bluuebunny/crossref_metadata_2025 huggingface dataset:
Viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Egyptian soft cheeses and ice‐cream
Authors: Nassib Taha A , Zin El‐Din Mohamed , El‐Sharoud Walid M
Published: March 2003

Changes occurring in the viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Domiati cheese, Kariesh cheese and ice‐cream were examined. A significant decrease in numbers was observed after whey drainage during the manufacture of Domiati cheese, but Salmonella remained viable for 13 weeks in cheeses prepared from milks with between 60 and 100 g/L NaCl; the viability declined in Domiati cheese made from highly salted milk during the later stages of storage. The method of coagulation used in the preparation of Kariesh cheese affected the survival time of the pathogen, and it varied from 2 to 3 weeks in cheeses made with a slow‐acid coagulation method to 4–5 weeks for an acid‐rennet coagulation method. This difference was attributed to the higher salt‐in‐moisture levels and lower pH values of Kariesh cheese prepared by the slow‐acid coagulation method. A slight decrease in the numbers of Salmonella resulted from ageing ice‐cream mix for 24 h at 0°C, but a greater reduction was evident after one day of frozen storage at −20°C. The pathogen survived further frozen storage for four months without any substantial change in numbers.
Structure—Function Studies of the Eighth Hydrophobic Domain of a Serotonin Receptor
Authors: Hurley Joyce H. , Bloem Laura J. , Pavalko Fred , Liu Jian , Tian Mingting , Simon Jay R. , Yu Lei
Published: March 2003

Abstract : The most prominent structural feature of the G
protein‐coupled receptor superfamily is their seven hydrophobic domains, which
are postulated to form membrane‐spanning α helices. Some members of the
G protein‐coupled receptor family, specifically several serotonin (5‐HT)
receptors, possess eight hydrophobic domains. The importance of this extra
hydrophobic domain, located at the N terminus of the receptor, is unknown.
This question was addressed by deleting the extra hydrophobic region from the
5‐HT2C receptor and comparing its function and topology with those
of the wild‐type receptor. Immunofluorescence microscopy was used to determine
the location of the N terminus of the epitope‐tagged wild‐type and mutant
receptors. The N terminus of both receptors was extracellular, suggesting that
the extra hydrophobic domain does not change the topology of this receptor and
is unlikely to be a membrane‐spanning α helix. Radioligand‐binding
studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild‐type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.
Transgenic Analysis of Rds/Peripherin N‐Glycosylation
Authors: Kedzierski Wojciech , Bok Dean , Travis Gabriel H.
Published: March 2003

Abstract : Rds/peripherin is an integral membrane glycoprotein that
is present in the rims of photoreceptor outer segment disks. In mammals, it is
thought to stabilize the disk rim through heterophilic interactions with the
related nonglycosylated protein rom1. Glycosylation of rds/peripherin at
asparagine 229 is widely conserved in vertebrates. In this study, we
investigated the role of rds/peripherin N‐glycosylation. We generated
transgenic mice that expressed only S231A‐substituted rds/peripherin in their
retinas. This protein was not glycosylated but formed covalent dimers with
itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin
also interacted noncovalently with rom1 homodimers to form a heterooligomeric
complex. The glycosylated rds/peripherin ·· rom1 complex bound
to concanavalin A‐Sepharose, suggesting that the glycan is not directly
involved in the interaction between these proteins. In double transgenic mice
expressing normal and S231A‐substituted rds/peripherin, the mRNA‐to‐protein
ratios were similar for both transgenes, indicating no effect of
N‐glycosylation on rds/peripherin stability. Finally, expression of
nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of
outer segment nondevelopment in retinal degeneration slow (rds‐/‐)
null mutants. These observations indicate that N‐glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.
Presenilins Interact with Armadillo Proteins Including Neural‐Specific Plakophilin‐Related Protein and β‐Catenin
Authors: Levesque G. , Yu G. , Nishimura M. , Zhang D. M. , Levesque L. , Yu H. , Xu D. , Liang Y. , Rogaeva E. , Ikeda M. , Duthie M. , Murgolo N. , Wang L. , VanderVere P. , Bayne M. L. , Strader C. D. , Rommens J. M. , Fraser P. E. , George‐Hyslop P. St.
Published: March 2003

Abstract : Missense substitutions in the presenilin 1 (PS1) and
presenilin 2 (PS2) proteins are associated with early‐onset familial
Alzheimer's disease. We have used yeast‐two‐hybrid and coimmunoprecipitation
methods to show that the large cytoplasmic loop domains of PS1 and PS2
interact specifically with three members of the armadillo protein family,
including β‐catenin, p0071, and a novel neuronal‐specific armadillo
protein—neural plakophilin‐related armadillo protein (NPRAP). The PS1 :
NPRAP interaction occurs between the arm repeats of NPRAP and
residues 372‐399 at the C‐terminal end of the large cytoplasmic loop of PS1.
The latter residues contain a single arm‐like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.
A Combined Analysis of Regional Energy Metabolism and Immunohistochemical Ischemic Damage in the Gerbil Brain
Authors: Ueda Hirokazu , Tagawa Kunio , Furuya Eisuke , Matsumoto Masayasu , Yanagihara Takehiko
Published: March 2003

