Experimental Biology and Medicine

β-Diethylaminoethyl diphenylpropylacetate hydrochloride (SKF No. 525-A) reduced significantly plasma total cholesterol and aortic cholesterol in dogs. Chronic daily administration of the SKF No. 525-A to dogs for 5 months resulted in marked fatty infiltration of the liver, which was rapidly reversible upon withdrawal of the compound.

Additional experimental evidence of dissimilarity of canine distemper (CDV) and measles (MV) viruses are presented: 1. Ferrets inoculated with repeated doses of MV-adjuvant mixture and which developed M antibodies proved as susceptible as control animals to challenge with CDV. 2. Paired convalescent sera from measles patients showed significant measles antibody rises, while CD antibodies remained essentially absent. 3. Growth of MV in HeLa cells was not inhibited by presence of CD antiserum or normal serum, while MV antiserum apparently reduced virus multiplication. Presence of measles antiserum did not interfere with propagation of CDV. 4. By 14th day after MV infection, serologically negative monkeys developed high antibody titers for MV but none for CDV. It remains to be determined whether results obtained are attributable to particular virus strains used, or apply also to other isolates of MV and CDV.

Authors thank Marie Murrin and Hubert Klaver for technical assistance and E. Manoogian for help in preparation of manuscript.

Effects of lactogenic hormone (LGH) alone and in combination with growth hormone (GH) and L-thyroxine (T4) upon lactation in the rat have been studied. All rats were nursed on day 14 of lactation with aid of 1 USP unit of oxytocin (OXT) under standardized conditions. One mg LGH alone did not show galactopoietic effects when injected daily from day 7–14. Under combined action of 1 mg LGH and 1 mg GH/day from 7–14, litter weight gain and amount of milk on day 14 have been significantly increased. Addition of 3.0 μg T4/100 g body weight/day from day 7–14 to LGH plus GH resulted in a more highly significant increase in milk yield in comparison to oxytocin alone than did treatment with LGH plus GH. The increase in mean milk yield of 84% over untreated controls by 4 hormones is the highest so far attained in laboratory.

1) Bovine γ globulin is a satisfactory antigen for sensitizing rats when given intraperitoneally simultaneously with Bordetella pertussis vaccine. Intestinal lymph of animals so sensitized contains increased amount of histamine when collected 6 minutes after intravenous injection of shocking dose. The amount is roughly proportional to degree of shock. Rate of flow of lymph increases 8 to 25 times during the 12 minutes immediately after administration of shocking dose. 2) Lymph from mesenteric lymphatic system contains 0 to 0.02 μg histamine/ml, an amount similar to that in plasma. 3) A limited number of studies suggest that rats treated with polymyxin B sulfate and distilled water before shock were only partly depleted of histamine.

1) An improved L. casei assay procedure for serum folic acid activity is described. Its range of usefulness over previous procedures is increased because of greater sensitivity, better accuracy in lower ranges, stability of test organism and micromethod applicability. 2) With this improved assay, the overlap of serum folic acid activity levels among normal subjects and folic acid deficient patients is so great that this determination has no value as an index of folic acid deficiency.

Technical DDD fed to dogs in 200 mg/kg doses once daily for 3 days resulted in histologically normal adrenal glands which responded to ACTH with the expected increase in blood flow, but demonstrated a low resting secretion rate of 17-hydroxycorticosteroids, which did not increase after administration of ACTH. Dogs fed a similar 3 doses and allowed to continue normal diet for 18 days showed adrenals with definite changes in the cortex which did respond to ACTH with the expected increase of steroid secretion but showed usual increase in blood flow. These data are interpreted to mean that the vasodilating factor of commercial ACTH is separate from steroid formation and/or releasing factor(s) and possibly the vasodilating factor may act on medullary or extracapsular vasculature. Capacity of the cortical parenchymal cells to form and/or release 17-hydroxycorticosteroids, as influenced by DDD, does not parallel histological changes observed in standard hematoxylin and eosin preparations.

1) Chlorpromazine and stelazine interact with heparin and CSA to form products of visible turbidity. This turbidity is reduced by strandin, and reappears on readdition of phenothiazine. 2) Chlorpromazine reduces turbidity of the strandin-lysozyme interaction product, turbidity of which may be regenerated by subsequent addition of strandin. It also destroys metachromasy of the toluidine blue O-strandin system. 3) Equilibrium dialysis of chlorpromazine solutions and chlorpromazine-strandin solutions indicates good retention of chlorpromazine in the system containing strandin. 4) Strandin alters U.V. absorption spectrum of chlorpromazine and, more markedly, that of stelazine. 5) Ultracentrifuge studies indicate presence of slow moving peak in the strandin-chlorpromazine system, which may represent strandinchlorpromazine interaction product.

1. The penicillin moiety, 6-aminopenicillanic acid, has antibacterial activity but is considerably less active than penicillin. 2. Resistance to penicillin in one group of bacteria could be explained logically by presence of penicillinase activity, i.e., conversion of penicillin to the inactive penicilloic acid. 3. Resistance to penicillin in a second group of bacteria could be explained logically by presence of penicillin acylase activity, i.e., conversion of penicillin to the relatively inactive 6-aminopenicillanic acid. 4. In a third group of bacteria resistant to penicillin, neither penicillinase nor penicillin acylase activity could be detected. Still another metabolic mechanism other than penicillinase or penicillin acylase activity must be present in selected bacteria.

Protopam (pralidoxime) Chloride (or Iodide) and TMB4 (1, 1′-trimethyl-ene-bis(4-formylpyridinium bromide) dioxime) when injected intraperitoneally in the mouse at 50% of LD₅₀, raise the intraperitoneal lethal dose of anticholinesterase, Phospholine (echothiophate) Iodide, by factors of 50 and 280 respectively. When the same oximes are given orally 30 to 60 minutes before Phospholine the intraperitoneal LD₅₀ is increased by factors of 23 and 450. Graphs relating log dose of oxime and log dosage ratio of Phospholine are linear over a considerable range. The therapeutic indices for arbitrary increase of 10 times in LD₅₀ of Phospholine are 23 and 9 for Protopam and 114 and 120 for TMB4 by oral and intraperitoneal administration respectively. The possible nature of the antagonism is discussed.

