bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Coupling of the γ‐Aminobutyric AcidB Receptor with Calcium Ion Channel and GTP‐Binding Protein and Its Alteration Following Solubilization of the γ‐Aminobutyric AcidB Receptor
Authors: Ohmori Yoshiyuki , Hirouchi Masaaki , Taguchi Jun‐ichi , Kuriyama Kinya
Published: December 2006

Abstract: The coupling mechanism of the γ‐Aminobutyric acid (GABA)B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP‐binding protein was examined using a crude synaptic membrane (P2) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P2 fraction, [3H]GABA binding to the GABAB receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), a calmodulin antagonist, did not affect on the calcium ion‐induced enhancement of GABAB receptor binding. These results suggest that the GABAB receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABAB receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5′‐(3‐O‐thio)triphosphate (GTPγS), guanosine 5′‐(β,γ‐imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (‐)‐baclofen. Although the affinity of (‐)‐baclo‐fen for the GABAB receptor was decreased in the presence of GTP, pretreatment of the P2 fraction with islet‐activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (‐)‐baclofen induced an increase of GTPase activity in the P2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABAB receptor may also be functionally coupled with IAP‐sensitive GTP‐binding protein. Treatment of the P2 fraction with sodium deoxycholate resulted in the highest solubilization of GABAB receptor among various detergents examined. The solubilization, however, completely eliminated the stimulating effects of calcium ion and calcium ion channel blockers as well as the inhibitory effects of GTP and GTP analogues on GABAB receptor binding. Furthermore, the increase of GTPase activity induced by GABA and (‐)‐baclofen was also eliminated following the solubilization. These results suggest that functional coupling of the GABAB receptor with the calcium ion channel and GTP‐binding protein such as Ni or No may be easily destroyed following the solubilization.
Analysis of Rat Hindlimb Muscle Proteins by Two‐Dimensional Gels Following Spinal Cord Injury
Authors: Lee John C. , Braun Angela M.
Published: December 2006

Abstract: Proteins from extensor digitorum longus (EDL), plantaris (PLN), and soleus (SOL) muscles of adult, female rats were examined by high resolution two‐dimensional gel electrophoresis up to 4 weeks following spinal cord transection. The electrophoretograms were analyzed by computer imaging and densitometry. Reproducible and significant changes in the relative concentrations of several proteins in each muscle type were detected. Whereas changes involving the largest number of proteins were observed in SOL, changes in EDL and PLN were also detected. In SOL, approximately 50% of the altered proteins increased in concentration and the remaining decreased: Actin and myosin light chains LCF‐1 and LCF‐2 were among those proteins whose concentrations increased, whereas myosin light chains LCS‐1 and LCS‐2 were among those proteins whose concentrations decreased. The present findings regarding the reversal in myosin light chain composition provide biochemical support for previously published data on changes in contractile properties of muscles following spinalization. In EDL, the relative concentration of only one protein was elevated in a time‐dependent manner. The concentrations of two protein species in PLN were increased following cord transection. These findings provide new biochemical markers on the effects of spinal cord on gene expression in specific hindlimb skeletal muscles.
Uptake of Kynurenine into Rat Brain Slices
Authors: Speciale Carmela , Schwarcz Robert
Published: December 2006

Abstract: The transport of [3H]kynurenine ([3H]KYN) into slices from rat tissue was examined in vitro. Brain accumulated KYN seven to eight times more effectively than any of several peripheral organs. Of all the organs tested, only the brain exhibited a sodium‐dependent component of the uptake process. After an incubation period of 1 h, sodium‐dependent transport amounted to 60% of total uptake. Both processes were abolished by prior sonication of the tissue and significantly inhibited by inclusion of metabolic blockers in the incubation medium. Time resolution showed that the sodium‐independent uptake occurred rapidly and reached saturation within 30 min. In contrast, sodium‐dependent transport was linear for at least 2 h of incubation. Brain regional analysis revealed a sevenfold difference between the areas of highest (cortex) and lowest (cerebellum) uptake. With the exception of cerebellar tissue, the ratio between sodium‐dependent and sodium‐independent processes was consistent among brain regions. Kinetic analyses were performed on striatal slices and revealed a Km of 927 μM and a Vmax of 18 nmol/h/mg of protein for the sodium‐dependent process, and a Km of 3.8 mM and a Vmax of 38 nmol/10 min/mg of protein for the sodium‐independent transport. The transporters were equally amenable to inhibition by KYN and tryptophan, indicating that KYN entry into the cell may be mediated by neutral amino acid uptake sites. No strict stereoselectivity existed, but L enantiomers were clearly more active than the D forms. Studies in ibotenate‐lesioned striata showed a 70%decrease in the sodium‐dependent process and a concomitant 60%increase in the sodium‐independent transport, suggesting differential cellular associations of the two uptake sites. Cellular uptake of KYN may constitute a critical step in the disposition of KYN metabolites such as quinolinic acid and kynurenic acid in the brain.
Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes
Authors: Santoro Claudia , Hall Linda M. , Zukin R. Suzanne
Published: December 2006