Abstract : By combining immunohistochemical technique with microassay methods, we analyzed regional energy metabolism in vulnerable and tolerant areas of gerbil brains during evolution of neuronal damage after bilateral common carotid artery occlusion for 10 min with subsequent reperfusion. Four animals were used for each reperfusion period. Based on the information from the immunohistochemical examination, we dissected out vulnerable and tolerant subregions of the hippocampus, cerebral cortex, and thalamus from freeze‐dried 20‐μm‐thick sections, and measured the levels of creatine phosphate (P‐Cr), adenine nucleotides, guanine nucleotides, and purine bodies by HPLC, and the levels of glucose, glycogen, and lactate by an enzyme‐immobilized column method. There were no significant differences in the levels of metabolites between vulnerable and tolerant subregions of control brains. After reperfusion, both vulnerable and tolerant subregions recovered preischemic metabolic profiles by 2 days. Although the regional differences between vulnerable and tolerant subregions were minimal at each reperfusion period, there were delays in the recovery of P‐Cr, ATP, and/or total adenine nucleotides in all vulnerable subregions. A decline of P‐Cr, ATP, and GTP levels without change in %ATP, AMP, or purine bodies occurred after reperfusion for 3 days, coinciding with the development of immunohistochemical damage by the immunoreaction for microtubule‐associated protein 1A. The results supported the notion that subtle but sustained impairment of energy metabolism caused by mitochondrial dysfunction in the early reperfusion period might trigger delayed neuronal death in vulnerable subregions.
Involvement of the Carboxyl Terminus of the Third Intracellular Loop of the Cannabinoid CB1 Receptor in Constitutive Activation of Gs
Authors: Abadji Vasiliki , Lucas‐Lenard Jean M. , Chin Chen‐ni , Kendall Debra A.
Published: March 2003

Abstract: The human cannabinoid receptor CB1 functionally couples primarily to Gi‐, but also to Gs‐mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl‐terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist‐independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs‐ and not a Gi‐mediated pathway. Treatment with the CB1‐specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP‐55,940 to the L341A/A342L receptor was not markedly different from that for the wild‐type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.
Aluminum Enhances Iron Uptake and Expression of Neurofibrillary Tangle Protein in Neuroblastoma Cells
Authors: Abreo Kenneth , Abreo Fleurette , Sella M'Liss , Jain Sushil
Published: March 2003

Abstract: Aluminum (Al) and iron (Fe) have been implicated as playing a toxic role in the pathologic lesions of Alzheimer's disease. In the following report we describe the uptake and toxicity of Al, the effect of Al on Fe uptake, and the expression of neurofibrillary tangle (NFT) protein in murine neuroblastoma cells (Neuro 2A). Significant cell Al uptake and inhibition of cell growth were seen in Neuro 2A cells at 24, 48, 72, and 96 h after plating in medium containing Al transferrin (Al‐Tf) and Al citrate. Al‐loaded Neuro 2A cells showed increased rates of 59Fe and 125I‐Tf uptake and total cellular Fe content at 24, 48, 72, and 96 h after plating compared with control cultures. Significant increases in NFT protein staining were detected in Al‐exposed cells at 72 and 96 h in culture compared with controls. The intensity of NFT staining in Al‐loaded cells was directly proportional to the time in culture. There was no difference in malonyldialdehyde levels measured in control versus Al‐loaded Neuro 2A cells. These results suggest that the accumulation of Al in Neuro 2A cells resulted in increased uptake of Fe, inhibition of cell growth, and expression of NFT protein, partially mimicking the pathological hallmarks of Alzheimer's disease. This model system may also be applicable for Al‐induced dialysis dementia, because the Al concentrations at which cell toxicity occurred can be found in dialysis patients.
Repeated Cocaine Alters Glutamate Receptor Subunit Levels in the Nucleus Accumbens and Ventral Tegmental Area of Rats that Develop Behavioral Sensitization
Authors: Churchill Lynn , Swanson Chad J. , Urbina Mary , Kalivas Peter W.
Published: March 2003

