A Modified Pyridine-Silver Stain for Teased Preparations of Motor and Sensory Nerve Endings in Skeletal Muscle

This technique has been developed especially to stain sensory receptors which have been localised intramuscularly by electrophysiological means. Rat intertransverse caudal muscles, removed immediately after death, are fixed for 24 hr in a freshly prepared mixture of absolute ethyl alcohol, 4.5 ml; distilled water, 5 ml; and concentrated HNO_a, 0.1 ml. After a further 24 hr in 10 ml of absolute ethyl alcohol containing 0.1 ml of ammonia solution (sp. gr. 0.88), the muscles are washed in distilled water for 30 min and placed in full strength pyridine for 2 days. They are then washed for 24 hr in distilled water (changed 5-8 times) and left in 2% AgNO₃, in the dark for 3 days at 25 C. Following reduction in 10 ml of 5% formic acid containing 0.4 gm of pyrogallol for 6-24 hr, the specimens are washed briefly in distilled water and stored in pure glycerol. The nerve endings can then be teased out and mounted in glycerol, under cover glasses ringed with a waterproof cement. The advantage of this method is that it gives consistently good staining of receptors and motor end-plates in small muscles of the rat     

Fractionation of native and denatured transforming deoxyribonucleic acid from Bacillus subtilis

1. Native DNA from Bacillus subtilis was fractionated by stepwise elution from methylated albumin, the transforming activity being confined to two out of four fractions. Partial separation of DNA active in transformation for the arginine marker from that showing activity for the histidine and tryptophan markers was achieved. 2. Partial denaturation of DNA at 90 degrees and 93.5 degrees resulted in the preferential destruction of transforming activity for the histidine and tryptophan markers. 3. Denaturation of DNA at 100 degrees followed by chromatography on methylated albumin yielded five fractions, two of which exhibited residual activity. Redenaturation at 100 degrees resulted in the interconversion of four out of the five fractions. Redenaturation of fractions labelled with (15)N and (2)H suggested the presence of a specific component that did not readily take part in the interconversions.     

Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver

1. The activities of hydroxymethylglutaryl-CoA synthase and lyase in rat liver were found to be two- to 15-fold greater than those reported by other authors under similar conditions. 2. When expressed on the basis of body weight, no appreciable differences were found between the activities of hydroxymethylglutaryl-CoA synthase in whole homogenates of livers from normal and starved rats. The synthase activity increased by 70% and 140% in livers of alloxan-diabetic rats and rats fed on a high-fat diet respectively. 3. Hydroxymethylglutaryl-CoA lyase activity showed no significant increases in starvation or alloxan-diabetes, but a 40% increase was found in fat-fed rats. 4. Less than 12% of the activities of both enzymes were found in the cytoplasmic fraction of normal liver. The cytoplasmic activity doubled in alloxan-diabetes and starvation; on feeding with a high-fat diet the increase, though significant, was less marked. 6. The intracellular distribution of glutamate dehydrogenase indicated that the changes in the cytoplasmic activities observed were not due to leakage from the mitochondria. 7. Feeding with a normal or high-fat diet after 48hr. starvation caused within 24hr. a decrease in the cytoplasmic activity of hydroxymethylglutaryl-CoA synthase to values lower than those found in rats fed on a corresponding diet for a longer period of time. 8. Acetoacetyl-CoA deacylase activity in liver was about 20% of that of hydroxymethylglutaryl-CoA synthase and was primarily located in the cytoplasm. Starvation or alloxan-diabetes did not alter the acetoacetyl-CoA deacylase activity. 9. It is concluded that variations in the concentrations of enzymes involved in acetoacetate synthesis play no major role in the regulation of ketone-body formation in starvation and alloxan-diabetes. The changes in the cytoplasmic activities of hydroxymethylglutaryl-CoA synthase and lyase suggest that acetoacetate synthesis can occur in the cytoplasm. This may play a role in the disposal of surplus acetyl-CoA arising in the cytoplasm when lipogenesis is inhibited.     

