Experimental Biology and Medicine

These experiments were undertaken to determine whether intarvin—glyceryl margaric ester—yields extra sugar in the completely phlorhizinized dog. Ringer 1 has shown that propionic acid is quantitatively converted into dextrose in such dogs, and if the β oxidation of saturated normal fatty acids holds, one might expect each molecule of margaric acid to yield one molecule of propionic acid during oxidation in the body. In the phlorhizinized animal this should appear as sugar. 26.8 gm. of intarvin, if pure, could yield 10 gm. of dextrose, assuming that one molecule of the fat would yield one molecule of glycerol and three molecules of propionic acid, and that the glycerol and propionic acid could yield two molecules of dextrose. This calculation takes into consideration the 12 per cent mineral oil added to intarvin but does not allow for the fact that the fat probably is a mixture of about 85 per cent margaric acid and 15 per cent stearic acids combined with glycerol.

Phlorhizin was administered either in olive oil twice a day, or in 1.2 per cent Na₂CO₃ every eight hours, subcutaneously. Dogs were fasted for three days before phlorhizination, and urine discarded until a constant D:N ratio was obtained for a 24 hour control period, then the animal was fed intarvin or butter mixed with lean meat hash and the urine followed for the following 24 hours or until the D:N ratio returned to the figure obtained for the control period. Toluene or NaF (1 per cent) were the preservatives used, nitrogen was determined by Kjeldahl's method, sugar by the Allihn method, total acetone bodies by the VanSlyke method, and organic acids by the method of Van Slyke and Palmer. The dogs were most irregular in acetone body formation, and the results in this field are not felt to be of much value.

As ragweed appears to be the chief cause of hay fever in this country it seemed desirable to attempt to separate the substance in the pollen which is responsible for the sensitization of hay-fever patients.

A number of the constituents have been characterized by Heyl, but this author did not attempt to test systematically the various fractions which he isolated. Recently Coca and assistants subjected ragweed extract to the action of trypsin. On dialysis most of the nitrogenous consituents diffused through the membrane while the residue retained its activity; therefore the authors conclude that the active substance is not a protein.

Having found that 3 per cent ammonia was a useful solvent for the preparation of horse dander and other proteins used in clinical work, we also employed it for the extraction of ragweed pollen. Ammonia possesses certain advantages. It is a weak alkali with little racemizing or hydrolyzing effect and the protein obtained by precipitation of the ammonia extract with acetone is readily freed from alkali by drying in vacuo over sulfuric acid.

As the thick hard capsule is said to form 65 per cent of the pollen (Heyl) it was thought that extraction would have to be preceded by thorough grinding, but it was found that ammonia penetrated the capsule and completely extracted the active substance.

The preparations were tested by the cutaneous reactions on patients sensitive to ragweed. The varying sensitiveness of patients had to be considered in the interpretation of reactions.

The following results were obtained with the ammonia extract:

The hydrogen ion concentration of the blood in epilepsy has been determined in thirty-eight cases. In order to be able to ascertain the significance of any changes found, we have also studied the hydrogen ion concentration in the blood of twenty-eight normal individuals. In conjunction with the work of Hastings and Cullen, who have also made a certain number of observations on the pH of normal individuals, we find that the range for the latter is from 7.36 to 7.43. In contrast to this we find that the blood of treated and untreated epileptics ranges from 7.25 to 7.50. The blood of any given case of epilepsy shows a wider range of blood reaction than does the blood of a given normal individual. Chart No. 1 illustrates the extent of the range of blood pH determination in all the cases of epilepsy studied as contrasted with the range of variation observed in all the normal individuals studied. One case of hysteria, three cases of migraine and three cases of Jacksonian epilepsy, and one unclassified case show a range of blood hydrogen ion concentration which is similar to the range observed in normal people.

Recently the writer recorded the occurrence of fifty mice having in each case a solitary kidney. 1 They constituted 16 2/3 per cent of a group of 300 autopsied animals, descendants of animals that were subjected to light doses of x-ray irradiation. The treatment was apparently associated with the production of hereditary abnormalities of the eyes, feet and other parts of the body.

Several matings have now been made where in each case both parents had but one kidney, and the number of young per litter was considerably less than the average for animals with normal kidneys. Several of the young animals died during the first week of life and were promptly destroyed by their mother. The examination of a single litter of seven mice apparently throws considerable light on the high mortality shown in such animals and is considered of sufficient interest to warrant a brief report. In this instance, however, the male parent had but one kidney and was blind in the right eye, while the mother, although apparently normal, was heterozygous for such defects.

Two of the young of the above parents were found dead about 24 hours after birth. They had but recently died and at autopsy apparently showed complete absence of both kidneys. No other gross defects were noted except that one of the animals had a defective left eye. The remaining five animals were active and apparently in a good state of nutrition. Four of these were males with apparent'ly no external deformities, and one was a female with a defective left eye. Three of the animals were killed about 24 hours after birth, and the point of interest is that the female with the defective left eye showed bilateral absence of kidneys and one male had only a very small remnant of renal tissue on one side of the body.

With no prejudice regarding the results to be obtained we have exposed five series of normal rabbits to the quartz mercury arc lamp daily for periods of three to seven weeks and observed at autopsy the weights of certain endocrine glands in relation to the body weights of the animals. It was expected that this survey might orient us for further studies in the biophysics of ultra violet light. The survey was made possible by the cordial cooperation of Doctors Brown, Pearce, and Van Allen, who placed at our disposal unpublished figures from similar examinations on a large number of normal rabbits covering a period of two years. Many controls are required because of individual and especially seasonal variations in the gland/body weight ratio.

Light colored or albino male rabbits, with ears shaved and backs clipped, in groups of eight (two controls), were exposed to the unfiltered radiations of a quartz mercury arc lamp for 30 minutes daily at a distance of one meter. This arbitrary dosage was probably too large at first and relatively ineffective during the final periods, when pigmentation had occurred. It caused a marked erythema with some desquamation, followed by moderate pigmentation. At the expiration of the chosen periods the rabbits were etherized and bled from the aorta. The following glands were weighed and examined in gross and microscopically: pineal, hypophysis, external parathyroids, thyroid, thymus, adrenals, and testicles. The weights of these glands were recorded in milligrams per kilogram net body weight after removel of the gastro-intestinal tract and its contained food and feces, and the expression of bladder urine.