1. Carbonyl reagents such as hydrazine, hydroxylamine and isoniazid inhibit purified preparations of pyridoxal kinase and of glutamic decarboxylase from beef brain, but the kinase is 100 to 1000 times more sensitive. 2. Administration of hydrazine to rats decreases activity of these 2 enzymes in liver and brain; kinase levels are affected more severely. 3. In contrast to their effects in vitro, isoniazid, hydroxylamine, or pyridoxal oxime produced no significant inhibition of kinase or decarboxylase activity in vivo. Pyridoxal oxime appears rapidly catabolized in vivo since its injection results in extensive excretion of 4-pyridoxic acid and its lactone. Unlike free hydroxylamine, injection of pyridoxal oxime does not produce convulsions or extensive methemoglobin formation. 4. Similar administration of pyridoxal azine resulted in rapid and extensive decrease of pyridoxal kinase levels in liver, kidney, and brain. No effects on glutamic acid decarboxylase in brain were observed. 5. These observations demonstrate that individual carbonyl reagents inhibit quite different enzymes in vivo, and indicate that appropriate derivatives of such agents may show enhanced selectivity in their inhibitory actions.

1) The virulence for mice of INHR laboratory strains of H37Rv was increased when cortisone was administered. 2) Cortisone also increased the pathogenicity for mice to some extent of 2 of 4 scotochromogens and 3 of 9 photochromogens tested. Seven Battey strains were unaffected.

INH is stable for several weeks in aqueous and buffered solution at pH values below 8 and at low temperature. It is unstable in human and rabbit plasma whether added to pure plasma or present after oral or parenteral administration. Instability is enhanced by increased temperature, but is quite marked at refrigerator temperature. The presence of a factor in blood causes changes in chemical state of INH so that it no longer reacts with reagent used in the assay method.

When β-3-thienylalanine, an analogue of phenylalanine, is fed to rats for appropriate time before and after antigen injection it will markedly inhibit antibody response. This analogue added to an antibody synthesizing tissue culture system gave no inhibition of antibody synthesis under these conditions. It appears that unless β-3-TA is fed during induction period, its inhibitory effect will not be manifested. It is possible then that β-3-TA is active only in inhibiting the “setting up” of the antibody forming mechanism.

A streptomycin-dependent mutant was isolated from a virulent strain of S. typhimurium for evaluation as a living vaccine in mice. In vivo and in vitro studies indicated that this strain was unsafe as an immunizing agent due to reversions to streptomycin independence. A mutant of the streptomycin-dependent strain was obtained requiring purine, a nutritional factor absent in mouse peritoneum. Reversions of this double mutant strain to the purine-streptomycin-independent state could not be detected. The results of experiments comparing this living vaccine with heat-killed preparation indicated that the living vaccine was not superior in protecting mice.

Suitable conditions for assay for 5-nucleotidase activity in lamb skeletal muscle tissue are described. The activity of this enzyme was significantly greater in tissues from lambs affected by white muscle disease than in tissues from normal lambs.

The authors acknowledge efficient technical assistance of Pauline Fleetwood.

Intradermal injection of crude sonic extracts and fractions obtained by starch electrophoresis of group A streptococci produced a spreading erythema in a variable percentage of rabbits. Recurrence of the lesion developed in about 10% of rabbits after abatement of primary lesion. The variability in rabbit response made it difficult to characterize the active principle and to determine the underlying mechanism of the reaction.

Erythrocytes produced in rats under stimulus of human urinary erythropoietin have a normal survival time in circulation. This and other evidence suggest that erythropoietin administration results in production of normal erythrocytes. This evidence is compatible with the concept that erythropoietin is part of the normal mechanism controlling erythropoiesis.

1) Radioactivity of S³⁵O₄ labeled cells can be measured directly with cells collected on Millipore filters. 2) Following a single intravenous injection of S³⁵O₄ rat peritoneal mast cells take up the radioactivity; the maximum is reached by 48 hours. No loss of radioactivity is evident in the following 5 days. Radioactivity in other free cells of peritoneal fluid attains a maximum at 4 hours and then falls rapidly to low levels.

The local Shwartzman reaction was elicited in the rabbit oral cavity and skin with endotoxin prepared by phenol-water extraction procedure from a strain of oral Veillonella. Lipid-carbohydrate-protein products prepared by same procedure from viridans streptococci, or a solution of physiologic saline did not produce this reaction in either tissue. Lesions obtained in oral mucosa with each substance were grossly and histologically similar to lesions obtained with the same material in skin.

Adrenalectomy in young female rats resulted in a significant reduction in rate of eruption of incisors which averaged 28% below that of unoperated controls during 6 weeks of measurement. Daily administration of cortisone produced a significant acceleration in rates of adrenalectomized rats to levels above that of nontreated intact controls. It is suggested that the severe reduction in rate of eruption of the continuously erupting incisor observed by others following hypophysectomy is due to loss of effect of both thyroid and adrenal hormones.

Injection of 15 μg of a haptenazoprotein, such as p-aminobenzoic acid diazotized to hen egg albumin, in Freund's adjuvant, into footpads of guinea pigs produces delayed hypersensitivity in 5 days and circulating antibody in 10-11 days after sensitization. Injection of 1 μg of such conjugate in saline produces delayed hypersensitivity but no detectable circulating antibody. Specificity of delayed hypersensitivity is oriented toward the protein; that of circulating antibody, toward the hapten. When a primary dose of 1 μg in saline of protein, azoprotein or heterologous hapten-homologous protein is followed by a secondary dose of hapten-homologous protein, antibody response to the hapten is accelerated. A primary sensitizing dose of hapten, such as p-aminobenzoic acid (PABA) + protein such as hen egg albumin (HEA), followed by a secondary dose of the same hapten attached to heterologous protein, does not result in accelerated response to the hapten. These phenomena are explained if delayed hypersensitivity is considered an early step in antibody synthesis.

Technical assistance of Jane Nishio, Aspascia Cobure, and Leroy F. Peel is gratefully acknowledged.

Tissue fixed globulin has been demonstrated in interstitial tissue and basement membranes of various guinea pig organs following inoculation of guinea pigs with saline extracts of rabbit tendon. These animals had a significantly diminished growth. It is likely that the fixed globulin represents “auto-antibody” induced by heterologous antigen.

1) In rabbits given thyroxine or propylthiouracil, external balance studies for magnesium were performed, and Mg²⁸ used to measure exchangeable body content of magnesium. In 8 rabbits given 2 intravenous injections of thyroxine sodium (0.3 mg/kg) no significant changes were noted in body weight, magnesium balance, or exchangeable magnesium content, although a decrease in serum magnesium concentration occurred. In another group of 8 rabbits the tissue relative activity of Mg²⁸ was determined following 3 intravenous injections of thyroxine (1.0 mg/kg) given within 4 days. The relative activity was increased in liver, skin, appendix, and heart. 2) Eight rabbits given propylthiouracil orally showed a decrease in exchangeable body content of magnesium at end of third week. A significant decrease was found also in relative radioactivity of liver and bone. 3) The value for exchangeable body content of magnesium appears related to rate of turnover of Mg28 in bone and muscle, the 2 largest body stores of magnesium.

Mg²⁸ was supplied by Brookhaven Nat. Lab. on allocation from U. S. Atomic Energy Comm.

A study was made of RNA, water, and collagen contents of adenohypophyses of rats of various age groups from 35 days to one year old, sacrificed by decapitation or an overdose of ether. There were no significant differences in level of RNA in tissue from one age group to another. Concentration of anterior pituitary nucleic acid was not influenced by manner of sacrifice for most age groups studied. Contents of water and collagen in the tissue were similar in young and old rats.

Daily administration of 1 μg EB plus 3 mg P to mature ovary-thyro-para-thyroidectomized rats for 19 days stimulated mammary gland growth (DNA) comparable to ovariectomized rats receiving same treatment. Replacement therapy with 10 and 20 μ AT 10/100 g/day, respectively, in combination with 1 μg EB plus 3 mg P increased total DNA significantly. No benefit was observed by increasing ovarian hormones to 2 μg EB plus 6 mg P. Combined administration of AT 10 at above mentioned levels and 1.5 μg and 3 μg T4/100 g depressed DFFT significantly resulting in reduced total DNA.

Immunization with purified Vi antigen elicited incomplete Vi antibody in the Cinnamon strain, and complete Vi antibody in BALB/c mice. Incomplete Vi antibody alone accounted for protection afforded by serum from Cinnamon mice against challenge with fully virulent typhoid bacilli. Absorption tests verified the specificity of incomplete Vi antibody and protection it afforded.

Variola virus was lethal for mice by intracerebral route when relatively high doses were inoculated. Lower doses produced erratic death patterns. Virus proliferated in mouse brain reaching peak titer between 48 and 72 hours. Amount of virus at peak titer was dependent on quantity of virus inoculated. Ten consecutive passages of the virus in mouse brain were successfully carried out. The most important factor which appeared to influence successful serial passage of the virus was level of titer attained in preceding passage. This titer was, in turn, dependent on size of inoculating dose, time of harvest and age of mouse. The passaged virus was identical with parent strain with respect to a number of properties examined.

Intravenous administration of Triton to rats causes a marked elevation of plasma triglycerides. If the rats have been poisoned with carbon tetrachloride 2 hours previously, accumulation of triglycerides in the plasma following Triton administration is markedly reduced. The existence of an hepatic triglyceride secretory mechanism is inferred from the work of others, and it is postulated that carbon tetrachloride poisons this triglyceride secretory mechanism. Triglycerides, which would normally be secreted into plasma, are retained in the liver.

From comparative study of changes in adrenal ascorbic acid and in adrenal and plasma corticosterone concentration induced by unilateral adrenalectomy in the rat, adrenal ascorbic acid depletion appears more closely related in time to release than to synthesis of corticosterone, since maximal depletion did not correspond to peak of corticosterone concentration in the gland (30 minutes postoperatively), but was achieved at the same time (60-120 minutes p.o.) as its peak concentration in plasma, when rate of release of the hormone presumably reached equilibrium with its rate of removal or inactivation. In view of reported instances of dissociation between the 2 phenomena, the observed temporal relationship should be interpreted with caution.

The authors are indebted to Miss R. C. Wood for able technical assistance.

Diastolic regurgitation of opaque contrast material can occur through normal mitral valve when the period of diastasis exceeds a specific time interval. This period of diastasis in dogs varies from 0.22 to 0.60 second, average of 0.39 second. When diastasis is shorter than this interval no regurgitation occurs, but it always occurs when prolonged beyond the critical period.

(1) Extracts of arteriosclerotic aortas of female Sprague-Dawley breeder rats were assayed for lactic dehydrogenase, glucoses-phosphate dehydrogenase, and 6-phos-phogluconic dehydrogenase. (2) Extracts obtained from moderately and severely arteriosclerotic aortas contained significantly less lactic dehydrogenase than extracts of aortas grossly normal or only slightly affected. TPNH production by combined action of glucose-6-phosphate dehydrogenase and 6-phosphogluconic dehydrogenase was significantly depressed in extracts from moderately and severely arteriosclerotic aortas. No significant differences were observed in levels of 6-phos-phogluconic dehydrogenase.

Ultracentrifuge analysis confirms that polyanion-complex precipitation method for human serum β-lipoproteins is adequate and does not denature the lipoproteins when either rice starch sulfate or mepesulfate are used as precipitating agent. The method is applicable to isolation of β-lipoproteins from several but not all species of animals.

A method for determination of ceruloplasmin in serum is presented in which rate of oxidation of p-phenylenediamine is measured spectrophotometrically or photometrically. Correction for catalytic oxidation by free metals present is made by reading the test against a serum blank in which enzymatic activity is inhibited by azide. The effects of pH and temperature on the reaction have been investigated and normal values obtained by our method. Ethylenediaminetetraacetate significantly inhibits enzymatic oxidase activity of serum.

When glycine-1-C¹⁴ is injected into rats bearing Ivalon sponges implanted 2 weeks previously, significant radioactivity is found in the various protein fractions (including collagen) of the sponge connective tissue after 6 hours. Adrenalectomy at the time of implantation exerted some influence upon the extent of incorporation into some of the fractions, but administration of hydrocortisone for 2 days to either intact or adrenalectomized animals had no effect. When studies of glycine uptake by sponge-biopsy connective tissue proteins were made in rats of various ages, a steady decrease in rate of incorporation into all fractions was observed as age of the host increased from one month to one year.

