After Temporary Middle Cerebral After Occlusion
Authors: Dogan Aclan , Rao A. Muralikrishna , Hatcher James , Rao V. L. Raghavendra , Baskaya Mustafa K. , Dempsey Robert J.
Published: March 2003

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex (P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex (P < 0.05) and 17% in the subcortex (P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia‐reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.
Gö 6976 Is a Potent Inhibitor of Neurotrophin‐Receptor Intrinsic Tyrosine Kinase
Authors: Behrens M. Margarita , Strasser Uta , Choi D. W.
Published: March 2003

Abstract : We report here that addition of the protein kinase C
inhibitor Gö 6976 blocked neurotrophin‐induced
signaling and autophosphorylation of neurotrophin‐specific tyrosine kinase
(Trk) receptors, either Trk B in cortical neurons or Trk A in GT1‐1‐trk9
cells. The effect of Gö 6976 on Trk
autophosphorylation was not inhibited by 100μM orthovanadate,
suggesting that the block was not due to the activation of tyrosine
phosphatases. Moreover, addition of 10‐100 nM concentrations of Gö 6976 inhibited either Trk B or Trk A intrinsic kinase activity in cell‐free assays. Gö 6976 also blocked the ability of brain‐derived neurotrophic factor to promote cortical neuronal survival and the ability of nerve growth factor to promote PC12 cell survival and differentiation. These results suggest that Gö 6976, besides its known inhibitory effects on lipid‐ and calcium‐dependent isoforms of protein kinase C, can also inhibit neurotrophin signaling by directly inhibiting the instrinsic Trk.
Nuclear Translocation of Glyceraldehyde‐3‐Phosphate Dehydrogenase Isoforms During Neuronal Apoptosis
Authors: Saunders Paul A. , Chen Ren‐Wu , Chuang De‐Maw
Published: March 2003

Abstract : Treatment with cytosine β‐d‐arabinoside
(AraC ; 300 μM) induced a time‐dependent accumulation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase‐3. Moreover, Asp‐Glu‐Val‐Asp‐fluoromethyl ketone (DEVD‐fmk), a CPP32‐selective antagonist, dosedependently suppressed AraC‐induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil‐DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC‐treated cells. The more alkaline isoforms, 1‐3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4‐6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h ; a 4‐h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside‐induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC‐induced neuronal death.
Brain‐Derived Neurotrophic Factor Levels in the Nervous System of Wild‐Type and Neurotrophin Gene Mutant Mice
Authors: Kolbeck Roland , Bartke Ilse , Eberle Walter , Barde Yves‐Alain
Published: March 2003

Abstract: Although brain‐derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain‐derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody cross‐reacts with neurotrophin homodimers other than brain‐derived neurotrophic factor, although reactivity was detected with brain‐derived neurotrophic factor/neurotrophin‐3 heterodimers. As both nerve growth factor and neurotrophin‐3 are known to affect the development of a variety of neurons expressing the brain‐derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin‐3 (nt3) gene. Marked differences were observed between mutants and wild‐type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin‐3 is a unique regulator of brain‐derived neurotrophic factor levels in the newborn mouse CNS.
Transforming Growth Factor β1‐Regulated Cell Proliferation and Expression of Neural Cell Adhesion Molecule in B104 Neuroblastoma Cells
Authors: Luo Jia , Miller Michael W.
Published: March 2003

