Experimental Biology and Medicine

The alleged ability of sodium rhodanate (thiocyanate) to peptize heat-coagulated egg albumin led Bancroft and Rutzler 1 to elaborate a general theory, based upon an early observation of Claude Bernard, 2 that anesthesia is the result of a reversible coagulation of the colloids of the brain-cells, and that the action of narcotics in general can be antagonized by peptizing agents such as NaCNS and NaI. Their principle has been so extended in its application that now such pathological conditions as drug addiction, 3 histamine poisoning, 4 and insanity 5 , 6 are held by them to result from disturbances in the colloidal state of the nervous system.

The action of NaCNS as a narcotic antagonist was studied, to determine whether the action postulated for it could be observed in the case of a simple cell. The material was the egg of Arbacia punctulata. selected for its convenient size, ready availability, and because of the great amount of information amassed concerning it. 7

Preliminary determinations were made to determine the toxic and lethal effects of different concentrations of ethyl urethane and sodium thioeyanate. In all cases freshly shed eggs of a single female were selected, using the criteria suggested by Just 8 for obtaining normal eggs. The eggs were washed twice to remove the perivisceral fluid, pipetted into sea-water solution, beakers covered, and after requisite time, washed and inseminated in sea-water. Controls were run for each determination. The solutions were made from purest reagents obtainable, with sea-water acting as natural buffer. The criterion of viability was the development of gastrulae and pluteii. When striking abnormalities were observed in the larvae the concentration was designated as toxic; when development was inhibited, as lethal.

It was the purpose of this study to determine the relationship of plasma protein concentration to the plasma volume of non-edematous dogs. The value of plasma albumin determinations in studying fluid exchange has been amply demonstrated. It was consequently determined to perform simultaneous analyses of plasma albumin and globulin concentrations and of plasma volume in dogs that were undergoing certain experimental procedures. The method of Koch and McMeekin 1 was used for determining the plasma protein levels of the first 2 dogs and a micro-kjeldahl procedure involving distillation into N/70 H₂SO₄ and subsequent titration with N/20 alkali was used for the other 2 dogs. The circulating plasma volumes of the first 2 dogs were kindly determined for me by Mr. John Morcan, who used the brilliant vital red technique as described by Whipple and coworkers. 2 Those of the other 2 dogs were performed by the author, utilizing the spectrophotometric procedure for analyzing brilliant vital red described by Clarke and Graff. 3

Changes in the plasma albumin concentration proved to be the most significant ones and consequently they alone will be mentioned in this report.

Two dogs in the anemia colony in this laboratory were studied weekly over a period of 2-3 months. In one of them, both the plasma volume and the plasma albumin concentration remained fairly constant. In the other, as the plasma albumin concentration decreased, the plasma volume decreased. As the plasma albumin concentration increased the plasma volume increased.

Two other dogs (not anemic∗) were placed upon a low protein diet developed by Weech and Goettsch 4 and studied weekly for about 2 months. This diet consisted mainly of carrots and contained essential salts and apparently all of the recognized vitamins.

The writer 1 demonstrated the presence of complement fixing bodies in the blood serum of individuals infected with Endamoeba histolytica and subsequently 2 , 3 , 4 , 5 , 6 reported the results obtained with a complement fixation test in cases of human amebiasis and in normal individuals, together with the technique of the test. These observations have been confirmed by Spector, 7 Fernandez, 8 Heathman, 9 and Sherwood and Heathman. 10

The observations here reported were undertaken to throw further light upon the phenomenon of complement fixation in amebiasis, using the dog, an animal that Faust 11 has shown is easily infected with Endamoeba histolytica and in which the lesions produced by this parasite are comparable to those occurring in intestinal amebiasis in man. Alcoholic extracts of cultures of Endamoeba histolytica were used as antigens.

Tests were made upon 29 dogs infected with Endamoeba histolytica and in all but one (96.5%) a positive complement fixation reaction developed in the blood serum. In the dog giving a negative reaction death occurred 7 days after colonic inoculation from a very severe amebic colitis. Of the dogs giving a positive reaction, 21 (75%) gave a 4+ reaction; 3 (10.7%) gave a 3+ reaction; 2 (7.1%) gave a 2+ reaction; and 2 (7.1%) gave a 1+ reaction.

Of the 28 dogs giving a positive reaction 22 died and 5 were sacrificed, 3 while still suffering from intestinal amebiasis while 2 had recovered from the infection. Of the 22 dogs that died, all showed typical amebic ulcerations in the colon and in all motile forms of Endamoeba histolytica were found in the lesions. The 3 dogs killed while still suffering from the infection showed both healed and active amebic lesions in the colon and motile Endamoeba histolytica were present in the active lesions.

This communication reports the results of tests upon the differences in filtrability of the Tunnicliff coccus of measles under in vivo and in vitro growth conditions. The question whether microorganisms ordinarily regarded as non-filtrable can become filtrable and vice versa, under special conditions of growth, is of more than academic interest. Kendall, 1 with the use of his “K” medium, claims that it is relatively easy to cause non-filtrable organisms to become filtrable and filtrable to become microscopically visible and non-filtrable.

Different environmental conditions cause some microorganisms to undergo rather marked changes in size and shape of the individual cell. Particularly is this true for certain parasitic varieties capable of cultivation in vitro, where they are distinctly larger and often differently shaped than they are in vivo. Adaptation to the artificial environment is the accepted explanation for the morphological variation. The reaction of the culture medium, quality of nutritives, absence of the living host influence and the osmotic pressure are undoubtedly responsible for this in vitro metamorphosis.

Experiments. Full-grown, healthy guinea pigs were intracardially injected with 1 cc. quantities of a heavy saline suspension from a 24-hour blood-agar growth of pure cultures of the Tunnicliff coccus of measles. The cultures used for animal inoculation were isolated in 1926 by Duval and Hibbard 2 directly from the blood of human measles upon a modified Noguchi plasma medium. The morphology was that of a Gram positive diplococcus corresponding in size to the ordinary pneumococcus.

To investigate the problem of photo sensitization in pellagrins, need has been felt for quantitative in vitro methods. The observations here described were made with the hope of aiding in the development of such methods.

As a sensitizing agent yellow eosin (National Analine and Chemical Company) has been used. The light source was a nitrogen-filled tungsten filament lamp of 1500 watts, operated at 115 volts by alternating current. The suspension of erythrocytes was contained throughout exposure to light and measurement of degree of hemolysis in standard Pyrex test tubes of 16 mm. outside diameter with walls 1.5 mm. thick. Equal exposure of each tube was insured by placing the tubes in a device to be described in detail elsewhere. This apparatus moved tubes in a circle 30 cm. in diameter about the light source at the approximate center, while the tubes were immersed in a constant temperature waterbath at 37.5° C. and received light through plane windows of plate glass 3 mm. thick. The layer of tap water between the tube and the plate glass was 2 mm. where the tube's circumference came closest to the window. Adequate provision for aeration and maintenance of suspension was provided. Human blood from finger prick was allowed to fall into 0.85% NaCl solution made sufficiently alkaline to give a pink color with phenol red by the addition of 0.1 N NaOH and washed by centrifugation 3 times in specimens of the same solution. This cell suspension was added in 0.2 cc. quantities, measured from pipettes of the between-marks type into 5 cc. quantities of the various solutions which were under study. The final concentration of erythrocytes was about 15,000 per mm³; it was kept close to this figure by adjusting the concentration of the heavy suspension as was indicated by photometer readings of the trial dilutions.

In the course of experiments to test the relative pathogenicity for kittens of a number of strains of Endamoeba histolytica in culture, rectal injection proved unsatisfactory because of the small percentage of kittens which became infected. The technique of rectal injection was simply to withhold food on the day of injection, and inject the sediment from a rich culture by means of a rubber-tipped pipette which was inserted gently through the anus to a distance of about 3 inches. The kitten was then held head downward for a few minutes before being released. In one series the anus was sutured for 24 hours, without producing any better results. A preliminary enema, as used by Kessel 1 was not given because of the desire to avoid even microscopic trauma as far as possible and to avoid early evacuation of the injected material.

Feeding cysts by mouth produced no better results with the one strain with which the method was employed. This method was also impracticable because some strains did not form cysts in culture.

In considering a better route of infection, injection directly into the cecum after laparotomy, as practiced by Rees 2 was not adopted because it was not desirable to pass a needle directly through the wall of the cecum. It was felt that this might create a small lesion in which a few amoebae or bacteria might be left in withdrawing the needle. Faust's method 3 of inoculating dogs directly into the ileum through the anus was not feasible because the colon of the cat is acutely flexed in its upper third, and because there was too much danger of trauma.