There is no evidence of diurnal variation of plasma NEFA levels in rats. Hypophysectomized, adrenalectomized and thyroidectomized animals show significantly lower plasma NEFA values than normal rats either when food is provided ad lib. or after fasting. After 16 hour fast, both normal and operated rats show marked increase in plasma NEFA levels. It is probable that the mechanism involved in NEFA response to fasting is not controlled by pituitary, thyroid or adrenal hormones.

A procedure for estimation of PAM and related alkylating agents in blood by its reaction with NBP is given. This procedure is applicable for study of the reaction with and persistence in blood of these agents in vitro and in perfusion systems.

1. Isografts of intact immature vaginas grew maximally in castrated male mice with ovarian transplants and minimally in intact males with similar grafts. Growth was intermediate in castrated or intact males without ovarian grafts. 2. Vaginal grafts showed one or 2 estrous phases in castrated males with ovarian grafts between 4 and 5 months after grafting but otherwise remained in late proestrus. Such activity did not occur in castrated males without ovarian transplants or in intact males with or without ovarian transplants, vaginal grafts in these hosts remaining in diestrus.

Effect of a single dose of 5 mg of estradiol cyclopentylpropionate on lipid metabolism in 3-week-old cockerels was studied. Cholesterol and phospholipids of serum, liver and aorta were determined daily during 7 days following injection of hormone. to evaluate time response of birds to this estrogen. Early and significant rise of serum and liver cholesterol and phospholipids was observed. Increase of aortic lipids was slower to appear, but significant changes in these parameters were also noted.

Bile salts and detergents cause “lysis” of fibrin s by a simple solution of fibrin. Fibrin i is not dissolved by bile salts and detergents. Fibrin i should be used in fibrinolytic assays for plasmin or plasmin activators.

A technically simple agglutination test for detection and titration of an unidentified anti-staphylococcal factor, presumably antibody, in human and animal sera is described. By this test variable titers have been found to occur naturally in sera of animals and of man. Agglutinins are present in large amount in pooled human gamma globulin and in insignificant amounts in a single agammaglobulinemic serum. Titers of this factor can be increased by treatment of both animals and man with a staphylococcal vaccine. The suggestion is made that this factor is an antibody on the basis of a) its presence in antibody bearing fraction of blood b) its heat stability c) ability to adsorb it semiquantitatively with coagulase-positive staphylococci and d) ability to elevate titers in rabbits and man by use of staphylococcal vaccine as antigen. Preliminary observations do not enable us to identify the factor being measured by the agglutination test nor assess its clinical significance.

Mouse leukemia (L1210) was grown successfully when introduced by intracerebral route. Brain tissue was rapidly infiltrated and the disease became systemic 2-3 days following I.C. inoculation. Deaths occurred somewhat sooner than when the disease was inoculated S.C. Administration of antifolic agents (amethopterin and 3′,5′-dichloroamethopterin) increased survival time.

Method is described whereby polyethylene tubes, permanently implanted in aorta, may be used routinely for repeated direct measurement of arterial pressure in un-anesthetized rats.

Conversion of mevalonic acid to non-saponifiable material by rat liver homoge-nates under conditions where conversion is a function of ATP level existing at time of mevalonic acid addition was inhibited markedly by preincubation with protamine or polylysine. Inhibition was less or absent following preincubation with trypsinized protamine, bovine serum albumin or a number of small molecular weight bases. The results are interpreted as indicating that a combination of high molecular weight and strong basicity are essential for inactivation of a polyanion previously shown to be essential for maintenance of ATP levels in liver homogenates.

1. Metabolites of Rose Bengal could not be demonstrated in human bile, serum or urine or in extracts from rat liver by a variety of systems involving paper and column chromatography and electrophoresis. 2. Conjugation with cystine-S³⁵ or glucuronic acid was not demonstrable. 3. It was concluded that Rose Bengal is not conjugated by the liver in the same form reported for bilirubin (glucuronides) or bromsulfalein (mercapticles).

1) The protected decapeptide carbobenzoxyglycyl - S - benzyl - l - cys-teinyl - l - tyrosyl - l - isoleucyl - l - gluta-minyl - l - asparaginyl - S - benzyl - l - cysteinyl - l - prolyl - l - leucylglycinamide was synthesized by coupling of the p-nitrophenyl ester of carbobenzoxyglycine with S-benzyl-l - cysteinyl - l - tyrosyl - l - isoleucyl - l -glutaminyl - l - asparaginyl - S - benzyl - l -cysteinyl - l - prolyl - l - leucylglycinamide. Removal of the protecting groups followed by oxidation to the disulfide gave glycyloxytocin which was isolated by countercurrent distribution. 2) The pharmacological effects of this peptide derivative of oxytocin were investigated by testing it for avian depressor, pressor, and rat uterine-contracting activities. The sample of glycyloxytocin possessed only a slight degree of these 3 activities. It was found to exert a marked inhibitory effect on the avian depressor activity of oxytocin. On the other hand, the glycyloxytocin did not show inhibitory action against oxytocin on the isolated rat uterus or against oxytocin or vasopressin in the assay for pressor activity in the rat. 3) Thus, the attachment of a glycyl residue to the free amino group of oxytocin causes almost complete loss of its avian depressor, rat uterine-contracting, and pressor effects. In addition, it is of considerable interest that this peptide derivative of oxytocin exerts a marked inhibitory action upon the avian depressor effect of the hormone.

Two triglyceride emulsions prepared for intravenous use in man have been examined for ability to release histamine from tissue mast cells in rat peritoneal cavity. The emulsions themselves had no direct action upon smooth muscle. Intraperitoneal injection of these materials caused rupture of the mast cells. The fluid recovered from the peritoneal cavity produced bronchiolar constriction in the pithed guinea pig and contraction of guinea pig ileum in vitro, indicating a significant histamine content. It is suggested that histamine release may play an important role in development of the occasional “anaphyloctoid” response following intravenous infusion of triglyceride emulsions in patients.

1. X-irradiation of rachitic epiphyseal cartilage with 250,000 R resulted in small decrease in calcifiability, loss of β-metachromatic granules from some cells of the proliferative zone, and a slight general decrease in staining of the hypertrophic zone. X-irradiation with 500,000 R resulted in marked decrease in calcifiability, loss of most of the β-metachromatic granules from some cells of the proliferative zone, and loss of β-metachromatic granules from some cells of the hypertrophic zone. X-irradiation with 1,000,000 R resulted in almost complete loss of both calcifiability and stainability with toluidine blue. 2. Toluidine blue staining of fresh sections of epiphyseal cartilage was employed for demonstration of metachromatic substances. 3. Once the process of calcification had been initiated, the inactivating effect of irradiation on calcifiability was considerably reduced. 4. A change in mucopolysaccharide resulting in increase of free electronegative groups is a factor associated with calcifiability of epiphyseal cartilage.

Free testosterone, and its methyl, ethyl, vinyl and ethynyl derivatives were tested as antagonists of estrone-induced uterine growth and vaginal smear changes. Methyl testosterone was most potent of the compounds; there were no major potency differences among remainder. These findings are in contrast to 19-nor derivatives, which are considerably more potent and among which the ethyl is most potent.

A series of steroidal spirolactones, of interest for their aldosterone antagonistic actions, were tested as estrogen antagonists in a mouse uterine growth test. The 2 activities did not appear to be correlated. Progestational activity similarly showed no direct relationships. Structural requirements for estrogen antagonistic potency are discussed. SC-8109 and 2 other 19-nor derivatives of SC-5233, 3-(3-oxo-17β-hy-droxy-4-androsten-17α-yl) propionic acid γ-lactone, were estrogen antagonists.

A cytotoxic effect is described which occurs when leukemic guinea pig plasma is added to normal guinea pig spleen cells growing in primary tissue culture. The nature of the cytotoxic factor is unknown.

Adrenal weight-body weight ratios of 89 male and 102 female cats were determined. The ratio for males is 0.147 g/ kg; for females, 0.162. The difference is statistically significant, and is due to a difference in average body weight of the 2 groups. A similar difference in ratios is seen between large and small male and large and small female cats.

Sodium deoxycholate and hot and cold phenol were used to extract nucleic acid from Rous sarcoma. The hot phenol technic in some experiments yielded a RNAse-sensitive product that caused non-transmissible focal lesions in chick fibroblast tissue cultures, but no extracts initiated a transmissible infection in tissue cultures, chicks or embry-onated eggs. The same hot and cold phenol technics did yield infective nucleic acid from West Nile. Mengo, and Semliki Forest virus preparations. Rous sarcoma virus was rapidly inactivated by deoxycholate.

Addition of 25-1600 ppm triparanol to the diet of turkey poults lowered the ratio of plasma cholesterol to phospholipid. Incidence of beta-aminopropionitrile-induced dissecting aneurysms was not lessened by triparanol. Degree of growth depression due to triparanol was proportional to logarithm of dose.

(1) Pretreatment of new mice with CrP³²O⁴, in well tolerated doses for topical prevention of tumor growth and spread was applied in various topographic areas, i.e., pleural cavity, scalp, abdominal wall and leg. (2) Reduction of tumor growth in all areas and of its spread in pleural and scalp were directly proportional to dose of radioactivity and inversely to the period between isotope injection and tumor inoculation. (3) These findings showed that amount of radioactivity stored in pretreated tissues at time of tumor inoculation was the major factor of the isotope's preventive effect. However, previous results(1) suggested also another effect of pretreatment, that of tissues increased resistance to invasion with tumor cells. (4) It was concluded that topical pretreatment with CrP³²O₄ may be attempted for prevention of postoperative growth and spread of tumor cells.

Pancreatic hypertrophy, poor growth and lowered food efficiency result from feeding raw soybean meal to rats as sole source of dietary protein. Addition of 4 specific amino acids to raw soybean diet corrects poor growth and reduced food efficiency but does not prevent pancreatic hypertrophy. These results support the concept that decreased growth rate and protein efficiency caused by feeding raw soybean meal are due to direct stimulation of the pancreas resulting in excessive loss of critical amino acids contained in pancreatic enzymes excreted in feces.

A technic for using latex (polystyrene) particles in a serological test with anaerobic diphtheroids is described. Optimum conditions under which latex particles may be used with this bacterial antigen-antibody system are reported. Evidence is presented to indicate a possible immunological relationship between various anaerobic diphtheroids.

The authors wish to acknowledge with thanks the capable assistance of Leona Caroline, Helen Lyons, and Dr. Liborio Garcia.

Rate of absorption of erythrocytes from peritoneal cavity of rats was found to be similar to that reported in the literature. Absorption was almost completely blocked by phosphorylated hesperidin. Evidence was presented suggesting that the block is achieved at the membranes separating peritonal cavity from lymphatics of the diaphragm. Hyaluronidase increased rate of absorption only slightly; however, this increase was statistically significant.

Five inbred strains of mice (C₅₈, CBA, RIII, DBA and BALB/C) and 5 F₁ hybrid combinations were studied with respect to effects of single injections of various concentrations of goldthioglucose on body weight gain and lethal toxicity. Gain in body weight and mortality following a single injection of goldthioglucose was dependent on strain of mice and amount injected. In general, there was a greater increase in weight gain when larger doses of goldthioglucose were administered. The C₅₈, RIII, DBA and BALB/C strains of mice showed a relatively variable weight gain and mortality as compared to the CBA strain over the dose range of administered goldthioglucose. Mice of the latter strain were unique in that they showed a uniform response with respect to these 2 parameters. The F₁ hybrid mice showed a weight gain response and a LD/100 dose comparable to that of the CBA strain.

1) Lymphocytic leukemia, of sporadic occurrence in random-bred Princeton mice (3.1% m 1050 adults), was transmitted to weanlings by intraperitoneal injection of lymphoid organ suspensions and maintained by serial passage. Spleens and regional lymph nodes were commonly enlarged but lungs were not involved. 2) A high level leukemia line from inbred Princeton mice, originally established by Dr. Clara Lynch, was similarly maintained in random-bred weanlings. Neoplastic cells also proliferated in lymphoid tissue, though with evidence of increased toxicity, and also invaded the lungs. Pulmonary lesions as well as generalized ones were regularly produced by intranasal injection of cells from high leukemia line but not from the low level one. 3) Necrotic lesions of liver appeared in one passage series with each of the 2 lines and murine hepatitis viruses of low virulence were recovered.

