The potential importance of streptomycin (Schatz et at., 1944) as a therapeutic agent has made the study of this antibiotic an urgent problem. In order to expedite progress the Abbott, Eli Lilly, Parke Davis, and Upjohn research laboratories have cooperated in a joint attack on the problem, and the Departments of Chemistry and Horticulture of the University of Illinois have contributed to certain phases of the work. One of the first problems to be investigated was that of developing an assay method which would be satisfactory, not only for beers, but also for solutions of streptomycin containing organic solvents and reagents. Waksman and coworkers have used the agar-dilution method in defining the currently accepted Escherichia coli streptomycin unit (Schatz et al., 1944). The agar-diffusion method has been recommended for an assay of potency (Waksman, 1945) and has been applied to the determination of streptomycin in body fluids (Stebbins and Robinson, 1945). A slide-cell technique for the assay of body fluids also has been described (Heilman, 1945). Since considerable difficulty was encountered in obtaining consistent results with the agar-diffusion method, it seemed desirable to make our experiences available to other workers in the field. In this paper we are presenting studies on the important sources of variation and the procedure currently employed in our laboratories for the assay of streptomycin. PROCEDURE Medium. Bacto-streptomycin assay agar, dehydrated, was prepared by the Difco Laboratories, Inc., in collaboration with our group, to provide a supply of a standard, uniform medium for the assay of streptomycin.1 The medium is prepared for use by dissolving 25.5 g per 1,000 ml in doubly distilled water. After sterilization the medium may be stored at 2 to 4 C until used. 1 The authors gratefully acknowledge the cooperation of Mr. H. G. Dunham of the Difco Laboratories, Inc., in making this medium available.
Results of this investigation indicate that the suckling rat treated with phenylacetate should be a useful new model for studying the pathogenesis of phenylketonuria and neuronal development. Both cerebellar and retinal neurons of postnatally treated rats are vulnerable to the adverse effects of phenylacetate. Morphological changes observed in the cerebellum, retina, and optic nerve of treated animals during the fourth to twenty-first days of life consist of regional reduction in the size of cerebellar vermis lobules IV, V, VIa, and IX, 35 to 40% reduction in thickness of the molecular layer, accumulation of cerebellar external granular cells and retinal neuroblastic cells, fewer parallel fibers in the cerebellar cortex, and fewer myelinated axons in the optic nerve.
A comparison was made of cerebellar dendritic development in the normal rat and in a new model of phenylketonuria, the phenylacetate-treated suckling rat. Golgi stain analysis of the Purkinje cells shows striking regional variations in the dendritic growth. These variations were observed in both the control and phenylacetate-treated animals and were especially striking before 15 days of life. Quantitative analysis of the dendritic tree revealed, in the phenylacetate-treated rat, a significant reduction in the total number of dendritic branches. However, the individual terminal dendritic length was largely unaltered. These effects of phenylacetate differ from those of deafferentation, and starvation. Results of this investigation clearly define the harmful effects of phenylacetate on developing neurons and are compatible with the clinical observation that brain damage in phenylketonuria occurs mainly during the first few years of life, the critical period of neuronal development.
Abstract— The distribution of B6 vitamers in subcellular fractions of cerebral cortex was examined. No pyridoxine and only traces of pyridoxamine could be detected in cerebral cortex. Significant quantities of pyridoxal were found in the cytoplasmic fraction. No vitamin B6 was detected in the subcellular fractions carrying microsomes, myelin, vesicles, and small membranes settling above the 1‐0 M‐sucrose gradient. Pyridoxamine phosphate was the predominant form of vitamin B6 in cerebral cortex of mature rats and guinea pigs, being present in almost twice the concentration of pyridoxal phosphate. More of the latter compound was present in the cytoplasm than in the mitochondria. In contrast, pyridoxamine phosphate was compartmentalized in extraterminal and intraterminal mitochondria. Of especial interest was the finding that there was a significant amount of pyridoxamine phosphate attached to the presynaptic membrane and a small but detectable amount in the fraction containing postsynaptic membranes.
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