Abstract: Increased glutamate transmission in the nucleus accumbens and ventral tegmental area has been proposed as a mechanism underlying sensitized behavioral responses to repeated cocaine administration. GluR1, GluR2/3, and NMDAR1 subunits of glutamate receptors were quantified from immunoblots in these brain nuclei in rats at 24 h and 3 weeks after discontinuing 1 week of daily cocaine injections. Motor behavior was monitored after the first and last injections of daily cocaine, and those rats that showed >20% increase in motor activity after the last compared with the first injection were considered to have developed behavioral sensitization. The subjects that developed behavioral sensitization showed a significant increase in GluR1 levels in the nucleus accumbens at 3 weeks but not at 24 h of withdrawal. Conversely, sensitized animals showed a significant increase in NMDAR1 and GluR1 levels in the ventral tegmental area at 1 day but not at 3 weeks of withdrawal. None of these increases occurred in the rats exposed to daily cocaine that did not develop behavioral sensitization (<20% increase in motor activity), and no changes were measured in the level of GluR2/3 in any treatment group. The functional importance of the increases in glutamate receptor subunit levels is suggested by the fact that the changes were present only in rats that developed behavioral sensitization to repeated cocaine administration.
Magnetic Resonance Spectroscopy Studies on Changes in Cerebral Calcium and Zinc and the Energy State Caused by Excitotoxic Amino Acids
Authors: Thatcher N. M. , Prior M. J. W. , Morris P. G. , Bachelard H. S.
Published: March 2003

Abstract: Under control conditions, superfused hippocampal slices
exhibited a significantly higher phosphocreatine (PCr)/ATP ratio than cortical
slices; the evidence suggests that this is due to lower concentrations of ATP,
rather than higher concentrations of PCr. Glutamate caused relatively rapid
decreases in PCr and ATP levels to ∼45%, accompanied or immediately
followed by an increased free intracellular calcium concentration
([Ca2+]i) and the release of Zn2+ in the
cortex. In the hippocampus PCr and ATP decreased further to ∼20% of
control values, but the changes in [Ca2+]i and
Zn2+ content were slower. This is in contrast to the effects of
depolarisation, which produced the same rapid changes in the energy state and
[Ca2+]i, with no detectable Zn2+, in both
tissues. NMDA causes effects similar to those of glutamate in the cortex
(decreases in the energy state, increased [Ca2+]i, and
release of Zn2+). Pretreatment of the cortex for 1 h with the NMDA
blocker MK‐801 prevented all of the observed effects of NMDA. In contrast,
pretreatment with MK‐801 had no detectable effect on the increase in
[Ca2+]i or the decreases in PCr and ATP caused by glutamate, although it prevented the release of zinc. The results are discussed in relation to the function of the NMDA subtype of glutamate receptor in excitotoxicity.
Differences in the Activation of the Mitochondrial Permeability Transition Among Brain Regions in the Rat Correlate with Selective Vulnerability
Authors: Friberg Hans , Connern Cathal , Halestrap Andrew P. , Wieloch Tadeusz
Published: March 2003

Abstract: Mitochondria from different regions of the brain were
prepared, and the activation of the mitochondrial permeability transition
(MPT) by calcium was investigated by monitoring the associated mitochondrial
swelling. In general, the properties of the MPT in brain mitochondria were
found to be qualitatively similar to those observed in liver and heart
mitochondria. Thus, swelling was inhibited by adenine nucleotides (AdNs) and
low pH (<7.0), whereas thiol reagents and alkalosis facilitated swelling.
Cyclosporin A and its nonimmunosuppressive analogue
N‐methyl‐Val‐4‐cyclosporin A (PKF 220‐384) both inhibited swelling
and prevented the translocation of cyclophilin D from the matrix to the
membranes of cortical mitochondria. However, the calcium sensitivity of the
MPT differed in mitochondria from three brain regions (hippocampus > cortex
> cerebellum) and is correlated with the susceptibility of these regions to
ischemic damage. Depleting mitochondria of AdNs by treatment with
pyrophosphate ions sensitized the MPT to [Ca2+] and abolished regional differences, implying regional differences in mitochondrial AdN content. This was confirmed by measurements showing significant differences in AdN content among regions (cerebellum > cortex > hippocampus). Our data add to recent evidence that the MPT may be involved in neuronal death.
Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N‐Terminal Truncation of mGluR1α
Authors: Robbins M. J. , Ciruela F. , Rhodes A. , McIlhinney R. A. J.
Published: March 2003

Abstract: The metabotropic glutamate receptor mGluR1α in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide‐bonded dimer on sodium dodecyl sulphate‐polyacrylamide gels. Dimerization of mGluR1α takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N‐terminal domain of mGluR1α, truncated just before the first transmembrane domain (NT‐mGluR1α), we show that the N‐terminal domain is secreted as a soluble disulphide‐bonded dimeric protein. In addition, the truncated N‐terminal domain can form heterodimers with mGluR1α when both proteins are cotransfected into HEK 293 cells. However, mGluR1α and its splice variant mGluR1β did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N‐terminal domain of mGluR1α is sufficient for dimer formation, other domains in the molecule must regulate the process.
Protein Synthesis Blockade Differentially Affects the Degradation of Constitutive and Nicotinic Receptor‐Induced Tyrosine Hydroxylase Protein Level in Isolated Bovine Chromaffin Cells
Authors: Fernández Elizabeth , Craviso Gale L.
Published: March 2003

Abstract: Continuous incubation of bovine adrenal chromaffin cells with the nicotinic receptor agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) causes a twofold increase in the steady‐state level of catalytically active tyrosine hydroxylase (TH) protein by 3‐4 days. The present study examined the processes that control the time course of enzyme induction. In cells exposed to DMPP for 36 or 54 h, incorporation of [3H]leucine into TH was increased 1.9‐ and 2.2‐fold, respectively, compared with control (non‐DMPP‐treated) cells. The increase correlated with a twofold rise in TH mRNA level, indicating the absence of translational control of TH synthesis by DMPP. Also absent was an effect by DMPP on the rate of degradation of TH protein because pulse‐chase analysis estimated a half‐life for TH of 26 ± 5 h in DMPP‐treated cells, a value that was (a) essentially the same as that estimated in control cells (29 ± 3 h), (b) within the same range as that estimated by approach to steady state (t1/2 = 19 ± 4 h), which measured the decline of TH protein content from the DMPP‐induced steady‐state level back to the basal value during deinduction with the nicotinic antagonist hexamethonium, and (c) consistent with the time course of accumulation of TH protein to a new steady‐state level in response to DMPP. However, different rates of degradation for TH protein were observed in control and DMPP‐treated cells under conditions in which protein synthesis was blocked. In control cells incubated with 100 μM puromycin or 20 μM cycloheximide for 3 days, the level of catalytically active TH protein failed to decline and exhibited a half‐life of ≥250 h. This finding indicated that TH protein was stabilized. TH protein level also failed to decline when cells were incubated for 3 days with a concentration of the transcription inhibitor α‐amanitin that caused a >90% loss of TH mRNA. Thus, degradation of constitutively expressed TH protein appears to be controlled by processes dependent on ongoing transcription and translation. In contrast, the increased amount of TH induced by DMPP was not stabilized but instead underwent a decline to the basal level following addition of puromycin or cycloheximide. It is important to note, however, that the decline occurred at a slower rate (t1/2≥ 45 h) than that measured during deinduction. Taken together, these data suggest that alterations in the rate of degradation of TH protein may play a role in controlling TH level when TH synthesis is blocked but not when TH synthesis is increased, such as during nicotinic receptor stimulation.
Molecular Characterization and Imidacloprid Selectivity of Nicotinic Acetylcholine Receptor Subunits from the Peach‐Potato Aphid Myzus persicae
Authors: Huang Yao , Williamson Martin S. , Devonshire Alan L. , Windass John D. , Lansdell Stuart J. , Millar Neil S.
Published: March 2003

Abstract: The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach‐potato aphid Myzus persicae. Two M. persicaeα‐subunit cDNAs, Mpα1 and Mpα2, have been cloned previously. Here we report the isolation of three novel α‐subunit genes (Mpα3‐5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect α subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non‐α subunits are clearly different from vertebrate neuronal β and muscle non‐α subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co‐expression of M. persicae nAChR α‐subunit cDNAs with the rat β2 in Drosophila S2 cells resulted in high‐affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the α subtype. This is the first demonstration that imidacloprid selectively acts on Mpα2 and Mpα3 subunits, but not Mpα1, in M. persicae.
Roles of Thapsigargin‐Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons
Authors: Yao Chih‐Jung , Lin Chii‐Wann , Lin‐Shiau Shoei‐Yn
Published: March 2003

Abstract : The roles of the intracellular calcium pool involved in

regulating the Ca2+ profile and the neuronal survival rate during

development were studied by using thapsigargin (TG), a specific inhibitor of

endoplasmic reticulum (ER) Ca2+‐ATPase in cultured cerebellar

granule neurons. Measuring the neuronal [Ca2+]i directly

in the culture medium, we found a bell‐shaped curve for

[Ca2+]i versus cultured days in cerebellar granule

neurons maintained in medium containing serum and 25 mM

K+. The progressive increase in [Ca2+]i of

the immature granule neurons (1‐4 days in vitro) was abolished by TG, which

resulted in massive neuronal apoptosis. When the [K+] was lowered

from 25 to 5 mM, neither the progressively increasing

[Ca2+]i nor the survival of immature granule neurons was

significantly changed over 24‐h incubation. Similarly, TG caused a dramatic

decrease in the [Ca2+]i and survival rate of these

immature neurons when switched to 5 mM K+ medium.

