摘要:

Abstract:Sodium‐dependent3H‐labeled nucleoside transport was studied using a mixed population of dissociated brain cells from adult rats. The accumulation of [3H]adenosine during brief (15‐s) incubation periods was significantly greater in the presence of 110 μMNa+than in its absence. This occurred at substrate concentrations that ranged from 0.25 to 100 μM. Similar findings were observed for the rapid accumulation of [3H]uridine. Kinetically, the rapid accumulation of [3H]adenosine in both the absence and the presence of Na+was best described by a two‐component system. In the presence of Na+, theKTandVmaxvalues for the high‐affinity component were 0.9 μMand 8.9 pmol/mg of protein/15 s, and those for the low‐affinity component were 313 μMand 3,428 pmol/mg of protein/15 s, respectively. In the absence of Na+, theKTvalue for the high‐affinity component was significantly higher (1.8 μM). [3H]Uridine accumulation was best described kinetically by a one‐component system that in the presence of Na+hadKTandVmaxvalues of 1.0 mMand 2.6 nmol/mg of protein/15 s, respectively. As was found for [3H]adenosine, in the absence of Na+, theKTvalue was significantly higher (1.8 mM). The sodium‐dependent transport of [3H]adenosine was inhibitable by ouabain and 2,4‐dinitrophenol. Of the three nucleoside transport inhibitors tested, only nitrobenzylthioinosine demonstrated high affinity and selectivity in blocking the sodium component. Thus, high‐affinity sodium‐dependent nucleoside transport systems, in addition to facilitated diffusion systems, exist on

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10900.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation–flotation centrifugation, temperature‐induced phase separation with Triton X‐114, and lectin affinity chromatography were used separately as well as in combination. The two‐step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening>6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface‐specific monoclonal antibodies than glycoproteins immobilized on lectinagarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent‐labile epitope of G4 (neuron–glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N‐CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180‐kilodalton isoform of N‐CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10901.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Cellular and subcellular distributions of axolinin, the 260‐kilodalton (kD) microtubule‐associated glycoprotein originally purified from squid axons, in various squid tissues such as optical lobes, bundles of small nerve fibers (fin nerves), giant stellate ganglia, skin, muscle, liver, and gill, were immunologicaly studied using monoclonal antibodies specifically recognizing the polypeptide chain of axolinin. The following results were obtained: (1) Axolinin is confined to squid neurons and skin; (2) axolinin is localized in the axon whereas another 260‐kD microtubule‐associated protein, MAP B, is localized in the cell bodies; and (3) axolinin is localized mainly in the peripheral part of the axoplasm of the squid giant axon. The last result has confirmed our previous conclusion obtained using polyclonal antisera against axolinin, which contain antibodies recognizing not only axolinin‐specific epitopes but also nonspecific epitopes. The physiological importance of the localization of axolinin in axons and the skin is discussed based on its possible relationship to excitability

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10902.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:An early increase in ornithine decarboxylase (ODC) activity and polyamine levels in rat cerebral capillaries was previously implicated in the mediation of blood–brain barrier (BBB) breakdown in cold‐injured brain. A time course study in rat cerebrum indicated that cold injury evokes a biphasic increase in ODC activity and polyamine levels in perilesional cortex. ODC activity rose sharply (fourfold) within 1 min, remained elevated for 5 min, and then returned to the basal level by 10 min. A transient rise in polyamine concentration followed in the rank order of putrescine>spermidine>spermine. A secondary rise in ODC activity commenced in perilesional tissue at 2–6 h and peaked (8.8‐fold) at 48 h. Major increases in the content of putrescine (330%), spermidine (103%), and spermine (50%) developed at 48–72 h. α‐Difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, suppressed the evoked increase in ODC activity and abolished the associated increase in content of polyamines, findings indicating that the accumulation of polyamines in cryoinjured brain reflects enhanced synthesis resulting from an ODC‐mediated increase in putrescine content. Cycloheximide and actinomycin D were without effect on the early increase in ODC activity but inhibited the delayed increase in ODC activity, an observation suggesting that the initial increase in activity reflects an activation of a cryptic ODC via a posttranslational process, whereas the delayed increase in activity results from ODC synthesis mainly under transcriptional control. Because membrane phospholipid degradation, release of diacylglycerol and free arachidonic acid, and prostaglandin formation are early events in cold‐injured brain, we assessed the effects of verapamil (a calcium channel blocker), dexamethasone (which inhibits arachidonic acid release), and aspirin (a cyclooxygenase inhibitor). These agents resembled DFMO in that they inhibited the early (2‐min) and delayed (24‐h) increase in ODC activity and polyamine concentrations and concurrently attenuated BBB breakdown in the perilesional cortex, as monitored by fluorescein transport. Exogenous putrescine nullified the protective effect of verapamil, dexamethasone, and aspirin on BBB breakdown following cryogenic injury. These results implicate Ca2+influx via calcium channels, phospholipid hydrolysis, and prostaglandin synthesis in cryogenically induced stimulation of ODC activity and further strengthen the evidence linking polyamines to BBB breakdown. Changes in ODC‐regulated polyamine synthesis in brain cells may play an important role in other aspects of the pathophysiol