Abstract: Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD= 8.8 ± 1.7 nM; Bmax= 2.3 ± 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a μ‐selective ligand: KD= 150 ± 34 nM; Bmax= 3.0 ± 0.6 pmol/mg of protein. Trypsin or heat treatment (100°C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by >80%. The rank order of potency of drugs at this site was levorphanol > DHM > normorphine > naloxone « dextrorphan; the μ‐specific peptide [d‐Ala2,Gly‐ol5]‐enkephalin and δ‐, κ‐, and μgm‐ligands were inactive at this site. The other site was labeled by (‐)‐[3H]ethylketocyclazocine {(‐)‐[3H]EKC}, a κ‐opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD= 212 ± 25 nM; Bmax= 1.9 ± 0.2 pmol/mg of protein). (‐)‐[3H]EKC binding could be displaced by κ‐opioids but not by μ‐, δ‐, or μgm‐opioids or by the κ‐peptide dynorphin(1–17). Specific binding constituted ∼70% of total binding at 1 nM and ∼50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the δ‐ligands [3H][d‐Ala2,d‐Leu5]‐enkephalin and [3H][d‐Pen2,d‐Pen5]‐enkephalin was undetectable in this preparation. These findings demonstrate the presence of morphine (μ‐like) and EKC (κ‐like), but not δ like, binding sites in Drosophila and indicate that vertebrate opioid peptides are inactive in this preparation. To determine whether opioid sites in Drosophila nervous tissue mediate a functional response, morphine sulfate was administered to Drosophila larvae throughout development. Morphine produced a dose‐dependent delay in pupation of larvae and in eclosion of adults and significantly reduced the ability of larvae to survive to the adult stage. Together, these results indicate that morphine elicits a physiological response in Drosophila larvae and suggest a possible function for opioid receptors in the Drosophila nervous system.
Characterization with Antibodies of the γ‐Aminobutyric AcidA/Benzodiazepine Receptor Complex During Development of the Rat Brain
Authors: Vitorica Javier , Park Dongeun , Chin Gwendolynne , de Bias Angel L.
Published: December 2006

Abstract: The postnatal development of the γ‐Aminobutyric acidA/benzodiazepine receptor (GABAR/BZDR) complex of the rat brain has been investigated using the monoclonal antibody 62‐3G1 and the polyclonal rabbit antiserum A, specific for the 57,000 and 51,000 Mr, receptor subunits, respectively. Both GABAR and BZDR binding activities co‐precipitated during all postnatal ages. Adult rats showed a main 51,000 Mr [3H]flunitrazepam photoaffinity‐labeled peptide, whereas newborn rats showed several photolabeled peptides of higher Mr. All the photolabeled peptides could be immunoprecipitated with each antibody regardless of the age of the rats. These results suggest that the physical coupling between the GABAR and the BZDR is already present in newborn animals and it is maintained afterwards during development. Glycosidase and peptidase treatments of the immunoprecipitated GABAR/BZDR complex indicated that all the [3H]flunitrazepam‐photolabeled subunits are different peptides, although they seem to conserve a high degree of homology. In addition to the age‐dependent heterogeneity, the results also suggest that for each age, there is heterogeneity in the subunit composition of the GABAR/BZDR complex.
Effects of Protein Kinase C Down‐Regulation on Secretory Events and Proopiomelanocortin Gene Expression in Anterior Pituitary Tumor (AtT‐20) Cells
Authors: Vyas Sheela , Bishop John F. , Gehlert Donald R. , Patel Jit
Published: December 2006

Abstract: To elucidate the role of the diacylglycerol‐protein kinase C (PKC) pathway in β‐endorphin synthesis and secretion in anterior pituitary corticotrope tumor cells (AtT‐20), a procedure for down‐regulating PKC activity in the cells was developed. Treatment of AtT‐20 cells with 12‐0‐tetradecanoylphorbol 13‐acetate (TPA) led to an increase in [3H]phorbol 12,13‐dibutyrate binding to PKC in the membrane fraction of these cells 30 s after its addition to the culture medium. Thereafter, a decrease in both [3H]phorbol 12,13‐dibutyrate binding and PKC‐specific phosphotransferase activity occurred in a time‐ and dose‐dependent manner in both the cytosolic and membrane fractions. For example, treatment of the cells with 100 nM TPA for 24 h resulted in an almost complete depletion of PKC activity. Immuno‐reactive β‐endorphin secretion was found to be stimulated two‐ to fourfold in the control cells after incubation with corticotropin‐releasing factor (10‐7M), forskolin (10‐6 M), or TPA (10‐7M) for 4 h. In cells rendered PKC deficient, TPA‐stimulated immunoreactive β‐endorphin release was abolished, forskolin‐stimulated release was unaffected, and corticotropin‐releasing factor‐stimulated release was depressed. Treatment of control cells with any one of the three stimulatory agents led to an increase in proopiomelanocortin mRNA levels, and these responses were also depressed after TPA pretreatment. The results suggest that physiological processes thought to be entirely cyclic AMP dependent, such as corticotropin‐releasing factor‐elicited secretion, may be partially dependent on PKC‐mediated biochemical events.
Role of Excitatory Amino Acid Receptors in K+‐and Glutamate‐Evoked Release of Endogenous Adenosine from Rat Cortical Slices
Authors: Hoehn Katja , White Thomas D.
Published: December 2006

Abstract: : K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+‐ but not glutamate‐evoked adenosine release. Tetrodotoxin decreased K+‐ and glutamate‐evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto‐5′‐nucleo‐tidase by α,β‐methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto‐5′‐nucle‐otidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate‐evoked release of adenosine. Glutamate‐evoked adenosine release was diminished 50 and 55% by the TV‐methyl‐D‐aspartate (NMDA) receptor antagonists, DL‐2‐amino‐5‐phosphonovaleric acid and (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801), respectively. The remaining release in the presence of MK‐801 was diminished a further 66% by the non‐NMDA receptor antagonist, 6,7‐dinitroquinoxaline‐2,3‐dione, suggesting that both NMDA and non‐NMDA receptors were involved in glutamate‐evoked adenosine release. Surprisingly, K+‐evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+‐evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.
Binding of the Neuronal Protein B‐50, but Not the Metabolite B‐60, to Calmodulin
Authors: Coggins Philip J. , Zwiers Henk
Published: December 2006

Abstract: : Protein kinase C phosphorylates the neurone‐specific protein B‐50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B‐50 phosphorylation‐inhibiting N‐terminal fragment and a large C‐terminal metabolite B‐60 (B‐5041‐226). The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho‐B‐50. In an attempt to unify two aspects of B‐50 biochemistry, we have examined the interaction of B‐50 binding to calmodulin and B‐50 proteolysis. HPLC‐ and affinity‐purified B‐50 bound to calmodulin, but purified B‐60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B‐60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B‐50 protease. B‐60 derived from dephospho‐B‐50 also failed to bind calmodulin. The results demonstrate a direct connection between B‐50 binding to calmodulin and B‐50 proteolysis. The position of the proposed calmodulin‐binding domain within intact B‐50 is discussed in light of the failure of calmodulin to bind B‐60.
Optimal Duration of Experimental Period in Measurement of Local Cerebral Glucose Utilization with the Deoxyglucose Method
Authors: Mori Kentaro , Schmidt Kathleen , Jay Thérèse , Palombo Ernesta , Nelson Thomas , Lucignani Giovanni , Pettigrew Karen , Kennedy Charles , Sokoloff Louis
Published: December 2006

Abstract: The time course and magnitude of the effects of product loss on the measurement of local cerebral glucose utilization (LCGU) by the 2‐[14C]deoxyglucose (DG) method were studied by determination of LCGU in 38 rats with 25–120 min experimental periods after a [14C]DG pulse and in 45 rats with experimental periods of 2.5–120 min during which arterial plasma [14C]DG concentrations (CP*) were maintained constant. LCGU was calculated by the operational equation, which assumes no product loss, with the original set of rate constants and with a new set redetermined in the rats used in the present study; in each case the rate constants were those specific to the structure. Data on local tissue 14C concentrations and CP* were also plotted according to the multiple time/graphic evaluation technique (“Patlak Plot”). The results show that with both pulse and constant arterial inputs of [14C]DG the influence of the rate constants is critical early after onset of tracer administration but diminishes with time and becomes relatively minor by 30 min. After a [14C]DG pulse calculated LCGU remains constant between 25 and 45 min, indicating a negligible effect of product loss during that period; at 60 min it begins to fall and declines progressively with increasing time, indicating that product loss has become significant. When CP* is maintained constant, calculated LCGU does not change significantly over the full 120 min. The “Patlak Plots” reinforced the conclusions drawn from the time courses of calculated LCGU; evidence for loss of product was undetectable for at least 45 min after a pulse of [14C]DG and for at least 60 min after onset of a constant arterial input of [14C]DG.
In Vivo Spectrophotometric Determination of Striatal Acetylcholinesterase Activity: The Modulation Induced by the Antidepressant Amitriptyline
Authors: Chappey Olivier , Testylier Guy , Gourmelon Patrick , Galonnier Monique , Bourre Jean Marie , Fatome Marc , Scherrmann Jean Michel , Viret Jacques
Published: December 2006

Abstract: A new technology called in vivo spectrophotometry was applied to the quantitative determination of the variations in local acetylcholinesterase (AChE) activities. Repeated measurements of the enzyme activities in the same live animal allowed the study of the in vivo inhibition of AChE by amitriptyline. Interactions between AChE and this tricyclic antidepressant were investigated at the striatal level in anesthetized rats. In this anesthetized model, AChE assays were shown to be stable for ∼8 h. The dose‐effect relationship was explored in the 2.5‐ to 50‐mg/kg amitriptyline range. A reversible inhibition was observed after acute amitriptyline administration. The maximum of inhibition appeared between 90 and 210 min after the intoxication and reached up to 22% for the 50‐mg/kg dose. The threshold dose was established as 8 mg/kg. Evidence for an indirect interaction between tricyclic antidepressant and AChE was demonstrated when the total integrity of the biological system was preserved.
In Vivo Translocation and Down‐Regulation of Protein Kinase C Following Intraventricular Administration of Tetanus Toxin
Authors: Aguilera José , Yavin Ephraim
Published: December 2006

Abstract: A single intraventricular injection into adult rats of 100 mouse lethal doses of tetanus toxin (TeTox) produces a marked intracellular redistribution of Ca2+/phosphatidylserine (PtdSer)‐dependent protein kinase C (PKC) activity. Changes are particularly pronounced in hypothalamus, hippocampus, and spinal cord structures. Translocation of PKC from the inactive cytosolic compartment to a membrane‐bound active form is followed by a time‐dependent reduction in both total activity and enzyme protein. The down‐regulation of PKC activity in the hypothalamus is accompanied by a marked increase in a Ca2+/PtdSer‐independent kinase activity, predominantly in the cytosolic fraction. Our data identify PKC as a possible indirect target for TeTox and suggest that down‐regulation of the enzyme may provide a clue for tetanus neurotoxicity.
Characterisation of Binding Sites for δ‐Dendrotoxin in Guinea‐Pig Synaptosomes: Relationship to Acceptors for the K+‐Channel Probe α‐Dendrotoxin
Authors: Muniz Zilda M. , Diniz Carlos R. , Dolly J. Oliver
Published: December 2006

Abstract: With use of biologically active 125I‐labelled δ‐dendrotoxin, a putative K+‐channel ligand, homogeneous, noninteracting, high‐affinity acceptors (KD= 0.32 ± 0.07 nM; Bmax= 0.33 ± 0.04 pmol/mg) were observed in synaptosomes from guinea‐pig cortex. This binding was antagonised noncompetitively by α‐dendrotoxin, an inhibitor of certain fast‐activating, voltage‐gated K+ channels. Chemical cross‐linking of the δ‐dendrotoxin‐acceptor complex in synaptosomes yielded two specifically labeled polypeptides with molecular masses of 69 and 82 kilodaltons. Although α‐dendrotoxin prevents the labelling of both these bands, it cross‐linked only a single protein with a molecular mass of 69 kilodaltons. It is concluded that δ‐dendrotoxin interacts with a distinct site on the oligomeric acceptors for α‐dendrotoxin.
Purification of a Phosphatidylinositol 4‐Phosphate Kinase from Bovine Brain Membranes
Authors: Moritz Albrecht , De Graan Pierre N. E. , Ekhart Peter F. , Gispen Willem H. , Wirtz Karel W. A.
Published: December 2006

Abstract: A phosphatidylinositol 4‐phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six‐step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl‐cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP‐agarose. The enzyme preparation was nearly homogeneous and was purified 5,600‐fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.
Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain
Authors: Smith Susan M. , Nahorski Stefan R.
Published: December 2006

Abstract: The regional distribution of inositol 1,4,5‐trisphosphate (InsP3), inositol 1,3,4,5‐tetrakisphosphate (InsP4), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [3H]InsP3 binding indicated similar positional and stereospecificity as observed in other tissues, with 100‐fold selectivity for lnsP3 over InsP4. Similarly, high‐affinity [32P]InsP4 binding also showed a high degree of positional specificity, with a 1,000‐fold selectivity for InsP4 over InsP3. Initial characterisation of [3H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca2+ concentrations and inhibited by Ca2+ levels of >1 mM. [3H]‐Ryanodine binding was also enhanced by β,γ‐methylene‐adenosine 5′‐trisphosphate and caffeine and inhibited by magnesium and ruthenium red (Ki= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca2+‐induced Ca2+ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [3H]InsP3 and [32P]InsP4, an effect most probably due to nonspecific complex formation. Regional binding of InP3, InsP4, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP3 sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High‐affinity InsP4 sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP3 > InsP4 > ryanodine.
Differential Effects of a Phorbol Ester on Carboxypeptidase E in Cultured Astrocytes and AtT‐20 Cells, a Neuroendocrine Cell Line
Authors: Klein Robyn S. , Flicker Lloyd D.
Published: December 2006