Following maturation, the granule neurons became less sensitive to TG for both

[Ca2+]i and neuronal survival. However, TG can protect

mature granule neurons from the detrimental effect of switching to a 5

mM K+ serum‐free medium by decreasing

[Ca2+]i to an even lower level than in the respective

TG‐free group. Based on these findings, we propose that during the immature

stage, TG‐sensitive ER Ca2+‐ATPase plays a pivotal role in the

progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.
The Relationship of Goα Subunit Deamidation to the Tissue Distribution, Nucleotide Binding Properties, and βγ Dimer Interactions of Goα Subunit Isoforms
Authors: McIntire William E. , Dingus Jane , Wilcox Michael D. , Hildebrandt John D.
Published: March 2003

Abstract : The distribution and properties in brain of the α

subunits of the major bovine brain Go isoforms, GoA,

GoB and GoC, were characterized. The

αoA and αoB isoforms arise from alternative

splicing of RNAs from a single αo gene, whereas

αoC is a deamidated form of αoA. All three

Go isoforms purify from brain with different populations of

βγ dimers. This variable subunit composition of Go

heterotrimers is likely a consequence of their functional differences. This

study examined the biochemical properties of the αo isoforms

to see if these properties explain the variable βγ composition of

their heterotrimers. The brain distribution of αoB differed

substantially from that of αoA and αoC, as

did its guanine nucleotide binding properties. The unique subunit composition

of GoB can be explained by its expression in different brain

regions. The αoA and αoC showed slight

differences in guanine nucleotide binding properties but no preference for

particular βγ dimers when reassociated with a heterogeneous

βγ pool. The αoC protein occurred in a constant

ratio to αoA throughtout the brain, but was a much larger

percent of total brain αo than previously thought, ~35%.

These results suggest that αoA is a precursor of

αoC and that the association of Goα subunits with different βγ dimers reflects the function of an adaptive, G‐protein signaling mechanism in brain.
Effects of Serine Mutations in Transmembrane Domain 7 of the Human Norepinephrine Transporter on Substrate Binding and Transport
Authors: Burgess Krisstina S. Danek , Justice Joseph B.
Published: March 2003

Abstract : Two serine residues in the β‐adrenergic receptor

(β‐AR) have been proposed to form hydrogen bonds with the catechol moiety

of the ligand and contribute to the activation of the receptor. These

conserved serine residues in the dopamine (DA) and norepinephrine transporters

(DAT and NET, respectively) have also been shown to affect substrate transport

in the rat DAT. In the present work, hydrogen bonding interactions between the

corresponding serine residues in the human NET (hNET), 354 and 357, and the

hydroxyl groups on the substrate were systematically evaluated by examining

the transport and binding properties of DA and several single hydroxyl

analogues of DA at wild‐type and serine‐to‐alanine‐substituted transporters. A

comparison of [3H]nisoxetine binding at the serine 354 mutant, in

which KD increased 70‐fold from the wild‐type value, with

the binding of DA, m‐tyramine (m‐TYR), and

p‐tyramine (p‐TYR) at mutant 354, where the increase in

Ki was less dramatic, revealed that serine 354 is more

influential in inhibitor than substrate binding. The binding of m‐TYR

and p‐TYR at the serine 354 and serine 357 mutants did not show a

direct interaction between one serine and one substrate catechol hydroxyl

group. DA, m‐TYR, and p‐TYR binding affinity did not deviate

from the wildtype value at the serine 357 and double mutant transporters. At

these two transporters, however, the Km of DA uptake

increased, suggesting that the roles of serine 357 and serine 354 in substrate

transport are different from their roles in binding. The

K′m for induced efflux of DA decreased at the serine

357 mutant compared with the wild‐type, whereas the

K′m at the serine 354 mutant was the same as that of

the wild‐type. Further investigation of the role of substrate hydroxyls in the

transport process revealed no difference between the transport of

m‐TYR or p‐TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the β‐AR does not exist at the hNET.
Laminin 1 Attenuates β‐Amyloid Peptide Aβ(1‐40) Neurotoxicity of Cultured Fetal Rat Cortical Neurons
Authors: Drouet Béatrice , Raymond Martine Pinçon , Chambaz Jean , Pillot Thierry
Published: March 2003

Abstract : A growing amount of evidence indicates the involvement of extracellular matrix components, especially laminins, in the development of Alzheimer's disease, although their role remains unclear. In this study, we clearly demonstrate that laminin 1 inhibits β‐amyloid peptide (Aβ)‐induced neuronal cell death by preventing the fibril formation and interaction of the Aβ peptide with cell membranes. The presence of laminin at a laminin/Aβ peptide molar ratio of 1:800 significantly inhibits the Aβ‐induced apoptotic events, together with inhibition of amyloid fibril formation. The inhibitory effects of laminin 1 were time‐ and dose‐dependent, whereas laminin 2 had less effect on Aβ neurotoxicity. A preincubation of laminin and Aβ was not required to observe the protective effect of laminin, suggesting a direct interaction between laminin 1 and Aβ. Moreover, laminin had no effect on the toxicity of the fibrillar Aβ peptide, suggesting an interaction of laminin with nonfibrillar species of the Aβ peptide, sequestering the peptide in a soluble form. These data extend our understanding of laminin‐dependent binding of Aβ and highlight the possible modulation role of laminin regarding Aβ aggregation and neurotoxicity in vivo.
Localization of the Noradrenaline Transporter in Rat Adrenal Medulla and PC12 Cells
Authors: Kippenberger Andree G. , Palmer David J. , Comer Alison M. , Lipski Janusz , Burton Lucille D. , Christie David L.
Published: March 2003