The electro-myograms making up the data from which these observations are reported were obtained from oscillographic records of amplified muscle action potentials. Ten adult human subjects were used. The action potentials were picked up by modified pore electrodes 1 which were inserted into the body of the muscle to be investigated. The active electrodes were insulated from all but a small area of the interior of the muscle, allowing fair isolation of antagonistic muscles. The amplified action potentials were photographed in a Westinghouse multi-element oscillograph. Action potentials could be simultaneously photographed from 2 different sources, usually the biceps and triceps brachii of the right arm.

A simple reaction time set-up was used. The subject was instructed to extend his forearm as quickly as possible following the disappearance of the glow of a low wattage neon bulb. The arm was supported horizontally on a rest allowing free movement about the elbow. The photographs included markers showing arm movement, time in 10 sigma intervals and a marker showing when the subject was given the ready signal and the stimulus. Reaction times were read to the first action potentials after the stimulus and to the first arm movement. The records show an average of about 58 sigma lapse between the first large action potential changes and the corresponding movement of the forearm. This time is fairly constant, varying in ordinary subjects by not more than ±8 sigma. There was no evidence of a preliminary tension in either the muscle which was to be used in the response or its antagonist preceding the stimulus and related to the warning signal.

The peptizing action of urea may be an important factor in increasing the solubility of these sparingly soluble compounds which are normally present in urine at about their saturation limit.

The widespread destruction of rabbits and grouse in Minnesota during 1925 and 1926 led to a consideration of tularemia in epizootic proportions as a cause of the periodic decimation of wild rabbits and possibly of grouse. Green and Wade 1 reported that ruffed grouse were susceptible to experimental infection with Pasteurella tularensis. Parker and Spencer 2 had previously considered tularemia as a possible disease of grouse and had produced a fatal infection in a blue grouse. Tularemia as a natural disease of birds was definitely established by Green and Wade, 3 when they isolated Pasteurella tularensis from a quail dying in the wild. Parker, Philip, and Davis 4 recovered Pasteurella tularensis from sage hens, found dead or killed, on an area in which these birds were known to be dying.

In the present paper is reported the isolation of Pasteurella tularensis from a sharp-tailed grouse and from a ruffed grouse in Minnesota. In the course of the wild-life disease investigation, routine examinations and study were carried out on game birds collected in various areas of Minnesota during the fall of 1932. Most of the specimens were collected by game wardens, through the cooperation of the State Department of Conservation.

Tularemia in the Sharp-tailed Grouse (Pedioecetes phasianellus). A sharp-tailed grouse, No. B-10419, collected by shooting on September 27, 1932, in Pine County, apparently well when killed, was received at the laboratory on September 28, and 46 ticks of the genus Hemophysalis were collected, principally from the bag in which the carcass was shipped. Necropsy of the grouse revealed no abnormal pathology.

1. Strychnine sulphate is quickly absorbed from the lumen of the normal bowel and from the peritoneal cavity. 2. Interference with the blood supply of a segment of bowel prevents or greatly delays the absorption of strychnine from the lumen. 3. complete arrest of blood flow to (arterial occlusion) or from the bowel (venous occlusion) probably precludes absorption through the misrepresenter vessels. 4. Evidence of transperitoneal absorption was demonstrated in some of these experiments but only through gut wall whose nutrition was seriously damaged. In the absence of gross perforation or rupture of the gut wall, transperitoneal absorption is slight even in the presence of a badly damaged wall.

The removal of a phrenic nerve to paralyze one-half of the diaphragm is now extensively performed in certain cases of pulmonary tuberculosis and in other conditions. Although the literature contains many statements to the effect that cough and expectoration are facilitated by phrenicectomy we have been unable to find any recorded experimental work dealing with such a result. Our experiments show that on the contrary the operation actually interferes with cough and diminishes the elimination from the lung of lipiodol injected into it.

The efficiency of the cough mechanism was measured by the elimination of lipiodol from the lungs before and after phrenicectomy. The rate of disappearance of the oil was determined by means of fluoroscopy and serial roentgen-ray films. Experiments were performed upon 10 dogs. In 2 dogs the time required for the complete disappearance of the oil from the lung was determined. In these 2 dogs a unilateral phrenicectomy was performed after all the oil had disappeared. After they had recovered from their operations the rate of disappearance of lipiodol was again determined and the effect of phrenicectomy upon this rate noted. In 8 dogs unilateral phrenicectomy was performed first and then equal amounts of lipiodol were injected into the right and left lower lobes.

Following the phrenicectomy it was noted under the fluoroscope that some of the oil injected into the lung on the side of the normal diaphragm was coughed up either during the injection or immediately thereafter. Fluoroscopic observations indicated that the cough mechanism was interfered with on the side of the paralyzed diaphragm.

The effects of superior vena cava obstruction vary according to the degree and location of the obstruction and the suddenness of its development. To determine the tolerance of animals to this condition, to measure the effect on venous pressure and to trace the paths of collateral circulation, experimental obstruction of the superior vena cava was produced in dogs.

The vein was exposed through an intercostal incision and completely occluded by dividing between ligatures. The usual aseptic technique was followed and the operations were performed under intratracheal ether anesthesia administered with positive pressure.

Obstructions were of 2 types, (1) above the junction of the azygos vein, and (2) including the azygos vein. Two attempts were also made to obstruct the superior vena cava below the junction of the azygos vein but both dogs died within a few minutes. Obstruction above the level of the azygos vein was produced in 7 dogs. One died within 24 hours and 3 died within 12 to 14 days as a result of a propagating thrombus or an empyema. Three recovered. Of the latter, one was sacrificed for study after 30 days. Another was later subjected to azygos vein obstruction and the third is alive after more than 5 months. One dog survived the immediate effects of a 2-stage obstruction of the superior vena cava and azygos vein but died at the end of 21 days from an infected bilateral pleural effusion.

The most striking general results of superior vena cava obstruction were cyanosis of the tongue and oral mucosa and injection of the conjunctivae. Somnolence, listlessness and slow deep respirations were also noted. The dogs with obstruction just above the right atrium, dying within a few minutes, had extreme cyanosis of the upper part of the body.

In the transmission of experimental trachoma to animals, perhaps the most significant and refractory difficulty is the complete lack of corneal involvement. Except in man where infection followed accidental or intentional transfer of trachomatous material. pannus formation has not accompanied folliculosis experimentally induced in a variety of animals. Since the appearance of cicatricial changes and pannus comprise the essential diagnostic differentiation between trachoma and folliculosis, the necessity is obvious for studying the conditions under which vascularization of the cornea is stimulated. While it is not yet clear that the data thus far obtained may be correlated eventually with pannus formation in trachoma, it is of interest that pannus may be evoked by various stimuli. From this point of view the experiments undertaken are here reported.

The experiments have been done on rabbits;† and because the results of a former study indicated that pannus may be a manifestation of bacterial hypersensitiveness, it was decided to study vascularization of the cornea as an allergic response. Rabbits have been sensitized to different bacteria; Staph. aureus, Bact. granulosis, Pneumococcus, diphtheroids and indifferent streptococci. Various methods for inducing hypersensitiveness of the cornea to bacteria were studied, but the most successful one was scarification of the cornea with one scratch reaching from below the sclero-corneal margin to the pupil, and then instilling into the conjunctival sac 2 drops of a young, actively growing culture. This treatment was repeated at weekly intervals.S. aureus proved to be the most effective sensitizing antigen, while B. granulosis was one of the least.

It has been observed that the injection of acid extract of cattle anterior pituitary produces in guinea pigs a condition comparable to that seen in Graves'disease in human beings. Loeb 1 has found a marked hypertrophy of the thyroid gland with diminution in the amount of colloid and a marked increase in the number of mitoses in the epithelial cells. Under the action of this substance the animals lose weight, and Siebert 2 has shown that there is an increase in their metabolic rate. Other effects corresponding to conditions found in Graves'disease have been established in subsequent investigations carried out in this laboratory. Inasmuch as tachycardia and nervous irritability are very prominent symptoms of Graves'disease in man it was of interest to study the effect of this extract of anterior pituitary on the heart rate and the reflex irritability of guinea pigs. Cursory observations of Loeb seemed to indicate that such an effect might possibly exist.

Twelve young male guinea pigs, approximately 200 gm. each, were chosen and normal records were obtained under a standard procedure using the electrocardiograph (lead one) to record the cardiac pulsations. Daily injections of 1 cc. of the acid extract were given to each of 6 guinea pigs, 3 of which had been thyroidectomized. The remaining 6 guinea pigs, 3 of which had been thyroidectomized, were used as controls, and were not injected. Records of the heart rate of all the animals were obtained on the 2nd, 4th, 9th, 11th, 16th, and 18th days. The heart rate of every animal in the group of injected non-thyroidectomized animals was faster on each occasion, after the second day of the injections, than that of any of the animals in the other groups.

Recent physiological evidence shows that the small, thinly myelinated fibers of peripheral nerves are concerned with the conduction of pain (Heinbecker, Bishop and O'Leary 1 ). Evidence for the participation of fiber elements in the tract of Lissauer of the spinal cord in the transmission of pain seems adequate (Ranson and Billingsley 2 ). It is necessary, therefore, to determine how large is the number of small, thinly myelinated fibers which enter the tract of Lissauer from the dorsal roots.

To examine this tract, the seventh lumbar segment of the spinal cord of the cat with attached dorsal roots was isolated and divided into lateral halves. One half was fixed in ammoniated alcohol for the pyridine silver technique, the other in 2% osmic acid by immersion. The osmic acid cross-sections revealed that Lissauer's tract contains closely packed, small, thinly myelinated fibers. The silver pyridine preparations showed from 1 1/2 to 2 times more fine axons than could be accounted for by the number of fibers apparent in the osmic acid sections. The additional axons were those of non-myelinated fibers.

Evidence secured by Ranson 3 through degeneration of the dorsal roots indicates that in the lumbosacral cord (cat) the tract has medial and lateral divisions; the lateral is primarily endogenous to the cord, the medial contains fibers which have entered from the dorsal roots. 4 Accordingly, in 7 cats, under ether anesthesia, the dorsal roots (levels L. 5 through S. 3) were ligated and cut (3) or crushed (4) between the spinal ganglia and the cord. The cats were killed after 20 to 100 days and the material prepared by the silver pyridine, 2% osmic acid and Marchi methods. The results show that a majority of the small, thinly myelinated fibers in the medial half of the tract of Lissauer degenerate (cord levels L. 6 and L. 7).

In a recent communication (Heinbecker, Bishop and O'Leary 1 ) the fibers in a skin nerve associated with the sensations of touch and of pricking pain were identified. Knowing that the fibers responsible for such pain are included in a well-defined group of small myelinated ones, and that those for touch fall in a different group of larger fibers, we have two points of reference for locating other functions. The differential susceptibility of nerve fibers to novocaine permits a further assignment of various functions mediated by nerves, to known fiber groups. Such fiber groups can be recognized by their potentials when stimulated, and in novocaine anesthesia the differential blocking of these potentials can be correlated with the differential disappearance of sensations.

Observations were made on 3 surgical patients to determine the order of disappearance and reappearance of various sensations and of skin temperature changes during low spinal anesthesia produced by dissolving novocaine crystals in spinal fluid. The changes noted were first a very early increase in skin temperature, after 2 to 3 minutes a loss of sensitivity to heat and cold, and then to cutaneous and pressure pains. The order of disappearance of these sensations was definitely distinct but the time interval between their disappearance was brief. After a distinctly wider gap there occurred a loss of motor function and after another short interval a loss of joint sense, sensation of pressure and touch. In recovery the order of sensibility return was reversed with the gaps between the return of different sensations much wider.

In vitro experiments to determine the effect of novocaine on the action potential of the cat's saphenous nerve definitely showed that the first potential to disappear was that derived from unmyelinated fibers.

The domestic fowl is subject to an infectious coryza, generally known as contagious catarrh, which under laboratory conditions remains localized in the nasal and orbital passages. In this respect it is unlike the coryza of laryngotracheitis, fowl pox, and fowl cholera from which there is commonly an extension of the etiological agent to other loci with the production of characteristic lesions.

Contagious catarrh was recently studied in Holland by deBlieck, 1 who isolated an organism resembling the B. influenzae of man from exudate streaked on an aerobic blood agar plate. Pure cultures of the bacterium when injected intranasally in normal fowl regularly produced a nasal discharge.

In the present investigation an uncomplicated coryza was initiated and subsequently maintained in a flock of disease-free birds by the intranasal injection, through the palatine cleft, of exudate originally obtained from naturally infected fowl. A nasal discharge, the only consistent symptom, generally appeared after an incubation period of 24-48 hours and continued, on the average, for 11 days.

The etiological agent of the coryza was not established by the injection either of bacteria isolated from aerobic plates or of exudate filtered through Berkefeld V and N candles. The bacteria were the usual mucous membrane inhabitants, among them several strains of a hemophilic bacillus similar to B. influenzae.

It was subsequently found, however, that the fluid from blood agar cultures of exudate which had been filtered through certain Berkefeld V candles contained a small, non-motile, Gram-negative bacillus apparently in a pure state. The organism grew sparsely in fluid blood at the base of slanted agar but failed to colonize on the slant or on the surface of aerobic blood agar plates. Colonization was later initiated by sealing the plates with modeling clay.

In experiments already reported 1 it was shown that while “mad itch” virus regularly caused rapidly fatal infections in cattle, cats, rabbits, mice and guinea-pigs, it produced in swine, when injected subcutaneously, only a temporary illness. Serum from swine that had recovered from this infection neutralized both the “mad itch” and pseudorabies viruses∗ as was shown by injection of mixtures of viruses and serum into susceptible guinea pigs. These facts indicated that the 2 viruses were closely related if not identical, but their relationship could not be determined conclusively at that time because serum of animals immune to pseudorabies was not available.

Schmiedhoffer 2 has reported that pseudorabies virus was without effect when injected into swine although von Ratz 3 observed the disease occurring naturally in wild swine. In our hands the pseudorabies virus has produced a febrile disease of a few days'duration. After recovery the serum from such swine neutralizes the virus.

With serum available from swine that have recovered from pseudorabies and with serum from swine recovered from “mad itch”, cross neutralization tests to determine the relationship of the 2 viruses have been made and are given in Table I.

The data show that serum from swine recovered from either “mad itch” or pseudorabies neutralizes both viruses. The conclusion to be drawn, therefore, is that the inciting agents of “mad itch” and pseudorabies are immunologically identical. The differences in the experimental disease produced by the 2 viruses 1 must then be considered only as variations induced by 2 strains of the same virus. In recognition of the identity of “mad itch” as occurring in this country and pseudorabies as occurring in Hungary and other European countries, in any future publications the “mad itch” virus will be designated as Pseudorabies Virus (Iowa Strain).

An antiviral substance which has the ability to neutralize the virus of herpes simplex has been found to be present in the blood early in the life of a few individuals. As age advances, though not necessarily because of maturation, more individuals acquire the antiviral property. A peak is reached in early adult life, after which apparently a decline sets in.

A group of sera from children whom we may assume carried no antiviral substances for poliomyelitic infection were shown likewise to be devoid of antiviral substances toward herpetic infection. Finally, herpes virus neutralizing properties have been discovered in the blood of monkeys which have reacted to poliomyelitis virus.

In a previous communication1 the striking resemblance of the bacterial flora of the upper respiratory tract of the chimpanzee to that of human beings has been described. To what extent these animals possess a similar flora in their native environment, or to what extent the organisms present represent new acquisitions after landing in the United States is not known.

The chimpanzee is highly susceptible to the common cold, whether naturally communicated by contact with infected human beings or artificially induced by bacterium-free filtrates of naso-pharyngeal washings from subjects suffering from acute colds. One of the striking phenomena of these chimpanzee colds is the great increase both in numbers and in area of distribution during the early stages of the cold, whether spontaneous or experimental, of the common pathogens of the upper respiratory tract such as H. influenzae, pneumococcus and S. hemolyticus.

In particular, we have noticed from the beginning of our study of the common cold in apes, during periods of infection, a change in the number and character of the colonies of H. influenzae on rabbit's blood agar plates. During the healthy period cultures from the naso-pharynx and nose give few colonies of H. influenzae from the naso-pharynx and usually none from the nose. The colonies themselves are small and opaque. During the period of infection numerous colonies of H. influenzae are obtained both from the naso-pharynx and the nose. Furthermore, the colonies themselves are of an obviously different character, very large, translucent and moist.

A more careful study of this phenomenon has revealed facts of great interest and importance. In April, 1932, an outbreak of spontaneous colds occurred among our chimpanzees.