Abstract: Cultured astrocytes have been shown to secrete various neuropeptides and the neuropeptide processing enzyme, carboxypeptidase E (CPE). The secretion of CPE enzymatic activity from astrocytes has been shown previously to be increased approximately twofold by treatment with tetradecanoylphorbol 13‐acetate (TPA), a phorbol ester. In this study, metabolic labeling with [35S]Met was utilized to examine the effect of TPA on the biosynthesis of CPE protein in cultured astrocytes and in AtT‐20 cells, a pituitary‐derived cell line. Treatment of astrocytes with 0.1 μg/ml TPA for 24 h caused an 80% increase in the level of radiolabeled CPE in both the media and the cells, indicating that the synthesis of CPE was stimulated by the TPA. AtT‐20 cells also secreted more radiolabeled CPE in response to TPA, but this increase was offset by a proportional decrease in the cellular level of radiolabeled CPE, and synthesis of CPE was not stimulated in this cell line. Northern blot analysis demonstrated that 0.1 μg/ml TPA elevated CPE mRNA by approximately 50% in cultured astrocytes but not in AtT‐20 cells. Quantitative in situ hybridization studies demonstrated that the TPA‐induced increase in CPE mRNA expression was largely due to increases in the number of cells expressing CPE mRNA, although for astrocytes from some brain regions the average level of CPE mRNA per cell was also elevated by TPA. These results suggest that astrocytes can be induced to express CPE, which is consistent with a role for astrocytes in intercellular signaling.
β‐Amyloid Protein Precursor and τ mRNA Levels Versus β‐Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain
Authors: Oyama Fumitaka , Shimada Hiroyuki , Oyama Rieko , Titani Koiti , Ihara Yasuo
Published: December 2006

Abstract: To learn whether or not the levels of β‐amyloid protein precursor (APP) and τ mRNAs are related to the formation of β‐amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto‐chemically for β‐amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β‐amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β‐protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β‐amyloid and/or tangle formation in the aged human brain.
Interactions Between the Glutamate and Glycine Recognition Sites of the N‐Methyl‐d‐Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies
Authors: Grimwood Sarah , Wilde Geraint J. C. , Foster Alam C.
Published: December 2006

Abstract: The N‐methyl‐d‐aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P2 membranes we have investigated the effect of glutamate recognition site ligands on [3H]glycine (agonist) and (±)4‐trans‐2‐car‐boxy‐5,7‐dichloro‐4‐[3H]phenylaminocarbonylamino‐1,2,3,4‐tetrahydroquinoline ([3H]l‐689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l‐[3H]glutamate (agonist), dl‐3‐(2‐carboxypiperazin‐4‐yl)‐[3H]propyl‐1 ‐phosphonate ([3H]‐CPP; “C‐7” antagonist), and cis‐4‐phosphonomethyl‐2‐[3H]piperidine carboxylate ([3H]CGS‐19755; “C‐5” antagonist) binding to the glutamate site. “C‐7” glutamate site antagonists partially inhibited [3H]l‐689,560 binding but had no effect on [3H]glycine binding, whereas “C‐5” antagonists partially inhibited the binding of both radioligands. Glycine, d‐serine, and d‐cycloserine partially inhibited [3H]CGS‐19755 binding but had little effect on l‐[3H]‐glutamate or [3H]CPP binding, whereas the partial agonists (+)‐3‐amino‐1‐hydroxypyrrolid‐2‐one [(+)‐HA‐966], 3R‐(+)cis‐4‐methyl‐HA‐966 (l‐687,414), and 1‐amino‐1‐carboxycyclobutane all enhanced [3H]CPP binding but had no effect on [3H]CGS‐19755 binding, and (+)‐HA‐966 and l‐687,414 inhibited l‐[3H]glutamate binding. The association and dissociation rates of [3H]l‐689,560 binding were decreased by CPP and d‐2‐amino‐5‐phosphonopentanoic acid (“C‐5”). Saturation analysis of [3H]l‐689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [3H]l‐689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [3H]l‐689,560 is discussed.
Catecholamines Are Present in a Synaptic‐Like Microvesicle‐Enriched Fraction from Bovine Adrenal Medulla
Authors: Annaert W. G. , Liona I. , Backer A. C. , Jacob W. A. , De Potter W. P.
Published: December 2006

Abstract: “Synaptic‐like microvesicles” are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β‐hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic‐like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]‐Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β‐hydroxylase. Immunoelectron microscopy of synaptic‐like microvesicle fractions showed an intense labeling for synaptophysin on 60‐90‐nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic‐like microvesicles of the chromaffin cell.
Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis in Human Neuroblastoma Cells
Authors: Fisher Stephen K. , McEwen Edward , Kunkle Christopher , Thompson Angela K. , Slowiejko Diana
Published: December 2006

Abstract: To examine the possibility that NaF enhances phosphoinositide‐specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (Gp), we have evaluated the contribution of Gp activation to NaF‐stimulated phosphoinositide hydrolysis in human SK‐N‐SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC50 of ∼ 8 mM). Inclusion of U‐73122, an aminosteroid inhibitor of guanine nucleotide‐regulated PIC activity in these cells, resulted in a dose‐dependent inhibition of NaF‐stimulated inositol lipid hydrolysis (IC50 of ∼ 3.5 μM). When added to digitonin‐permeabilized cells, NaF or guanosine‐5′‐O‐thiotriphosphate (GTPγS) resulted in a three‐ and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF‐stimulated inositol phosphate release were inhibited by inclusion of 10 μM U‐73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate that in SK‐N‐SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of Gp.
Complex Interactions Among Second Messenger Pathways, Steroid Hormones, and Protooncogenes of the Fos and Jun Families Converge in the Regulation of the Nerve Growth Factor Gene
Authors: Jehan Frédéric , Neveu Isabelle , Naveilhan Philippe , Brachet Philippe , Wion Didier
Published: December 2006

Abstract: Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated via the protooncogene c‐fos. Activation of the protein kinase C pathway and 1,25‐dihydroxyvitamin D3[1,25(OH)2D3] has also been reported to increase the pool of NGF transcripts in L929 fibroblasts. Here we show that activation of the cyclic AMP second messenger pathway antagonized the effect of phorbol 12‐myristate 13‐acetate (PMA) or serum on NGF synthesis, whereas it enhanced that of 1,25(OH)2D3. A positive effect was also observed when serum, PMA, and 1,25(OH)2D3 were added together, but dexamethasone reduced this enhancement. There was no close correlation between the increase in c‐fos mRNA and that in NGF mRNA, suggesting that expression of the c‐fos protooncogene is not necessarily followed by induction of the NGF gene. Rather, these two genes are simultaneously, and not sequentially, induced after forskolin treatment. It appears that regulation of the NGF gene depends on a repertoire of multiple regulatory AP‐1 complexes arising from activation of the second messenger pathways. This suggests that NGF gene expression is under the control of a complex interplay among second messenger pathways, protooncogenes, and steroid hormones such as 1,25(OH)2D3 and glucocorticoids.
Medication Costs: The Role Physicians Play with Their Senior Patients
Authors: Sue Beran Mary , Laouri Marianne , Suttorp Marika , Brook Robert
Published: December 2006

OBJECTIVES: To determine how often primary care physicians discuss medication costs with their senior patients and what cost‐reducing strategies they employ.

DESIGN: Cross‐sectional, random‐sample mail questionnaire.

SETTING: State of California.

PARTICIPANTS: Six hundred seventy‐eight of 1,098 (62%) internal medicine and family practice physicians selected from the American Medical Association Masterfile.

MEASUREMENTS: Main outcomes included frequency of cost discussions with senior patients in the previous 30 days and choice of cost‐reducing strategy when a senior expresses financial difficulty with medication costs.

RESULTS: Forty‐three percent of physicians reported discussing medication cost with at least half of their senior patients in the previous 30 days. Patients initiated most of these discussions. Forty percent reported that, at least one time in the previous 30 days, they had not discussed cost but wished they had. The most common reason given was “I ran out of time” (36%). Physicians with high perceived knowledge of medication costs were more likely to discuss cost (odds ratio (OR)=3.49, 95% confidence interval (CI)=1.66–7.3) versus low perceived knowledge, but this trend was not seen in physicians who scored high on actual knowledge of medication costs (OR=0.78, 95% CI=0.43–1.43) versus low actual knowledge. The most common cost‐reducing strategies were generic substitution (33%) and offering samples (25%).

CONCLUSION: The frequency of medication cost discussions between physicians and senior patients is low, and when it occurs, is often initiated by patients. Physicians' perception of their knowledge of medication costs may be an important factor in initiating cost discussions.
The Association Between Serum Folate Levels and Periodontal Disease in Older Adults: Data from the National Health and Nutrition Examination Survey 2001/02
Authors: Yu Yau‐Hua , Kuo Hsu‐Ko , Lai Yu‐Lin
Published: December 2006

OBJECTIVES: To assess the relationship between serum folate levels and periodontal disease in older adults.

DESIGN: Population‐based cross‐sectional study.

SETTING: National Health and Nutritional Examination Survey 2001/02.

PARTICIPANTS: Eight hundred forty‐four dentate elderly subjects aged 60 and older (mean age 70.6) who completed a periodontal examination and laboratory test for serum folate levels.

MEASUREMENTS: Periodontal examination, including probing depth and attachment loss, was performed. Periodontal disease was defined as having at least 10% of sites with clinical attachment loss of more than 4 mm and at least 10% sites with probing depth of more than 3 mm. Serum folate levels were measured using a commercially available radioprotein binding assay kit.

RESULTS: After controlling for demographics, educational level, body mass index, bleeding on probing, and probing sites, the odds ratio for periodontal disease was 0.74 (95% confidence interval=0.59–0.93) for each standard deviation increase in natural‐log‐transformed folate levels. After additionally controlling for levels of vitamin B12 and homocysteine, chronic diseases (hypertension, diabetes mellitus, heart disease, and stroke), and health behaviors (smoking status and alcohol consumption), the negative association between folate level and periodontal disease remained statistically significant and essentially unchanged. There was no effect modification of sex on the association between serum folate levels and periodontal disease.

CONCLUSION: A low serum folate level was independently associated with periodontal disease in older adults. The results suggest that serum folate levels, important indicators of periodontal disease in older adults, may provide an important clinical target for intervention to promote oral health.
Treatment and Prevention of Depression After Surgery for Hip Fracture in Older People: Randomized, Controlled Trials
Authors: Burns Alistair , Banerjee Sube , Morris Julie , Woodward Yvonne , Baldwin Robert , Proctor Rebekah , Tarrier Nicholas , Pendleton Neil , Sutherland Deborah , Andrew Glynne , Horan Mike
Published: December 2006

OBJECTIVES: To evaluate the effect of a psychiatric intervention in treating depression (treatment study) and the effect of a psychological treatment in preventing depression (prevention study) after hip fracture in older people.

DESIGN: Two linked randomized, controlled trials.

SETTING: Orthopedic units in Manchester, England.

PARTICIPANTS: Two hundred ninety‐three older people who had undergone surgery for a fractured hip: 121 in the treatment study and 172 in the prevention study.

MEASUREMENTS: The Geriatric Depression Scale and Hospital Anxiety and Depression Scale for mood, functional tests for mobility and pain measures.

RESULTS: There was a slight reduction in depressive symptoms in the active arm of the treatment study. In the prevention study, there was no significant difference in incident depression between the psychological intervention and treatment as usual. There were no differences in the functional and pain outcomes.

CONCLUSION: The results from these two randomized, controlled trials show that, after hip fracture surgery, no statistically significant benefits can be achieved from a psychiatric intervention in people who are depressed or a psychological intervention to prevent the onset of depression.
Preoperative Immunosuppressive Therapy and Surgery as a Treatment for Anal Furunculosis
Authors: KLEIN ARNAUD , DENEUCHE AYMERIC , FAYOLLE PASCAL , HIDALGO ANTOINE , SCOTTI STEFANO , ZYLBERSTEIN LUCA , DESBOIS CHRISTOPHE , TESSIER DOMINIQUE , MOISSONNIER PIERRE , VIATEAU VÉRONIQUE
Published: December 2006

Objectives—To evaluate the efficacy of combining preoperative immunosuppressive therapy with surgical excision for treatment of anal furunculosis (AF) in dogs.

Study Design—Retrospective study.

Animals—Dogs (n=25) with stages 1–4 AF.

Methods—Preoperative immunosuppressive therapy was either cyclosporine A (CyA) alone or in combination with ketoconazole (Group 1; n=18), or azathioprine combined with prednisolone (Group 2; n=7). Surgical excision of residual draining tracts, cryptectomy, and anal sacculectomy were performed. Only dogs with postoperative clinical follow‐up exceeding 9 months were retained for the study.

Results—Both immunosuppressive protocols were effective in reducing progression of AF. Subsequent draining tracts excision, cryptectomy, and anal sacculectomy were bilateral (12 dogs) or unilateral (13 dogs of which 4 had bilateral anal sacculectomy). Postoperative recovery was uneventful, except for 2 dogs that had wound breakdown. Recurrence was not observed in any of the dogs that had bilateral surgical excision and or in 9 dogs that had unilateral excision.

Conclusion—Preoperative immunosuppressive therapy, combined with bilateral surgical resection of affected tissue consistently, resulted in resolution of AF. Four dogs that had recurrence had unilateral excision despite initial bilateral involvement, suggesting that all diseased tissue should be excised.

Clinical Relevance—These preliminary results suggest that immunosuppressive therapy before surgical excision for AF yields minimizes recurrence in dogs.
INVITED REVIEW—Morphologic and Functional Features of the Canine Cruciate Ligaments
Authors: DE ROOSTER HILDE , DE BRUIN TANYA , VAN BREE HENRI
Published: December 2006

Objective— To review the gross, microscopic, and functional anatomy of the cranial cruciate ligament (CCL) in dogs.

Study Design— Literature review.

Methods— Reports of the anatomy and function of the cruciate ligaments in dogs were retrieved by search of the 1975–2005 PubMed database.

Results— The CCL has an important biomechanical function resisting cranial drawer, hyperextension, and internal rotation and acts to fine tune and guide the stifle through its rolling and sliding motion. It has a complex architecture, and distinct geographic regions within the ligament have different functional roles depending on the angle and loading conditions. Collagen type I is the main component of the extracellular matrix; the fibrils have a crimped structure. The cruciate ligaments are almost completely covered by synovium, protecting them from synovial fluid. Cruciate blood supply is mainly of soft tissue origin. The intraligamentous network is relatively limited whereas the core of the middle third of the CCL is even less well vascularized. Neurohistologic studies are very limited in the dog. Various mechanoreceptors and proprioceptive receptors have been identified within the substance of the cruciate ligaments.

Conclusions— CCL structural characteristics play an important part in its complex behaviour with the crimped pattern of the collagen fibrils being an important determinant of its biomechanical properties. In contrast to reports of managing CCL rupture, there are few reports describing the microanatomy and neurovascular morphology of the cruciate ligaments.

Clinical Relevance— Cruciate disease is likely multi‐factorial. Improved understanding of CCL degradation leading to CCL rupture is critical to development of new diagnostic tests for cruciate disease in dogs. Appropriate intervention during the early stages of disease process might preserve CCL structural properties by preventing further collagen degradation. Accurate knowledge of functional and fiber bundle anatomy is imperative for reconstruction and restoration of normal stifle joint physiology. Reconstructive goals should alleviate existing instability and mimic normal kinematics. Knowledge of the exact function of the CCL in the neuromuscular control around the stifle joint could possibly explain osteoarthritis progression after CCL damage.
Coil Occlusion of Residual Shunts After Surgical Closure of Patent Ductus Arteriosus
Authors: FUJII YOKO , KEENE BRUCE W. , MATHEWS KYLE G. , ATKINS CLARKE E. , DEFRANCESCO TERESA C. , HARDIE ELIZABETH M. , WAKAO YOSHITO
Published: December 2006

Objective— To describe use of coil embolization to occlude residual flow through a patent ductus arteriosus (PDA) after incomplete surgical ligation.

Study Design— Clinical study.

Animals— Dogs (n=4) with continuous murmur after surgical ligation of PDA.

Methods— After PDA ligation, residual ductal flow through the PDA was visible on color‐flow Doppler examination and left ventricular end‐diastolic diameter remained increased. Coil embolization by an arterial approach was performed to achieve complete occlusion of the PDA.

Results— Embolization coils were delivered without complications and hemodynamically successful occlusion was achieved. Doppler‐visible flow resolved in 2 dogs within 3 months after embolization. Left ventricular end‐diastolic diameter indexed to body weight decreased in all dogs.

Conclusions— Transcatheter coil embolization appears to be a safe and minimally invasive procedure for complete occlusion of residual PDA flow after incomplete surgical ligation.

Clinical Relevance— Transcatheter coil embolization should be considered for correction of hemodynamically significant residual shunts in dogs that have incomplete PDA occlusion after open surgical ligation.
Incidence and Breed Predilection for Dystocia and Risk Factors for Cesarean Section in a Swedish Population of Insured Dogs
Authors: BERGSTRÖM ANNIKA , NØDTVEDT ANE , LAGERSTEDT ANNE‐SOFIE , EGENVALL AGNETA
Published: December 2006

Objectives— To estimate the incidence and breed predilection for canine dystocia using data from insurance claims. The risk factors for cesarean section (CS) were assessed for bitches with dystocia.

Study Design— Retrospective, longitudinal study.

Sample Population— Insurance claims records (1995–2002) from a Swedish animal insurance database (Agria), including ∼200,000 bitches.

Methods— The overall incidence rate of dystocia in insured bitches was calculated by dividing the number of reimbursed dystocia claims with the number of dog years at risk. Subsequently, incidence rates were stratified by breed, region, and habitat. The proportion of bitches with a dystocia claim that had CS were calculated, and risk factors for CS were assessed using a logistic regression model.

Results— Between 1995 and 2002, 3894 (2%) of 195,931 Swedish bitches included in the study had a reimbursed insurance claim for dystocia. The overall incidence rate of dystocia was 5.7 cases/ 1000 dog years at risk. Some breeds like the Scottish terrier were at increased risk of dystocia. Among bitches with dystocia, 63.8% were treated by CS.

Conclusion— Dystocia in the bitch is more common than reported earlier. The risk of developing dystocia varies by breed, and a high percentage (63.8%) of affected bitches undergo CS.

Clinical Relevance— Breeders and veterinarians could use this information to better predict which bitches are likely to experience dystocia and/or CS.
Confidence Intervals and P‐Values for Meta‐Analysis with Publication Bias
Authors: Henmi Masayuki , Copas John B. , Eguchi Shinto
Published: December 2006

Summary We study publication bias in meta‐analysis by supposing there is a population (y, σ) of studies which give treatment effect estimates y∼N(θ, σ2). A selection function describes the probability that each study is selected for review. The overall estimate of θ depends on the studies selected, and hence on the (unknown) selection function. Our previous paper, Copas and Jackson (2004, Biometrics60, 146–153), studied the maximum bias over all possible selection functions which satisfy the weak condition that large studies (small σ) are as likely, or more likely, to be selected than small studies (large σ). This led to a worst‐case sensitivity analysis, controlling for the overall fraction of studies selected. However, no account was taken of the effect of selection on the uncertainty in estimation. This article extends the previous work by finding corresponding confidence intervals and P‐values, and hence a new sensitivity analysis for publication bias. Two examples are discussed.