The changes in weight, per cent, compared with the figures for 15 to 36 normal control rabbits examined at the same season of the year are shown in the accompanying table.

This investigation was undertaken in order to ascertain the effect of supplementing a basal diet with dried samples of ungerminated, germinated and green corn. The object of the experiments was to see whether any growth-promoting substances were produced in the course of germination and “greening”.

Experiments with ten groups of rats on diets: (1) regular (normal); (2) S + “A” + “B” (3) S + “B” (4) S + “A” (5) S + “A” + ungerminated corn; (6) S + “B” + ungerminated corn; (7) S + “A” + germinated corn; (8) S + “B” + germinated corn; (9) S + “A” + green corn; (10) S + “B” + green corn—were carried out simultaneously under such conditions as to make the results, from a comparative standpoint, suggestive. During the first month each group of rats (four to a cage) receiving either ungerminated, germinated or green material, was given 4 gm. of such material per day; during the second month this was increased to 8 gm. and during the third month to 12 gm. The results at the end of the first month showed that aside from the group on the “regular” diet (1) the one showing the most notable increase in weight is group 10 (synthetic + “B” (yeast) + green). Since neither 6 (synthetic + “B” + ungerminated) nor 8 (synthetic + “B” + germinated) shows corresponding gains, the most obvious explanation is that a “growth” of vitamin (A?) or an increase in the quantity present, has taken place during “greening”. This view would fall in line with suggestions made by several workers in the field (for example, Drummond2 and Wilson'). But another explanation offers itself as a result of the following experiment:

The addition of cystine, cysteine or their derivatives to the control rations of rabbits caused a prompt and marked increase in the amount of sulfur eliminated in the urine. When these sulfur compounds were withdrawn the elimination of sulfur dropped to the control level, and in some instances even below this. This decrease in sulfur took place in the first two days of the period following the cystine sulfur feeding, and in most cases the first day. On the day following this marked depression of the sulfur elimination there was a distinct increase of sulfur in the urine. The cystine sulfur was not completely eliminated in the period during which it was fed, except in cases of phenyl acetyl benzyl cysteine. The increased sulfur output following the depression of sulfur elimination is considered as a lag. It would seem that the R-SH type of sulfur compound has some specific effect upon the mechanism of sulfur excretion. This effect is observed both with reduced sulfur and oxidized sulfur, but not with conjugated SO₄. In three cases, however, there was an increase in conjugated SO₄—namely 6 mg. for phenyl acetyl benzyl cystine and phenyl hydantoin cystine, and 10 mg. for acetyl benzyl cysteine. This “deferred lag” was unforeseen and must be taken into special account in our next series of this program. The excess elimination of sulfur in some of the tests may be charged to thils “deferred lag”-but not altogether. In the case of phenyl acetyl benzyl cysteine the increased sulfur elimination exceeded the cystine sulfur added to the ration by one-half its amount. This could not come from any previously ingested R-SH added to the animal's normal ration. There seems to be positive evidence that ingestion of R-SH causes a sulfur elimination in addition to the sulfur immediately taken in so that the per cent of cystine sulfur as given in the table may include some endogenous reduced sulfur.

As part of a general experimental study of the epidemiology of certain bacterial infections of domestic animals, experiments have recently been made with rabbits with Bact. lepisepticum.

Rabbits developing spontaneous snuffles at the Rockefeller Institute have been examined bacteriologically during life and at autopsy and have been found to carry in their nasal passages Bact. lepisepticum as the predominating organism. This microbe has been classed with the “pasteurella” or hemorrhagic septicemia group and is known as the bacillus of rabbit septicemia. The constant association of the organism with snuffles and its appearance simultaneously with spontaneous snuffles was considered as sufficient evidence to justify the employment of Bact. lepisepticum in an attempt to produce snuffles experimentally.

Efforts in this direction were followed by confusing and varied results until young rabbits bred and raised at the Rockefeller Institute from snuffles-free and Bact. Cepisepticulm-free parents were employed. These young rabbits which had not been exposed to snuffles and had always been free from Bact. lepisepticum were found regularly to respond to inoculation in a characteristic manner.

Cultures of Bact. lepisepticum, grown in broth, were dropped without trauma into the external nares by means of a 2 cc. Luer syringe. Two strains were employed for inoculation.

The less virulent strain was given to one series of five rabbits. Two of these rabbits developed snuffles; the five controls remained normal.

The more virulent strain was given to three series of 6, 12, and 22 rabbits respectively. The following tabulation shows the result:

Of the first series of six rabbits, three died, two developed chronic snuffles and survived, and one which had shown no evidence of infection remained well. Of the second series of 12, three died, eight developed chronic snuffles and survived, and one which had shown no evidence of infection remained well.

It was stated in the previous communication that following intranasal inoculation of Bact. lepisepticum into rabbits, a certain number of fatalities ensued. These animals died of pneumonia.

The time of onset of symptoms varied from 24 hours to three days following the inoculation. The rabbits developed signs of respiratory distress, appeared very ill, and showed serous or purulent discharges from the nares. Death ensued in from two to seven days as a rule.

Gross autopsy inspection showed fluid in the pleural cavity varying from a few drops to several cubic centimeters. In the acute cases the fluid was slightly cloudy; in the more prolonged cases it tended to be purulent. The appearance of the pleuræ varied in a corresponding manner from mere dullness to surfaces covered with fibrinous exudate. The lungs usually showed massive areas of consolidation; in early cases, on section, moist and hemorrhagic; in later cases, granular and dry. In some of the older cases the small bronchi exuded pus.

Histologically, the acute cases showed a pleural exudate consisting of fibrin, coagulated serum, and necrotic leucocytes. The pneumonic process seemed to start around the blood vessels where the adjacent alveoli were filled with fibrin, serum, and many red blood corpuscles. Elsewhere little change in the alveoli was seen. The blood vessels were surrounded by sheaths of fibrin containing many polymorphonuclear, mononuclear, and eosinophilic cells. The bronchi were normal. In more advanced cases the alveoli were filled with leucocytes in which the mononuclear type predominated. Finally, the involvement became general; usually an entire lobe was affected. The bronchi contained leucocytes and abscess-like formations occurred.