Numerous investigators have reported that a specifically immunized animal eliminates intravenously injected antigen more rapidly than does a normal animal. 1 , 2 , 3 Evidence has been presented to show that the precipitin antibody is of importance in the disposition of the foreign material. 4 Virtually nothing has been established, however, as to whether the circulating or a fixed-tissue precipitin plays the major role in this elimination. The attempts to determine the relative importance of the circulating precipitin and the fixed-tissue precipitin in the removal of the injected antigen have been hindered by the lack of suitable methods for quantitatively estimating this antibody in an antiserum. Recently, we 5 proposed a procedure, which we called the “neutralization method,” for quantitatively titrating the precipitin against crystalline egg albumin. This method seems to overcome the difficulty in estimating precipitin and appears advantageous for investigating the relative importance of the circulating and fixed-tissue antibody in eliminating antigen.

We have been able by the neutralization method to determine quantitatively the effect on the circulating precipitin of the intravenous injection of antigen into an immunized rabbit. The amount of antibody, expressed in milligrams of nitrogen per cc. of the serum, present just before and one hour after injecting a known amount, in milligrams of nitrogen, of crystalline egg albumin has been found experimentally. The total circulating precipitin of the rabbit has been calculated by multiplying the amount of precipitin determined per cc. of serum by the number of cc. in the animal's plasma volume. The plasma volume has been considered, from determinations we have recently offered, 6 to approximate 3.4% of the body weight of the rabbit. Data, suggesting the essential importance of the circulating precipitin in removing the intravenously injected antigen, are presented in Table I for 8 rabbits of a longer series studied in this regard.

The present investigation was undertaken to determine whether the chemical condition of the fat of a ration influenced the ability of an experimental animal to maintain health. McAmis, Anderson and Mendel 1 and Burr and Burr 2 have described a disease in rats due to the absence of fat in an otherwise complete diet. The syndrome is characterized by failure in growth, changes in the skin, especially of the tail and feet, and kidney degeneration. Burr and Burr 3 later reported that the disease could be prevented or cured by the feeding of linoleic acid. Evans and Lepkovsky 4 confirmed Burr and showed that glycerides of saturated fatty acids do not cure nor prevent the disease, but fatty acids containing more than one double bond have remarkable ability to improve fat-free diets. The conclusion was that certain fatty acids, essential for wellbeing, cannot be synthesized by the rat, although the rat is able to produce body fat on diets deficient in these essential fatty acids. Most of the work on fat feeding has been concerned with neutral fat or pure fatty acid. There have been a few contributions on the effects of fats altered in various ways. Powick 5 and Fridericia 6 showed independently that rancid fat destroyed the vitamin A of a ration when the fat came in contact with the vitamin-containing material.

The investigation to be reported deals with the effect on dogs of fat subjected to oxidative rancidity. A basal diet of purified foodstuffs planned to be adequate in all known dietary essentials was prepared: casein 16%, sucrose 55%, fat 25%, agar 2.5%, salt mixture 1.44%, and cystine .06%. It was very low in vitamins and was supplemented with Harris yeast concentrate and oscodal,† a cod liver oil concentrate.

1. The disease described as nutritional encephalomalacia of chicks is not associated with significant alterations in cell plasma ratio, plasma or blood volume. 2. During the early growth period of the chick, the growth of the plasma and blood follow closely the growth of body weight. The blood and plasma volume per kilo, aside from individual variations, remain constant throughout the early growth period.

In this communication a comparison is made between certain secretory, motor and vascular phenomena produced by pilocarpine administered into the cerebral ventricles of monkeys, and the corresponding responses to the drug when given subcutaneously.

A series of 15 green monkeys (Lasiopyga callithricus) has been used. On 6 of them, fractional gastric analyses have been made following the administration by stomach tube of 100 cc. of a farina test-meal. Several experiments have been conducted on each animal to demonstrate the action of the drug pilocarpine (hydrochloride), when administered at various times in the course of digestion of the test-meal. The drug has been given in varying doses subcutaneously and by the intraventricular route.

The intraventricular injection of pilocarpine, in doses of 5-10 mg. per kilo of body weight, invariably caused a sudden and complete cessation of the gastric secretion of free HCl, at whatever stage in the digestion the drug was given. The samples obtained after pilocarpine injection consistently failed to show any free HCl acid.

The total acidity curve usually paralleled the'free acid'curve but at a higher level. After intraventricular injection of pilocarpine, the total acid generally fell pari passu with the'free'acid. This fact, together with the lessened volume of recoverable gastric contents, indicates that the phenomenon is not merely one of neutralization by mucus or regurgitated bile. In one monkey with a low type of acid curve the level of total acidity merely ceased to rise. The'free'acid in this case fell to zero.

If a subcutaneous injection of histamine (1-2 mg. per kilo) was given in the achlorhydric phase following intraventricular pilocarpine administration, the “acid tide”—both ‘free’ and ‘combined’—was rapidly restored and reached a high level.

Berg and Jobling 1 reported the occurrence of peptic ulcers in dogs following the deprivation of bile. These findings were corroborated by Kim and Ivy 2 and Bollman and Mann. 3 The incidence of ulcers was approximately 60%.

During the past few years, in connection with other investigations, we have examined a number of animals deprived of their pancreatic secretions by means of fistulas, ligation of the pancreatic ducts, and pancreatectomy, and have been impressed by the infrequent occurrence of peptic ulcers in these animals compared to dogs in which bile was excluded. On the other hand, Elman and Hartmann 4 found ulcers in the duodenum in all of the animals of a series of 6 dogs with pancreatic fistulas existing from 13 days to 18 days. They described the lesions as ulcers, although microscopic examination revealed only defects in the continuity of the mucosa, not involving the deeper muscular layers of the intestine. These investigators attributed the lesions to the loss of the neutralizing effect of pancreatic juice upon gastric acidity, and minimized the importance of bile as a factor in the development of peptic ulcers in dogs.

The following observations were made upon a series of 14 dogs deprived completely of pancreatic juice by means of fistulas made according to the technique of Rous and McMaster, as adapted by Elman and McCaughan. 5 Twelve of the animals received sodium chloride by mouth or intravenously; 2 received no special form of treatment and were killed after 25 days and 31 days respectively. Some of the animals were killed while they were still in good condition. Others developed symptoms typical of pancreatic insufficiency 6 and died spontaneously.

Investigators have reported many studies on gastrointestinal pH in animals, using various methods of determination—hydrogen electrodes, quinhydrone electrode, and colorimetry. The results have all been somewhat open to question because of the possible inaccuracy of these methods in a medium such as intestinal contents. For the range of pH encountered in such work, the glass electrode seems to be the method of choice. We have used glass electrodes prepared by the methods of MacInnes and Dole 1 or Robertson. 2 The electrical apparatus developed for this work was that described by Rose-bury. 3

Four hundred and fifty-five albino rats were used. Groups were placed on various diets: (I) a “normal” diet which included corn, wheat, barley, oats, soy bean, milk, meat scrap, alfalfa, NaCl, and CaCO₃; (II) diet I + 10% lard; (III) diet I + 30% lard; (IV) bread; (V) potato; (VI) suet; (VII) lean beef; (VIII) McCollum's 4 rachitic diet 3143; (IX) diet VIII + 2% cod liver oil; (X) Steenbock's 5 diet 2965; (XI) diet X + 2% cod liver oil; (XII) Zucker's 6 diet 401; (XIII) diet XII + 2% cod liver oil; (XIV) diet XII +15% cod liver oil. The rats on the last 3 and the suet diets showed consistent malnutrition.

The rats were killed by a blow on the head or by chloroform, and the abdomen opened immediately. The stomach, the small intestine in 3 approximately equal lengths, the cecum, and the colon were quickly tied off and removed and the contents gently expressed under oil. The contents were then diluted sufficiently with water and the pH determined.

Plotting histograms of the incidence of the data revealed that if calculated as pH the occurrences closely approximated the normal bell-shaped curve.

A comparison was made of infants and children with eczema and an essentially normal group of similar age distribution without eczema or known allergic disturbance, in respect to the concentration in the serum of total protein, albumin, globulin, cholesterol and lipoid phosphorus. The protein determinations were made by the gravimetric method of Barnett, Jones and Cohn, 1 so modified as to permit the separate measurement of the albumin and globulin fractions, the latter by difference, † Cholesterol was determined by the method of Myers and Wardell. 2 The lipoid phosphorus was determined by a combination of the methods, slightly modified, of Bloor 3 (preliminary extraction), Benedict and Theis 4 (determination of phosphorus). The protein osmotic pressure was calculated from the figures for albumin and globulin.

