Experimental Biology and Medicine

Although it has been demonstrated that a carnivorous animal can be kept alive and maintained in activity for considerable periods on an exclusive diet of meat it is not known whether growth as well as maintenance can proceed on a regimen entirely free from both fats and carbohydrates. Hammarsten has stated that omnivora and herbivora cannot survive on such a ration. The few experiments on record in relation to this problem have without exception been conducted on a wrong plan, the food mixtures being inadequare in respect to one or more essential factors. Our successful experiences in growing rats on foods extremely poor in fats 1 and in carbohydrates 2 respectively encouraged us to test diets containing only minimal quantities of both. The mixtures included protein 95 per cent., inorganic salts 5 per cent., along with a supply of vitamins A and B in the form of tablets of alfalfa (0.4 gm.) and dried brewery yeast (0.2 gm.) daily. On such diets, when casein furnished the protein component, animals have already grown to three times their weight at the beginning of the trial. The vitamin-bearing substances were the only noteworthy sources of either fat or carbohydrate, and supplied 4-8 per cent. of the food eaten. Whether rats will attain adult size and normal function on such diets, furnishing protein as the almost exclusive source of energy and tissue substance, remains to be determined further. If future experiments prove as successful as those here described various problems of nutrition and physiological function can be approached from new experimental standpoints.

The experiments were made on white rats, one group of rats being fed a diet of pasteurized milk and a second group being fed a milk and yeast diet. The rats receiving the yeast made more satisfactory growth gains than did the rats receiving no yeast. Inasmuch as milk has been shown to be low in the water-soluble “B” vitamine, which is present in high concentration in yeast, it would seem that yeast may be found to be an important dietary adjunct for use in baby feeding.

This investigation embraces three types of complement-fixing substances: (1) those elicited in rabbits due to injection of purified proteins; (2) those produced in the same animals due to injection of bacteria, and (3) those found in the serum of syphilitic patients. The antigens employed in the first two cases were specific, while in the case of the syphilitic sera, non-specific; four different Wassermann antigens being employed with each serum. Three fixation temperatures—water-bath, room and ice-box—were resorted to. Some phases of this investigation are still in progress and in this preliminary report, the work with the purified proteins only will be reported, although our findings indicate that the rate of fixation of complement is the same, no matter what type of fixing antibody is used.

Two purified proteins were employed: Edestin obtained from hempseed and phaseolin obtained from the kidney bean. These were kindly furnished by Dr. Thomas B. Osborne. Two rabbits were immunized with edestin and two with phaseolin. In order to elicit quantitative differences in the antibody production in the rabbits, four modes of immunization were resorted to. The edestin rabbits were injected intravenously according to “Immunization Methods No. I and No. 2,” respectively, described by Kahn and McNeil in another paper. 1 The phaseolin rabbits were injected intraperitoneally. One rabbit received 100, 150, 200, 250 and 300 mgm. of phaseolin at 24-hour intervals, and the other 100, 150 and 200 mgm. of this protein at 24-hour intervals.

The complement fixation experiments were carried out in one tenth quantities of regular Wassermanns, otherwise in the usual manner, with 2 units of complement, 2 units of amboceptor and 0.1 C.C. of a standard 5 per cent. suspension of sheep-cells. The respective antigens were prepared by weighing out 10 mgm. of the protein and dissolving these in 10 C.C. of N/1000 NaOH to which was added 0.05 C.C. of N/IO NaOH.

In the course of investigation on precipitin and complement fixing antibodies produced by injections of edestin, it was observed that, while the serum of a rabbit immunized with this protein showed the presence of precipitin antibodies, it did not show any complement fixing antibodies when employing the usual 2 units of complement in the fixation tests. It appeared reasonable at first to accept this finding as evidence of the lack of relation between these two types of antibodies. It seemed, however, that possibly the employment of 2 units of complement in the tests might give a sufficient excess of this ingredient to render a serum negative for complement fixing antibodies, although a reasonable number of such antibodies might still be present in the serum. With this possibility in mind, a series of complement fixation tests were carried out, using complement gradations of 1¼ units, 1½ units, 1¾ units and 2 units. The quantity of serum employed was 0.01 c.c.

It was observed that the same serum which gave negative results when 2 units of complement were employed, gave weak positive results with 1¾ units of complement; stronger positive results with 1½ units; and still stronger with 1¼ units of complement. In the last case the serum showed slight anticomplementary properties, which disappeared after about 10 minutes' incubation in the water bath.

