Research Methods in Neurochemistry

Section I Properties of Intact Neural Tissues.- 1 Biochemical Study of Tumors of the Nervous System.- I. Introduction.- II. Human Brain Tumors.- A. Autopsy Specimens.- B. Surgical Specimens.- III. Experimental Tumors in Laboratory Animals.- A. Spontaneous Tumors in Animals.- B. Radiation Induction of Tumors.- C. Experimental Tumors Induced by Hydrocarbon Carcinogens.- D. The Use of Neuro-oncogenic Viruses.- E. Resorptive Neuro-oncogens.- F. Transplantable Tumors.- G. Clinical Recognition of Tumors in Small Animals.- H. Procurement of Samples.- IV. The Use of Tissue Culture of Tumors for Biochemical Study.- A. Background.- B. General Characteristics of Brain Tumor Cells in Culture.- C. Problems in Comparison of Tumor Cell Cultures with Normal Cells.- D. Specific Applications for Tissue Culture.- E. Technical Considerations in the Study of Tissue Culture Samples.- V. Technical Approaches to the Study of Spontaneous or Experimental Tumors.- A. Gross Tumor Analyses.- B. In Vitro Metabolic Studies.- C. Homogenate Fractionation.- D. Quantitative Histochemistry, Serial Section Method.- E. Quantitative Histochemistry, Microdissection Methods.- F. Quantitative Cytochemistry.- G. The Closed-System Procedure for Energy-Flux Estimation.- H. Kinetics of Cell Cycle.- VI. Comments on the Expression of Data.- References.- 2 Neurochemical Study of the Cerebrospinal Fluid.- I. Introduction.- A. Location, Formation, and Drainage of CSF.- B. Insulation of CSF.- II. Sampling the CSF.- A. Lumbar Fluid.- B. Cisternal Fluid.- C. Cortical Fluid.- D. Ventricular Fluid.- III. Perfusion of CSF Spaces.- A. Ventriculocisternal Perfusion.- B. Perfusion of Subarachnoid Spaces.- C. Advantages and Disadvantages of Perfusion Experiments.- IV. Results and Discussion.- A. Biochemical Heterogeneity of CSF.- B. Biochemical Changes in the CNS and Their Reflection in the CSF.- C. Blood as Potential Origin of Substances in CSF.- D. Origin of Substances in the Lumbar Fluid.- E. Some Suggestions for Further Research.- Summary.- References.- 3 Measuring Protein Synthesis and Degradation Rates in CNS Tissue.- I. Introduction.- II. General Considerations.- A. Terminology.- B. Precursor Selection.- C. Sacrifice.- D. Tissue Processing.- E. Protein.- F. Amino Acid Separations.- III. Methods for Measuring Protein Synthesis Rates.- A. Pulse Labeling.- B. Infusion.- C. Multiple Injections.- D. Massive Precursor Injection.- E. Pellet Implantation.- F. Feeding.- G. In Vitro Determinations.- H. Protein Accumulation.- I. Inhibition of Synthesis.- IV. Methods for Measuring Protein Degradation.- V. Criteria and Expectations.- References.- Section II Components of Neural Tissues—Enzymes and Amines.- 4 Enzymes Involved in Catecholamine Metabolism: Tyrosine Hydroxylase, Aromatic Amino Acid Decarboxylase, Dopamine ?-Hydroxylase, Phenylethanolamine N-Methyltransferase, Catechol O-Methyltransferase, Aldehyde Dehydrogenase, and Alcohol Dehydrogenase.- I. Introduction.- II. Tyrosine Hydroxylase.- A. Assay.- B. Potential Sources of Interference.- III. Aromatic L-Amino-Acid Decarboxylase.- A. Assay.- B. Determination of the Separated Amines.- IV. Dopamine ?-Hydroxylase.- A. Assay.- V. Phenylethanolamine N-Methyltransferase.- A. Assay.- VI. Catechol O-Methyltransferase.- A. Assay.- B. Potential Sources of Interference.- VII. Aldehyde Dehydrogenase.- A. Assay.- VIII. Alcohol Dehydrogenase.- A. Assay.- References.- 5 Enzymatic-Isotopic Assay of Histamine, Histidine, Histidine Decarboxylase, and Histamine Methyltransferase.- I. Introduction.- II. Development of Method.- III. Materials.- A. Radioisotopes.- B. Enzymes.- C. Microfuge Tubes.- IV. Combined Microassay of Histamine, Histidine, Histidine Decarboxylase, and Histamine Methyltransferase.- A. Preparation of Samples.- B. Histamine Assay.- C. Histidine Assay.- D. Histidine Decarboxylase Assay.- E. Histamine Methyltransferase Assay.- F. Protein Assay.- V. Problems and Difficulties in the Enzymatic-Isotopic Technique.- A. Stability of [14C]- and [3H]S-Adenosylmethionine and Histamine Methyltransferase.- B. Nonspecific Binding of Histamine to Tissue.- C. The Effect of Drugs on the Activity of HMT.- D. Overlap in Counting Channels of the Liquid-Scintillation Counter.- E. Postmortem Loss of Brain Histamine.- F. Variations in the Brain Histamine Concentration with Strain of Rat.- VI. Application of Methods.- A. Brain Regional Studies.- B. Subcellular Studies.- C. Release from Brain Slices.- D. In Vitro Studies of Histidine Decarboxylase and Histamine Methyltransferase.- VII. Discussion.- References.- 6 Analysis of Amines by Mass Spectrometry: Identification and Quantitation of Trace Amines at the Picomole Level.- I. Introduction.- A. Synthesis and Metabolism of the Trace Amines.- B. Trace Amines in Urine and Tissue.- II. Sensitive Analytical Procedures.- A. Fluorimetric Quantitation of Dansyl Derivatives.- B. Radio-Dansyl Assay.- C. Radio-Enzymatic Assay.- D. Gas Chromatography-Mass Spectrometry.- E. Electron-Capture Gas Chromatography.- F. High-Resolution Mass Spectrometric Integrated-Ion-Current Procedure.- III. The Chromatographic, Direct-Probe, High-Resolution Mass Spectrometric Integrated-Ion-Current Procedure.- A. Choice of Derivative.- B. Isolation of the Amine Fraction.- C. Chromatographic Separation.- D. Qualitative Analysis.- E. Quantitative Analysis.- F. Automatic Ion-Current Integration.- IV. Applications.- A. Distribution of Phenylethylamine, m- and p-Tyramine, and Tryptamine.- B. Effect of Some Drugs on the Trace Amines.- C. Metabolic Studies.- D. Identification and Quantitation of Amino Acids, Other Amines, and Some Drugs.- V. Conclusion.- References.- Section III Components of Neural Tissues: Proteins, Lipids.- 7 Highly Purified Neurophysin Proteins Free of Hormonal Activities.- I. Introduction.- II. Preparation of Neurophysins.- A. Purification of Neurophysin by Chromatographic Procedures.- B. Purification of Neurophysin by Continuous Preparative Polyacrylamide-Gel Electrophoresis (PAGE).- III. Conclusions.- References.- 8 The Preparation of Nerve Growth Factor.- I. Introduction.- II. Assays.- A. In Vitro Neurite Proliferation or “Halo” Assay.- B. Immunoassays.- C. Receptor Competition Assays.- III. Isolation Procedures.- A. Mouse Submandibular Gland.- B. Snake Venom.- IV. Concluding Remarks.- References.- 9 Radioactive Immunoassay for Nerve Growth Factor.- I. Introduction.- A. Choice of Assay.- B. Types of Antigen Available.- II. Two-Site Assay of NGF.- A. Preparation of Immunoadsorbent Paper.- B. Preparation of Labeled Antibodies.- C. Assay Technique.- D. Parameters Affecting the Assay.- E. Application of Assay to 7S Species of NGF.- III. Results Using Immunoassay.- A. Plasma NGF.- B. Submaxillary Gland NGF.- IV. Conclusions.- V. Addendum.- References.- 10 Recent Methods for the Separation and Analysis of Central Nervous System Glycoproteins.- I. Introduction.- II. Solubilization of Membranes for Affinity Chromatography of Glycoproteins.- III. Affinity Chromatography of Membrane Glycoproteins on Immobilized Lectins.- A. General Considerations.- B. Purification of Lectins.- C. Preparation of Sepharose-Bound Lectins.- D. Affinity Chromatography on Immobilized Lectins.- IV. Analysis of Glycoprotein Constituents by GLC.- A. Release of Constituent Sugars from Heteropolysaccharides.- B. Separation and Quantitative Determination of O-Methyl Glycosides.- C. Analysis of Sugar Constituents of Glycoproteins and Glycolipids by GLC.- D. Summary of Practical Procedure.- References.- 11 Preparation of Proteolipids.- I. Introduction.- II. Preparation of Crude Proteolipid.- A. General Aspects.- B. Extraction and Washing.- C. Modification of the Extraction and Washing Procedures.- D. Evaporation.- E. Emulsification.- F. Centrifugation.- G. Further Purification of Crude Proteolipid.- III. Preparation of Proteolipid Apoprotein.- A. General Aspects.- B. Removal of Lipids.- C. Conversion to a Water-Soluble Form.- D. Properties of the Apoprotein.- IV. Alternative Procedures.- A. Solvent Partition Procedures.- B. Chromatographic Procedures.- C. Salt Denaturation.- D. Drying in a Two-Phase System.- V. Separation of Proteolipids from Brain Subcellular Fractions.- A. Myelin.- B. Other Subcellular Fractions.- VI. Analytical Procedures.- A. Lipids.- B. Proteins.- C. SDS-Polyacrylamide-Gel Electrophoresis.- D. Molecular Weights.- References.- 12 Methods for Isolation and Analysis of Gangliosides.- I. Introduction.- II. Structures and Distribution.- III. Isolation and Purification.- A. Procedure Based on Folch Partitioning.- B. Procedure Based on DEAE-Sephadex and Silicic Acid.- C. Precipitation with Trichloroacetic Acid-Phosphotungstic Acid.- D. Notes on Alternate Procedures.- IV. Resolution.- A. Thin-Layer Chromatography.- B. Column Chromatography.- V. Analytical Procedures.- A. Colorimetric and Fluorimetric Procedures.- B. Gas-Liquid Chromatography.- VI. Structure Determination.- References.- 13 Preparation and Determination of Cerebrosides.- I. Introduction.- II. Isolation and Purification.- A. Extraction and Preliminary Treatment.- B. Purification.- C. Bulk Isolation.- III. Analytical Procedures.- A. General Discussion.- B. Determination of the Whole Molecule.- C. Determination of Various Components.- IV. Synthesis of Radioactive Cerebrosides for Tracer Study.- A. Labeled at Galactose.- B. Labeled at Sphingosine.- C. Labeled at Fatty Acid.- References.