Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

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.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

B7-2胚胎性癌细胞诱导分化过程中的表型转变

胚胎性癌细胞(简称EC细胞)作为一类肿瘤(畸胎瘤)的干细胞近年受到广泛的重视,从胚胎学、肿瘤学和分子生物学等许多学科领域都应用它作为实验材料,离体诱导分化研究是其中的一个方面。B 7-2 EC细胞是我们从129品系小鼠的自发睾丸畸胎瘤中分离克隆得到的一株多能EC细胞,它在同种同基因小鼠