1) Adult garter snakes (Thamnophis spp.) inoculated intraperitoneally, overwintered WEE virus for at least 139 days. There was a steady decline in virus, with only 1 of 14 showing a viremia after 92 days hibernation. With warm spring weather, (139 days after virus inoculation) 9 of 14 animals exhibited a viremia with blood titers varying from 10^-1 to 10^-3.5. 2) Two of 4 garter snakes inoculated with WEE virus and kept at 4°C, for 17 days harbored the virus in the blood, but not the brain. One snake harbored the virus in the brain but not in the blood. No virus was detected in either blood or brain after 17 days at 4°C in one of the snakes.

Daily subcutaneous injections of 200 units Acetylcholinesterase into rats caused stimulation of erythropoiesis demonstrated by significant reticulocytosis within 7 days and increased erythrocyte counts after 14 days. Hematocrit values did not change significantly.

The histology of testis in albino rats submitted to hypoglycemic comas was studied. Comas were produced by either insulin or tolbutamide. No changes were seen in prepubertal animals, but in adults, injuries were seen in the seminiferous epithelium, coning of cellular and intercellular vacuoles, sloughing, nuclear pyknosis and multinucleated cells. Congestion and oedema were invariably present in the interstitium, though no injury was seen in any other testicular structure. Lesions increased in number and intensity with repetition of comas and recovery after comas was incomplete. Hypoglycemia is considered the specific cause of the injuries.

Previously 1 I reported results of experiments on carbon dioxide fixation by strains of Actinomyces and Lactobacillus in which the data indicated a high incorporation of carbon dioxide into the methylene carbons of succinic acid. The data were supported by isolation of beta-alanine formed from succinic acid by the Schmidt degradation done at 40 to 70°C. Recently studies in this laboratory were initiated to determine the mechanism of this reaction in strains of Actinomyces. Although the initial experiments reproduced the previous results, isolation of the methylene carbons of succinic acid as ethylenediamine was preferred since it would eliminate several chromatographic steps and simplify the procedure. Although I had been unsuccessful previously in obtaining ethylenediamine by the procedure of Phares and Long 2 the use of a better grade fuming sulfuric acid and high temperatures permitted adequate formation of ethylenediamine. With some dismay, it was found that the carbon dioxide formed from the ethylenediamine was totally inactive. Subsequently, it was found that failure to adequately trap sulfur dioxide fumes formed by the reaction 3 accounted for the reduced specific activity in the carboxyl carbons. However, the close correlation of the specific activity of the isolated beta-alanine with that predicted is unexplained. The present results clearly show a net fixation of CO₂ are valid. It is, therefore, concluded that anaerobic fixation of CO₂ into succinic acid by Actinomyces primarily occurs via a Wood-Werkman reaction. In air, the oxidation of some intermediate of glucose metabolism results in formation of CO₂ and consequently reduces the specific activity found in the succinic acid.

I regret the error in the analyses and the concomitant circumstances which lead to a misinterpretation of the data. I would like to apologize for any inconvenience the use of such data may have caused.

1. Natural and synthetic selachyl alcohol is devoid of hemopoietic stimulatory activity in normal rats. 2. Batyl alcohol, derived from hydrogenation of selachyl alcohol, enhances proliferation of all myeloid elements but does not stimulate synthesis of hemoglobin. This effect is manifested in normal rat recipients by erythromicrocytosis, reticulocytosis, thrombocytosis, granulocytosis, and increased numbers of both erythrocytic and granulocytic elements in their marrows. 3. Batyl alcohol is active both orally and parenterally. The hemopoietic properties of this substance are not dependent on optical activity. 4. It is suggested, although not proved, that batyl alcohol may play an important role in regulatory control of hemopoiesis.

1) Passive serum sickness was produced in mice using homologous antibodies, obtained from immune ascitic fluid, in the form of soluble antigen-antibody complexes. Lesions were similar to those obtained with rabbit antibody-antigen complexes, but, in addition, amorphous eosinophilic material was seen in many glomerular capillary loops. Incidence and intensity of lesions using homologous antibody was greater than with equal amounts of heterologous rabbit antibody. 2) Homologous antibody was more effective in producing lethal anaphylaxis than rabbit antibody when both were given as soluble complexes.

In the human erythrocyte, menadione is capable of supporting oxygen consumption. In this respect, it can replace autoxidizable dyes such as methylene blue that have been previously used. Oxygen consumption, as a measure of the pentose pathway, is inhibited by dicumarol.

Serum leucine aminopeptidase (LAP) activity during third trimester of pregnancy was significantly increased in 11 patients with documented twin pregnancy, when compared with 25 patients with a single fetus of same gestational age.

Concentration of ESF in the urine of Thalassemia patients increases with severity of anemia. Subjects with hemoglobin levels below 4 g % may reasonably be expected to provide urine with a high titre of human ESF. A biological assay depending upon the single unit obtained by the additive changes in hematocrit and reticulocyte values of rats has been developed which produces a cheaper and more reliable method of screening samples for ESF activity than the starved rat red cell Fe⁵⁰ uptake procedure. The latter method, however, is more sensitive and there-fore more useful in following the purification of ESF preparations of established potency.

The incidence of susceptibility of inbred mice to 2 transplantable neoplasms, L1210 leukemia and adenocarcinoma 755, was not influenced by variation in the dietary fats. Diets deficient in fat likewise had neither inhibiting nor stimulating activity in the pathogenesis of the 2 neoplasms.

The authors are indebted to Dr. Clyde Culbertson and Dr. Irving Johnson, Eli Lilly and Co., for help in supplying mice and transplantable tumers, and to Mr. James F. Glore, Dept. of Illustration at the Medical Center, for preparation of graphs. The capable technical assistance of Pearl Driver, Betty Corya, Shirley Stockslager, and Donna King is also appreciated.