There were 31 children in the eczema group and 25 in the control group. The results—excluding lipoid phosphorus in which no differences of interest were found in the two groups—can be summarized by giving the mean, the 10 percentile and 90 percentile values, in order.

1. Total protein. Eczema: 7.13; 6.13; 7.95 (gm. %). Controls: 6.82; 6.01; 7.73.

2. Albumin. Eczema: 5.29; 4.62; 5.93. Controls: 4.72; 4.05; 5.37.

3. Globulin. Eczema: 1.84; 1.26; 2.66. Controls: 2.10; 1.25; 3.17.

4. Albumin: globulin ratio. Eczema: 3.19; 1.96; 4.14. Controls. 2.52; 1.45; 3.77.

5. Protein osmotic pressure. Eczema: 32.2; 28.0; 35.9 (mm. Hg.). Controls: 29.0; 25.3; 32.8.

6. Cholesterol. Eczema: 195.7; 187; 139 (mgm. %). Controls: 173.1; 132; 194.

The more striking differences observed were: (1) Marked elevation of the serum albumin, albumin-globulin ratio, and protein osmotic (oncotic) pressure in the serum of the eczema group; (2) Marked elevation of the serum cholesterol in the eczema group, 40% of which showed figures in excess of 200 mg. %; (3) Slight, possibly not significant, lowering of the serum globulin in the eczema group; (4) Moderate elevation of the total protein in the eczema group, due mainly to the high albumin fraction; (5) No difference between the 2 groups in respect to lipoid phosphorus.

Under conditions simulating a chronic focal infection, S. aureus, S. hemolyticus, and Proteus sp. are capable of inducing a marked neutropenia which has been sustained for 5 months in animals implanted with Staphylococcus and Proteus.

A better understanding of the mechanism of the reflex regulation of heart rate and blood pressure is to be gained from a study of the afferent nerve impulses from the circulatory system and of the efferent impulses to the heart and blood vessels. Previous communications have described the nature of the nervous discharge from the arch of the aorta and from the carotid sinus, 1 , 2 and its relation to blood pressure. On the efferent side, sympathetic impulses concerned in maintaining the tone of the blood vessels have been recorded. 3 The present report is concerned with the activity of the sympathetic fibers to the heart.

One of the small nerve twigs running to the heart from the stellate or inferior cervical ganglion in a cat under urethane anesthesia was freed from the surrounding tissue and cut close to the heart, all other cardiac sympathetic fibers remaining intact. The nerve was then slung onto electrodes and the action potentials, after amplification, were recorded by means of an oscillograph. Figure 1A shows a typical discharge. It will be observed that the impulses tend to come in bursts but in general we have not been able to identify the frequency of these volleys with the heart rate or respiratory rhythm, although it has been shown 3 that in the case of sympathetic nerves carrying fibers to the blood vessels, there is usually a grouping that is synchronous with either the respiratory or cardiac rhythm. The records further show that under the conditions of these experiments there is normally a ”tonic” sympathetic discharge to the heart.

The relation of the discharge in the cardiac sympathetic fibers to the heart rate is shown by a comparison of Figures 1A and B. In A, the frequency of the heart beat was 132 per min.

The rate of cornification of the superficial epidermis of Triturus is markedly variable and probably independent of thyroid control, but the molting of the cornified layers is dependent on the thyroid. Experiments indicate that a change in cutaneous circulatory conditions is probably an essential factor in the molting mechanism.

1. Albino rats kept on vitamin B complex-free diet survived on an average longer after infection with Trypanosoma equiperdum than controls kept on a complete diet. 2. Lack of vitamin B₁ seems to be the important factor although a slight effect was also observed with animals receiving B₁ but no G. 3. No change was found in the plasma of deficient animals which could account for the phenomenon observed. 4. Although feeding of vitamin B, after the injection of trypanosomes cured polyneuritis it did not restore the normal reactivity towards the infection before death. 5. Rats kept on a vitamin A free diet and infected with Trypanosoma equiperdum apparently died somewhat earlier than controls kept on a complete diet.

In a previous report the precocious development of sexual characters following homeoplastic hypophyseal implants in cockerels was discussed. 1 The present report concerns the effects of daily administrations of hebin, a purified gonad-stimulating hormone prepared from sheep pituitary glands, on the juvenile sex characters of cockerels. 2

Light brown Leghorn cockerels ranging from 21 to 47 days at the beginning of the experiment received daily injections of hebin over a period ranging from 14 to 36 days. The daily dosages administered per bird varied from 4 to 32 rat units. Treated and control birds were weighed and head furnishings measured at regular intervals. Treated individuals, with but one exception, remained active and in good condition throughout the duration of the experiments.

The first effect to be noticed was a pronounced stimulation of head furnishings, in some instances, as early as 48 hours after injections began. These became turgid and reddish in color and revealed steady growth throughout so that at the conclusion of experiments they were usually perceptibly larger than controls—in some instances very considerably, so. Bird No. 250, 28 days old when the experiment began, received 20 rat units daily for 21 days. The day the experiment began its comb measured 2.0⁺ cm. in length and 0.8⁺ cm. in height, while 21 days later its comb measured 7.3 cm. in length and 3.7 cm. in height. The largest control comb measured but 4.3 cm. in length and 1.8 cm. in height at the conclusion of the experiment, though its size at the beginning was similar to that of the treated individual. Some of the older individuals were known to crow and attempt treading, a reaction not exhibited by normals until considerably older.† Plumage and spurs revealed no noticeable modifications.

The precocious development of sexual characters following homeoplastic hypophyseal implants in juvenile Leghorn females was previously discussed by Domm. 1 In a second series the effects of daily subcutaneous injections of hebin on these characters were studied. 2

Light brown Leghorn females ranging from 21 to 47 days at the beginning of the experiment received daily injections of hebin over a period ranging from 14 to 36 days. The daily dosages administered per bird varied from 4 to 32 rat units. Head furnishings were measured and birds weighed at regular intervals as in experiments on cockerels. All treated females remained active and in good condition. In none of the experiments did there seem to be a significant difference in weight between treated and control, both groups showing consistent gains throughout.

Here, as in the male, the first effect to be noticed was a phenomenal growth of head furnishings. This was definitely noticeable within 48 hours in individuals receiving higher concentrations. In such experiments the head furnishings revealed a continuous high rate of growth throughout, becoming large and masculine in character. Bird No. 249, 28 days old when the experiment began, received 20 rat units daily for 21 days and was killed on the day following the last injection. Its comb measured 1.7 cm. in length and 0.5 cm. in height when experiment began and 4.5 cm. by 2.3 cm. on the day the bird was killed. The best control increased from 1.3 cm. length and 0.4 cm. height to 2.1 cm. by 0.7 cm. during this period. The growth of head furnishings in treated females frequently approximates that of similarly treated males. The comb in such individuals is stout of blade and erect whereas that of similar size in normals, only found in much older individuals, shows a thin lobby blade. Plumage, spurs, and behavior were apparently unaffected.

The rise in blood sugar following the discontinuation of insulin administered to an anorexia case suggested that a temporary hyperglycemia and even a glycosuria might be induced in this way in non-diabetic subjects. Accordingly, 5 other non-diabetic patients were given insulin, starting with Units V before, each meal, and the dosage increased at intervals of a few days until one patient was receiving Units XVII and another Units XXV 3 times a day. The injections were given 15 to 30 minutes before meals. Blood sugars 2 hr. after meals were usually normal, sometimes above normal. Hypoglycemic symptoms were rarely encountered. Upon discontinuation of the insulin all of these patients showed a hyperglycemia and 4 of them a temporary glycosuria. Glucose tolerance curves were done on 3 of the 6 patients and showed a diminished tolerance, even in one case where there was failure to produce a glycosuria.

For example, a female patient, age 34 years, entered the University Hospital June 2, 1932, following a pleurisy with effusion; she had lost weight and her appetite was poor. On June 4 a 50 gm. sugar tolerance test was normal, fasting blood sugar was 87 mg., the maximum at the half hour was 159 mg. Insulin was started at noon on this day with an initial dosage of Units V 3 times a day with the patient on a general diet. A second glucose tolerance test was done June 18 when the patient was receiving Units X 3 times a day; blood sugar reached a maximum of 231 mg. after 1 hr. The patient had been receiving insulin Units XXV 3 times a day on July 10 and insulin was discontinued the following day.

Certain theoretical considerations in respect to the energetics of heat production as defined 1 for states of changing weight are of special interest but should also prove of rather great practical importance. In view of the fact that the hyperbolic term in equation (6) of the foregoing paper has dampened out and become negligible in the case of human growth beyond the age of twenty, this relation assumes the simpler form,

for heat production in calories per Kg. per unit of time, the various factors possessing the same significance as before.

It is clear from (7) that U will not alter greatly during any period in which fails to change rapidly—a condition, as we have seen, which is actually reached on the average during the second and third decades of life. For constant values of , U will also remain constant; but an important question arises as to the conditions under which U, as representative of heat output, will be most economical. So long as is positive there cannot be a true minimum, although U may, of course, with low rates of reproduction become as small as p, E _c and A′ will allow. If, however, no restriction be placed upon the sign of , a rather significant result may be at once attained by differentiating (7) with respect to t and equating to 0, whence,

a condition which leads to the result that U, the unit rate of energy output in the form of heat will actually become and remain a minimum (for constant values of ρ, E _c and A') as long as the unit rate of change in weight is negative, and equal to .

Attention is thus immediately directed to the possibilities of studying heat production during periods of starvation.

From theoretical considerations in conjunction with data on heat production during prolonged fasting it has been shown 1 that the optimal relative rate of starvation at which a human individual would conserve his body reserves to the utmost ought to be closely in the neighborhood of 0.0064 Kg./Kg./Day. It is now proposed to examine this result in the light of several observations on the point.

By far the most accurate of the data at hand are those reported by Benedict, 2 but in Figure 1 we have also included the curves of Succi for several fasts, as well as that for Beauté. The ordinates are in log₁₀ (Weight) = .4343g, the abscissae in days. There is, here, a striking similarity not only in the almost identical shape of these curves, but also in their slope, especially after the 4th or 5th day of fasting. This slope appears so decidedly linear that purely graphical analysis could hardly detect any significant curvature. We shall call this the “apparently linear” phase.

Taking the data on Levanzin by Benedict for each day from the 8th to the 30th inclusively and analyzing this apparently linear phase by least squares, we find that the slope is 0.00645 Kg./Kg./Day, a numerical result completely confirming that given above and already reached by an independent method previously described. 1

Straight lines have also been drawn through the other examples and indicate the expected slope, 0.0064. These likewise pass excellently through their respective points in every case, notwithstanding the diverse conditions and times of observation.

But, even though the foregoing value for an “optimal” rate of starvation is actually witnessed in practice—we must avoid the conclusion that starvation would necessarily continue indefinitely in this way. Indeed, as it will appear in a subsequent paper, the “linear” phase in such experiments discloses merely a temporary, though an admirably close approach to what are fundamentally “ideal” conditions of starvation for the individual subject and for the kinetic system his organism represents.

In several previous communications 1-5 we have outlined a concept in which the phenomena of growth and metabolism are viewed as separate forms of a single underlying mode of motion. A scaffolding is thus provided by which each of these major processes may be studied more directively. But our descriptions up to this point have been restricted to the special case of human growth and metabolism, and it is therefore proposed, since the methods and the theory are sufficiently general, to consider an example of the cognate phenomena in a population of unicellular organisms. In order, however, to apply the present theory, it is essential that we possess simultaneous information upon the two chief processes in action, namely, growth and coincidental heat production. Such data, for our purposes, need to be more than ordinarily accurate, else analysis is labor in vain. Considering the difficulties at hand, there is small wonder that suitable observations are few; indeed, in the bacteriological field there are no data which are more worthy of attention and study than those reported within the past few years by Bayne-Jones and Rhees. 6 We shall employ the data on growth and simultaneous heat production as recorded for their experiment 2 in order to illustrate the general method of application of our own results.

In experiments described by Smith 1 it was shown that liver tissue extracted with alcohol until fat-free has a biological value inferior to that of whole liver, as shown by a subnormal growth rate. In the present experiments an attempt was made to gain information as to the manner in which whole liver exerts its favorable influence. A combination of the efficiency quotient method of Palmer and Kennedy 2 and the paired-feeding method used by Mitchell and Carman 3 was used. Male albino rats on a complete diet containing 20% extracted liver, 1 supplemented by 0.5 gm. of dried whole liver or 1.5 gm. of raw liver daily, and whose food intake was limited to that of control animals, grew at a faster rate and had lower efficiency quotients than did the controls. Animals receiving the supplements and having no restrictions in food intake consumed more food, but made greater gains and had somewhat lower efficiency quotients than those whose food intake was limited. When the diet was supplemented with an equivalent amount of alcohol-extracted liver (0.5 gm.), the animals with limited and with unrestricted food consumption made no greater, or but slightly greater, gains than did the control animals and also had similar efficiency quotients. In all, 8 experiments (24 animals) with the whole liver and 4 (12 animals) with the extracted-liver supplements were carried out. A typical experiment with each of the supplements is shown in Table I.

It appears, therefore, that the increased growth rate resulting from feeding whole liver is due to some influence other than an increased food consumption, and that the factor responsible for the growth-promoting effect is removed by alcohol.

The respiratory quotients of minced brain stem, cerebellum and medulla of fed rats were studied in the Warburg apparatus, The tissues were suspended in Ringer solution containing 0.1% glucose and buffered with phosphate at pH 7.4. The results are presented in Table 1.

Further work is in progress in an attempt to determine the ability of the brain to oxidize various food stuffs.

It is well known that large doses of viosterol cause a hypercalcemia and increased elimination of calcium from the body and good evidence is accumulating to the effect that the source of the excess of the calcium under such conditions is from the bone, at least when the diet is calcium poor. 1 , 2 , 3 Since Aub 4 has shown that the metabolism of lead parallels the metabolism of calcium, large doses of viosterol should promote the elimination of lead from the body. Experiments to determine the effect of large doses of viosterol on lead excretion in cats and guinea pigs and on lead storage, and the course of lead poisoning in guinea pigs were carried out. Throughout the series Viosterol Squibb 250D was used. All assays for lead were made by the Fairhall method. The guinea pigs were kept on a normal diet of oats, hay, cabbage, or lettuce. The cats were given low calcium diets consisting of meat and fish or vegetables. Viosterol dosage in the 2 cat experiments was 20 drops daily and in the case of the guinea pigs, 6 drops daily in 2 experiments and 10 drops daily in one.

In both cat experiments the excretion of lead was immediately increased after the administration of viosterol. In the first experiment the viosterol cats excreted more lead than the control group in each of 5 three-day periods. The difference between the groups was greatest in the second period when the viosterol cats showed a lead elimination 10 times greater than the control group.

In the second cat experiment the average daily excretion of lead per cat rose sharply in the first 3 day period after viosterol was begun and reached a peak in the fourth period.

The absorption rates of galactose and mannose were studied in rats and dogs using one hour as the absorption period. The rats were not fed during the 48 hours before being used but water was given as usual. One gram (2 cc. of a 50% sol.) of the sugar was given by stomach tube. Animals were killed by a blow on the head after which a ligature was tightly tied about the neck to prevent loss of sugar by mouth. The abdomen was then opened and ligatures placed about the oesophagus and lower end of ileum. This portion of the gastrointestinal tract was removed and the unabsorbed sugar washed out with warm water for quantity determinations.

In using closed loops of ileum in dogs 2 gm. of the sugar were given (10 cc. of a 20% sol. introduced by needle and syringe). At the end of an hour the content was aspirated and the loop washed by injecting warm physiological salt solution and aspirating. More complete description of these procedures has been published. 1

Sugars of definite rotation (d-galactose + 80.5°, d-mannose + 14.250°) were used and concentrations made by weight were checked by polariscope. Galactose determinations were made by the Shaffer-Hartman method for determining glucose, consequently 10% had to'be added to the findings to approximate the actual amount of galactose present. This method can not be used with any degree of accuracy for mannose estimations. A satisfactory method consists in reducing Benedict's qualitative solution and then plating the copper out and weighing the copper electrodes. 2

The results are shown in Table 1. While there is some difference in the comparative rates at which these 2 sugars were taken up by dogs and rats, the findings would seem to justify the conclusion that galactose is absorbed approximately twice as fast as mannose.

Local vital stains were applied to the prospective head region in the late yolk plug stage of Amblystoma punctatum by Vogt's 1 method. From the records obtained by making camera lucida drawings of the position and extent of the stained areas in the stages following, composite diagrams were constructed showing the arrangement of the parts of the prospective ectoderm involved in the formation of the nose, lens, ear, balancer, gills and stomodaeum. Two of these, Harrison stages 13 (slit blastopore) and 22 (shortly after closure of neural folds), are shown in the figures.

The wheeling about of the head epidermis due to the dorsal convergence of the neural material which lies almost out at the equator of the embryo in the gastrula stage, takes place in Amblystoma punctatum in a manner similar to that observed by Vogt 2 and Goerttler 3 on European salamanders. There is a forward and downward growth of the ectoderm of the head region anteriorly in such a way that the material at the middle of the transverse fold comes to lie just in front of the stomodaeum and ventral to the eye—a shifting of the material nearest the dorsal midline in an anterior and ventral direction while that originally ventral moves caudally and dorsally.

The results obtained for Amblystoma are much less diagrammatic than those Röhlich 4 gives for Triton but the arrangement of the anlagen is essentially the same in both forms. The position of the lens agrees with that given by both Manchot 5 and Röhlich for Triton. In Amblystoma however the nasal ectoderm seems to extend up on the outer side of the neural fold (stage 15) for a short distance and the ear ectoderm not quite to reach the fold. The latter may also be slightly farther anterior in Amblystoma than in Triton.

When the force which is exerted by a muscle is plotted, on the ordinates, against the speed with which it shortens, on the abscissae, the curve relating the two variables is, in general, a straight line sloping towards the x axis. This relationship between force and velocity means that the force exerted by a muscle diminishes as its speed of contraction increases. This result has appeared in the work of Gasser and Hill, 1 Fenn 2 and in our own experiments soon to be published at length elsewhere. The purpose of this paper is to report a fact which emerges from our data which seems to throw light on the cause of the force-velocity relation.

Our experiments were made on the gastrocnemius muscle of the decerebrate cat stimulated through the tibial nerve by a single maximal, break, shock of a Harvard inductorium. The muscular response was, therefore, a twitch. The muscle contracted against an inertia disc (radius 8.65 cm.) the moment of inertia of which was 290,400 gm.-cm. 2 The displacement of the disc was recorded optically. Time was imprinted on the record in intervals of .001″ by means of a slotted disc which was rotated by a synchronous motor in such a way as to interrupt a thin beam of light from a 500 watt lamp. The displacement of the inertia disc was measured from the photographic record with a cathetometer for each .001″ and from these measurements, by taking first and second differences, the angular velocity and angular acceleration of the disc were computed. Although this record was measured to each .001″, we have used in this computation, the interval of .01″. The force, calculated from the moment of inertia and the angular acceleration of the disc, is, therefore, known for each time interval (.01″) during the contraction cycle.

The finding that carotene is provitamin A for certain species-raises the question as to whether a direct determination of the carotene content of plant tissue, coupled with information with regard: to the vitamin A potency of isolated carotene, could be substituted for the extended biological assay. Euler, Demole, Karrer and Walker 1 report that a carotene determination in plant material discloses the magnitude of its vitamin A activity.

For the determination of carotene a colorimetric method was used, based on the original procedure of Willstatter and Stoll 2 but modified after critical study by one of us. 3 The vitamin A technique was essentially that employed by Sherman and has been referred to in an earlier publication. 4

Alfalfa samples 45, 46 and 49 were taken from adjacent parts of the same field. Nos. 45 and 46 were dried in a mechanical drier by artificial heat within a few hours after cutting and No. 49 was sun-dried in the field. Samples 61 and 62 were artificially-dried and sun-dried, respectively, but from different parts of the country. A stock solution of carotene from carrots was prepared by dissolving a weighed amount of the crystalline substance (M.P. 169-171°, corr.) in ethyl laurate and olive oil. A dilution was made with olive oil every 2 weeks and in order to insure a constant level of carotene intake a colorimetric determination was made before each dilution. Hydroquinone was added to all solutions to the extent of 1 mg. per cc. as an antioxidant.

Table 1 displays the feeding levels which are in a comparable range as to rat growth response.

One of the authors reported the remarkable antispasmodic properties of benzyl benzoate and of the benzyl esters for smooth muscle, 1 and the local anesthetic properties of benzyl alcohol. 2 Numerous attempts were made by different investigators to prepare and study analogous benzyl esters and derivatives of benzyl alcohol, with the object of improving the therapeutic value of the original substances. Thus, Hirschfelder 3 pointed out that hydroxy-benzyl alcohol, or saligenin, also produced local anesthesia and was water-soluble. While phenmethylol and saligenin were both potent local anesthetics they had also a mildly relaxant or antispasmodic effect on smooth muscle preparations. Starting with saligenin, we synthesized a series of halogenated and other derivatives, and studied them especially as to their local anesthetic properties and their antispasmodic effects on smooth muscle.

The following compounds were prepared: amido saligenin, nitro saligenin, nitroso saligenin, mono-chlor saligenin, di-chlor saligenin, mono-brom saligenin, di-brom saligenin, mono-iodo saligenin, di-iodo saligenin, chlor-brom saligenin, brom-iodo saligenin, brom-nitro saligenin, nitro saligenin benzoate and saligenin nitro benzoate. All these compounds are solids and the majority are soluble in water only to a limited extent. Di-iodo saligenin is a very poorly soluble substance, saturated solutions of which give concentrations of only 1 : 20,000.

The local anesthetic effects of these compounds were studied on sensory nerve endings of frogs'legs, on the conjunctivae of cats and rabbits and by the wheal method in guinea pigs. Minimal concentrations of the drugs, as well as the duration of the anesthesia, were taken into consideration when comparing the relative efficiency of the different compounds.

It has been shown that the radiations from the cold quartz mercury lamp are powerfully antirachitic. Direct irradiation for 3 seconds daily at a distance of 5 inches from the burner prevented the development of rickets in rats fed on a rickets-producing diet. Direct irradiation for 10 seconds daily for 2 weeks. at a distance of 5 inches from the burner, brought about complete healing of rickets in severely rachitic rats. Advanced healing occurred in one week in rats irradiated daily for 45 seconds or longer. Under the conditions mentioned in the text, exposure of ergosterol dissolved in olive oil to the radiations from a cold quartz mercury lamp for 1, 5, 10, 20, and 30 minutes at a distance of 5 inches from the burner resulted in the antirachitic activation of all of the solutions, of which 0.002mg. of irradiated ergosterol and 0.001 cc. of irradiated olive oil together prevented and cured rickets in rats. The minimum protective and curative doses were not determined.

The sex hormones of the anterior pituitary are not sex specific, but stimulate the secretion of the male as well as the female sex glands. However, for the same dose of hormone the effect on the female is far more striking than the effect on the male. In connection with other work in this laboratory, and prompted by the recent work of Funk and Harrow, 1 we carried out experiments to increase the stimulating effect of the anterior pituitary hormone on the male sex glands by simultaneous injections of male hormone.

These experiments were done on our laboratory albino rats. Eight rats of approximately equal age and weight were divided into 4 groups of 2 each; (1) control; (2) injected with anterior pituitary hormone; (3) injected with male hormone; (4) injected with both anterior pituitary and male hormone. All injections were made intramuscularly and twice daily. At autopsy the whole genital tract was exposed and the seminal vesicles and testes inspected macroscopically, and weighed.

The solution of the anterior pituitary was prepared from fresh glands and standardized by the Zondek-Aschheim technic to contain 100 mouse units in each cc. This solution was a mixture of the maturing and the luteinizing fractions as determined by previous assay on female rats.

The solution of the male hormone was prepared from fresh testes and tested on the castrated rooster for its ability to restore secondary sex characteristics. Each cc. contained approximately 1/2 cock unit. A cock unit is the amount of hormone necessary to cause an increase of 20% in the size of the comb and wattles in the course of 10 days. The solution of male hormone was purposely made quite dilute to preclude the possibility of any great amount of activity originating from this solution.

The present experiment dealing- with the addition of small traces of oil of gaultheria (true wintergreen oil) to the otherwise normal diet of mice, has given some interesting data. The mice used were individuals belonging to a highly inbred stock (the D strain). This stock is a branch of the dilute brown strain which has an unbroken line, mainly of brother-to-sister descent, extending over 25 years (Little and Murray). The inbreeding was initiated by Little and the stock is usually referred to as the Little Dilute Browns. My branch had passed through several individual laboratories before coming into my own. At present my dilute brown stock is in the tenth generation of pedigreed brother-to-sister matings.

