ELECTROPHORESIS OF HISTONES ON POLYACRYLAMIDE GELS

Histories extracted from various organs of the rat have been fractionated by electrophoresis on Polyacrylamide gels. The most convenient reagents to form the gel were selected and the effects of varying concentrations of these reagents on the resolution of histones were investigated. Optimum conditions for the separation of histones were found to be the same as those for starch gel electrophoresis. More consistent results were obtained with the use of Polyacrylamide gels because these gels were equilibrated to constant pH and ionic strength prior to electrophoresis and the gels were electrophoretically destained to demonstrate the histone bands.

FOOD CONSUMPTION AND RECTAL TEMPERATURE IN RATS DURING THIAMINE DEFICIENCY AND REPLETION

Rectal temperature in male rats fell slowly and gradually from ad libitum and pair-led control levels throughout a thiamine depletion period. During this period, food consumption dropped suddenly and sharply to a minimal level. A single oral dose of 50 μg of thiamine hydrochloride produced, within 4 hours, a significant rise (to less than control levels) in rectal temperature and an increase in food consumption within 24 hours. The increase in temperature was independent of the ingestion of food since diet was withheld during the 4 hours following thiamine administration. Subsequent feeding of control diet (containing thiamine) had not further increased the "4-hour" temperature after 24 hours. With continued feeding of control diet, rectal temperature rose to control levels after 3 days. On subsequent withdrawal of dietary thiamine from the deficient group, temperature and food consumption fell as before. When the animals were again repleted with 50 μg thiamine and deficient diet was continued, temperatures rose to the same level reached after the first thiamine administration. A third deprivation and repletion produced identical results.Food restriction alone, in pair-fed control groups, induced an initial elevation of rectal temperature above ad libitum control levels as temperatures in the deficient group were falling, and an eventual decrease below ad libitum control levels only after prolonged food restriction. It is suggested that the initial fall in body temperature in thiamine-deficient rats is not simply a terminal result of food restriction per se, but may reflect alterations in metabolism due to the deficiency.

THE CYTOLOGICAL LOCALIZATION OF HUMAN CHORIONIC GONADOTROPIN

The indirect fluorescent antibody technique was used to determine the cellular site of human chorionic gonadotropin (H.C.G.) in normal and toxaemic placentas, and in choriocarcinomas. In placental tissue specific fluorescence was located in the syncytial cells of the chorionic villi but not in the cytotrophoblast cells. In choriocarcinomas specific fluorescence was seen in the syncytial giant cells. No distinct difference could be demonstrated between normal and "toxic" placentas. Differences in the content of H.C.G. in placentas at various stages of pregnancy were noted. Maximum amounts were demonstrated in tissue of less than 14 weeks gestation. Lesser quantities could be seen in more mature placentas and some specific fluorescence could be seen in most full term placentas.

SYNTHESIS AND STORAGE OF ACETYLCHOLINE IN NERVOUS TISSUE

A discussion of the factors involved in the physiological synthesis of acetylcholine in nerve endings, and of the action thereon of hemicholinium No. 3.

CHOLINERGIC TRANSMISSION AND ACETYLCHOLINE OUTPUT

Evidence is presented refuting the possibility that pyruvylcholine might be a transmitter in cholinergic transmission processes.The physiology and pharmacology of acetylcholine release is discussed, with special reference to recent work.Experiments on the output of acetylcholine from electrically stimulated ileum preparations are described. It is suggested that two output mechanisms may exist: one important at low firing rates, which yields a constant output of transmitter per unit time and is morphine-sensitive; the other brought in at higher frequencies of excitation, which yields a more nearly constant volley output, and is morphine-resistant.

REDUCING SUBSTANCES IN THE BLOOD OF NORMAL AND ALLOXAN-TREATED FISH

Blood sugar changes in fish, after the intravenous injection of alloxan, were determined by a modified Folin method and by an enzymatic method using glucose oxidase. The rest reduction was calculated as the difference between the two methods. The blood rest reduction levels were normally 15 mg% in toadfish and 11 mg% in bullheads (glucose 19 and 22 mg% respectively). One hour after alloxan administration, the rest reduction had increased on the average several times while the glucose level was only slightly above normal. From a maximum at 1 hour the rest reduction level dropped while the glucose level began to rise. Considerable variation in the response to alloxan occurred.The reducing substances in the blood were studied by paper chromatography. In normal blood, glucose and another compound (Rf value approximately one-half that of glucose) were detected by alkaline silver nitrate. No increase in reducing material in alloxan-treated samples could be shown on the chromato-grams by the methods used.

ABSORPTION OF INJECTED OXIMES

Concentrations in the blood of rabbits of 1,1′-trimethylene bis (4-formylpyridinium bromide) dioxime (TMB4), of 2-formyl-1-methylpyridinium chloride oxime (PAM Cl), and of 2-formyl-1-methylpyridinium methanesulphonate oxime (P2S) were measured at short intervals during the period immediately following intravenous or intramuscular injection. Dose for dose the concentration in the blood of TMB4 was found to be at least twofold higher than the concentration of either of the others. The rate of absorption from an intramuscular injection was the same for all three oximes, the peak concentration being reached in approximately 9 minutes.Other experiments, in which P2S was injected by the intrapulmonary route, showed no advantage in this species as regards speed of absorption over the intramuscular route.

CYTOPLASMIC INHIBITOR OF DEOXYRIBONUCLEASE I FROM RAT LIVER TISSUE

A cytoplasmic inhibitor has been found effective against the mitochondrial deoxyribonuclease I from rat liver tissue. This inhibitor is similar in properties to most DNAase inhibitors reported so far in the literature. Attempts at identification have led to the conclusion that this inhibitor is a β-globulin. It is a non competitive inhibitor.

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

THE PURIFICATION OF BEEF THYROGLOBULIN WITH THE USE OF GEL FILTRATION

A method is described for preparing a 19 S thyroglobulin component from a saline beef thyroid extract by chromatographic fractionation on Sephadex G-200. By employing Sephadex G-200 it was possible to separate a saline extract of minced beef thyroid gland into several fractions, the first of which (fraction I) contained approximately 60% of the protein and 96% of the iodine. Spectrophotometric, ultracentrifugal, and immunochemical methods revealed that fraction I possessed, in addition to the 19 S thyroglobulin protein, a 25 S component as well as serum proteins. A narrow band of fraction I appeared to be free of both the 25 S component and the serum proteins. The material in this subfraction had a sedimentation constant of S20,w = 19.1 and contained 1.2 mg of iodine per 100 mg of protein.