Abstract: The expression and activity of factors influencing early
neuronal development are altered by ethanol. Such factors include growth
factors, for example, platelet‐derived growth factor and basic fibroblast
growth factor (for cell proliferation), and cell adhesion molecules (for
neuronal migration). One agent, transforming growth factor β1
(TGFβ1), may affect both events. We tested the hypothesis that ethanol
alters myriad TGFβ1‐mediated activities [i.e., cell proliferation and
neural cell adhesion molecule (N‐CAM) expression] using B104 neuroblastoma
cells. TGFβ1 inhibited the proliferation of B104 cells as evidenced by
decreases in cell number and [3H]thymidine ([3H]dT)
incorporation. TGFβ1 induced sustained activation of extracellular
signal‐regulated kinases (ERKs), which are part of the family of
mitogen‐activated protein kinases (MAPKs). Treatment with PD98059 (a MAPK
kinase blocker) abolished TGFβ1‐regulated inhibition of [3H]dT incorporation. TGFβ1‐mediated growth inhibition was potentiated by ethanol exposure. Ethanol also produced prolonged activation of ERK, an effect that was partially eliminated by treatment with PD98059. On the other hand, TGFβ1 up‐regulated N‐CAM expression, and this up‐regulation was not affected by treatment with PD98059. Ethanol inhibited the TGFβ1‐induced up‐regulation of N‐CAM expression in a concentration‐dependent manner. Thus, TGFβ1 affects ERK‐dependent cell proliferation and ERK‐independent N‐CAM expression in B104 cells. Both activities are sensitive to ethanol and may underlie the ethanol‐induced alterations in the proliferation and migration of CNS neurons.
Expression of Cytokines by Human Astrocytomas Following Stimulation by C3a and C5a Anaphylatoxins
Authors: Sayah Sakina , Ischenko Alexander M. , Zhakhov Alexander , Bonnard Anne‐Sophie , Fontaine Marc
Published: March 2003

Abstract: C3a and C5a anaphylatoxins are two proinflammatory
peptides generated during complement activation that act through distinct
Gi protein‐coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α, and transforming growth factor‐β mRNA expression was studied by quantitative RT‐PCR. Whereas IL‐1β, tumor necrosis factor‐α, and transforming growth factor‐β mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL‐6 mRNA level. The amount of IL‐6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti‐C3aR or anti‐C5aR antibodies completely blocked the IL‐6 mRNA increase. The IL‐6 response was also investigated at the protein level, but IL‐6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin‐mediated transcriptional activation of IL‐6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.
Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Müller Glial Cells
Authors: Goureau O. , Régnier‐Ricard F. , Courtois Y.
Published: March 2003

Abstract: Retinal Müller glial cells express the inducible isoform (‐2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS‐2 [NOS‐2 (‐/‐)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN‐γ‐stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS‐2 (‐/‐) mice and because inhibition of NOS‐2 induction by transforming growth factor‐β or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP‐ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells in responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.
CDK‐5‐Mediated Neurofilament Phosphorylation in SHSY5Y Human Neuroblastoma Cells
Authors: Sharma Monica , Sharma Pushkar , Pant Harish C.
Published: March 2003

Abstract: Cyclin‐dependent kinase‐5 (CDK‐5) has been shown to play important roles in neuronal development and neurogenesis. In vitro studies indicate a role of CDK‐5 in phosphorylation of neurofilaments (NFs). In this study, we have chosen the human neuroblastoma cell line SHSY5Y as a model system to study the in vivo phosphorylation of NF proteins by CDK‐5. Upon differentiation of SHSY5Y cells with retinoic acid, we found that the phosphorylation of high molecular mass (NF‐H) and medium molecular mass (NF‐M) NFs increased, whereas the CDK‐5 protein level and kinase activity were unaffected. The role of CDK‐5 in the phosphorylation of cytoskeletal proteins was studied by using antisense oligonucleotides (ONs) to inhibit the expression of the CDK‐5 gene. We found that inhibition of CDK‐5 levels by antisense ON treatment resulted in a decrease in phosphorylation of NF‐H that correlated with a decline in neurite outgrowth. These results demonstrate that CDK‐5 is a major proline‐directed kinase phosphorylating the human NF‐H tail domain.
Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons
Authors: Nakazawa Hiroyuki , Murphy Timothy H.
Published: March 2003

Abstract : L‐type voltage‐sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L‐type VSCCs following synaptic stimulation, using the long‐wavelength Ca2+ indicator fluo‐3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by ~60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L‐type VSCCs mediated ~50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs ; ~15 mV) or single action potentials were much less effective at activating L‐type VSCCs. Antagonist experiments indicated that activation of the NMDA‐type glutamate receptor leads to a long‐lasting somatic depolarization necessary to activate L‐type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage‐clamp pulses indicated that nuclear Ca2+ transients mediated by L‐type VSCCs were produced by sustained depolarization positive to ‐25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non‐L‐type VSCCs. Our results suggest that action potentials, in combination with long‐lived synaptic depolarizations, facilitate the activation of L‐type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.
Endogenous δ‐Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108‐15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C
Authors: Zhang Zhe , Xin Shun‐Mei , Wu Guo‐Xiang , Zhang Wen‐Bo , Ma Lan , Pei Gang
Published: March 2003

Abstract : The p38 mitogen‐activated protein kinase (MAPK) cascade

transduces multiple extracellular signals from cell surface to nucleus and is

employed in cellular responses to cellular stresses and apoptotic regulation.

