Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

Binding of the Novel Serotonin Agonist 8-Hydroxy-2-(Di-n-Propylamino) Tetralin in Normal and Alzheimer Brain

Binding of [3H]8-hydroxy-2-(di-n-propylamino) tetralin, a putative ligand for the 5-hydroxytryptamine (5-HT, serotonin) 1A recognition site, was measured in neocortex from postmortem human brain. The substance was found to bind to a saturable site with a KD value and pharmacological profile similar to that of rat. Binding to membranes from normal human temporal cortex was found to significantly correlate (inversely) with age. A significant reduction in binding, reflecting decreased density of recognition sites, was observed in the frontal cortex of patients with Alzheimer's disease (48% loss). This region in the dement brains showed unaltered presynaptic 5-HT function (5-HT and 5-hydroxyindoleacetic acid content) whereas 5-HT concentration was reduced in the temporal cortex.     

High-Resolution Proton Magnetic Resonance Analysis of Human Cerebrospinal Fluid

High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

利用人摄金蛋白(METALLOTHIONEIN)启动子和牛乳突瘤病毒在哺乳动物细胞中表达乙型肝炎表面抗原

用人Metallothioenin-Ⅱ启动子、乙型肝炎表面抗原基因,SV40早期基因的编接位点和多聚A位点构成了表达组件,然后插入到经改造过的BpV-1质粒中。所得质粒pdMTsAg-5转染小鼠C127细胞得到转化克隆。Ausria Ⅱ检测证明13株中有12株能产生HBsAg。对其中一株进行HBsAg收率观察,隔天收获为292.6～525.8μg/升,每天收获为200.9～369.0μg/升,可连续收获60天以上。经重金属离子诱导后,收率增至583～854.4μg/升。培养液经超滤浓缩和两次密梯离心后,可集中为一个狭窄的峰,顶峰的Ausria Ⅱ cpm为1.05×10~7,密度为1.20g/ml。     

利用单克隆抗体鉴定人甲胎蛋白(HAFP)抗原结构可分性

本文利用两株针对HAFP分子不同抗原决定簇的单克隆抗体,鉴定HAFP酶解片断的抗原抗体反应性质,并同完整HAFP分子进行比较。结果表明,酶解片断上失去了一株单克隆抗体所对应的分子部份,完整保留着另一株单克隆抗体所识别的抗原决定簇,从而证实HAFP分子某些抗原结构之间具有可分割性。     

体外分化的人体巨噬细胞抑制天然杀伤(NK)细胞的研究

本文继先前工作后,进一步应用正常健康人外周血单个核细胞(PBMNC)经塑料培皿粘附技术把单核细胞分离出来,经培养进一步纯化,随后动态观察培养0,2,4,6和8天的单核-巨噬细胞的形态变化和对新鲜分离同种异基因个体PBMNC中NK活性的影响。实验表明,体外分化6天和8天的巨噬细胞质/核比例和胞浆内空泡显著增加,细胞直径约为0天时的2倍。这些细胞和PBMNC之比为0.5:1时,引起了NK细胞活性的50%以上抑制(4小时~(51)Cr标记K 562肿瘤的同位素释放试验)。这种抑制效应不为过氧化氢酶(Catalase 4000单位/毫升)和前列腺素合成酶的抑制剂(Indom 1×10~(-5)M)所阻断。实验证明,同种异基因个体的NK细胞不能识别巨噬细胞表面抗原,从而排除了巨噬细胞和K562肿瘤抗原竞争的可能性。实验还表明,巨噬细胞对NK活性的抑制是不受HLA约束的。应用高频超声振荡破碎巨噬细胞膜方法和免疫调变技术进一步提示,人体巨噬细胞对NK活性的抑制与巨噬细胞体积无关,而与体外分化所赋有的固有特性和它们分泌的免疫调节分子有关。     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.