Abstract : The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells and the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49‐residue segment (NATM2) of the extracellular region (residues 184‐232) of bovine NAT. Affinity‐purified NATM2 antibodies specifically recognized an 80‐kDa band in PC12 cell membranes by western blotting. Bands of a similar size were also detected in membranes from human neuroblastoma (SK‐N‐SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells stably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue showed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a minor fraction of the NAT was present at the cell surface. Subcellular fractionation of PC12 cells showed that relatively little NAT colocalized with plasma membrane, synaptic‐like microvesicles, recycling endosomes, or trans‐Golgi vesicles. Most of the NAT was associated with [3H]noradrenaline‐containing secretory granules. Following nerve growth factor treatment, NAT was localized to the growing tip of neurites. This distribution was similar to the secretory granule marker secretogranin I. We conclude that the majority of NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.
Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria
Authors: Berman Sarah B. , Hastings Teresa G.
Published: March 2003

Abstract : Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson’s disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase‐generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson’s disease and methamphetamine‐induced neurotoxicity.
Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes
Authors: Vega‐Agapito Victoria , Almeida Angeles , Heales Simon J. R. , Medina José M. , Bolanños Juan P.
Published: March 2003

Abstract : The biosynthesis of the physiological messenger nitric

oxide (•NO) in neuronal cells is thought to depend on a

glial‐derived supply of the •NO synthase substrate arginine.

To expand our knowledge of the mechanism responsible for this glial‐neuronal

interaction, we studied the possible roles of peroxynitrite anion

(ONOO‐), superoxide anion (O2•),

•NO, and H2O2 in

L‐[3H]arginine release in cultured rat astrocytes. After 5 min of

incubation at 37°C, initial concentrations of 0.05‐2 mM

ONOO‐ stimulated the release of arginine from astrocytes in a

concentration‐dependent way ; this effect was maximum from 1 mM

ONOO‐ and proved to be ~400% as compared with control cells.

ONOO‐‐mediated arginine release was prevented by arginine transport

inhibitors, such as L‐lysine and NG‐monomethyl‐L‐arginine,

suggesting an involvement of the arginine transporter in the effect of

ONOO‐. In situ xanthine/xanthine oxidase‐generated

O2• (20 nmol/min) stimulated arginine release to a

similar extent to that found with 0.1 mM ONOO‐, but this

effect was not prevented by arginine transport inhibitors. •NO

donors, such as sodium nitroprusside,

S‐nitroso‐N‐acetylpenicillamine, or

1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1

‐ium‐1,2‐diolate, and H2O2 did not significantly modify

arginine release. As limited arginine availability for neuronal

•NO synthase activity may be neurotoxic due to

ONOO‐ formation, our results suggest that ONOO‐‐mediated

arginine release from astrocytes may contribute to replenishing neuronal

arginine, hence avoiding further generation of ONOO‐ within these cells.
A Novel Microtubule‐Associated Protein‐2 Expressed in Oligodendrocytes in Multiple Sclerosis Lesions
Authors: Shafit‐Zagardo Bridget , Kress Yvonne , Zhao Meng Liang , Lee Sunhee C.
Published: March 2003

Abstract : Elucidation of the mechanisms involved in the regeneration of oligodendrocytes and remyelination is a central issue in multiple sclerosis (MS) research. We recently identified a novel alternatively spliced, developmentally regulated oligodendrocyte‐specific protein designated microtubule‐associated protein‐2+13 [microtubule‐associated protein‐2 expressing exon 13 (MAP‐2+13)]. MAP‐2+13 is expressed in human fetal oligodendrocytes during process extension and myelination but is minimally expressed in normal mature CNS. To test the hypothesis that MAP‐2+13 is reexpressed in regenerating oligodendrocytes in MS lesions, we examined the brains of MS patients for the expression of this protein. By immunocytochemistry using a series of monoclonal antibodies specific for MAP‐2+13, we determined that MAP‐2+13 expression was up‐regulated in all 31 lesions from 10 different MS brains. MAP‐2+13 was expressed in regenerating oligodendrocytes associated with demyelinated lesions, with the highest counts found in regions of extensive remyelination. By electron microscopy, MAP‐2+13 was localized to oligodendrocytes engaged in remyelination, evident by their process extension and association with thinly myelinated (remyelinated) and demyelinated axons. These results suggest a hitherto unsuspected role for this microtubule‐associated protein in oligodendrocyte function during development and myelin repair.
A Novel Rat Tetraspan Protein in Cells of the Oligodendrocyte Lineage
Authors: Birling Marie‐Christine , Tait Steven , Hardy Rebecca J. , Brophy Peter J.
Published: March 2003

Abstract : The tetraspanin/transmembrane 4 superfamily gene

superfamily encodes proteins that span the plasma membrane four times.

Tetraspan proteins are implicated in proliferation, motility, and

differentiation in various cell types, and in some cells they may link plasma

membrane proteins into signalling complexes. Using a subtractive cDNA library

prepared from oligodendrocytes and their progenitor cells, we have identified

Tspan‐2 as a member of this superfamily. In situ hybridization analysis

revealed robust expression in cells of the oligodendrocyte lineage in

comparison with the Plp gene, a well‐characterized marker for myelin‐forming glia in the CNS. Rat Tspan‐2 mRNA is restricted to the nervous system and is detectable by northern blot shortly after birth in the CNS. Subsequently the gene is up‐regulated strongly between postnatal day 3 and 10, and expression levels continue to rise up to postnatal day 22. These data indicate that Tspan‐2 is likely to play a role in signalling in oligodendrocytes in the early stages of their terminal differentiation into myelin‐forming glia and may also function in stabilizing the mature sheath.
Transcriptional Regulation of Mouse Type 1 Inositol 1,4,5‐Trisphosphate Receptor Gene by NeuroD‐Related Factor
Authors: Konishi Yoshiyuki , Ohkawa Noriaki , Makino Yasutaka , Ohkubo Hiroaki , Kageyama Ryoichiro , Furuichi Teiichi , Mikoshiba Katsuhiko , Tamura Taka‐aki
Published: March 2003

Abstract: The type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box‐I element, which is located —334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E‐box consensus sequence, is involved in the up‐regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS‐related basic helix‐loop‐helix (bHLH) factors bind to the box‐I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS‐related bHLH factors, neuronal differentiation factor (NeuroD)‐related factor (NDRF), specifically bound to the box‐I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co‐expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue‐specific regulation of IP3R1 gene expression in the CNS.
Protein Kinase C and the Opposite Regulation of Sodium Channel α‐ and β1‐Subunit mRNA Levels in Adrenal Chromaffin Cells
Authors: Yanagita Toshihiko , Kobayashi Hideyuki , Yamamoto Ryuichi , Takami Yasunari , Yokoo Hiroki , Yuhi Tomoaki , Nakayama Tatsuo , Wada Akihiko
Published: March 2003

Abstract : Our previous [3H]saxitoxin binding and

22Na influx assays showed that treatment of cultured bovine adrenal

chromaffin cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or

phorbol 12,13‐dibutyrate (PDBu), an activator of protein kinase C (PKC),

decreased the number of cell surface Na channels (IC50 = 19

nM) but did not alter their pharmacological properties ; Na channel

down‐regulation developed within 3 h, reached the peak decrease of 53% at 15

h, and was mediated by transcriptional/translational events. In the present

study, treatment with 100 nM TPA lowered the Na channel

α‐subunit mRNA level by 34 and 52% at 3 and 6 h, followed by restoration

to the pretreatment level at 24 h, whereas 100 nM TPA elevated the Na

channel β1‐subunit mRNA level by 13‐61% between 12 and 48 h.

Reduction of α‐subunit mRNA level by TPA was concentration‐dependent

(IC50 = 18 nM) and was mimicked by PDBu but not by the

biologically inactive 4α‐TPA ; it was prevented by H‐7, an inhibitor of

PKC, but not by HA‐1004, a less active analogue of H7, or by H‐89, an

inhibitor of cyclic AMP‐dependent protein kinase. Treatment with

cycloheximide, an inhibitor of protein synthesis, per se sustainingly

increased the α‐subunit mRNA level and decreased the

β1‐subunit mRNA level for 24 h ; also, the TPA‐induced

decrease of α‐subunit mRNA and increase of β1‐subunit

mRNA were both totally prevented for 24 h by concurrent treatment with

cycloheximide. Nuclear run‐on assay showed that TPA treatment did not alter

the transcriptional rate of the α‐subunit gene. A stability study using

actinomycin D, an inhibitor of RNA synthesis, revealed that TPA treatment

shortened the t½ of α‐subunit mRNA from 18.8

to 3.7 h. These results suggest that Na channel α‐ and

β1‐subunit mRNA levels are differentially down‐ and up‐regulated via PKC ; the process may be mediated via an induction of as yet unidentified short‐lived protein(s), which may culminate in the destabilization of α‐subunit mRNA without altering α‐subunit gene transcription.
Brevetoxin‐Induced Autocrine Excitotoxicity Is Associated with Manifold Routes of Ca2+ Influx
Authors: Berman Frederick W. , Murray Thomas F.
Published: March 2003