The involvement of the p38 MAPK cascade in opioid‐ and opioid receptor‐like

receptor‐1 (ORL1) receptor‐mediated signal transduction was

examined in NG108‐15 neuroblastoma × glioma hybrid cells. Stimulation of

endogenous δ‐opioid receptor (DOR) or ORL1 resulted in

activation of p38 MAPK. It also induced the activation of extracellular

signal‐regulated kinases (ERKs), another member of the MAPK family, with

slower kinetics. Activation of p38 MAPK was abolished by selective antagonists

of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a

specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant

effect on opioid‐induced ERK activation, indicating that p38 MAPK activity was

not required for ERK activation, though its stimulation preceded ERK

activation. Inhibition of protein kinase A (PKA) strongly diminished p38

activation mediated by DOR or ORL1 but had no significant effect on

ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation

of p38 while inhibiting activation of ERKs. Taken together, our results

provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor‐mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.
Toxicity of Pyroglutaminated Amyloid β‐Peptides 3(pE)‐40 and ‐42 Is Similar to That of Aβ1‐40 and ‐42
Authors: Tekirian T. L. , Yang A. Y. , Glabe C. , Geddes J. W.
Published: March 2003

Abstract : An N‐terminal truncated isoform of the amyloid

β‐peptide (Aβ) that begins with a pyroglutamate (pE) residue at

position 3 [Aβ3(pE)‐42] is the predominant isoform found in senile

plaques. Based upon previous in vitro studies regarding Aβ N‐terminal

truncated isoforms, it has been hypothesized that Aβ3(pE)‐x isoforms may

aggregate more rapidly and become more toxic than corresponding Aβ1‐x

peptides. However, the toxicity and aggregation properties of Aβ3(pE)‐42

and Aβ3(pE)‐40 have not previously been examined. After initial

solubilization and 1‐week preaggregation of each peptide at 37°C and pH

7.4, the toxicity of 5‐50 μM Aβ3(pE)‐42 was similar to that of Aβ1‐42. Moreover, the toxicity of Aβ3(pE)‐40 paralleled that induced by Aβ1‐40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged Aβ1‐42, Aβ3(pE)‐42, Aβ3(pE)‐40, and Aβ1‐40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N‐terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of Aβ.
Regional and Temporal Alterations in DNA Fragmentation Factor (DFF)‐Like Proteins Following Experimental Brain Trauma in the Rat
Authors: Zhang Chen , Raghupathi Ramesh , Saatman Kathryn E. , LaPlaca Michelle C. , McIntosh Tracy K.
Published: March 2003

Abstract : DNA fragmentation, an early event in neuronal death

following traumatic brain injury, may be triggered by the 40‐kDa subunit of

DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45‐kDa

subunit of DFF (DFF45), and activation of DFF40 may occur as a result of

caspase‐3‐mediated cleavage of DFF45 into 30‐and 11‐kDa fragments. In this

study, the intracellular distribution of DFF45 and DFF40 was examined

following lateral fluid percussion brain injury of moderate severity (2.4‐2.7

atm) in male Sprague‐Dawley rats. In the cytosolic fraction (S1) of the

injured cortex at 2 and 24 h postinjury, significant decreases in the

intensities of DFF45‐like proteins at 45‐ and 32‐kDa bands and a concomitant

increase in the 11‐kDa bands were observed (p <0.05 vs. uninjured

controls). A significant decrease in the intensities of the 32‐kDa band in the

nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h

postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was

observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and

in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the

hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction

(p <0.05) and recovered by 24 h postinjury, whereas DFF40 was

significantly decreased in the S1 and increased in the P1 fraction at both 2

and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase‐3‐DFF45/DFF40‐DNA cleavage observed following traumatic brain injury.
Presence of Multiple Functional Polyadenylation Signals and a Single Nucleotide Polymorphism in the 3′ Untranslated Region of the Human Serotonin Transporter Gene
Authors: Battersby Sharon , Ogilvie Alan D. , Blackwood Douglas H. R. , Shen Sanbing , Muqit Miratul M. K. , Muir Walter J. , Teague Peter , Goodwin Guy M. , Harmar Anthony J.
Published: March 2003