Castration of male guinea pigs produced a decrease in activities of DPN and TPN 17β-hydroxysteroid dehydrogenases of the kidneys. Subcutaneous injection of testosterone propionate for 14 days restored activities of kidney enzymes. Extension of injections to 65 days gave no further changes. Dehydrogenases of liver were not affected by castration or androgen administration. Ratio of DPN to TPN dehydrogenase was approximately 2:1 in liver and 1:4 in kidney. DPN enzyme activity of liver was approximately 10 times that of kidney.

Strains of mice are being produced that are isogenic with inbred stocks except for the hemoglobin locus. With mice of such strains, experiments were carried out to ascertain whether peripheral blood of normal mice contain cells capable of repopulating the marrow of irradiated recipients. In 3 of 5 recipients injected intravenously with ~ 100 million nucleated peripheral blood cells after 600 r, increasing numbers of graft-derived erythrocytes were noted within 60 days after treatment, indicating the presence in peripheral blood of cells that are able to give rise to mature erythrocytes.

In animal species in which T-1824 is firmly bound to the plasma proteins, absorption of dye by the reticulo-endothelial system and other tissues during the “mixing period” is not a significant source of error in measurement of plasma volume with this dye. With T-1824, as well as with other suitable test substances, back-extrapolation of the time concentration curve on a semi-log plot provides satisfactory correction for loss during the mixing period.

The F_cells factor in the splenectomized cat is 0.78 (s.d. ± .01), but with spleen intact the F_cells factor is highly variable as shown by the striking effects of adrenaline on the F_cells value in the normal anesthetized cat. Evidence is presented indicating that species differences in the F_cells value may be fundamentally related to body size.

With the use of Cr³¹ and T-1824, cell and plasma volumes are measured in sympathectomized - splenectomized dogs. Average cell volume, plasma volume, and blood volume are 32.2 ml/kg, 54.8 ml/kg. and 87.0 ml/kg respectively for the male dogs; and 30.2 ml/kg, 52.5 ml/kg, and 82.7 ml/kg respectively for the females. These sex differences are not statistically significant. A comparison between sympathectomized-splenectomized dogs and splenectomized dogs shows higher cell and blood volumes (significant at the 5% level) for the former group. Analysis of variance demonstrates significant individual differences in blood volume among the male sympathectomized-splenectomized dogs. The F_cells factor shows neither significant individual difference nor sex difference, and is unchanged by sympathectomy.

In 6 out of 25 experiments on sympathectomized - splenectomized dogs, the posthemorrhage F_cells factor decreases to below the control range. The constancy of F_cells factor following hemorrhage in splenectomized dogs is partially due to sympathetic activity.

In connection with a study of replacement of Vit. E in vivo by certain synthetic antioxidants, liver uptake and intracellular distribution of the Vit. E-active compound N,N’- diphenyl - p - phenylenediamine (DPPD), following its administration to rats, has been determined by a method based on spectrophotometric measurement of the color developed by oxidation with ferric chloride in ethanol. Over half of the DPPD recovered was present in the soluble fraction of liver cells, one-quarter was associated with microsomes, and a small fraction recovered from mitochondria. This distribution pattern is similar to that reported for total lipids, and indicates that the intracellular location of UPPD may be passively determined by its lipid solubility. Alpha-tocopherol. on the other hand, was found predominantly in mitochondria.

During water diuresis stop flow-analysis the nephron volume is less than during osmotic diuresis stop flow analysis, there is a demonstrable increase in osmolal U/P in the distal nephron samples, and sodium U/P minimum occurs near the PAH peak. During water diuresis stop flow analysis active proximal reabsorption of sodium is demonstrable or para-aminohippurate is not a valid marker of proximal nephron samples.

In pento-barbitalized dogs, partial clamping of abdominal aorta, always lowered C_k, C_er and C_K/C_er. For these changes to occur mean arterial pressure had to fall below 75 mm Hg. Extraction ratio of PAH was not lowered by hypotension. The calculated renal plasma flow was markedly reduced and reactive hyperemia probably did not occur after clamping. The fraction of calculated quantity of K delivered to kidney that was excreted was much less during hypotension. Hypotension, insufficient to lower C_er, markedly lowered Na excretion but not C_K. In agreement with Davidson, elevation of rate of Na excretion, reduced the effect of hypotension on C_K and allowed C_K/C_er to rise above one. The need to consider another factor in addition to urinary Na in the effect of hypotension on K excretion is pointed out.

1. Prolonged centrifugation can alter an organism's growth. 2. After centrifligation at 20 and 28°C, fruit fly larvae do not require as much oxygen for growth. 3. It is suggested that the animal has adapted by developing a greater efficiency for growth.

Adaptation of orbital bleeding procedure to rapid, serial blood sampling of mice and other small laboratory animals has been made. Blood (0.2 ml) is collected in disposable, heparinized micropipettes by rupturing the fragile opthalmic venous plexus with tip of blood collection tube. The tube may be sealed and centrifuged for hematocrit determination and plasma separation for enzyme or other biochemical studies. Bleeding procedure for a mouse requires less than 30 seconds, and 100 mice can be bled and processed by a technician in about 1I/2 hours. Appropriate illustrations demonstrate technical details of procedure.

Our experiments indicate that the nucleotides of different tissues vary greatly in lability. When dog muscle was removed rapidly and minced in ice cold trichloroacetic acid, its nucleotide pattern resembled that seen in fresh blood, i.e., ATP greatly exceeded ADP or AMP. Delay in mincing the tissue caused some decrease in ATP with increases in ADP and AMP. On the other hand, dog or rabbit intestinal mucosa was so labile that usual procedure of removing tissue and transferring it immediately to cold trichloroacetic acid was much too slow. Injecting cold trichloroacetic acid into the live tissue was necessary to give a nucleotide pattern which resembled patterns seen in other tissues. Incubation under circumstances tried, were unsuccessful in restoring tissue nucleotide patterns. Extrapolation of foregoing results suggests that studies involving either distribution of nucleotides in tissues or determination of tissue nucleotides after in vitro incubation must indicate degree of lability of these tissue nucleotides.

