Claude F. Baxter

Laboratory contaminants in lipid chemistry: Detection by thin-layer chromatography and infrared spectrophotometry and some procedures minimizing their occurrence.

Many sources of contamination for lipid preparations exist in the laboratory. These contaminants can be detected using thin-layer chromatography (TLC) and infrared spectroscopy. Numerous components that are potential contaminants and can lead to false analyses were demonstrated by TLC in laboratory soaps, cleaners, hand creams and lotions, hair tonics, laboratory greases, floor waxes, oil vapors, tobacco smoke, hydrocarbon phases for gas-liquid chromatography, etc. Procedures preventing introduction of contaminants are presented including descriptions of equipment and precautions to eliminate or minimize contamination. These are useful in isolation of pure polar and nonpolar lipids.

Some Metabolic Studies of γ-Aminobutyric Acid.

Summary In agreement with previously reported enzymatic studies, experiments with intracerebrally administered L-glutamic-U-C14 in intact mice showed that carbon chain of L-glutamic acid is a source of the carbon chain of GABA formed in brain and that thiosemicarbazide decreases the rate of conversion of cerebral glutamic acid to GABA. It was shown that carbon of GABA can enter the tricarboxylic acid cycle at the succinate level in intact rats in experiments with intraperitoneally administered GABA-1-C14 employing the isotope trapping technic. Experiments in which GABA-1-C14 was injected intracerebrally to mice, or incubated with minces of mouse brain, indicated that utilization of GABA in brain probably takes place by the same mechanism as in the whole animal. The results of present and past biochemical studies can be correlated with relevant physiological data.

Elevation of γ-Aminobutyric Acid in Rat Brain with Hydroxylamine.

Summary 1. Intraperitoneal injection of hydroxylamine resulted in elevation of levels of γ-aminobutyric acid in 8 brain areas of the rat. 2. These elevated levels of γ-aminobutyric acid were attained even after rats were pretreated with methylene blue to reduce methemoglobin formation. 3. The effect of a single injection of hydroxylamine persisted for at least 5 hours. 4. The potential value of this procedure for physiological study has been discussed.

Demonstration of Thiosemicarbazide-Induced Convulsions in Rats with Elevated Brain Levels of γ-Aminobutyric Acid.

Summary In confirmation of biochemical data for whole brain, injection of thiosemicarbazide produced convulsive seizures in rats concomitantly with decreases in levels of γ-aminobutyric acid in 9 areas of the brain. However, rats injected with both hydroxylamine and thiosemicarbazide had convulsive seizures when levels of γ-aminobutyric acid were either elevated or normal in different areas of brain. Seizures produced by administration of thiosemicarbazide are not related to a generally decreased level of γ-aminobutyric acid in the brain.

Effect of Altered Blood Plasma Osmolalities on Regional Brain Amino Acid Concentrations and Focal Seizure Susceptibility in the Rat

Abstract: Blood plasma hypo‐ or hyperosmolality alters significantly the concentration of some amino acids in brain tissues of the medial septum and hippocampus of adult Sprague‐Dawley rats. With some notable exceptions, brain amino acid concentrations decreased under hypoosmotic conditions and increased under hyperosmotic conditions. Osmotic changes and brain amino acid changes appear to be related to each other in an almost linear fashion. A comparison of rats and toads indicates that the patterns of changes in brain amino acid concentrations in response to a hypoosmotic plasma osmolality were almost identical for both species. Changes achievable under hyperosmotic conditions were considerably greater in toads. When rats with kindled epileptogenic foci were made hypoosmotic by water‐loading, seizure thresholds decreased dramatically. Our data suggest a possible relationship between the hypoosmotically induced biochemical changes in brain tissues (especially some amino acid neurotransmitters and neurotransmitter precursors) and the hypoosmotically induced increase in seizure susceptibility.

Liquid scintillation counting of C14-labeled amino acids on paper, using trinitrobenzene-1-sulfonic acid, and a modified combustion apparatus

Methods for the separation of amino acids from tissue extracts by two-dimensional paper chromatography or by paper electrophoresis, have become well established in many laboratories. A number of one- and two-dimensional scanning devices have been developed for purposes of determining quantitatively the C14 radioactivity in amino acid spots on paper [1–4]. However the counting efficiency of these devices is low even when so-called 4π geometry is employed and both sides of the paper are counted simultaneously.

UPTAKE AND METABOLISM OF GLUTAMATE BY ISOLATED TOAD BRAINS CONTAINING DIFFERENT LEVELS OF ENDOGENOUS AMINO ACIDS

—The uptake of [U‐14C]glutamate into the amphibian brain was studied in vitro using brains from toads (Bufo boreas) adapted either to a fresh water (FWA) or an hyperosmotic saline (HOA) environment. Initial rates of 14C‐glutamate uptake showed a single apparent Km of about 0·2 mm. Uptake by HOA brains was slower than that by FWA brains, reflecting perhaps a non‐competitive type of inhibition by the higher content of glutamate in the HOA brains. Although the glutamate content of HOA brains was maintained during prolonged incubation at twice the level found in FWA toads, other metabolic parameters measured in the two types of brain preparations were surprisingly similar. Tissue to medium concentration ratios of greater than 3000:1 were generated by both FWA and HOA brains. In both brain systems the clearance of glutamate from the medium was accompanied by a rapid conversion of the amino acid to glutamine and its release into the medium. In both the FWA and HOA toad brain systems some [U‐14C]glutamate was metabolized to aspartate and GABA; in both systems the specific radioactivity (SA) of glutamine in the tissue was from two to four times greater than that of glutamate; also the SA of glutamine released into the medium was higher by several orders of magnitude than the SA of glutamine in brain tissues.

Rapid visualization of proteins in isoelectric focusing gels: Isolation of brain tubulin subspecies

A method for visualization of unmodified proteins in polyacrylamide isoelectric focusing (IEF) gels is described. The proteins appear as white, translucent bands when disc IEF gels are placed in water. The gel can be scanned with a scanning spectrophotometer, or the protein bands can be excised using a simple apparatus, which is described, and the protein eluted. The method is fast, selective for proteins, sensitive, and quantitative. It has been used to isolate tubulin species separated by as little as 0.5 mm in disc IEF gels.

γ‐Aminobutyric acid and seizure susceptibility in areas of normal cut brain cortex*

IN mammalian organisms, y-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) am found exclusively in the central nervous system (CNS), predominantly in the gray matter er 01.. 1951). Hydrazides depress the levels of GABA and GAD and at the same time increase seiaae susceptibility (U, 1957; K I L w and Bm. 1957). GABA is known to have inhibitory e m in various biologically excitable system (Eum and JASPER, 1959) and, wben applied diractly to the exposed cortex of cats, it has stopped thiosemicarbazide-induced seizure activity (DASGUPTA ct d.. 1958). Elevated levels of cerebral GABA produced by the administration of suitabk dosa of hydroxylamine appeared to have some sedative action in rats (BAJCCER and ROERTS, 1959) and raduDed susceptibility to electrically-induced seizures in cortical areas of cats brain ( J ~ E L B W O er uf.. 1959). In view of these findings it a p p r e d desirable to examine the relatiomhip b e m n normally occurring levels of GABA in different areas of the cerebral cortex and the Seinve susceptibility of these areas. METHODS

Are Age-Related Changes in Receptor Activity an Expression of Altered Membrane Fluidity?

Among the various theories of aging, the membrane hypothesis of aging is particularly appealing (Sun and Sun, 1979; Grinna, 1977). Cellular membranes function in mediating and regulating active transport of substances across the cell boundary as well as serving as an important permeability barrier. Enzyme activities and receptor functions are also modulated by the lipid composition of membranes. The nature of membranes may change during aging either as a result of oxidative damage (Tappel, 1973) or from a deterioration of homeostatic mechanisms that maintain proper lipid composition (Kates and Kuksis, 1980). In vitro and in vivo manipulation of membrane lipids may thus provide a model of aging.