Abstract: The human serotonin transporter (hSERT) gene is a candidate for involvement in the aetiology of affective disorders. In humans, multiple transcripts of the gene have been detected by northern blot analysis of brain and other tissues. We performed 3′ rapid amplification of cDNA ends to identify the common sites of polyadenylation in hSERT mRNA from human JAR cells and whole blood. Two major polyadenylation sites were identified: one 567 bp downstream of the stop codon, consistent with the usage of the polyadenylation signal AATGAA, and a second site 690 bp downstream of the stop codon. The putative polyadenylation signal upstream of this site contained a single nucleotide polymorphism (AG/TTAAC). However, allelic variation at this site did not influence polyadenylation site usage, and there were no significant differences in the abundance of the two alleles of this polymorphism between 329 control subjects, 158 individuals with major depression, and 130 individuals with bipolar affective disorder. This single nucleotide polymorphism in the 3′ untranslated region of the hSERT gene should provide a useful genetic marker in the evaluation of hSERT as a candidate gene influencing susceptibility to mood disorders.
Molecular Cloning of a Novel Member of the HSP110 Family of Genes, Ischemia‐Responsive Protein 94 kDa (irp94),Expressed in Rat Brain After Transient Forebrain Ischemia
Authors: Yagita Yoshiki , Kitagawa Kazuo , Taguchi Akihiko , Ohtsuki Toshiho , Kuwabara Keisuke , Mabuchi Takuma , Matsumoto Masayasu , Yanagihara Takehiko , Hori Masatsugu
Published: March 2003

Abstract: To identify genes induced by transient forebrain ischemia, we used the mRNA differential display technique in the four‐vessel occlusion model in rats. Some genes were identified as candidates that encode ischemia‐responsive protein, and one of them was cloned as ischemia‐responsive protein 94 kDa (irp94) from the rat hippocampal cDNA library. Sequence analysis suggested that rat irp94 was a transcriptional variant or a homologue of mouse apg‐2 and human heat shock protein (hsp) 70RY and a member of the HSP110 family, because IRP94 was >90% identical to APG‐2 and HSP70RY and ∼60% identical to the other members of the HSP110 family. Although irp94 mRNA was constitutively expressed in the normal hippocampus, it was clearly enhanced 4‐24 h after ischemia for 10 (1.9‐fold increase) and 15 min (3.4‐fold increase). These changes mainly occurred in neuronal cells, as judged by the localization of irp94 mRNA using in situ hybridization histochemistry. On the other hand, hyperthermic stress did not enhance irp94 mRNA expression, suggesting that irp94 expression was enhanced under ischemic stress and not related to the heat shock signaling mechanism. Our study suggested that irp94, a novel member of the HSP110 family, might play an important role in the environment altering neuronal functions, especially after transient forebrain ischemia.
Growth Factor‐Mediated Stabilization of Amyloid Precursor Protein mRNA Is Mediated by a Conserved 29‐Nucleotide Sequence in the 3′‐Untranslated Region
Authors: Rajagopalan Lakshman E. , Malter James S.
Published: March 2003

Abstract: Using a cell‐free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29‐nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin‐like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5′‐untranslated region (UTR) and coding region of β‐globin and the entire 3′‐UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild‐type globin‐APP mRNA containing an intact APP 3′‐UTR and mutant globin‐APP mRNA containing a mutated 29‐nucleotide element decayed with identical half‐lives (t1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild‐type mRNA (t1/2 = 10 min), but caused only a moderate decrease in the half‐life of transfected mutant mRNA (t1/2 = 40 min). We therefore conclude that the 29‐nucleotide 3′‐UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.
Role of Corticotropin‐Releasing Factor Receptor‐1 in Opiate Withdrawal
Authors: Iredale Philip A. , Alvaro John D. , Lee Younglim , Terwilliger Rosemarie , Chen Yuhpyng L. , Duman Ronald S.
Published: March 2003

Abstract: Previous studies indicate that corticotropin‐releasing factor (CRF) contributes to the anxiety‐like and aversive states associated with drug‐induced withdrawal. The present study extends this work by analyzing the CRF receptor subtype involved in withdrawal responses. First, the influence of a selective CRF receptor‐1 (CRF‐R1) antagonist, CP‐154,526, on opiate withdrawal behavior was examined. Pretreatment with the CRF‐R1 antagonist significantly attenuated several behavioral signs of naltrexone‐induced morphine withdrawal, including writhing, chewing, weight loss, lacrimation, salivation, and irritability, measured during the first hour of withdrawal. Next the expression of CRF‐R1 was determined as a second measure of the involvement of this receptor in opiate withdrawal. Naltrexone‐induced morphine withdrawal resulted in down‐regulation of CRF‐R1 mRNA in several brain regions, including the frontal cortex, parietal cortex, striatum, nucleus accumbens, and amygdala, but not in the hypothalamus or periaqueductal gray. Expression of CRF‐R2, the other major CRF receptor subtype, was not down‐regulated significantly by withdrawal in any of the regions examined, although morphine alone significantly increased levels of this receptor subtype. Taken together, the behavioral and receptor regulation findings indicate that CRF‐R1 is the primary mediator of the actions of the CRF system on opiate withdrawal, although it is possible that CRF‐R2 contributes to the response.
Mercury Induces Cell Cytotoxicity and Oxidative Stress and Increases β‐Amyloid Secretion and Tau Phosphorylation in SHSY5Y Neuroblastoma Cells
Authors: Olivieri G. , Brack Ch. , Müller‐Spahn F. , Stähelin H. B. , Herrmann M. , Renard P. , Brockhaus M. , Hock C.
Published: March 2003

Abstract: Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer’s disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, β‐amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 μg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p < 0.001). Preincubation of cells for 30 min with 1 μM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury‐induced GSH loss. Similarly, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 μg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p < 0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of β‐amyloid peptide (Aβ) 1‐40 and 1‐42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Aβ 1‐40 showed maximal (15.3 ng/ml) release after 4 h, whereas Aβ 1‐42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p < 0.001). Preincubation of cells with melatonin resulted in an attenuation of Aβ 1‐40 and Aβ 1‐42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p < 0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.
Dehydroevodiamine·HCl Prevents Impairment of Learning and Memory and Neuronal Loss in Rat Models of Cognitive Disturbance
Authors: Park Cheol Hyoung , Lee Young‐Jae , Lee Sang Hyung , Choi Se Hoon , Kim Hye‐Sun , Jeong Sung‐Jin , Kim Sung Soo , Suh Yoo‐Hun
Published: March 2003

Abstract: We previously reported that dehydroevodiamine·HCl (DHED) has anticholinesterase and antiamnesic activities. To verify the effects of DHED on cognitive deficits further, we tested it on the scopolamine‐induced amnesia model of the rat using the passive avoidance and eight‐arm radial maze tests. A single (20 mg/kg p.o.) and repeated (10 mg/kg p.o.) administrations of DHED could significantly reverse the latency time shortened by scopolamine (1 mg/kg i.p.) to control level. The impaired spatial working memory induced by scopolamine (1 mg/kg i.p.) was also improved significantly by a single injection (6.25 mg/kg i.p.) and repeated administrations of DHED (10 mg/kg p.o.) in the eight‐arm radial maze test. In addition, we examined the effects of DHED on the memory impairment and the histological changes of the brain after unilateral electrolytic lesion of the entorhinal cortex (EC) and middle cerebral artery occlusion in rats. The cognitive deficits caused by EC lesion and middle cerebral artery occlusion were improved significantly by repeated administrations of DHED (6.25 mg/kg i.p.) after EC lesion or ischemic insult once a day for 7 days in the passive avoidance test. Histological analysis showed that the neuronal loss in the DHED‐treated group was notably reduced in the hippocampal area (CA1) of ischemic rats and in the dentate gyrus and hippocampal area (CA1 and CA3) of EC‐lesioned rats compared with the nontreated group. The infarction area was decreased significantly by a single administration of DHED (6.25 mg/kg i.p.) 30 min before ischemic insult for 6 h. These results suggest that DHED might be an effective drug for not only the Alzheimer’s disease type, but also the vascular type of dementia.
Mechanisms of Agonism and Inverse Agonism at Serotonin 5‐HT1A Receptors
Authors: McLoughlin David J. , Strange Philip G.
Published: March 2003