This work is still being continued, but the results obtained thus far indicate that the employment of 2 units of complement in complement fixation tests is too great an excess of this ingredient for correct results in some cases, and that the employment of lesser quantities of complement, properly controlled, would serve as a finer measure of the complement fixing power of a given serum.

In a series of studies on the rate of destruction of antisyphilitic complement fixing substances by heat, recently reported by Kahn and Boyd, 1 it was observed that in practically all cases these substances were destroyed when subjected to temperatures ranging from 60 to 65 degrees C. These results were obtained by heating a number of fractions of syphilitic sera at different temperatures in the water bath and running regular Wassermann tests with each fraction. It was felt, however, that the results obtained with the complement-fixing substances present in the sera of syphilitic patients could not be applied to specific complement fixing substances obtained after protein injections, in view of the fact that, in the former case, the antigens employed were non-specific. This has led us to study the rate of destruction by heat of specific complement-fixing antibodies.

The mode of immunization as well as the complement fixation tests were conducted as indicated in the first paper of this series. The tests were carried out in each case with unheated serum and the same immune serum heated to varying temperatures, beginning with 5 minutes at 56 degrees C. and ending with I hour at 65 degrees C. It was soon found, however, that these temperatures did not lessen the antibody content of the rabbit serum, and that the thermal destructive point of these complement fixing antibodies existed apparently at a higher temperature level.

The sera were then diluted 1-10 with saline in order to raise the protein coagulation level (Eberson) and placed in the water bath for 2 hours at 65 degrees C.; I hour at 70 degrees C.; and 1/2 hour at 75 degrees C.-without any apparent effect on the antibody content.

Various methods have been used in order to overcome the difficulties attendant upon the carrying of a minute organism, such as a protozoön, through the numerous reagents necessary for imbedding in paraffine preliminary to microtome sectioning. It has been found that the phenomenon of coagulation in blood plasma makes it of use in work of this type. In carrying out this method, a few drops of blood plasma is placed on a depression slide under a binocular microscope and the specimen at once placed in it with a pipet. After the plasma coagulates, which will take place in a very few minutes, the specimen will be found to be firmly imbedded in a resistant fibrin clot which can be taken through the various reagents, imbedded in paraffine, sectioned in any desired plane and finally mounted on a slide. In brief, the clot containing the specimen may be treated as a regular piece of tissue of the same size.

The orientation of the specimen in the clot may be accomplished before the clot is fully formed or at the time when the clot is in the clearing fluid just prior to imbedding. In the clearing fluid the clot becomes transparent and can be examined under a microscope and the imbedded specimen located in it. The clot can then be trimmed so as to indicate the orientation of the specimen.

Plasma of various animals can be used. I have generally used frog plasma and have secured it by the method previously described. 1

Blood plasma has also been found to be useful in holding small animals firmly in a certain position so that they can be dissected. In this connection it may be noted that the dissection can proceed as far as desired and then no matter how fragile the dissected parts may be, additional plasma can be added and the fragile dissected part or parts imbedded in a clot in the same manner as described above.

The serum of the domestic fowl has been found to protect mice and guinea pigs against infection with the pneumococcus. The protective power of the serum was demonstrated by giving mice, intraperitoneally, 1 c.c. of the serum and giving the infecting culture by the same route four to six hours later. The following protocol illustrates the plan of the experiment and the results obtained.

Further experiments showed that the serum protects against all serologic types of pneumococci and that there is a particular protective substance for each type. Thus, serum adsorbed with Type I organisms no longer protects against this type but protects against Type I1 cultures as well as before the adsorption and vice versa. This relation exists between all of the types.

A peculiar relationship was found to exist between Type II strains and the subtypes of this group. Thus, serum adsorbed with Type II organisms failed to protect against Type II culture but still protected against the cultures of Types II A and II B. Serum adsorbed with Type II A organisms loses its protective power for II A and II cultures but not for II B cultures. Also, serum adsorbed with II B organisms no longer protects against II B and II cultures but still protects against II A cultures. Hence, in terms of the protective substances in chicken serum, Types II A and II B are main groups with Type II as a subgroup to them both.

Fractionation of the serum by means of ammonium sulphate and dialysis showed that the protective substances adhere quantitatively to the water-insoluble globulin.

At the request of Dr. H. M. Smith, commissioner of fisheries, certain studies have been made at the Woods Hole Laboratory of the Bureau of Fisheries on the factors influencing fish migration.

In testing reactions to various salts a simple apparatus was devised that seems to eliminate some of the drawbacks of the well-known Shelford apparatus.