The reversal of phenylarsenoxide inhibition of keto acid oxidation in mitochondrial and bacterial suspensions by lipoic acid and other disulphides

1. Inhibition of pyruvate oxidation in suspensions of Aerobacter aerogenes cells and of isolated mitochondria from rat heart and liver by phenylarsenoxide is prevented by an excess of lipoic acid, whereas inhibition due to certain bivalent cations is not. 2. In both systems inhibition persists when the bacteria and mitochondria are recovered and resuspended in fresh media in the absence of the inhibitor. Persistent inhibition due to preincubation with phenylarsenoxide, but not with the metal ions, is reversed by lipoic acid and by certain other disulphides. 3. 2,3-Dimercaptopropan-1-ol prevents the inhibition of pyruvate oxidation by phenylarsenoxide and by bivalent cations in both mitochondria and bacterial cells. 4. In aerobic suspensions of mitochondria and bacteria disulphides such as lipoic acid are reduced rapidly to dithiols. Reduction is inhibited by Co(2+), Ni(2+), Cd(2+) and Zn(2+), but not by phenylarsenoxide. 5. It is concluded that the inability of lipoic acid to prevent the action of the metal ions on pyruvate oxidation is due to the inhibition of its reduction to the effective dithiol.     

Studies onAzotobacter species in soil

Summary Methods for countingAzotobacter species in soil have been examined. The highest counts were obtained from soil suspensions shaken in sterile distilled water containing 10-g glass beads and plated on to glucose agar. Mannitol has been rejected as a suitable substrate in agar media because it gives lower counts of Azotobacter than glucose, an effect which is further enhanced by drying the agar plates. A clear medium free from precipitated phosphate and CaCO₃ is recommended for the agar-plate method; the Azotobacter count is affected by the phosphate concentration.The agar-plate and dilution-tube methods were compared; the latter is less accurate but more convenient when many soil samples have to be examined.     

Substitution of Manganese for Tomato Juice in the Cultivation of Lactic Acid Bacteria

Tomato juice was separated by chemical and physical methods into various active fractions, as measured by growth response and acid production by numerous lactic acid bacteria. Incineration of the treated extracts with little apparent loss in activity established the fact that the stimulatory component was of inorganic composition. Of the various cations tested, manganese was the only element that produced biological activity comparable to that of the original extract. Of the 71 strains of lactic acid bacteria tested, 63 strains showed a definite requirement for manganese or tomato juice.     

CYTOTAXONOMY OF TWELVE SPECIES OF HIBISCUS SECTION FURCARIA

Menzel, Margaret Y. (Florida State U., Tallahassee), and F. D. Wilson. Cytotaxonomy of twelve species of Hibiscus section Furcaria. Amer. Jour. Bot. 50(3): 262–271. Illus. 1963.—Metaphase-I chromosome numbers and pairing in 88 accessions showed that H. cannabinus, H. costatus, and H. surattensis are diploid (n = 18); and H. acetosella, H. aculeatus, H. bifurcatus, H.furcellatus, H. meeusei, H. radiatus, H. rostellatus and H. sabdariffa are tetraploid (n = 36), with similar low multivalent frequencies, hence probably allotetraploids each combining 2 well-differentiated genomes. No intraspecific variation in ploidy was found. Fertile, vigorous F₁ hybrids between H.furcellatus and H. bifurcatus showed complete chromosome pairing (n = 36), confirming a close relationship between the parents. Two African strains of H. diversifolius were octoploid (n = 72) with low multivalent frequency and hence probably contain 4 differentiated genomes. At least 4, perhaps 5 or 6, differentiated genome groups are represented in tropical Africa, and at least 2 in the American tropics.     

Production of the Milk Agent in Cultures of Mouse Mammary Carcinoma

Thin sections of tissue cultures grown from tumors of the RIII high-breast-cancer strain mice were studied in the electron microscope. These tissues contain an abundance of particles whose morphology is consistent with biophysical measurement of the milk agent. These particles, found only extracellularly in our cultures, are formed at the cell membrane. The process of formation, as reconstructed from sections, appears to include a thickening and protrusion of the cell membrane which then evolves gradually into a dense sphere and separates from the cell in much the same manner as does influenza virus. The contents of the newly formed body are later rearranged to form a nucleoid within a membranous sac.