Abstract: Mechanisms of agonist and inverse agonist action at the serotonin 5‐HT1A receptor have been studied using the modulation of guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTPγS) binding in membranes of Chinese hamster ovary (CHO) cells expressing the receptor (CHO‐5‐HT1A cells). A range of agonists increased [35S]GTPγS binding with different potencies and to different maximal extents, whereas two compounds, methiothepin and spiperone, inhibited both agonist‐stimulated and basal [35S]GTPγS binding, thus exhibiting inverse agonism. Potencies of agonists to stimulate [35S]GTPγS binding in membranes from CHO‐5‐HT1A cells were reduced by adding increasing concentrations of GDP to assays, whereas changes in sodium ion concentration did not affect agonist potency. The maximal effect of the agonists was increased by increasing sodium ion concentrations. The affinities of agonists in ligand binding assays were unaffected by changes in sodium ion concentration. Increasing GDP in the assays of the inverse agonists increased potency for spiperone to inhibit [35S]GTPγS binding and had no effect for methiothepin, in agreement with the sensitivity of these compounds to guanine nucleotides in ligand binding assays. Potencies for these inverse agonists were unaffected by changes in sodium ion concentration. These data were simulated using the extended ternary complex model. These simulations showed that the data obtained with agonists were consistent with these compounds achieving agonism by stabilising the ternary complex. For inverse agonists, the simulations showed that the mechanism for spiperone may be to stabilise forms of the receptor uncoupled from G proteins. Methiothepin, however, probably does not alter the equilibrium distribution of different receptor species; rather, this inverse agonist may stabilise an inactive form of the receptor that can still couple to G protein.
Protein Kinase C Activation Reduces Microglial Cyclic AMP Response to Prostaglandin E2 by Interfering with EP2 Receptors
Authors: Patrizio Mario , Colucci Mariantonella , Levi Giulio
Published: March 2003

Abstract: We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12‐myristate 13‐acetate (PMA) or 4β‐phorbol 12,13‐didecanoate, which activate PKC, but not with the inactive 4α‐phorbol 12,13‐didecanoate, substantially reduced cAMP accumulation induced by 1 μM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (∼85%) was obtained after 10‐min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol‐ or forskolin‐stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10‐9‐10‐5M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17‐phenyl‐PGE2 (10‐6‐10‐5M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11‐deoxy‐16, 16‐dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose‐dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC‐activating agents present in the pathological brain may prevent the cAMP‐mediated microglia‐deactivating function of PGE2.
Entrapping of Impermeant Probes of Different Size into Nonpermeabilized Synaptosomes as a Method to Study Presynaptic Mechanisms
Authors: Raiteri Maurizio , Sala Roberta , Fassio Anna , Rossetto Ornella , Bonanno Giambattista
Published: March 2003

Abstract: Small molecules present during brain tissue homogenization are known to be entrapped within sub‐sequently isolated synaptosomes. We have revisited this technique in view of its systematic utilization to incorporate into nerve endings impermeant probes of large size. Rat neocortical synaptosomes were prepared in the absence or in the presence of each of the following compounds: 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), tetanus toxin (TeTx) or its light chain (TeTx‐LC), pertussis toxin (PTx), anti‐syntaxin, or anti‐SNAP25 monoclonal antibodies. Release of endogenous GABA and glutamate was then evoked by high K+ depolarization. GABA and glutamate overflows were inhibited by entrapped BAPTA and in synaptosomes prepared by homogenization in the presence of varying concentrations of TeTx or TeTx‐LC. When synaptobrevin cleavage in synaptosomes entrapped with TeTx was monitored by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by western blotting, the extent of proteolysis was found to correspond quantitatively to that of release inhibition. GABA and glutamate overflows were increased by entrapped PTx; moreover, (‐)‐baclofen inhibited amino acid overflow more potently in standard than in PTx‐containing synaptosomes. The overflows of GABA and glutamate were similarly decreased following incorporation of anti‐syntaxin or anti‐SNAP25 antibodies. Synaptosomal entrapping may be routinely used to internalize membrane‐impermeant agents of different size in studies of presynaptic mechanisms.
Cytokines that Signal Through the Leukemia Inhibitory Factor Receptor‐β Complex in the Nervous System
Authors: Turnley Ann M. , Bartlett Perry F.
Published: March 2003

Abstract: Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as “suppressor of cytokine signalling” proteins, which inhibit the JAK/STAT but not the mitogen‐activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.