Bact. lepisepticum was demonstrable in the circulating blood, in the pleural fluid, and lungs.

It has, therefore, been possible to induce experimentallly in rabbits the several manifestations of the native respiratory disease called snuffles, pleuropneumonia, and septicemia.

A review of the laboratory records of the Class in Surgery, S. 101-102 (1921-1924) shows 63 operations upon the thyroid with data incomplete in 30 and complete in 32. Of the latter, 16 were superior thyroid ligations following hemithyroidectomies, 3 inferior thyroid ligations following hemithyroidectomies, 11 bipolar ligations combined with the removal of one lobe, one bipolar ligation without thyroidectomy and one excision of adventitia from a portion of the carotid artery. It is believed that a superior polar ligation in a hemithyroidectomized dog, in a measure, corresponds with the ligation of the two superior poles in a human hyperplastic goitre. The experimental bipolar ligation may be analogous to the ligation of all four poles in the human gland. The changes in the thyroid remnants under these conditions are thought to be suggestive of the findings in selected human ligations and thyroidectomies.

Of the unipolar ligations less than 20 per cent remained normal while more than 80 per cent revealed hyperplasia of the interstitial epithelial cells or adenomatosis. Of the latter, approximately one-third showed hemorrhage into the thyroid capsule or into the gland or congestion or replacement fibrosis to some degree. After bipolar ligations, the residual thyroid tissue appeared hyperplastic or atrophic in abut equal numbers of instances. A few of the latter animals died suddenly within two days after operation.

Parathyroids appeared on section in 21 of the above 32 cases. Of these, approximately one-third remained normal while two-thirds showed congestion or replacement fibrosis. Halsted's observation of degenerative changes in the parathyroids following inferior polar ligations appeared in part borne out in the few instances at hand. In one instance colloid was noted with epithelial cells and acini closely resembling thyroid tissue.

In a number of preparations of residue antigen obtained from various micro-organisms, chemical analyses have been carried out to find whether or not the presence of proteins, etc., could be definitely excluded. To this end, total nitrogen has been determined, together with amino-nitrogen before and after acid hydrolysis of the preparation.

The bacteria were grown in large agar plates, washed off with saline and treated in the manner previously described in detail, 1 and the resulting material dried in the dessicator.

Specific precipitin tests were given by saline solutions of the dry material with immune rabbit sera, but insufficient quantities were available to attempt to correlate the dilution at which the test became negative, with the chemical composition. For analysis, the entire specimen was carefully weighed, brought into solution in a few cc. of water, and diluted to a definite volume, usually in a 10 cc. volumetric flask, aliquot portions being taken for separate determinations.

Total nitrogen was determined by the micro-kjeldahl method, titrating with 0.02 N solutions, and the amino-nitrogen determinations were carried out by the micro-Van Slyke method.

Phosphorus was determined by Tisdall's 2 method, after wet ashing.

In those preparations in which sufficient material was obtained for duplicate analyses, agreement was fairly uniform. However, we do not believe that absolute accuracy can be claimed for all the determinations, particularly those of amino-nitrogen after hydrolysis, because of the small quantity available for use, and the relatively low content of nitrogen. The values for total nitrogen are probably the most accurate.

It will be observed that there is a rather considerable variation in each type of nitrogen in the different lots. Considering the results as a whole, one sees that the low and variable total nitrogens point to the presence of a nitrogenous substance associated with a non-nitrogenous compound.

1. According to Leo Loeb (1910) alkali may under certain conditions have a preserving effect on the blood cells of Limulus. 1 If we add a drop of Limulus blood to an excess of an isotonic sodium chloride solution, those amebocytes which come into direct contact with the solution soon send out sharp pseudopodia and lose their granules and become hyaline. Similarly in a watery solution of very weak alkali the cells soon lose their granules and are dissolved; but if both substances are combined, a small amount of alkali being added to the isotonic sodium chloride solution, the cells are preserved much better and their granules may be protected against destruction for a considerable period of time. Alkali may have the same preserving effect even if added to isotonic solutions of anelectrolytes like glucose.

2. This effect of alkali on ameboid cells observed by this method is apparently contrary to the effect of alkali which Loeb and the writer observed by means of the tissue culture method. 2 If the latter procedure is used, alkali causes in the main a more rapid extension and solution of the cells, although even here a preserving effect is noticeable under certain conditions.

Considering this apparent contradiction in the effects of alkali observed, it was necessary to repeat and if possible to extend those earlier experiments, especially in view of the fact that so far they have apparently not been repeated by other investigators.

3. In carrying out such experiments at Woods Hole, last summer, we could confirm the preserving effect of the addition of alkali to isotonic solutions of sodium chloride on the amebocytes of Limulus. We found furthermore that alkali has likewise a preserving effect if added to isotonic solutions of glucose, to which a small amount of sodium chloride has been added.

In previous publications 1 we have shown, (1) that a small amount of acid added to an isotonic sodium chloride solution exerts a certain preserving effect on the amebocytes in a drop of Limulus blood surrounded by an excess of the solution. (2) That in tissue cultures of amebocyte tissue there occurs not only an active migration of the amebocytes into a sodium chloride solution, which has been made slightly acid, but that under certain conditions the addition of acid may increase the duration and intensity of this migration, which is followed by, or associated with, a formation of tissue-like structures not unlike those produced by the mesenchyme of vertebrates. 2 (3) That this effect of acid is due to its action on the cells, which consists in an increase in contraction and consistency of the protoplasm. (4) Inasmuch as we had previously shown that there exists a parallelism between the effect of various agencies on the state of contraction and consistency of the amebocytes on the one hand, and on the other hand, on the character of ameboid movement, the shape of pseudopods, the mode of movement of the granuloplasm, the power of resistance to the stereotropic tendency on the part of these cells, we found in accordance with these observations under the influence of acid a sharpening and lengthening of the pseudopods and a tightening of the granuloplasm and a greater state of contraction of the cells. (5) We found that it is possible, through grading the amount of acid added, to grade correspondingly the effects of the acid on the consistency of the cells in such a way that all gradations could be obtained from the extreme condition of round contracted cells, too hard to carry out ameboid movements, and contracted cells sending out very long, often multiple thread pseuclopodia, to a medium state in which the plasticity of the cell is greater and the ordinary tongue pseudopods are formed, and finally to the other extreme condition of cells which are very soft, which spread out readily and soon degenerate (Loeb & Blanchard).

During the past few months Winter and Smith, 1 Best and Scott, 2 Funk and Corbitt, 3 Collip, 4 Perry, 5 and Dubin and Corbitt 6 have reported the presence of a substance in yeast and in plants and vegetables, which causes a fall in blood sugar upon injection into normal rabbits. In view of these results the possible presence of this substance in various fruits suggested itself to us.

Various methods of extraction were used with oranges, grape fruit and lemons with the following results:

1. In every case a blood sugar increasing and a blood sugar decreasing principle was clearly demonstrated.

2. The type of curve obtained was dependent upon the varying amounts of either substance present.

3. Predominance of the toxic fraction (blood sugar increasing substance) invariably produced a preliminary rise in the blood sugar which was followed by a fall below normal. The period of delay in the fall of blood sugar varied with the amount of the toxic substance present.

4. By methods of purification, which wit1 be reported later, it is possible to separate to a very large degree the hypoglycemia-producing principle from the hyperglycemia-producing substance; at least by these methods the double effect described when both substances are present, can be eliminated and a continuous rise or fall of the Flood sugar can be obtained.

5. The hypoglycemia-producing principle and the hyperglycemic substance are present in the juice, pulp and rind of the fruits we have examined.

6. The hypoglycemia-producing principle acts similar to insulin. No delayed action has been observed as described for “Glucokinin”.

The above findings appear to be in agreement with those reported by Dubin and Corbitt for vegetable extracts. At present it is impossible to state whether the slightly delayed fall in blood sugar of normal rabbits is a qualitative or a quantitative phenomenon of the toxic fraction.

In a previous communication on the nature of specific hemolysins, one of us¹ has dealt on the complexity of erythrocytes as antigen and endeavored to demonstrate that the cell stroma is the main, if not the sole source of hemolysinogens. In this paper, as a supplementary study, we have undertaken to demonstrate that isohemagglutinogens, in a similar way, originate in the cell stromata.

To obtain cell stromata in a more or less pure condition and individually from each of the four different types of bloods we used the same technique as discussed in our previous communication. In short it consists of: First, isolating the four types of erythrocytes. Samples of blood, left over from Wassermann reactions were used for this purpose. Second, filtering through gauze and centrifugalizing the saline suspension of broken clots. This step was repeated until the supernatant fluid was free of serum ingredients. Third, hemolyzing the final sediment in 0.45 per cent saline solution and sedimenting stromata by centrifugalization. To get rid of hemoglobin further sedimentations were done in physiological saline to avoid deterioration of cell stromata.

The final product, when examined under the mitroscope, should show the cell stromata intact, more or less globular, varying in size from that of a blood platelet to half the size of an erythrocyte and devoid of hemoglobin. Presence of hemoglobin means deficient “dehemoglobinization”, which will necessitate repetition of step three until dehemoglobinized cells (cell stromata) look almost transparent.

Although of some general interest, the question of the heredity of species-specific properties in animals as indicated by serum reactions has not yet been investigated. While some difficulty exists in discriminating substances derived from closely related species by precipitins, it is easy to do so by the use of hemagglutinins.

After producing agglutinins in rabbits with the blood, for instance of the horse or donkey, one obtains, by absorption with the heterologous blood, solutions which act specifically on the blood of the species used for immunization. In this way the blood corpuscles of a species-hybrid, namely the mule, were examined by means of immune sera against horse, donkey, and mule blood. Of the several possibilities, such as conformity of the blood of the hybrid with that of one parent, a combination of the properties of both, or formation of new substances, the one which actually occurs is the inheritance of agglutinable substances of each parent.

It remains to be estabilished whether the results are different in the cross between the mare and the jack and the reciprocal cross.

We hope to extend the studies to cases of fertile hybrids.

Novasural is a preparation containing 33.9 per cent of mercury bound in a large molecule. It is given as a 10 per cent solution in doses of 1 to 2 cc. It was originally introduced as an antisyphilitic, but its marked action on urine output suggested its use as a diuretic. It was first used for this purpose by Zeiler. 1 Later studies have been published by Saxl and Herlig, 2 Mühring 3 and Nonnenbruch. 4 These writers agree as to its efficiency as a diuretic in selected cases, but fail to agree on the mechanism of its action.

In our cases novasural has been administered to patients with severe heart failure and edema in whom digitalis had failed to relieve the edema. It has been administered in 1 to 2 cc. doses intramuscularly and has been followed by a striking diuresis—the output often rising from 300 cc. to 2000 to 3000 cc. per diem.

The diuresis as a rule starts within two hours and attains its maximum in three to nine hours. The more serious the case, the more delayed is the time of the maximum diuretic effect. The action passes off in about 24 hours.

Studies have been made of the urine, blood, and when possible the edema fluid before, during and after the diuresis. During the diuresis the total output of chlorides in the urine is increased and the concentration is also increased. The total urea and ammonia in the urine may be increased, but the concentration is reduced. The blood chlorides are slightly reduced and the chlorides in the edema fluid follow closely those of the blood. The blood urea shows practicallly no alteration.

There has been marked clinical improvement in all cases and no untoward effects have been observed.

Macht and Lubin have called attention first in these Proceedings and again in a more extensive publication appearing elsewhere to the remarkable phytopharmacological properties of menotoxin, the toxin present in the blood and other secretions of women at the beginning of menstruation. It was pointed out that while this toxin was extremely poisonous for plant protoplasm it was comparatively very little poisonous for animal tissues, although differences in toxicity between normal and menstrual blood for animals have been found. This difference between the phytopharmacological and zoöpharmacological actions of menotoxin were shown to hold for microscopic organisms as well. Thus menstrual serum was quite toxic for bacteria such as B. coli which are generally classed with plants on the one hand, and the same serum was but slightly toxic for trypano-somes which are more closely related to animals, such as the flagellates, on the other. In this connection it was interesting to inquire into the toxicity of menotoxin for spermatozoa.

Fresh and actively motile spermatozoa were obtained from men and the effect of various toxins was studied on the same in hanging drop preparations. It was found that menstrual serum was not more toxic for spermatazoa than normal serum. The authors made further studies on the effect of various chemicals which seem to be closely related to menotoxin. As has been pointed out by Macht and Lubin, menotoxin exhibits chemical and pharmacological properties resembling oxycholesterol on the one hand, and the bile acids on the other hand. Experiments were made with emulsions of oxycholesterol in normal serum, with solutions of cholic acid and other closely related chemicals. It was found that neither oxycholesterol nor cholic acids had any marked toxic effect on spermatazoa. The results obtained with these various substances agreed with the findings in the case of menstrual serum and further substantiate the close relationship between the chemical nature of nienotoxin on the one hand and oxycholesterol and cholic acid on the other hand.

The effect of corpus luteum extracts were studied on the behavior of rats in the circular maze according to the methods described by the senior author elsewhere. Sixteen young adult, female rats were trained in the maze until they could solve the problem in the shortest period of time without committing any errors. They were then given injections subcutaneously or intramuscularly of “lutein”, a protein-free extract of corpus luteum of the sow, prepared by Hynson, Westcott and Dunning. It was found that injections of doses varying from 0.2 to 0.5 cc. of lutein in the rats the average weight of which was about 150 gm. produce a distinct stimulation in their muscular activity and cerebro-spinal efficiency. This effect was noticeable 15 minutes after injection of the extracts and persisted for 24 hours. Injections of the corpus luteum in ovariectomized rats produced a similar stimulation in the behavior of the animals. A few experiments performed with injections of extracts of whole ovary yielded the same results. Further details to appear elsewhere. (Journal of Comparative Psychology).

The work presented in this paper is the first of a series of studies which were undertaken with the view of determining the existence of a reflex path between the vestibular nerve and the autonomic or visceral nervous system. This investigation was prompted by a two-fold purpose: to establish, if possible, the existence of a tonic influence of the kineto-static labyrinth on the visceral nervous system, and to determine the physiological relationship between the two; and to establish the presence of a phenomenon which would be applicable to practical use from a clinical standpoint.

The labyrinth exerts a tonic influence on the general body musculature through its intercommunications with the somatic nervous system. The effects of the labyrinth on the visceral nervous system are not nearly as obvious nor as easily demonstrable as its effects on the somatic nervous system. For example, the stimulation of the vomiting center which occurs during the performance of the labyrinthine tests, or during an attack of seasickness, may be the result of cerebral impulses produced by the cumulative nausea, or as some authorities believe, may be produced by a direct reflex action of the hyperactivated labyrinth upon the center in the dorsal nucleus of the vagus.

That an interrelation exists between the visceral nervous system and the labyrinth is apparent when one considers that tinnitus is a common symptom accompanying disturbances of the viscera in other parts of the body. Woakesl considers that impulses from the stomach pass by way of the vagus nerve to the inferior cervical ganglion, where a communication is established with the nerves which regulate the amount of blood supply to the inner ear through the internal auditory artery The vascular changes thus produced, cause the tinnitus.

For certain quantitative biologic tests it is necessary to use a suspending medium in which the streptococcus will survive without increase or diminution in numbers for a considerable length of time.

Falk has recently reviewed the extensive literature on the subject of the inhibitory effect of the cations of various salts on the biologic activities of bacteria and other cells. He concludes that balanced solutions containing two or more salts in the proper proportions preserve the life of bacteria better than either distilled water or solutions containing single salts unless they are very dilute.

Robertson, Sia and Woo have recently found that 0.1 to 0.125 per cent gelatin in distilled water or Locke's solution greatly increases the length of life of the pneumococcus in these fluids.

With these facts as a basis, experiments with a hemolytic streptococcus obtained from an acute gangrenous infection of the thigh have been carried out with six different fluids: 1, distilled water; 2, “normal” saline; 3, Locke's solution; 4, a balanced solution containing 1 per cent sodium chloride and 0.05 per cent calcium chloride (“20-1”); 5, 0.2 per cent sodium citrate; 6, a balanced solution containing 0.5 per cent sodium chloride, 0.5 per cent potassium chloride, 0.5 per cent calcium chloride and 0.1 per cent magnesium chloride (Zeug's mixture).

Each fluid was prepared both with and without 0.1 per cent gelatin and titrated after autoclaving to pH 7.8. The streptococcus was grown in 1 per cent dextrose digest broth, washed twice and then suspended in the solution to be tested at a standard concentration, approximately one billion organisms per cubic centimeter. per cubic centimeter. This was diluted down seven times with the fluid to be tested, each dilution containing a tenth of the number in the previous dilution so that the final dilution was estimated to contain 100 organisms.

In a previous series of experiments¹ on cats, it was found that the action of morphine and heroin on respiration was practically limited to a selective depression of the expiratory mechanism, and that codeine produced only stimulation of the spinal cord. These experiments were repeated upon decerebrated and anesthetized dogs, and upon anesthetized rabbits, inducing active expiratory efforts by inhalations of a constant CO₂-air mixture, and recording them by means of a record of intrathoracic pressure, using an esophageal balloon or a pleural cannula; blood pressure and respiratory volume were also recorded.

The results confirm those obtained with cats, namely, morphine and heroin have a selective action on the expiratory mechanism of dogs and rabbits as well as cats, while no constant depressant action could be detected with codeine.

With morphine, dogs showed passive expiration after 2 to 6 mg. per kilo, as a rule, and larger doses had no further effect until circulation was depressed; occasionally very large doses—300 mg. per kilo—reversed this effect and produced increased reflex excitability, but convulsions did not occur, even in decerebrated animelas. Depth of respiration was often increased as the rate was slowed. With rabbits, expiratory depression began with 1 mg. of morphine and was usually complete after 3 to 5 mg., but depth was more frequently decreased than was the case with dogs. Larger doses sometimes reversed this effect, and caused increased reflex excitability, with a return of active expiration, but the circulation of the rabbit was not depressed as much by morphine as was that of the dog or cat.

With heroin, 0.05 mg. per kilo sometimes made expiration completely passive in the dog, and depth of breathing was usually increased as the rate was slowed.

In the experiments on cats previously reported, it was apparent that the action of morphine and heroin on respiration was limited to a depression of expiration, and that an extremely slow rate and respiratory failure depended on a primary depression of the circulation. These observations have been confirmed in every case in experiments on dogs, and in most experiments on rabbits, though in one experiment heroin caused temporary respiratory failure and asphyxial effects on the blood pressure of a rabbit, and circulatory depression was usually less marked in rabbits after morphine or heroin than in dogs or cats. The extremely slow regular breathing of advanced morphine or heroin poisoning never occurred, in dogs or rabbits, unless circulation was definitely depressed, and the appearance was that of vasomotor failure.

As respiratory stimulants, atropine, caffeine, and camphor were tried; digitalis was used as a circulatory stimulant; and inhalations of CO₂, of 7 to 10 per cent strength, were also used, when respiration was seriously depressed by morphine or heroin.

Atropine stimulated respiration only exceptionally, and the stimulation appeared as an increase in depth but never in rate. As a rule, it either had no effect, or it added to the respiratory and circulatory depression especially if large doses were given—2 to 10 mg. to a dog.

Caffeine was the most reliable and safest stimulant, often increasing both rate and depth, and sometimes bringing back active expiration, especially if it improved the circulation. Large doses did no harm unless they depressed the circulation, when respiratory failure usually followed.

Camphor produced striking stimulation of respiration and circulation in one experiment on a rabbit, in 20 mg. dose, but in all other attempts on rabbits and dogs it either had no effect, or produced circulatory depression and respiratory failure

We had not seen any confirmation of Tulloch's seriological classification of tetanus bacilli and as we had a number of strains of this organism, isolated from the stools of man, we thought it worth while to see if they could be differentiated into groups by the agglutination reaction. Tulloch not only sent us transplants of his four type cultures but has been most kind in answering our questions and has sent us an impure culture of a strain of tetanus bacilli that did not agglutinate with his type sera. This culture he received from France in 1918 but since no data accompanied it and since it was the only culture that did not agglutinate with his type sera he did not mention it in his work. This culture, which we have called Type V, has interested us very much for, as will be seen in the table, it is a type that we have found most frequently in Peking.

Agglutinating sera were easily prepared by the injection into rabbits of washed bacilli from actively growing 24 hour bouillon cultures. The first injection was subcutaneous and was followed at intervals of a week hy three intravenous inoculations. The animals withstood the treatment well and ten days after the last injection their sera agglutinated the type bacillus used in a dilution of 1/6000 or higher. The differentiation of the types is quite striking as the other organisms were not, as a rule, clumped in dilution as low as 1/100.

The results of our tests are given in the table where we have included the findings of Tulloch for comparison.

It will be noted that we have confirmed the seriological grouping of tetanus bacilli though we have not, in the small numbers of cultures studied, found Types I1 or IV.

The demonstration that human carriers of tetanus bacilli have antitoxin in their blood has opened up a number of problems, which we are attempting to solve on experimental animals. We had first to determine whether carriers could be produced in these animals and if so whether antitoxin would be formed. The results of this preliminary experiment we will report on here and subsequently we hope to give the results of our further studies.

That we would succeed in establishing tetanus bacilli in the digestive tract of guinea pigs was indicated by our finding that 27 per cent of our stock animals were already carriers and that these animals had antitoxin in their serum. On the other hand, we have failed to find a rabbit that carried these organisms nor have we detected tetanus antitoxin in the serum of a number of rabbits examined.

As we planned to use the animals for other experiments a large number of guinea pigs were selected whose stools were free from tetanus spores and whose serum showed no antitoxin. These animals were starved for a day and were then fed a large number of washed spores and bacilli from month old bouillon cultures which were added to their usual food. After the feeding the animals were placed in metal cages which were sterilized from time to time. In order to prevent, as far as possible, infection from the outside all bedding and, with the exception of green stuffs which were washed, all food was autoclaved before it was placed in the cages.

The results of the stool examinations made on these guinea pigs, approximately four months after the feeding of tetanus cultures, are summarized in Table I.

Dogs were infected with the cercaria of Schistosoma japonicum either by subcutaneous injection or by immersing the dogs in infected water. Eggs were first recovered from the feces of one heavily infected dog on the twenty-seventh day after infection, and from other dogs at various intervals up to about forty days. The dogs were sacrificed at various intervals after the appearance of eggs in the feces, from the twenty-eighth to the seventy-seventh day. Portions of intestine were excised, often while the dog was still alive under ether anesthesia, and were fixed in Zenker's solution or 10 per cent formalin. Blocks were embedded in paraffin and sections were stained with hematoxylin and eosin. From five dogs, killed on the 28th, 33rd, 40th, 63rd and 65th days after infection serial sections were cut from the blocks of tissue which showed the most intense pathology. I wish to emphasize here two features of the pathology which have not been described previously.

1. The earliest position of deposited eggs. On the twenty-eighth day after infection nearly all the deposited eggs were in the stroma of the mucosa; a few were in the submucosa. The eggs were in large or small groups containing up to 200 eggs in one group. The eggs of each group usually radiated out into the mucosa from a single point in the submucosa, and in many cases it could be seen that all the eggs were within distended venules and copillaries. They often extended nearly to the surface epithelium of the mucosa.

Male and female worms, always in copula, were found in distended veins in the submucosa. At this early stage there was no hemorrhage into the tissues and practically no tissue reaction about the deposited eggs.

In a previous note¹ it was stated that the threshold density of an electric current necessary for inhibition of growth in the ends of Obelia internodes turned toward the cathode was very nearly equal to 66 microamperes per square millimeter.

This threshold inhibition is reversible.² For orientation of the axis of growth a threshold current density of only 6 to 13 microamperes per square millimeter of cross section is necessary. This is therefore only about one-tenth as great as the current density threshold for inhibition. We may translate this result into its equivalent of threshold of applied difference of electric potential.

The living tissue of the stem and pieces of stem in Obelia have also been shown to be the seat of a relatively constant electric potential.³ More recently this potential has been measured by the use of a suitable type of electrode.⁴ The results show that a difference of potential exists between the inside and outside of the ecto-endoderm layer. The outside surface of the ectoderm being electro-positive to the inside surface of the endoderm. Similar potential differences are well known to occur in the skin of the frog, intestine and some other epithelial structures. The magnitude of the inherent potential in the ecto-endoderm of Obelia is greater in the apical growing point of the main stem of the colony than in the middle, and somewhat lower regions of the same stem of the same stem. The same statement applies to growing branches of the colony. The result of this difference in the magnitude of the electric potentials across the ecto-endoderm in apical and more basal parts of the stem, is that when the outer surfaces of the apical and basal regions of the stem are connected to a galvanometer, a current flows in the galvanometer circuit from apical to basal parts of the isolated stem.

Recently, H. W. C. Vines has shown that calcium in normal unclotted blood is present in two forms. He found that approximately one-third of the calcium is present in a combined or non-ionized state, while two-thirds are in the ionized form. He also observed that chronic septic infections do not heal rapidly unless the ionic calcium is increased. Vines observed that the administration of dried parathyroid increased the relative ionic calcium and lead to rapid healing of ulcers.

The present authors have also observed similar effects, using an extract of parathyroid made in the following manner: 30 gm. of finely divided ox gland are boiled in 500 cc. of dilute hydrochloric acid (10 parts per thousand), cooled, filtered, and then made up to 500 cc. again. This extract which we know as hydrochloric × seemingly contains the active principle of the parathyroid. Dogs which had been completely parathyroidectomized and which were showing symptoms of tetany, recovered. Sometimes on injection, the symptoms increased at first before recovery took place.

This extract we have also found beneficial in the treatment of chronic inflammations such as gastric, duodenal, and varicose ulcers as well as cystitis or osteomyelitis.

From this hydrochloric × the active constituent may be precipitated by phosphotungstic acid, but we have not been able to redissolve the active constituent from this precipitate.

The problem of the causation of the respiratory variations in blood pressure is an old one, but one that has resisted solution surprisingly long. Examination of the literature on the subject shows that diametrically opposite observations have been made upon the relation between the phases of respiration and the rise and fall of blood pressure. About half of the workers have found an inspiratory rise, and half an inspiratory fall.

In examining the factors heretofore mentioned in explaining the phenomenon it is apparent that the possibility that factors tending to change blood pressure might not be immediately effective, has been neglected. In other words, the possibility that the effects of inspiration might show up in expiration, has been neglected.

In experiments in which the inferior vena cava, the superior vena cava and the azygos vein were clamped separately and in combination, it was found that there was absolutely no sign of these changes on the arterial pressure trace for a period, on the average, of three heart beats. Very evidently there is a latent period of three heart beats in this process.

The changes in arterial pressure were similarly recorded when variations were artificially produced in the thoracic cavity. Similar latent periods were observed.

These experiments were repeated upon animals with denervated hearts and the same observations made, exc'luding a nervous factor.

It is evident that the effects of inspiration may show up entirely in expiration and the effects of expiration in the succeeding inspiration. The heart-respiration ratio determines in what phase of respiration the various changes will appear.

Many published arterial pressure tracings have been examined and it has been found that this explanation is quite valid in interpreting them.

In experiments on rickets, osteoporosis and xerophthalmia by my students and myself, white rats belonging to litters 1-150 were fed diets 1-467. In most of these white flour was an ingredient but some were made of purified food substances. It was found that a group of vitamins known as Vitamin A were all contained in the cod liver oil used; that commercially dehydrated spinach, or spinach dried in the laboratory at a low temperature, contained a growth promoting Vitamin A, and prevented xerophthalmia, but had no detectable antirachitic effect. Its effect in preventing osteoporosis was extremely small, and since the spinach contained calcium it was very difficult to decide whether the effect was due to calcium or due to vitamin. Extracts made from this spinach also failed to show antirachitic and antiosteoporotic effects. Vitamin B was derived from bakers'yeast. In most of these series of experiments, the antirachitic vitamin was kept low and constant while the calcium and phosphorus were varied. Results confirm those of Sherman and Pappenheimer as well as those of Osborne, Mendel and Park. It has not been possible so far, however, to show that rickets depended on any ratio of calcium to phosphorus, the most striking result being that low calcium produces osteoporosis and low phosphorus produces rickets.

Diagnosis of rickets was made by Dr. C. M. Jackson¹ in the earlier experiments. By comparing the diagnosis made by Dr. Jackson on gross and microscopic pathology with x-rays taken the same day, a basis of interpretation was made of x-rays, and x-rays were used for diagnosis in the later experiments. It is not possible to record all1 these experiments but the following is given as an example. Ca was added as CaSO₄ and P as KH₂ PO₄.

Tetanus toxin, diphtheritic toxin, botulinus toxin and endotoxin, obtained from the actinomyces gypsoides, were treated with castor oil soap to determine its effect upon these various toxins. Tetanus toxin and diphtheritic toxin were found to be completely detoxified when sufficient castor oil soap was added to reduce the surface tension below forty dynes. Guinea pigs treated with one hundred lethal doses of the respective toxins, with the soap, failed to develop symptoms.

Botulinus toxin appears to be more resistant to soap than the other two soluble toxins. This observation offers a possible explanation of the fact that neither diphtheritic nor tetanus toxins are absorbed in toxic form by way of the digestive tract; while botulinus toxin is readily absorbed by the digestive organs. The contents of the intestinal canal having a low surface tension, because of the bile and soaps present, probably destroy tetanus and diphtheritic toxins, but not botulinus toxin.

Nelson and Henrici¹ have shown that the actinomyces gypsoides, an organism very pathogenic for guinea pigs, possesses a potent endotoxin which kills rabbits and guinea pigs regularly. When shaken in a two per cent solution of castor oil soap, so as to effect a thorough “wetting”, this organism loses its pathogenicity, and its endotoxin is also completely destroyed.

Whether or not the effect of soap on bacterial toxins is entirely a surface effect, or is due, in part, to some other property of the soap, cannot be determined definitely at this time.

Control experiments have convinced us that the action of the soap upon the bacterial toxins is not due to the low hydrogen ion. The effect of soap on the antigenic properties of bacterial toxins will1 be discussed in a future paper.

It has been found possible to emulsify a highly cohesive and agglutinable strain with M/50 sodium oleate, which concentration was less than for any other of a number of salts used, which included M/16 MgCl, M/8 Na Citrate, M/2 NaCl, copper acetate and CaCl₂, which are given in the order of their emulsifying efficiency.

With threshold concentrations of certain of the emulsifying salts the organism spreads suddenly over the surface of the liquid. The phenomenon is well recognized to be one of surface tension depression.

It is probable that the emulsifying action of the oleate is brought about by its ability to diminish the abruptness at the interfacial boundary between the bacterium and the solution. This view accords with the fact that COONa radical of the oleate is truly soluble in the water, and the hydrocarbon radical is not. The obvious lipoidal nature of the wall of the bacterium in question makes probable the adsorption of the oleate anion; in fact, repeated washing restores the flocculability. These conditions make for diminution of abruptness at the interfacial boundary which always produces a marked lowering of surface tension. Measurements of potential difference indicate the recognized importance of this factor also, but in the more dilute concentrations of salts.

A closely related strain emulsifiable in NaCl was precipitated by CaCl₂ and an ion antagonism was shown between the Na and Ca cations in a ratio of 80:L The effect was not permanent. The ratio is of the same order of magnitude as in oil-water and water-oil emulsions and suggests a similar lipoid arrangement in the membrane of the organism.

Two-tenths of a cc. of insulin were injected intravenously. Blood samples were withdrawn at frequent intervals, and sugar determinations were carried out on the whole blood.

The blood sugar decreases rapidly and at a uniform rate. The minimum sugar value is indicated at about 18 minutes after the injection. The sugar content of the blood then rises, at a slower rate, reaching the initial value within about one hour after the injection.

Peculiarities were observed in the reaction of overweight subjects. The insulin hypoglycemia is not as intense as in a lean subject of the same weight, and recovery from it seems slower. While this phenomenon apparently corroborates older views on the subject of “fat and glycogen antagonism”, it is being investigated further, and other experiments are being undertaken on the relationship between fat and glycogen formation.

It is suggested that the varying degrees of adiposity, as such, underlie the differences in the reaction to insulin of animals used in its assay.

The experiments were carried out, in part, under a grant' from the Heckscher Research Foundation.

Considerable experimental evidence has in recent years been brought forth which indicates that the union of proteins with acids and bases takes place by chemical combination.¹ It has been suggested by Robertson² that the −COHNgroups in the protein molecule are responsible for the acid and base-combining capacity but this hypothesis has been recently questioned by ourselves.³ Kossel and Cameron⁴ pointed out many years ago that the acid-combining capacity of salmin is equal to the combining capacity of the guanidine groups in the arginine, and Bracewell⁵ has carried this idea further by showing in a number of proteins a close agreement between the acid-combining capacity and the sum of the free amino nitrogen and the amino group of arginine which does not react with HNO₂. The recent work of Hitchcock⁶ in which he determined the acid-combining capacity of gelatin and of deaminized gelatin affords an opportunity for checking this hypothesis and our calculations which are given in Table I show an excellent agreement between the calculated and the experimental results.

As an index to possible smooth muscle reactions, we have studied the pressure changes in the urinary bladder during canine anaphylactic shock, with parallel tracings of the changes in the arterial blood pressure. The dogs were sensitized to horse serum by Weil's method.¹ They were tested by intravenous injections of 0.5 to 2.0 cc. horse serum per kg. of body weight, about twenty-one days after the final sensitizing dose. The intracystic pressure was recorded by means of a glass catheter (perineal incision, male dogs). The abdomen was opened to avoid errors from changes in intraabdominal pressure. The following cystic reactions have thus far been recorded:

1. Typical Anaphylactic Shock. Arterial blood pressure falls precipitously to about 35 mm. Hg by the end of 45 seconds, gradually decreasing to about 25 mm. Hg by the end of 90 seconds. Recovery usually begins about the twelfth minute, the blood pressure being restored to normal in from 60 to 90 minutes. No recordable change in the intracystic pressure during the first 45 to 75 seconds. The intracystic pressure then gradually increases, usually reaching a maximum of from 25 to 50 mm. Hg by the end of two and a half minutes. Recovery immediately sets in, the intracystic pressure usually being reduced to normal by the seventh minute. No reactions were observed in normal controls.

Assuming that the cystic reactions thus recorded are reliable indices to smooth muscle reactions in other parts of the body, one must conclude that in typical canine anaphylactic shock the smooth muscle structures are not thrown into contraction until the shock is fully established, as determined by the fall in arterial blood pressure. The smooth muscle reactions, therefore, apparently play no rôle in the initiation of the shock.

Convulsions and immediate death were found to result from the administration of fluid to rabbits which had been dehydrated by intravenous injections of sucrose 5 to 10 days previously.

These animals had been used to determine the effect of water abstinence and increased water loss on the excretion of urea and the blood urea concentration. They were kept without food or water and bled 1 to 3 times daily throughout the experiment. Extremely high blood urea concentrations (100 to 450 mg. per 100 cc.) were reached in every animal and fluid ingestion resulted in a very much higher blood urea concentration.

Water to the amount of 200 cc. was administered to a rabbit which had undergone simple water abstinence with negative results. Convulsions were obtained in those rabbits which had received sucrose after the administration of 100 to 200 cc. of distilled water or 0.9 per cent sodium chloride.

The severity of the albuminuria and the height of the blood urea concentration was greatest in the rabbits which had been dehydrated previous to water abstinence by intravenous sucrose injections. Since it was in these only that convulsions were produced it would seem that some damage produced by sucrose was a factor. The cause of these convulsions and their association with kidney damage is being further investigated.