Abstract: Real‐time alterations in intracellular Ca2+
([Ca2+]i) were monitored in fluo‐3‐loaded cerebellar
granule neurons (CGNs) exposed to the brevetoxin PbTx‐1.
[Ca2+]i was measured using a fluorescent plate reader
(FLIPR), which measures simultaneously the mean intracellular Ca2+
change in a population of cultured cells in each well of a 96‐well plate.
PbTx‐1 produced rapid and concentration‐dependent increases in neuronal
[Ca2+]i with a potency nearly identical to that
determined previously for PbTx‐1‐induced neurotoxicity. The NMDA receptor
antagonists MK‐801, dextrorphan, and D(‐)‐2‐amino‐5‐phosphonopentanoic acid,
and tetanus toxin, an inhibitor of Ca2+‐dependent exocytotic
neurotransmitter release, effected significant reductions in both the
integrated fluo‐3 fluorescence response and excitatory amino acid release and
protected CGNs against PbTx‐1 neurotoxicity. The L‐type Ca2+
channel antagonist nifedipine produced a modest reduction in the fluo‐3
response but reduced substantially the plateau phase of the PbTx‐1 increment
in [Ca2+]i when combined with MK‐801. When nifedipine
and MK‐801 were combined with the Na+/Ca2+ exchanger
(reversed mode) inhibitor KB‐R7943, the PbTx‐1 increment in
[Ca2+]i was nearly completely attenuated. These data
show that Ca2+ entry into PbTx‐1‐exposed CGNs occurs through three
primary routes: NMDA receptor ion channels, L‐type Ca2+ channels,
and reversal of the Na+/Ca2+ exchanger. There was a
close correlation between reduction of the integrated fluo‐3 fluorescence
response and the level of neuroprotection afforded by blockers of each
Ca2+ entry pathway; however, simultaneous blockade of L‐type
Ca2+ channels and the Na+/Ca2+ exchanger,
although reducing the integrated [Ca2+]i response to a
level below that provided by NMDA receptor blockade alone, failed to
completely attenuate PbTx‐1 neurotoxicity. This finding suggests that in
addition to total [Ca2+]i load, neuronal vulnerability
is governed principally by the NMDA receptor Ca2+ influx pathway.
Characterization of Arginine Decarboxylase in Rat Brain and Liver
Authors: Regunathan S. , Reis D. J.
Published: March 2003

Abstract: We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylornithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.
Expression of Retinoid Receptors During the Retinoic Acid‐Induced Neuronal Differentiation of Human Embryonal Carcinoma Cells
Authors: Cheung William M. W. , Chu Patrick W. K. , Lung Cheuk H. , Ip Nancy Y.
Published: March 2003

Abstract: Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRα is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRα and RARs during the RA‐induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRα, RARα, RARβ, and RARγ were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up‐regulation of RXRα expression in NT2 cells. The induced RXRα was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRα expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase‐ and vasoactive intestinal peptide‐positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRα expression in NT2 cells and suggest that RXRα might play a crucial role in the cellular functions of human CNS neurons.
Affinity of (±)‐Pindolol, (‐)‐Penbutolol, and (‐)‐Tertatolol for Pre‐ and Postsynaptic Serotonin 5‐HT1A Receptors in Human and Rat Brain
Authors: Castro M E , Harrison P J , Pazos A , Sharp T
Published: March 2003

There is considerable interest in the use of drugs that selectively block presynaptic (somatodendritic) serotonin 5‐HT1A receptors for the

adjunctive treatment of major depressive disorder. The

5‐HT1A/β‐adrenoceptor ligands (±)‐pindolol,

(‐)‐tertatolol, and (‐)‐penbutolol are currently under clinical investigation,

and knowledge of their affinity at different populations of central

5‐HT1A receptors is needed. Here we have determined the affinity of

these drugs for presynaptic and postsynaptic 5‐HT1A receptors in

postmortem human and rat brain using receptor autoradiography and the

selective 5‐HT1A radioligand [3H]WAY‐100635. The binding

of [3H]WAY‐100635 was specific and saturable and showed high

affinity in the rat dorsal raphe nucleus and hippocampus

(KD = 1.5‐1.7 nM). In competition studies, the

three compounds had nanomolar affinity and produced monophasic displacement of

[3H]WAY‐100635 binding in all regions of both species.

(‐)‐Penbutolol and (‐)‐tertatolol had similar affinity for pre‐and

postsynaptic 5‐HT1A receptors in both rat and human brain. However,

in the human, but not the rat, the affinity of (±)‐pindolol in dorsal

raphe nucleus (Ki = 8.9 ± 1.1 nM) was

slightly but significantly higher than that in hippocampus

(Ki = 14.4 ± 1.5 nM in CA1). In summary,

our data show that (±)‐pindolol, (‐)‐tertatolol, and (‐)‐penbutolol are

all high‐affinity ligands at native human and rat 5‐HT1A receptors.

(‐)‐Penbutolol and (‐)‐tertatolol do not discriminate between the pre‐ and

postsynaptic 5‐HT1A sites tested in either species, but (±)‐pindolol showed a slightly higher affinity for the presynaptic site in human brain. Further work is needed to establish whether the latter difference is clinically relevant.