Breeding females of this stock are very prone to develop spontaneous adeno-carcinoma of the breast. Murray, who has made more observations on the incidence of neoplasia in individuals of this stock than anyone else, has found more than 1300 spontaneous tumors in the past 5 years. Of all the female mice which are used as breeders, fully 80% develop carcinoma. The common infections are the cause of death of the remaining 20%. It is Murray's opinion that if all breeding females of this stock would live long enough they would all develop carcinoma of the breast.

My sub-line shows parallel findings, differing only in minor details. Whereas Murray reports 1300 spontaneous tumors, I have had only 115. This is due to the fact that individuals of other stocks in my laboratory have been better breeders and consequently have filled up most of my available cages. The incidence of spontaneous tumors in the individuals, however, is as high as it has been in the Murray derivatives of the dilute browns.

Intraperitoneal or lymph sac (frog) injections of 1% solutions of ferric ammonium citrate and sodium ferrocyanide were made in 50 rats, 10 turtles and 100 frogs. The presence of either salt or both in the kidney of these animals, depending on whether one or the other or a mixture was injected, was revealed by the Prussian blue reaction. In the kidney of the rat the cellular excretion of ferric iron is sharply localized in a segment of the proximal convolution, 2-3 mm. long, beginning 6-8 mm. from the glomerulus (Figs. 2 and 3, portion marked by a bracket). The excretion of ferrocyanide is not similarly localized. To the extent that it may be excreted by the cells of the proximal convolution, all of them appear equally active. When the 2 salts are injected as a mixture, the locus of excretion of both is the same as that stated for the excretion of the ferric salt alone. A probable explanation for this peculiarity is to appear in another publication. The accuracy of the determination of the presence and extent of this locus is the result of macerating and mounting the renal unit (Fig. 3) of the variously treated animals parallel to each other on a glass slide and studying them unstained under the microscope.

The characteristic appearance of P. blue in finely particulate form in the cells of that portion of the proximal convolution active in the excretion of iron is shown by the darker, stippled-appearing cells of the tangential section (Fig. 4, “Y”). In the kidney of the turtle and frog, the cellular excretion of both ferric iron and ferrocyanide is shared equally by the cells of the entire proximal convolution and the appearance of P.

The pharmacological action and method of preparation of a substance obtained from various species of Ilex are described. The action of the drug resembles very closely that of the digitalis bodies. It produces first an increase in the amplitude of the heart by increased relaxation and increased systolic contraction, followed by decreased relaxation in diastole with slowing and finally systolic standstill. All attempts to obtain the substance in crystalline form were unsuccessful. The possibility of the substance being caffeine is considered.

Ganter, 1 Landsberger and Gnoinski, 2 Rosenak and Siwon 3 report favorable results of peritoneal lavage in nephrectomized rabbits and dogs. The end products of nitrogen metabolism dialyze into the fluid and can be removed with it. Bliss and his coworkers 4 report that the survival period of nephrectomized dogs treated with peritoneal lavage is from 13 to 16 days as compared with the survival period of untreated dogs, from 2 to 3 days. The experiments reported herein were undertaken to investigate the value of peritoneal lavage in cases of temporary acute renal insufficiency, manifested by attacks of uremia following poisoning by bichloride of mercury. A series of 15 rabbits was used. The animals were given equally large doses of bichloride of mercury (30 mg. per kilo of body weight) by stomach tube. By means of a small rubber catheter 200 cc. of an isotonic salt solution at body temperature were injected into the peritoneal cavity of the animals. This fluid was removed after 30 minutes by syphonage through the same catheter. This procedure was repeated several times at each sitting and the hemoglobin and nonprotein nitrogen content of the blood, as well as the body weight of the animals, were determined before and after each experiment. No hydremic changes in the blood could be observed. The weight of the animals decreased slightly during the course of the experiments.

Figure 1 shows the curves of the nonprotein nitrogen values in the blood of treated animal F. 22 and control animal F. 18. After each washing of the peritoneal cavity, the non-protein nitrogen content of the blood of rabbit F. 22 was considerably lower than before the lavage.

The oxygen consumption of the resting frog's heart is about one-fourth that of the working heart. 8 Ouabain, as one of the digitaloid drugs, brings the heart to a standstill and simultaneously increases the oxygen consumption. This effect is greater in Ringer's than in sucrose solution. In certain concentrations this effect may be masked by a decrease in work, which results from cessation of beating. The time for the action of this drug on the oxygen consumption is inversely proportional to its concentration and does not seem to be affected by the presence of ions.

Experiments on the effect of combined injections of various anterior pituitary or pituitary-like hormones on the weight and changes in immature rat ovaries have given suggestive results. Fevold, Hisaw, and Leonard 1 have shown that combinations of fractions of the anterior lobe of sheep produced effects which suggested the possibility of 2 sex hormones, a follicular stimulator and a luteinizer. Leonard 2 showed that a growth hormone (phyone, van Dyke) when injected simultaneously with a sex stimulating extract of the sheep anterior pituitary, inhibited the action of the latter. More recently, Evans, Meyer and Simpson 3 have combined injections of prolan with sex-free growth hormone and also with growths-free sex hormone and have obtained activation only of their growth hormone with the production of ovaries greater than can be predicted by the additive effects of either hormone injected separately. Prolan did not react on their growth-free sex hormone which led them to conclude they were dealing with a prohormone (growth hormone) and an activator (prolan).

We here present data concerning the effect of single and combined injections of prolan with the sex hypophyseal hormone of sheep glands prepared by the pyridine method. 1 Only the water soluble fraction of the pyridine extract was used. The prolan was prepared by the alcoholic precipitation method as described by Zondek. In a litter of at least 3 immature rats between the ages of 21-24 days, one received prolan in doses sufficient to produce at least several corpora lutea in the ovaries in 5 days, one received a known amount of the hypophyseal extract, and the other a combination of the two.

The study of carbohydrates by the destructive action of yeast and bacteria has been carried out along different lines. 1 One of us 2 has caused differential sugar destruction by exposing test solutions to suspensions of resting yeast cells and then to bacterial growth. The present investigation was undertaken to study quantitatively the destruction of various sugars by resting bacteria grown upon sugar-free medium and medium enriched with various sugars.

A typical strain of B. coli (No. 4348 from the American Type Culture Collection) was grown upon a medium prepared as follows : Beef juice broth rendered sugar-free by the usual incubation with B. coli, filtered and adjusted to pH 7.6, 1.5% agar added. This mixture, alone or enriched with sugars, (0.5%) was slanted in culture tubes 1 inch in diameter. The slants were heavily seeded so that the entire surface was covered with organisms, and allowed to incubate at 37.5° C. for 18 hours. The growth was then removed with a small volume of physiological saline and the “resting” organisms washed at least twice with saline by centrifuging. About 0.45 to 0.55 cc. of packed organisms were suspended in 4 cc. of a solution containing 2.85% Na₂HPO₄ and 1.005% NaH₂PO₄. The suspension was brought to a temperature of 37.5° C. in a water bath, 2 cc. of an aqueous solution of carbohydrate† at the same temperature added, the contents of the tubes thoroughly mixed and incubation carried out for 10 minutes in the water bath.

In spite of the extent to which masses of yeast cells have been used in determining the true glucose content of blood, quantitative data upon the various factors which may affect the reaction are meagre. There seems to be even less available information upon the destruction of glucose by bacterial cells. The present report concerns quantitative studies of some of the factors involved in the destruction of glucose by “resting” B. coli. The general methods were those described in the preceding paper.

The effect of acid and alkali upon the destruction of glucose was first investigated. No destruction was demonstrated if glucose was added to organisms suspended in 0.01 N hydrochloric acid, and very little if 0.001 N acid was used. In distilled water some destruction took place, but in 0.001 N sodium hydroxide this was more pronounced. If the strength of alkali was increased the amounts destroyed lessened, and in 0.02 N alkali no destruction of glucose could be demonstrated. It seemed clear, therefore, that a buffered solution of moderate alkalinity would furnish suitable conditions for carrying out studies of the decomposition of sugars and, accordingly, the alkaline buffered phosphate solution described in the preceding paper was adopted. It is the same one used in earlier work upon the destruction of glucose in blood filtrates by incubation with the B. coli.

The effect of varying the number of organisms and the concentration of sugar was next investigated. It could easily be shown that the amount of glucose destroyed paralleled quite closely the concentration of the bacterial suspension, but it was necessary to control conditions carefully to demonstrate the effect of varying concentrations of glucose in the presence of a constant concentration of bacteria.