Thyroidectomy of weanling rats kept under normal dietary conditions results in initial prolonged retardation in production of polyploid nuclei in the liver, followed by gradual resumption of polyploid cell formation over long periods. The effect appears to be mediated, at least in part, through the pituitary, and polyploid shifts are greater than can be explained on the basis of absolute or relative liver weight, or liver growth rate. The liver cytology appears to be more sensitive to thyroid deficiency than the general growth of the animal, the time of greatest sensitivity for this study having been observed at 122 days of age.

Survival and microbial population studies in mice with their intestinal flora reduced by neomycin indicate that these animals are much more susceptible to intravenous staphylococcal infections than their littermate controls.

(1) Routine I¹³¹ diodrast renograms frequently fail to demonstrate significant renal lesions. (2) Initial loading of tubular mechanism responsible for secretion of diodrast by competitive substance (for example, para-amino-hippurate) blocks concentration and secretion of I¹³¹ diodrast. (3) As tubules secrete the competing substance and plasma level gradually returns toward normal, the control kidney in cases of unilateral disease demonstrates, on successive injections of I¹³¹ diodrast, earlier appearance of uptake and secretion as well as greater quantitative uptake and secretion of I¹³¹ diodrast.

1) A technic has been described whereby the dog adrenal may be infused in situ without disturbing its arterial blood supply. 2) Utilizing this technic, infusion into left adrenal arteries of a solution containing high concentrations of potassium did not affect serum potassium concentration or urinary potassium excretion any differently than infusion of similar solution into a non-adrenal artery. It is concluded that the kaliuretic response to potassium infusions is not mediated by a direct action of potassium on the adrenal cortex.

Livers of mice infected with mouse hepatitis virus (MHV 1) were examined by conventional staining procedures and by electron and fluorescence microscopy using acridine orange fluorochrome and fluorescein-tagged antibody. Preparations stained with acridine orange showed abnormal accumulations of RNA-staining material in cytoplasm of hepatic cells. Inclusion bodies of similar size, shape, and location were stained selectively with fluorescein-tagged antibody. Observations by electron microscopy showed virus particles in cytoplasm which measured 90 ± 20 mμ in diameter.

The authors are indebted to Dr. R. H. Rigdon and his staff for invaluable technical assistance.

RF mice exposed to 100-300 r of whole-body X-rays at various ages from before birth to 180 days after birth showed age-dependent variations in susceptibility to leukemia induction. Susceptibility to induction of granulocytic leukemia was minimal when irradiation was carried out during gestation or shortly after birth, rising later to a maximum at about 70 days of age. Susceptibility to induction of thymic lymphomas was, likewise, apparently minimal during gestation but maximal shortly after birth, declining later in life at the time of thymic involution. Susceptibility to induction of ovarian tumors was relatively low in mice irradiated in utero.

We are grateful to F. F. Wolff, E. S. Ledford, and W. D. Gude for technical assistance and to Dr. A. W. Kimball for statistical analysis of data.

1) Mice were maintained for 51 days in an atmosphere of oxygen at total pressure of 197 mm Hg. This provides a normal inspired O₂ tension. CO₂ and N₂ pressures in the ambient atmosphere did not exceed 5 mm Hg for either gas. 2) Under these conditions animals usually did not encounter any difficulties. Several litters were born and in one case young were conceived, born and raised in virtual absence of inert gas. However, in several cases animals died within 48 hours after being subjected to this environment. Once past this period Only mechanical failure of the apparatus terminated these experiments. It is suggested that pulmonary atelectasis was responsible for these early deaths.

1) Sprague-Dawley female rats, age 50 days, were given 10 mg H³-3-methyl-cholanthrene dissolved in sesame oil by gastric tube. 2) In the first experiment selected tissues, urine and feces were analyzed for radioactivity after 24 hours. Sixty to 90% of administered radioactivity was found in gastric and intestinal contents or in feces; 4.4% in urine. Less than 2% was recovered in the tissues analyzed. Results obtained by combustion technic for measurement of total radioactivity were compared with those obtained by toluene extraction. Data indicate that most radioactivity in fat and mammary tissue could be extracted with toluene, whereas negligible quantities could be obtained by this method for liver, kidney, brain, uterus and skeletal muscle. 3) In the second experiment, 24-hour samples of urine and feces were analyzed for total radioactivity for 24-25 days. Significant quantities of tritium-labeled material appeared in urine for 13-17 days and in feces for 7-11 days. Thereafter, fecal and urinary excretion of radioactivity occurred intermittently in trace amounts.

We wish to thank Miss Beverly Cockrell for technical assistance.

1. Daily administration of DOCA for short periods caused an increased glycogenic response in liver of rats following intraperitoneal injection of glucose. Daily administration of DOCA for longer periods resulted in decreased glycogenic response. 2. DOCA induced inhibition of glycogen formation was accompanied by decrease in potassium content of both liver and blood serum. 3. By forcing an increase in potassium intake, the potassium content of both liver and blood serum was maintained at essentially normal levels and liver glycogen production was not significantly inhibited in rats that received DOCA. 4. These data indicated that DOCA induced inhibition of glycogen formation was secondary to potassium depletion.

Nutritional muscular dystrophy in the chick did not occur when an arginine deficient diet was fed even though the diet was low in Vit. E and sulfur amino acids. When arginine was included in the basal diet, a high incidence of muscular dystrophy was observed by 5 weeks of age, even when growth was restricted in the chicks receiving adequate arginine by paired feeding with the basal group.

Tissue cultured cells of human cancer cell line HEp #2 inoculated intravenously into newborn rats at a dose of 1 million or more cells resulted in growth of the heterologous tumor cells in lungs and sometimes in other organs and produced death by respiratory impairment in 4 to 5 weeks.

Concentration of corticosterone in blood of the rat varied as a function of time of day. Highest levels of corticosterone were found between 2 and 3 p.m., and 9 and 10 p.m. There was no diurnal variation in concentration of an unknown component of adrenal origin found in blood. These data supply additional evidence for presence of a diurnal rhythm in secretory activity of the adrenal cortex and support the concept that corticosterone is the glucocorticoid of physiological significance in the rat.

Samples of corticosterone and 17-hydroxycorti-costerone were supplied through courtesy of Merck, Rahway, N. J., and Upjohn Co., Kalamazoo, Michigan.