It consists (Fig. 1) of two troughs running almost parallel into a large trough which has a movable partition permitting the water to escape at its sides. By a simple arrangement of stopcocks it is possible to change the inflow of any substance from one trough to the other very quickly. It is also possible to secure conditions similar to those in the Shelford apparatus by using an intake at the end of the large trough, thus furnishing three intakes, two of them parallel to each other. In this case the movable partition is not used, the outflow being in the middle of the larger trough.

The apparatus used during the summers of 1919 and 1920 is 20 feet long. The dimensions of the receiving trough are 10 feet in length, 8_3/4 inches in depth and 8% inches in width. The two tributary troughs are each 10 feet in length, 4 inches in depth and 4_1/2 inches in width. The tributary troughs have been elevated varying degrees so as to cause the water to flow downwards into the receiving pool. In addition to the use of barrels as containers for the solutions used, in some experiments the inflow has been directly from the circulation pipes of the laboratory.

The fish used in the experiments included Fundulus heteroclitus, Funddulus mnjalis, and Clupea harengus. They were tested with sea water of various dilution, and concentrations, m/10 solutions of NaC1, KCl, MgCl₂, CaCI₂, MgSO₄, various combinations of those salts and also with sewage polluted water.

The discovery of the interesting pharmacological properties of benzyl benzoate and benzyl acetate first announced by the author in these Proceedings and the widespread therapeutic use of benzyl benzoate which immediately followed it, has naturally stimulated various observers to search for other benzyl compounds which might be available for clinical work. A number of such compounds have been since described, but none of these to the author's knowledge possessed together the two characteristics of benzyl benzoate, namely simplicity of chemical structure and efficiency of pharmacological action. The present author has also examined a number of benzyl preparations and found a majority of these unimportant in comparison with the original drug. In the present communication, however, it is proposed to describe briefly three benzyl preparations which have more than scientific interest in as much as at least two of these may be suitable for therapeutic application in special cases.

Benzyl benzoate is a synthetic compound, but is also found in nature and it has been suggested that for the production of the benzyl effect Cinnarnein might be used. Cinnamein is a mixture of esters, alcohols and other substances obtained from the balsams of Peru and Tolu. Among these are benzyl benzoate and benzyl cinnamate. Cinnamein is a drug possessing a pleasant aromatic odor and bitierish taste and is anesthetic to the tongue and lips. Pharmacological experiments with it showed distinct benzyl effects on smooth muscle organs. The toxicity of the preparation was, however, found to be greater than that of benzyl benzoate, for rats, guinea pigs and cats, the ratio of toxicity between cinnamein and benzyl benzoate being three to two. This drug was administered therapeutically to a number of cases.

Methods hitherto in use have proved unsatisfactory for recording murmurs, except such as are loud and low-pitched or of a sustained musical character and not very high-pitched. Einthoven's method, in which a carbon microphone is used, gives murmur records which are often more complicated than the sounds themselves, a circumstance which appears to be due to lack of damping in the moving parts of the microphone, especially of the carbon particles. In most microphones the diaphragms have a natural period too low for best results, but the undamped motion of the carbon particles is the most serious drawback. The methods not involving use of a microphone all suffer from a common defect. They are insensitive if the instruments are made of high natural period and if made sensitive they give records so distorted as to be almost valueless except for time-relation studies of the first and second sounds and of very intense murmurs.

To overcome these difficulties an electromagnetic telephone has been used to convert the sound oscillations into electrical oscillations. This telephone is provided with an air-damped diaphragm of high natural period. A shallow ring fastened to the telephone cap serves as “mouthpiece” and has a small lateral opening to maintain atmospheric pressure within it except for the sudden variations due to sound. This device is applied directly to the chest wall and the air column confined between the diaphragm and the chest wall is very short and has a high natural period.

The currents produced by the telephone are amplified by a four-stage amplifier used in such a manner as to give practically distortionless amplification. It is necessary to protect the vacuum tubes against extraneous sound and mechanical vibration.

Attempting to shorten the incubation period of botulinus toxin by injecting gradually increasing amounts of toxin into mice, one of us (Orr) has observed early that when the amount of culture filtrate injected reach 0.5 c.c. animals frequently died within a few minutes after injection. Further study of the nature of this intoxication has brought out that when B. botulinus is grown on suitable medium there are produced, in addition to the specific toxin, other poisonous products. If such a culture is filtered through a Berkfeld candle and the filtrate treated with alcohol these secondary toxic products remain in solution. The alcoholic extract equivalent to 0.5 c.c. of the original filtrate is fatal when injected intraperitoneally into mice of 17-22 grams.

The first symptoms are noted immediately after injection and consist of restlessness and marked contraction of the abdominal muscles. Within a minute or two the animal shows increased response to external stimuli especially to sharp sound. Shortly the animal becomes prostrated. The increased excitability persists a few minutes longer, the respiration increases in depth and decreases in frequency, and may become as infrequent as 5 inspirations a minute. The animal goes into coma interrupted by sharp convulsive seizures with contraction of the extensor muscles throughout the body. Death occurs in 5 to 15 minutes after the injection and during one of these convulsive seizures. If less than a lethal dose is injected the animal may exhibit all the symptoms described above but will recover completely in 2 to 4 hours.

The toxic substances responsible for these acute symptoms are quite distinct from the specific botulinus toxin and are not neutralized by the specific antitoxin. Besides we obtained similar products from the cultures of “atoxic” strains of B. botulinus, as well as from those of B. sporogenes, B. tetani and B. proteus when these organisms were grown on medium composed of minced meat broth. 1

Spathidium spathula is an holotrichous infusorian whose sole diet consists of smaller ciliates, chiefly species of Colpidium. If the truncated anterior end of a Spathidium, which is swimming forward and revolving on its long axis, happens to come into direct contact with a Colpidium, the latter usually becomes motionless at once and its protoplasm shows signs of pathological changes.

Paralysis and death of the prey is apparently brought about by the liberation of a specific substance from the oral region of Spathidium which is toxic to small ciliates. Various authors have ascribed this result to trichocysts about the peristome, but as a matter of fact none are present. However, a number of rodlike bodies can be demonstrated in the thickened rim of the peristome which may represent the seat of the poison. One is at liberty to interpret these bodies as “trichocyst material,” though the poisonous nature of trichocysts, even in Paramecium, remains to be demonstrated.

The paralysis of a Colpidium by a Spathidium results in a marked change in the latter's behavior, which up to this instant has comprised random swimming movements. If the Colpidium happens to remain against the truncated anterior end, the Spathidium forthwith proceeds by means of the thickened edges of the peristome to force its prey down through the slit-like mouth into the interior of the cell. On the other hand, if the Colpidium is not instantly rendered motionless and becomes removed a short distance from the oral region of its captor, the latter institutes a series of rapidly repeated avoiding reactions. This behavior has a tendency to keep the animal in the general vicinity of its quarry because each time the Spathidium starts away its forward progress is checked by the reaction.

One of us has shown that a dog placed in an ice-water bath up to the neck exhibits as one of the reactions to cold a concentration of the blood, losing approximately 10 per cent. of its blood fluid. Such extreme temperatures, however, even well-nourished dogs are unable to resist, the rectal temperature falling rapidly.

Investigating further the mechanism of regulation against cold we attempted to find first a bath temperature which, while not sufficiently cold to lower the body temperature, would arouse distinctly the regulation against cooling. Secondly, on dogs deprived of their nervous regulatory mechanism by section of the cord between the sixth and seventh cervical segment, it was attempted to determine whether the failure to resist the temperature of baths not too cold to be normally withstood is associated with inability to concentrate the blood.

It has been found that normal dogs will exhibit a fairly efficient regulation against cooling in a bath the temperature of which is maintained for a half hour or more at 20° C. In two such dogs there were net gains in body temperature as a result of exposure to the bath (respectively 0.1° C. and 0.4° C. in forty minutes). In a third animal the temperature fell 1.5° C. during the first twenty minutes but in the following thirty-five minutes showed a gain of 0.8° C., thus showing that the regulation against cooling was established after a delay.

In these three normal experiments the increases in blood solids were I, 2.1 and 1.8 per cent. respectively. The following table illustrates these points. In all of the experiments here reported the room was kept at a standard temperature of 22° C.

With the above should be contrasted the effects of similar baths upon three dogs with transection of the cervical cord, as shown in Table 11.

Experiments upon normal dogs, kept at rest in hot and cold baths up to the neck, have shown regular changes in total blood solids. Blood solids were determined simply by weighing a sample of 15-16 drops shed freely from the ear vein and drying to constant weight.

Exposure to hot baths for various intervals is illustrated by Table I, to cold baths by Table II.

It will be seen from the tables that normal dogs respond regularly to a moderately high environmental temperature by hemodilution and to a cold environment by hemo-concentration. Roughly, the change usually approximates 2 per cent. of the total blood weight, which means a 10 per cent. change in the fluid content of the blood.

The circulatory factor in regulation against overheating and cooling consists not merely in transferring of blood respectively to or from the body surface but also in actual shifting of water into or out of the blood stream. The response to a hot environment is peripheral vasodilation plus hernodilution; a cold environment evokes vasoconstriction plus hemoconcentration.

To simplify the above presentation the rectal temperature readings have been omitted; these showed in the case of the cold baths and sometimes in the case of the hot, that the bath conditions were too extreme for the animal to withstand in spite of the regulatory responses.

Confirmatory evidence has been accumulated since the above experiments were carried out.

A series of pedigree lines of Didinium nasutum, consisting of the progeny of a single exconjugant, were bred continuously under practically constant environmental conditions from December 16, 1919, to September 10, 1920.

A cytological investigation of the preparations made daily from stock left after each isolation, demonstrated the absence of a morphological micronucleus in vegetative, dividing and conjugating animals. In the race from which these pedigree lines were derived by conjugation, and also in other unrelated races of Didinium, micronuclei were easily demonstrable. It is clear, therefore, that the parent race was micronucleate, while one of its progeny, a single exconjugant (the original cell of the race under investigation) was amicronucleate. Moreover, this amicronucleate condition persisted throughout the life of the race, that is, through 652 generations.

At various intervals during the life history of this culture, periods showing a tendency for encystment and conjugation occurred, but the animals which encysted or conjugated invariably died, a fact undoubtedly related to their amicronucleate condition. Rhythmical periods of depression followed by increased vitality, which resembled those shown by Woodruff and Erdmann 1 to be associated with endomixis in Paramecium caudatum were also observed. Endomixis, however, if such occurs in the free living state of Didinium, could not be carried out in this race during the rhythmical periods owing to the lack of a definitive micronucleus.

The amicronucleate state without doubt arose from some irregularity during conjugation. Prandtl 2 in his study of the cytology of conjugation in Didinium found individuals in which all the micronuclei had become transformed into macronuclei, but he thought animals so endowed were incapable of living. The present study shows that they are viable but unable to undergo processes such as encystment, conjugation, and endornixis which are dependent largely upon micronuclear activity.

One year ago we read a paper before this society on the “Calcium in the Blood of Patients with Furunculosis and Acne.” We were aware that we obtained very large amounts of calcium in the blood of some of the patients, but were unable to detect our error. At that time we used the gravimetric method and with it were unable to detect any calcium in the filter paper used, although we made six attempts. This year, after perfecting ourselves in the titration method for calcium determination, as given by Halversan and Bergeim, 3 we found that some of the filter paper we had been using contained considerable amounts of calcium. We then decided to abandon the gravimetric method and have used the titration method in obtaining the results given here.

We have made nearly one hundred determinations of the amount of calcium in human blood in the following conditions:

The figures given are milligrams per 100 C.C. of blood plasma: Acne 12.1, 10.0, 9.0, 4.5, 6 8, 8.3, 11.3, 10.6, 10.6, 9.8, 8.3, 9.8, 6.0, 4.9, 11.0, 9.1, 13.2 (16.6, 16.5 repeated), 13.2, 15.9, 12.1, 7. I.

Furunculosis 6.9 (Dr. J. I .I, February 11; 5.9, March II), 9.4, 9.1, 12.0,8.0,8.1,9.8.

Normal 9.8 9.4, 7.9, 9.1 9.3, 8.3, 7.8.

Diabetes (8.7, January 27; 9.4, January 31; 7.9, February 9; I .I, February I I), 5.0, 10.0, (10.0, January 31; 3.4, February 28), 4.8,(7.5, February 15; 10.6, February 25; 11.1, March 2), 9.8, 8.7, (8.8, February 4; 10.2, February 16), 9.8, (7.2, February 8; 6.0, February 11; 9.1, March 2), 8.7, (11.0, February 9; 12.8, February 14), 12.5, 10.2, 9.8, 9.1, 11.0, (9.8, February 21; 10.6, February 25), (7.9, February 25; 5.3, March 7), 3.7, 10.0, 8.0, 8.0, 5.9, 11.1, 5.3, 16.7; 11.1.

In continuation of the unpublished studies of Jencks in this laboratory upon blood regeneration in the rat, it is desired to report the following data: The normal erythrocyte count of rat's blood ranges between 7.5 and 10.5 millions per cubic millimeter; and the hemoglobin content between 110 and 140, as determined by the Smith-Cohen method. Data from rats of varying ages and different sex appear to fall within these limits.

Single hemorrhages, equivalent to 2 per cent. of the body weight, and double hemorrhages, of 2 per cent. on two successive days, were carried out. With the latter procedure it was found possible to reduce the erythrocyte count and hemoglobin content to about one third normal.

On the usual mixed food regeneration of blood was complete in from 7 to 10 days after single hemorrhage, and in 10 to 14 days after double hemorrhage; erythrocyte count and hemoglobin content being taken as indices of regeneration. During starvation regeneration followed single hemorrhage in normal time, but was accompanied by heavy loss in body weight. In starvation following double hemorrhage, the animals usually succumbed before regeneration was complete. Splenectomized rats appear to regenerate in normal time on the normal diet. Rats reared upon the Osborne-Mendel fat-deficient diet showed normal erythrocyte count and hemoglobin content, and after double hemorrhage regenerated in normal time upon the same diet. On a diet deficient in either protein, vitamine, or mineral matter, blood regeneration was appreciably delayed, after double hemorrhage. Hence the data at present available suggest that although blood is parasitic upon the other tissues, any one of the three dietary factors just mentioned can be made a limiting factor in the rate of regeneration, if the hemorrhage is sufficiently severe.

We owe to the kindness of Doctor Bordet a strain of B. coli with which he carried on his studies, a certain quantity of the corresponding lytic agent and a typical mucoid strain of his modified coli. With this material we have observed the following facts.

1. The inhibition produced by the lytic principle on the growth of B. coli is greatly influenced by the reaction of the medium: faint in a slightly acid (P_H 6.8) or neutral (P_H 7.0) or even slightly alkaline broth (P_H 7.4), it is much stronger in a more alkaline medium (P_H 8.0 or 8.5).

2. We have isolated from the original strain of B. coli two types of organisms: the one, Type S, 1 is sensitive to the lytic agent; the other, Type R, 1 is much more resistant. These types are distinguished also by other characteristics: type S grows quickly in artificial medium and is non-motile; type R grows móre slowly, is extremely motile, much less phagocytable and more virulent. Both types ferment carbohydrates, saccharose excepted; type R decolorizes neutral red, type S does not. Both types keep their individuality even after passage through a guinea pig.

3. The original lytic agent was found to be specific; it acted exclusively on the coli with which the guinea pigs had been injected. By allowing this original lytic principle to act on broth cultures of our two types of B. coli, we have obtained two new filtrates. The first, resulting from dissolution of the sensitive strain S, is specific as is the original filtrate. But with the second, obtained from the resistant strain R, Doctor Wollstein of the Rockefeller Institute has found a marked action on Shiga, on Flexner and on Hiss dysentery bacilli.

The occurrence of rickets in white rats maintained under laboratory conditions has been well known to pathologists since the first publication of Morpurgo in 1900; and the essential identity of the lesions with those of human rickets has been established by the work of Morpurgo himself, of Schmorl, of Weichselbaum, and especially by the detailed histological studies of Erdheim. One of us (A. M. P.) also has had opportunity to become familiar with the disease in rats, in the course of an investigation of the possible influence of the thymus upon the teeth and skeletal system. In none of these investigations, however, were the dietary conditions of the rats standardized and controlled.

In continuing by means of feeding experiments upon rats the study of the mineral elements in nutrition which has engaged much of the attention of one of us for the past fifteen years, we have found a relatively simple diet which has led to the development of rickets in every one of the cases thus far examined; while complete protection was afforded by the addition of 0.4 per cent. of potassium phosphate (K₂HPO₄) to this diet, or (more strictly) by the introduction of this mount of potassium phosphate in place of a part of the calcium lactate which the rickets-producing diet contained.

In our experience healthy rats of families on normal diet when separated from their mothers at 28 to 30 days of age average about 40 grams in weight and a calcium content (of about 0.3 gram or 0.7 per cent. If then placed upon good diet the calcium content of the body increases in greater ratio than the body weight so that the percentage of calcium in the body rises continuously until at about four to six months of age the adult percentage of calcium—about I to 1.25 per cent. of the body weight—is reached, after which the weight of the body and the weight of calcium which it contains continues parallel until growth is complete.

In studying the growth characteristics of mucoid bacilli Thjotta observed that B. influenzœ will grow in blood-free broth containing the mucoid material from cultures of Friedlander's bacillus and other closely allied organisms. The growth accessory substance or substances which can replace blood and blood derivatives in the cultivation of Pfeiffer's bacillus Thjotta found in both the saline suspensions and watery extracts of the heat killed bacillary material. Furthermore, these bacterial emulsions and extracts can be boiled for ten minutes and filtered through Berkefeld candles without losing their growth-inducing properties. It was thought not unlikely that the growth-stimulating effect of these bacterial extracts might be due to substances belonging to the class of the so-called vitamines. To test this assumption, similar extracts were prepared from yeast cells which are known to be rich in growth accessory substances. These extracts, even in minute amounts, were found capable of promoting growth. Extraction of the growth accessory substances from another source, namely, green vegetables, was tried; extracts of fresh tomatoes, green peas and string beans were found remarkably active in stimulating growth. These active yeast or vegetable extracts when added to broth greatly accelerate growth of organisms such as Bacillus influenzœ and pneumococci, so that within five hours abundant growth is evident. In the case of pneumococcus, a seeding too minute to initiate growth in plain broth alone will amply suffice to induce abundant growth in the same medium if a small amount of extract containing these growth accessory substances is added. In the case of Bacillus influenzœ, when seeded from blood media, luxuriant growth occurs in plain broth containing yeast extract, while no growth takes place in the same broth without the addition of extract.

To 1 c.c. of blood serum in a 10 c.c. test-tube, add 4 c.c. of a 1 per cent. solution of ammonium oxalate. Let stand for 1/2 to 1 hour, and centrifuge for 5 minutes. Pour off the supernatant fluid. Wash the precipitate three times with distilled water, recovering the precipitate by means of centrifugalization. Add to sediment 1 c.c. distilled water and transfer to a vitreosil crucible. The test-tube is washed with distilled water and the washings also collected in the crucible. The water is slowly evaporated, and the precipitate burnt in a strong flame until CaO is formed. Dissolve the ash in 1/2 c.c. N/50 HCl, add 1 c.c. distilled water and titrate excess of HCl with N/100 NaOH, using phenolphthalein as an indicator. The amount of CaO can now be calculated.

Eight analyses may be made by this method in one hour. Remarkably uniform results are obtained upon repeated examinations of the same blood. For clinical purposes, this method is of great assistance.

The fact may be recorded that highly virulent strains of Treponema pallidum are capable of penetrating some portion of the genital mucosa of normal rabbits and setting up an infection without necessarily producing the first gross lesion at the portal of entry. This fact was recently determined in 9 rabbits and with two highly virulent strains of Treponema pallidum.

The experimental method employed was as follows: The sheath of the animal was drawn forward to form a pouch into which was instilled 0.05 c.c. of a testicular emulsion rich in spirochetes. About 30 seconds were allowed for the emulsion to spread before releasing the sheath. Most of the fluid then ran out and between 0.04 and 0.05 c.c. could be recovered showing that only a thin film of the emulsion was retained. Infection was first indicated by enlargement and induration of the inguinal lymphnodes and later by the development of a general lymphadenitis with syphilitic lesions in other parts of the body.

All animals thus far inoculated by this method have become infected. In some of them, enlargement and induration of the inguinal nodes was clearly recognizable within 24 hours after the application of the emulsion. Thus far (5 weeks) only one of the animals has developed a visible lesion on either the penis or the sheath, although several of them have characteristic lesions in the testicles and scrotum.

The observations on these animals are not yet complete and the full significance of the experiments cannot be ascertained until the course of the infection has been followed much longer. Similar experiments with other mucous membranes are in progress.

Neoplasia as a sequel to syphilitic infection is not uncommon in man but has never been recorded in an experimental animal. Recently, we have observed an atypical growth arising from the scar of an old syphilitic lesion in the scrotum of a rabbit which may prove to be a neoplasm.

The animal was inoculated in the scrotum, June 16, 1916. Small chancres developed and then underwent spontaneous regression. Several months later, there was a recurrence and the lesion in the left scrotum persisted for some time. In October, 1920, there was a slight diffuse infiltration of the left scrotum and a small nodule appeared at the site of the old chancre. Although it was known that the animal still harbored spirochetes, 1 none could be demonstrated by dark-field examination of material from the nodule and it was excised for histological examination. There was a prompt recurrence and with the growth of the second cutaneous lesion, the left inguinal glands became markedly enlarged and indurated. Again no spirochetes could be demonstrated and the lesion with one of the adjacent glands was excised under ether anesthesia. Histological examination of the cutaneous nodules and gland showed a growth which presented more the appearance of a neoplasm than of a syphilitic lesion. It was composed for the most part of atypical epithelioid cells undergoing active proliferation and exhibiting marked invasive tendencies.

Meantime there was a second recurrence and extension of the skin lesion over the mid line at the pubis with enlargement and induration of the right inguinal nodes. A deterioration in the physical condition of the animal was then apparent and progressed very rapidly, culminating in emaciation, weakness, severe anemia, loss of sphincter control with some spasticity of the hind legs and the formation of trophic ulcers about the anus and sheath.

The action of acetone was studied on cats and dogs under light ether anesthesia as well as on the isolated heart of the frog and turtle. In the experiments on intact animals acetone injected intravenously produced a fall of blood-pressure amounting in some cases to fifty per cent., especially in dogs when given in a concentration of fifty per cent. in saline solution, and about 2-3 c.c. per kilo. But in a number of experiments smaller quantities of acetone injected in a concentration of 25 per cent. in saline solution produced a similar effect on the circulation. This occurred almost uniformly in cats. Recovery was prompt, as a rule, even when 50 per cent. acetone was given, but occasionally low blood-pressure persisted for 2-3 minutes when larger amounts were injected. The fall of blood-pressure was due to cardiac depression, as was indicated by the fact that there was a corresponding diminution in the volume of the kidney shown by oncometric studies. That acetone in certain concentrations depresses heart action was also observed in our experiments on the isolated heart. Ten per cent. acetone in Ringer solution produced in all our experiments arrest of the heart, and very frequently the same effect was obtained with a five per cent. solution of acetone. Recovery, however, was observed in all experiments, even when the heart was perfused with ten per cent. acetone for several minutes. Not infrequently a stimulating after-effect was observed. That no permanent injury occurred was further shown by the fact that repeated perfusion of the same heart was followed by recovery. With low concentration of acetone no change in the heart action was observed.

Observations were also made on the behavior of the vagus mechanism under the influence of acetone.

Although taurin differs from that particular group of amino acids which constitute the building stones of the proteins in that it contains a sulphonic instead of a carboxyl group, it nevertheless closely resembles the amino acids in its chemical properties. Like the amino acids taurin can be quantitatively estimated when in pure solution either by treating it with HNO₂ and determining the nitrogen set free or by titration with alkali in the presence of formalin. It possesses amphoteric properties 1 and reacts with many of the amino acid reagents. Thus Paal and Zitelmann 2 found that taurin reacts with phenylisocyanate to give α-phenyl-ureidoethylsulphonic acid, Gabriel 3 and his associates prepared benzoyl- and phthalyl-taurin, and Bergell 1 has attempted to use β-naphthalinsulphotaurin as a means of estimating taurin in urine.

We have found that taurin like the carboxy amino acids 2 reacts with α-naphthylisocyanate to yield the corresponding hydantoic acid. The barium salt of a-naphthylureidoethyl sulphonic acid was prepared by adding to a concentrated solution of taurin which contained an equivalent amount of KOH, 1¼ equivalents of a-naphthylisocyanate and the mixture was shaken at frequent intervals for an hour. The insoluble dinaphthyl urea was filtered off and on acidifying with HC1 the solution thickened but the free hydantoic acid did not separate. On addition of BaC1₂ solution the barium salt of the hydantoic acid was precipitated. This was washed with small quantities of ice water and then with alcohol and was dried to constant weight. Estimations of C, H, and Ba gave (per gram of substance) the following results.

One of the pressing needs of biochemistry and allied sciences is an adequate supply of pure amino acids. While methods for the isolation and purification of these substances are well known, nevertheless the labor and expense involved are in many instances prohibitive except for the production of relatively small quantities. With the exception of glutamic acid no use for amino acids other than for scientific purposes has been found and manufacturers have as yet been unable to apply the benefits of quantity production sufficiently to make these substances cheap enough for extensive investigations.

It was shown some years ago by Ikeda 1 that the taste of the constituents of the seaweed Laminaria japonica is due to the content of glutamic acid. This is to a certain extent also true of meat extract and allied preparations. The demand for condiments of this type has led to the production of the mono-sodium salt of glutamic acid on a scale large enough to make it relatively cheap.

Our experiments with Ajinomoto (a commercial preparation of sodium glutamate sold by S. Suzuki & Co., Tokio, Japan) were carried out with a view of using this substance as a cheap as well as a convenient source for pure glutamic acid. Our results show that this preparation consists largely of the mono-sodium salt of glutamic acid. To isolate glutamic acid, Ajinomoto is dissolved in a small quantity of water (600 c.c. for each 100 grams) and HCl is added in an amount equivalent to the amino nitrogen present. Purified charocal is then added, the mixture brought to the boiling point, filtered hot and the filtrate placed in the ice chest to crystallize. The crystals are drained, washed several times with small quantities of ice water until free from chlorides and dried.