Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.     

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.     

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.     

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.     

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.     

Immunoassay of the Neuronal and Neuroendocrine Marker PGP 9.5 in Human Tissues

An inhibitory, coated-well immunoassay for the neurone-specific protein PGP 9.5 has been devised and used to measure the concentrations of the protein in human tissues. Concentrations of PGP 9.5 between 40 ng/ml and 10 micrograms/ml could be measured using this assay. In brain PGP 9.5 was present at 100.58 +/- 16.18 micrograms/mg protein. Of the other organs examined only kidney and testis showed significant concentrations of PGP 9.5 (3.97 +/- 0.87 microgram/mg protein and 3.25 +/- 0.36 microgram/mg protein, respectively). All other organs contained less than 2% of the brain level. The tissue levels determined by coated-well immunoassay confirmed the tissue specificity of PGP 9.5 originally determined by high-resolution two-dimensional gel electrophoresis.     

丁酸对体外培养的人鼻咽癌上皮细胞的影响

无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞     

Amino Acids in Plasma and CSF and Monoamine Metabolites in CSF: Interrelationship in Healthy Subjects

Plasma and cerebrospinal fluid (CSF) concentrations of amino acids were measured in 65 healthy volunteers (50 men and 15 women). The CSF levels of the monoamine metabolites homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), and 5-hydroxyindoleacetic acid (5-HIAA) were also determined. Sex differences were observed in both plasma and CSF amino acid levels as well as in the relationship between these concentrations. No significant correlations were observed between the CSF levels of HVA and 5-HIAA, and the concentrations of their precursor amino acids in either plasma or CSF. The MOPEG level in CSF correlated positively with the plasma concentrations of several amino acids.