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10903.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Although peripheral‐type benzodiazepine recognition sites have been demonstrated in the brain of various species, the precise identity and function of the peripheral benzodiazepine receptor have not been established yet. In light of the recent demonstration of the mitochondrial localization of this receptor and its potential role in intermediary metabolism, we investigated the relationship between the benzodiazepines and the enzyme pyruvate dehydrogenase (PDH), a component of the mitochondrial membrane. The results obtained in the present study demonstrate a specific interaction between PDH and the ligands for the peripheral‐type benzodiazepine receptor, which might account for their effects on cell growth and differentiat

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10904.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The solubility and reactivity of the Folch‐Pi proteolipid from bovine CNS have been studied in reverse micelles of sodium bis(2‐ethylhexyl)sulfosuccinate, isooctane, and water. Such a membrane‐mimetic system resembles the aqueous spaces of the native myelin sheath in terms of its physicochemical properties. Although the proteolipid is completely insoluble in water, it can be inserted into the water‐containing micellar system. In contrast, the lipid‐depleted protein failed to be incorporated into these organized assemblies. The lipid requirements for insertion of the proteolipid were studied, therefore, after delipidation by several precipitations with isooctane, a nondenaturing solvent. Novel extraction procedures and quantitative analyses by HPLC of the protein‐bound lipids revealed the persistence of a lipidprotein complex (6 ± 1 mol of lipid/mol of protein) displaying optimal micellar solubilization. Competition experiments carried out with brain lipids provide evidence for a preference of the myelin protein for sulfatide, phosphatidylinositol, and phosphatidylserine, in that order. The resulting proteolipid, although differing in relative composition, showed good solubility in the membrane‐mimetic system. In contrast, reconstitution experiments carried out with the lipid‐depleted protein resulted in weak lipid binding and poor micellar incorporation. These results suggest that the tightly bound acidic lipids may stabilize a protein conformation required for insertion into the

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10905.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The hydrodynamic behaviour of both the soluble and purified γ‐aminobutyric acidA(GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X‐100 or in 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable)t‐butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAAreceptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAAreceptor in the above‐mentioned detergents was calculated using the H2O/2H2O method. A value of Mr230,000–240,000 was calculated for the bovine pure GABAAreceptor purified in sodium deoxycholate/Triton X‐100 media. A value of Mr284,000–290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10906.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Addition ofd‐aspartate, a substrate for the high‐affinity transport of acidic amino acid transmitters, to suspensions of rat brain synaptosomes increased the rate of O2consumption, uptake of86Rb, and transport of 2‐[3H]deoxyglucose. Stimulation of all three processes was abolished in the presence of ouabain.d‐Aspartate had no effect on respiration in the medium in which NaCl was replaced by choline chloride. The ratio of the ouabain‐sensitive increase in86Rb uptake to that in O2consumption was 12 to 1, which gives a calculated86Rb(K+)/ATP of 2. It is concluded that electrogenic, high‐affinity transport of sodium‐d‐aspartate into synaptosomes stimulates the activity of the Na+/K+pump through an inc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10907.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The specific binding of [3H]kainic acid was investigated in membrane preparations from human parietal cortex obtained postmortem. Saturation studies revealed that binding occurred to a single population of sites with aKDof 15 nMand aBmaxof 110 fmol/mg of protein. The kinetically determined dissociation constant for these sites agreed well with that obtained from saturation analyses. Pharmacological characterisation of these sites gave a profile consistent with those reported for kainate receptor sites in animal brain. The integrity of kainate receptors was studied in several brain regions from six patients who had died of Alzheimer's disease and from six closely matched control subjects. No change in either the affinity or the number of kainate receptors was seen in any of the regions studied, despite the loss of neo‐cortical and hippocampal glutamatergic terminals in the Alzheimer's diseased brains, as previously reporte

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10908.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The present study was designed to investigate the direct response of fetal adrenomedullary cells to hypoxia, and the possible change in this responsiveness with maturation. Ovine fetal adrenomedullary cells, when exposed to 30 min of hypoxia induced by perfusing with Krebs–Henseleit solution equilibrated with 1% O2, released significantly greater amounts of total catecholamine into the perfusate, compared to basal conditions. After a 1‐h control period, a second 30‐min hypoxic episode stimulated a catecholamine response which was significantly smaller in magnitude than the first. Following the two hypoxic episodes, the cells were capable of responding to 50 mMKCl with a large increase in total catecholamine release. During the first hypoxic episode, the release of both norepinephrine and epinephrine was stimulated by equal magnitude. Fetal adrenomedullary cells obtained from fetuses at 100, 120, and 130 days gestation showed similar responsiveness to the same hypoxic stimulus, and these responses were not different from that observed in maternal adrenomedullary cells. On the contrary, responsiveness to KCl‐induced depolarization was greatest in cells obtained from fetuses at 130 days gestation when compared to that in the younger fetuses. This increased responsiveness to KCl was accompanied by a greater catecholamine store in the adrenal medulla of the fetuses at this gestational age. These results suggest that ovine fetal adrenomedullary cells can respond directly to hypoxia by releasing catecholamines. This direct responsiveness became desensitized after repeated exposure. Finally, a decrease in direct responsiveness to hypoxia associated with maturation could not be demon

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb10909.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY