摘要:

AbstractProtein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0(P0‐ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N‐linked glycosylation. The x‐ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x‐ray diffraction data on the orientation of P0‐ED, and we propose how this immunoglobulin‐like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β‐strand predictions for P0‐ED are virtually superimposable with those of the VHdomain of the phosphocholine‐binding immunoglobulin M603 of mouse, which has a similar number of residues as P0‐ED and a structure that has been solved crystallographically. The dimensions of P0‐ED from the space‐filling model, developed using PC‐ based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P0‐ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0‐ED from apposed membranes if the molecules were oriented so that the β‐ strands were approximately perpendicular to the membrane surface. The apposed P0‐EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P0‐ED regions analogous to the complementarity‐determining regions of immunoglobulins can account for the recognition of P0‐ED from apposed membranes in the incompletely swollen state. Two of the faces of P0‐ED that show charge complementarity could account for the homophilic interactions of P0‐ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N‐ linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P0‐ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P0at the membrane surface. Our model shows how the single immunoglobulin‐like domain of P0can account for distinct structural s

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07434.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract[125I]RTI‐55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI‐55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI‐55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI‐55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two‐phase processes. A curve‐fitting analysis of the data derived in saturation experiments found a high‐affinity site withKD=66 ± 35 pMand Smax= 13.2 ± 10.1 pmol/g of tissue and a low‐affinity site withKD=1.52 ± 0.55 nMand Bmaxof 47.5 ± 11‐2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI‐55>GBR‐12909>mazindol>WIN 35428>= methylphenidate>(−)‐cocaine>buproprion>(±)‐amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site withKD= 370 ± 84 pM.It appears that [125I]RTI‐55 should be useful in further studies of the regulation of cocaine binding sit

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07435.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractWe measured the activity of the a‐ketoglutarate dehydrogenase complex (α‐KGDHC), a rate‐limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and α‐KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of α‐KGDHC. As compared with the controls, mean α‐KGDHC activity, with added TPP, was significantly (p<0.005) reduced in AD brain in frontal (‐56%), temporal (‐60%), and parietal (‐68%) cortices, with the reductions (‐25 to ‐53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean a‐KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (‐39 to ‐83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between α‐KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on α‐KGDHC activity in the AD group (23–280% mean stimulation) as compared with the controls (‐4 to ±50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain. Reduced brain α‐KGDHC activity could be consequent to loss of neurons preferentially enriched in α‐KGDHC, a premortem reduction in TPP levels (which may have affected enzyme stability), elevated brain levels of the α‐KGDHC inhibitor ammonia, or an actual failure in the expression of the gene encoding the enzyme. We suggest that a defect in this key Krebs cycle enzyme could contribute to an impairment of cerebra

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07436.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractUsing a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean ± SEM fold increase in dialysate concentrations of aspartate was 38 ± 13 (n = 5) for moderate TBI and 74 ± 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 ± 26 and 144 ± 23 for moderate and severe TBI, respectively. Although these increases normalized within 20–30 min following moderate TBI, concentrations of aspartate and glutamate took>60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 ± 0.6 and 7.6 ± 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 ± 0.2 and 4.1 ± 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 ± 20 and 414 ± 66 μM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonistsN‐methyl‐d‐ aspartate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07437.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractThe possible existence of a dopamine D2receptor‐mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [3H]dopamine and superfused with D2dopamine receptor agonists or antagonists to determine if there was an effect on [3H]dopamine release. The D2receptor antagonist sulpiride increased both baseline [3H]‐ dopamine release and [3H]dopamine release induced by an increase in extracellular potassium concentration. The D2receptor agonists LY‐171555 and RU‐24213 did not reduce baseline [3H]dopamine release but completely inhibited [3H]dopamine release induced by an increase in [K±]o. This action of the D2agonists was blocked by sulpiride. These studies demonstrate the existence of D2receptor, possibly autoreceptor, regulation of dopamine release in the teleos

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07438.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractTo date three β subunits of the GABAAreceptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity‐purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower‐molecular‐mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system‐specific labeling of a 55‐kDa band that comigrated with the 55‐kDa band of the purified receptor. Quantitative immunolabeling of this 55‐kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippo

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07439.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractMetabolites of [3H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5α‐dihydroprogesterone (5α‐DHP) and 3α,5α‐tetrahydroprogesterone (3α,5α‐THP) in rat brain slices, 5α‐DHP and 20α‐ dihydroprogesterone (20α‐DHP) in mouse brain slices, and 20α‐DHP in monkey brain slices. In rat olfactory bulb slices, 5α‐DHP represented 25.2 ± 3.3% of total radioactivity and 3α,5α‐THP 17.5 ± 2.8%, whereas in rat medulla oblongata slices, 5α‐DHP was 31.3 ± 3.5% and 3α,5α‐ THP 5.4 ± 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12–20% of total radioactivity.‐The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5α‐DHP represented 16.6 ± 4.6% and 20α‐DHP 9.5 ± 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 ± 4.4% and 2.4 ± 0.5% of total radioactivity, respectively, were converted to 20α‐DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3α,α‐THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 ± 2.5 nMafter electroshock. Endogenous 3α,5α‐THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABAAreceptor. The significant interspecies differences in the brain progesterone metabolism should be considered

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07440.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractThe expression of facilitative glucose transporter (GLUT) isoforms in human astrocytic tumors was examined. Reverse transcriptase‐polymerase chain reaction of a surgically biopsied glioblastoma was carried out using the degenerative oligonucleotide primers corresponding to the sequences of the human facilitative glucose transporter family, and polymerase chain reaction products were hybridized with human GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5 cDNA probes. The results showed that a biopsied glioblastoma expressed GLUT1, GLUT3, and GLUT4 glucose transporter genes. Northern blot analysis of total RNA (10 μg) from a biopsied glioblastoma showed the transcripts of only GLUT1 and GLUT3, suggesting that the expression of insulin‐responsive glucose transporter GLUT4 mRNA is relatively low. Immunoblot analysis of biopsied glioblastoma tissues by polyclonal antibodies against the C‐terminal synthetic peptides of GLUT1, GLUT3, and GLUT4 showed a single band of each polypeptide. However, elevated expression of GLUT1 and GLUT3 glucose transporters was not observed in the glioblastoma. Astrocytic tumor tissues (n = 14) were also examined immunohistochemically. Reactive products for GLUT1 were observed in the luminal surface of capillaries in all cases, whereas tumor cells were positive for GLUT1 in only two of 14 cases. GLUT3 was positive in astrocytic tumor cells in all cases. Three of 14 cases expressed the GLUT4 protein, which was localized in the cytoplasm of tumor cells. These results suggest that the facilitative glucose transport may be altered in astrocytic tumor cells and thus display a significant change in glucose meta

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07441.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractNeurofibromatosis type 1 (NF‐1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF‐1 gene is similar in sequence to theras‐GTPase activator protein (ras‐GAP), and investigations have confirmed that the NF‐1 gene product (now known as neurofibromin) stimulatesras‐GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63‐bp exon inserted in the GAP‐related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon‐intron boundaries. RNase protection and RNA‐polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07442.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

AbstractAccumulation ofl‐kynurenine and quinolinic acid (QUIN) in the brain occurs after either ischemic brain injury or after systemic administration of pokeweed mitogen. Although conversion ofl‐[13C6]tryptophan to [13C6]‐QUIN has not been demonstrated in brain either from normal gerbils or from gerbils given pokeweed mitogen, direct conversion in brain tissue does occur 4 days after transient cerebral ischemia. Increased activities of enzymes distal to indoleamine‐2,3‐dioxygenase may determine whetherl‐kynurenine is converted to QUIN. One day after 10 min of cerebral ischemia, the activities of kynureninase and 3‐hydroxy‐3,4‐dioxygenase were increased in the hippocampus, but local QUIN levels and the activities of the indoleamine‐2,3‐dioxygenase and kynurenine‐3‐hydroxylase were unchanged. By days 2 and 4 after ischemia, however, the activities of all of these enzymes in the hippocampus as well as QUIN levels were significantly increased. Kynurenine aminotransferase activity in the hippocampus was unchanged on days 1 and 2 after ischemia but was decreased on day 4, at a time when local kynurenic acid levels were unchanged. A putative precursor of QUIN, [13C6]anthranilic acid, was not converted to [13C6]‐QUIN in the hippocampus of either normal or 4‐day postischemic gerbils. Gerbil macrophages stimulated by endo‐toxin in vitro convertedl‐[13C6]tryptophan to [13Ce]QUIN. Kinetic analysis of kynurenine‐3‐hydroxylase activity in the cerebral cortex of postischemic gerbils showed that Vmaxincreased, without changes inKm. Systemic administration of pokeweed mitogen increased indoleamine‐2,3‐dioxygenase and kynureninase activities in the brain without significant changes in kynurenine‐3‐hydroxylase or 3‐hydroxyanthranilate‐3,4‐dioxygenase activities. Increases in kynurenine‐3‐hydroxylase activity, in conjunction with induction of indoleamine‐2,3‐dioxygenase, kynureninase, and 3‐hydroxyanthranilate‐3,4‐dioxygenase in macro‐phage infiltrates at the site of brain injury, may explain the ability of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb07443.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY