Experimental Biology and Medicine

Dogs were prepared by injecting subcutaneously I gram of phlorhizin suspended in olive oil once every twelve hours until the G: N ratio was constant, then every 24 hours.

It has been observed by Lusk 1 that fasting and phlorhizin alone do not remove all glycogen from the body. This was confirmed for dogs prepared in the manner described. Such animals were narcotized with ether, nitrous oxide and other narcotics. Narcosis with ether and nitrous oxide was always followed by a large increase of the glycosuria with a rise in the G: N ratio. When ether and chloroform were used, there was as a rule, a fall in the output of nitrogen and acetone bodies and a fall in sugar following the initial rise, but with nitrous oxide the rise in sugar was unaccompanied by such changes. In one of the ether experiments also the fall in nitrogen was very slight and there was no lessening of the glycosuria following the initial increase.

An animal prepared in the usual way was given a cold bath for 20 minutes followed by 6 hours of moderate shivering. After this 1 mg. of epinephrine was given intravenously once every 6 hours.

At first there was a marked increase in the glycosuria, but ultimately the G : N ratio resumed the normal level and was little affected by epinephrine. Nitrous oxide narcosis was then induced as before. The extra sugar for :24 hours was now 1.8 g. as compared with a minimum of 7 g. in two previous experiments.

It is concluded that the “extra” sugar which is eliminated when phlorhizinized dogs are narcotized with ether and nitrous oxide has its origin in glycogen; that the increase in glycosuria incidental to narcosis is a phenomenon which can occur independently of any fall in nitrogen or decrease of the acetone body output.

The following report is based upon the difference in the response of forty-eight animals of different ages to a constant quantity of uranium nitrate.

Dogs have been employed in all of the experiments. The animals have varied in age from puppies of four months old, to animals of extreme old age, one of the animals having reached the age of twenty years.

All of the animals have received uranium nitrate in the dose of 6.7 mgs. per kilogram on two successive days. The uranium was given subcutaneously.

The animals were fed on raw meat and bread.

In a recent publication 2 it has been shown that when a constant quantity of uranium nitrate is given to young and full grown animals, that the age of the animal influences the total output of urine and also the composition of the urine. The total output of urine in a twenty-four hour period was greater in the adult animals. The percentage of glucose in the urine (Benedict determination) was greater in the adult animals than in the puppies.

In the urine of the puppies acetone was either absent or present in very small amount. The urine of the adult animals invariably contained acetone.

The present series of forty-eight animals shows the same differences in the total output of urine and in the composition of the urine as has been above referred to.

In addition, these animals of different ages show certain other characteristics which are apparently dependent upon the age of the animal.

1. Ten of the animals were puppies varying in age from four to eight and a half months. Only one of these animals developed a glycosuria within the first twenty-four hours following the initial injection of 6.7 mgs. of uranium. The percentage of glucose in the urine of this animal was 0.103 per cent.

In the course of complement fixation work we have noticed that the specimens of blood cells obtained from different sheep under exactly similar conditions occasionally show marked differences in their susceptibility to laking by specific lytic serum. When this was first noticed we were using two sheep as a source of blood, and as the sheep whose cells were more highly resistant was one which had been bled repeatedly and profusely it was natural to attribute the increased resistance of the cells to this.

We determined to investigate this question and also to find out whether the increased resistance was a specific resistance to laking by amboceptor and complement or was a general resistance such as would be shown by an increased resistance to laking by hypotonic salt solution, and also whether the sera of different sheep show similar differences in their tonicity.

Three sheep were used:—

T—an apparently normal young male sheep, newly obtained from the dealer.

F—a sheep which had been kept in the laboratory for six months and had been bled repeatedly and on occasions profusely.

P—a sheep which had been confined for two and one half years, had been bled occasionally and had received immunizing injections of typhoid bacilli for a period of about eighteen months.

F was slightly and P decidedly anemic. The blood of all three was obtained on the same day and the cells were washed with great care to handle the blood of all three animals in precisely similar ways. All three were made up into 5 per cent suspension and tested with diminishing quantities of hemolytic amboceptor (rabbit serum) and a fixed dose of 10 per cent complement (guinea pig) with the following result:

It is seen that the new sheep, T, was the most resistant to laking, the frequently bled sheep, F, almost as resistant, and the typhoid injected sheep, P, least resistant.

It is well-known that destruction of the brain causes increase of reflex action in frogs, especially when they are kept in the cold, and last year I reported that the effect of morphin, which causes in frogs tetanus indistinguishable from that of strychnin, was very markedly increased by decerebration, the effective dose in such frogs being about one tenth of that in normal frogs.

I wish to report now on the result of a study of the effect of decerebration upon the convulsant action of caffein. Caffein salts which are strongly acid cause a peculiar muscular stiffness which masks the central action. The alkaloid itself causes also a certain muscular stiffness but it is much less marked and is only troublesome with large doses. The alkaloid was therefore used in these experiments. As was the case with other convulsant drugs we found the action of caffein much greater in the cold. If the frogs are kept cold tetanus can be obtained with doses which are too low to markedly affect the muscles.

In contrast to morphin we found that both in the cold (2-5° C.) and in the warm (12-18° C.) there was no difference between normal and decerebrate frogs in their response to the convulsant action of caffein. In both tetanus was obtained in the cold in all frogs with doses of 0.2 mg. p. gm. and in most frogs with 0.1 mg. p. gm. At room temperature tetanus was constantly obtained in normal decerebrate frogs with doses of 0.6 mg. p. gm. but never in either with 0.3 mg. p. gm.

The experiments show that there is a difference of some sort between the actions of caffein and morphin on the central nervous system although the convulsions are identical in appearance.

At the February meeting of this Society one of us (G.) reported that after an intravenous or intramuscular injection of a sublethal dose of a solution of megnesium sulphate in dogs hyaline casts invariably appear in the urine. As a result of this observation a series of experiments was made to answer the question as to which of the ions of the injected salt is the cause of the appearance of the hyaline casts—the kation magnesium, or the anion SO₄, the sulphate radical. We have tested in the first place several magnesium salts as well as several sulphates. This led up to further experimentation with some salts which contain neither magnesium nor the sulphate radical. Briefly stated, our results are in general as follows: The injection of any compounds which have either magnesium or the sulphate radical as a component caused the appearance of hyaline casts in the urine. The injection of salts which had in their composition neither Mg nor SO₄, produced, however, no such effect. Of the magnesium salts, besides the sulphate, the chlorid, nitrate and the acetate were studied also. They all caused the appearance of hyaline casts in abundance. The acetate was perhaps less effective. Of the sulphates, besides the magnesium, also the salts of sodium, ammonium, and potassium were studied. All gave hyaline casts; the action of potassium, however, was less transparent. On the other hand the chlorids of sodium and of ammonium and the nitrate and the acetate of sodium produced no hyaline casts. The effects of potassium salts were apparently complicated by the profound action of these salts upon the heart and probably also by some direct action upon the kidneys. Of course, we could not attempt to give here any further details, nor enter upon a theoretical discussion of the possible significance of the reported facts.

Ingestion of levulose by a dog which has been phlorhizinized does not increase the metabolism; the respiratory quotient is not changed and levulose is converted into dextrose, for this alone appears in the urine and in increased quantity. Ingestion of glycocoll or alanin largely increases the metabolism in glycosuria, although they are not oxidized and are converted into glucose and urea. The conclusion is drawn that the preliminary cleavage products of carbohydrate break-down are not stimulants of metabolism, but that the increased heat production following carbohydrate ingestion is due to the mass action of sugar molecules. On the contrary, amino-acids or the oxy-or keto-acids produced from them act as stimuli upon the cells thereby increasing the heat production.

In several papers I have pointed out the frequency of polycythemia or polyglobulia in patients suffering with non-bleeding duodenal ulcer. I have also called attention to the presence of eosinpenia in such patients. In gastric ulcer occurs just the reverse anemia and relative eosinophilia.

Of eighteen operatively demonstrated cases of duodenal ulcer, in fifteen polyglobulia was found, while of twelve cases of gastric ulcer, polycythemia was noted only in one.

For details I refer to my article, which will soon appear in the Journal of the Am. Med. Sc.

The question arose, what has duodenal ulcer to do with polyglobulia. As several investigators who injected adrenalin in dogs and in men, have found an increase in erythrocytes from 30-70 and 100 per cent (this experimental polyglobulia lasting sometimes about thirty hours) and also a reduction in the number of eosinophiles, the idea has gained access to us, that the initial lesion of duodenal ulcer may be caused by hyperfunction of the adrenals.

As in none of our patients with duodenal ulcer high blood pressure was noted, the assumption was, that in such patients a slight excess of epinephrin may circulate in the blood in quantities not sufficient to cause hypertention, but enough to exert its hormone influence upon the duodenal mucosa.

I have succeeded in producing duodenal lesions and ulcers in eight dogs by subcutaneous and intravenous injections of adrenalin hydrochloride in a period of about one to two weeks daily (with one or two intermissions). Single doses did not exceed 3 mg. subcutaneously and 2½ mg. intravenously.

Quite a number of gynandromorphs and mosaics have appeared in our experiments with the mutant stocks of Drosophila. In some of these the male side or part is maternal, and in others paternal, and in one case both paternal and maternal. For the first type Boveri's hypothesis of partial fertilization will cover the result, for the second my own hypothesis of polysperm will explain the facts. In the third case the result must be due to mitotic dislocation at some early cleavage stage.

In order to test these three hypotheses, I crossed a female homozygous for the sex-linked characters, yellow body color and white eyes, to a male with the normal sex-linked allelomorphs, viz., gray body color and red eyes, but in addition the male carried a recessive non-sex-linked character, viz., ebony body color.

One gynandromorph appeared among many thousand offspring. It was male on one side (partially) and female on the other. Both sides had red eyes and gray (or dark gray) body color. An analysis of this case shows that the male side must have contained the sex chromosome of its father and a non-ebony autosome from the mother. In other words, the gynandromorph on the male side is like the father except that it carries in addition one of the autosomal characters of its mother. The result means that at some early division a sex chromosome failed to pass to one pole and became lost.

Since this explanation will cover also the first two types, and since neither the hypothesis for the first nor that for the second type will explain all three types the third hypothesis is to be preferred. It leads to the conclusion that gynandromorphs and mosaics may arise through a mitotic dislocation of the sex chromosomes.

1. Herrmann's theory of alteration, which is accepted to-day by many physiologists, assumes that the junction of dead and of living tissue is the seat of an electromotive force which acts in such a direction that the dead tissue is negative while the living tissue is positive. This theory was advanced by Herrmann against DuBois Reymond who tried to explain the currents produced by muscles and nerves by means of complicated structural assumptions. Herrmann's theory has the advantage of expressing in an extremely simple form a large number of physiological observations. It has met general recognition partly also because all the opposing theories were of a very unsatisfactory character. The arguments however which Herrmann advances to support his views are not so conclusive as to fully justify his views. Especially his observation that a definite time elapses between the cutting of the muscle and the appearance of the current of injury must not necessarily be due to a chemical alteration resulting from the injury. This time is so exceedingly short (5/1000 of a second) that a mechanical explanation rather suggests itself than a biochemical.

2. The conception that a difference of chemical processes in the dead and in the living tissue produces currents is also contradictory to physical laws. This was already pointed out by DuBois Reymond and his arguments seem still valid to-day. According to well-established physical principles an E.M.F. may be produced as a result of a chemical alteration which is brought by a current passing through the system, not however through a local chemical reaction which liberates ions.

3. Moreover certain experimental facts are in direct contradiction to Herrmann's theory; if a muscle is brought in contact at one end with distilled water and at the other end with a physiological NaCl solution it is found that the part in contact with the water is positive.

Ten grams of sodium benzoate were administered to a healthy man on a diet of milk, butter, and cane sugar, i. e., glycocoll-free. The urine was collected at two hour intervals, and the relation between the elimination of hippuric acid and urea studied. As compared with the corresponding control periods on the same diet, there was observed a diminution of the urea-nitrogen eliminated during the first six hours after the benzoate ingestion, a diminution corresponding to the nitrogen eliminated as hippuric acid-nitrogen.

If the hippuric acid-nitrogen be subtracted from the undetermined N (shown in parentheses), the undetermined N is comparable with that of the control periods. This indicates that in man as in rabbits and pigs, the glycocoll available for synthesis into hippuric acid may be derived at the expense of substances whose N normally appears in the urine as urea-nitrogen. At the end of six hours the greater part of the hippuric acid had been eliminated and the urea elimination had become normal again.

No free benzoic acid nor glycuronates could be detected in the urine, indicating a complete conversion to hippuric acid and a very rapid elimination. In order to ascertain whether the rapidity of elimination was influenced by the liquid diet of the preceding experiment, the work was repeated on the same subject on a mixed diet, and on a purine-free diet. In both experiments, the greater part (85-95 per cent) of the hippuric acid was eliminated within six hours. Another subject received six grams of sodium benzoate and eliminated the greater part of the hippuric acid within six hours. An amount of sodium hippurate equivalent to the benzoate fed was administered, and the elimination of the hippuric acid studied.

A year ago after centrifuging some eggs of Rana sylvatica it was noted that all of the survivors in one lot had accessory tail-like appendages. This seemed to be a sufficiently striking modification to merit some effort to learn just what was responsible for their occurrence. After many trials similar modifications were produced again this season. Eggs were treated in various stages from unsegmented eggs to the gastrula, and the different lots were given three different treatments.

In this note the earlier stages will not be considered. In a stage at which the blastopore had just become evident, or soon afterward became evident, the eggs which were subjected to a centrifugal force equivalent to 1,700 times gravity for two minutes were mostly killed. Accessory tail-like appendages developed in the survivors. 1,350 times gravity killed very few and accessory tail-like appendages developed in all of the survivors. 200 times gravity for ten minutes produced all normals in cases where the blastopore was not yet evident at the time of treatment. Where the blastopore was just evident all produced the accessory appendages. All survivors of similar treatments in the advanced gastrula and later stages were normal and accessory appendages were not produced in earlier stages than the blastula.

The accessory appendages, usually one to each animal although in some instances as many as four have been noted, occurred on different parts of the body. By far the commonest location was the mid-ventral region although they were in all positions from the anal region to the under side of the head. In many cases the position was more or less lateral, or even dorso-lateral, the appendage usually extending ventrally however. The appendages in the positions described were all distinctly tail-like having the characteristic myomeres seen in the normal tail and having a fin-likekeel on one or both margins.

In a preliminary communication 1 from this laboratory, the results of experiments with the double salt of tin tartrate were announced. Experiments performed since with tin tartrate have shown that like the double salts, it caused marked injury to the kidneys. Large amounts of albumin were present in the urine of rabbits when administered subcutaneously and intravenously but larger doses of the normal salt were required to produce this effect.

The amount of tin as the double salt necessary to produce a very marked albuminuria was 20 to 30 per cent less than in the form of tin tartrate. Observations on the action of stannous and stannic salts failed to show any noteworthy difference. The effect of concentration indicated marked differences both when injected intravenously or subcutaneously. Thus 20 milligrams of tin per kilo, injected intravenously, produced a moderate albuminuria when the amount of tin per c.c. was 1.5 milligrams; much greater amounts of albumin and large amounts of sugar when each c.c. contain 8 milligrams of tin; acute death when each c.c. contained 20 milligrams of tin.

Sodium tartrate and citrate in Locke's or in Ringer's solution or in defibrinated blood perfused through the isolated heart of the frog, dog and cat caused a decrease of cardiac activity which became more marked with increased concentrations. The action of the citrate was considerably greater than that of the tartrate, the ratio being about 5: 1 with weak solutions. The difference was even more marked when the action of more concentrated solutions was compared, an N/100 citrate was as active as N/10 tartrate, as observed on the frog's heart. The action of citrate and oxalate was compared by its effects in calcium-free solutions (Ringer and Locke minus calcium). The effect of citrate and oxalate was found to be the same in some experiments but in others the action of the oxalate was distinctly less toxic than that that of the citrate. Since the solubility in water of calcium citrate is twice as great as calcium tartrate, while calcium oxalate is practically insoluble, it is apparent that the action of these salts is not due to the precipitation of the calcium. Again since the equimolecular solutions, calcium tartrate and calcium citrate, have the same effect as C_aCl₂ it would also indicate that a possible decrease in ionization of the calcium in the tissues is not a factor which determines the action of tartrate, citrate and oxalate on the heart.

November 17, 1911, the writer began rearing a number of pure lines of Daphnia pulex from large females taken in out-door ponds. The females were reproducing parthenogenetically and no males or “winter” eggs were found in the pond.

Each line is propagated by selecting from the first brood of a young female on the day this first brood is released from the brood pouch. The selected young are placed each in an individual bottle with standard food and though examined daily are otherwise undisturbed until the first brood of the next generation appears, when the selections are made as before. Several of these lines have passed the 95th generation and one has just reached the one hundredth generation without the appearance of sexual forms in any generation. All the individuals of the first broods of each generation have been under more or less close scrutiny until they themselves reproduced. If any, or at any rate, many males had occurred they must certainly have been noticed. The method of rearing the daphnids (in individual bottles) has precluded the possibility of sexual reproduction even had males been abundant in the cultures.

There is no evidence of decreased vigor or loss of vitality in the lines. Hence it appears that there is not a necessary sexual cycle in the reproduction of this daphnid. Male daphnids, apparently of this species, have been collected at Cold Spring Harbor since this work was begun. These facts would lend evidence (if additional evidence were necessary) that the sexual cycle in Daphnia pulex is not an inherent necessary thing but that it is determined by environment.

Simocephalus, presumably Simocephalus vetulus, has been reared for 76 generations in one line, likewise without the appearance of sexual forms.

At the meeting of this Society on February 18, I reported the results of my preliminary attempts to prove the specificity of complement deviation in tuberculosis with Besredka's tuberculin, in some cases giving positive Wassermann reaction at the same time.

In this paper I would like to present the results of my experiment proving the possibility of independent existence of the two reactions in the same animal.

I produced an experimental orchitis in a series of rabbits, and on March 3d selected amongst them the animals giving positive W.R. All the animals were tested for Tb.R. but it was invariably negative.

When, however, infected with tuberculosis, part of these animals developed Tb.R. For a time both reactions were existing until W. R. disappeared in some animals spontaneously, in others, as shown on the table under the influence of salvarsan administered intravenously (0.04 gram per kilo), the Tb.R. persisting. The table on page 181 is the protocol of the experiment.

As a result of several accidents reported as being due to intraspinal administration of antimeningococcus serum containing trikresol, a number of tests were made on dogs to determine if possible whether the fatal results were due to the influence of trikresol in the serum, to the serum per se, or whether they were due to pressure.

The records were taken with the kymograph, the pressure being taken at the carotid artery. The injections were made into the vertebral artery, the femoral artery and vein, the carotid artery and into the spinal canal.

The results which are somewhat contradictory, were as follows:

An antimeningococcus serum which had produced rashes and other disturbances in patients, caused well-marked depressions in 6.5 c.c. doses. Preservatives were chloroform and 0.4 per cent trikresol. The injections were made into the femoral vein. A whole antipneumococcus serum preserved with chloroform tested in the same way produced death. 1

Experiments (by F. and A.) with antimeningococcus serum to which was added varying quantities of trikresol (from .I per cent to .4 per cent) gave no deleterious results when first mixed, but after standing one week in some cases depressions were obtained.

An injection of 2 C.C. of antimeningococcus serum prepared 19 days before the experiment by the addition of .4 per cent trikresol was made into the vertebral artery. A marked depression resulted. Six C.C. of this serum also injected into the vertebral artery caused immediate clotting and were followed by a convulsion with almost complete cessation of respiration.

Further experiments carried out (by F., A. and 2.) on six dogs gave the following results:

I. As a rule, there was apparently no marked disturbance of blood pressure in normal dogs immediately after the lumbar subdural administration 2 by gravity or careful gentle pressure of moderate doses of antimeningococcus sera containing .3 per cent trikresol.

In order to test the hypothesis that in the metamorphosis of the larvæ of the frog autolysis is the principal factor, estimation of the amino-acid content was made by the gasometric method of Van Slyke and by the formol titration method of Sörensen. Beckmann freezing point and conductivity determinations were also made since Bayliss, 1 Wells, 2 Sjöqvist 3 and others have shown that the curve of proteolysis follows that of increase of conductivity and lowering of the freezing point.

Individual tadpoles were used at first, the tails being cut from the bodies, dried in an air-blast at 40° C., pulverized in a mortar, dissolved in water to make a one per cent solution, the estimations being made upon the coagulable protein-free filtrates from boiling with basic lead acetate. About 0.40 g. dried material could be obtained from one individual, but this amount became in the neighborhood of 0.10 g. in specimens which were absorbing their tails. The determinations mentioned above gave no characteristic differences between absorbing and non-absorbing animals.

Attention was then directed to larger amounts and accordingly the dried material from a number of larvæ in the stages preceding metamorphosis was mixed and another mass was obtained from a number of individuals undergoing involution. The former gave 3.84 g. dried material, the latter 2.50 g. Upon these amounts, determinations were made as above, but again no perceptible differences in amino-acid content was found, nor were the freezing point and conductivity determinations different in the two cases. As typical examples, in conductivity experiments, with R = 500, a = 58.1 for non-absorbing and 50.3 for absorbing; for freezing points, D = 0.72 for non-absorbing and 0.73 for absorbing. For formol titration with 20 cm3 aliquots, non-absorbing gave 0.1cm3, absorbing 0.3 cm.3 of n/5 Ba[OH]₂, in average of several determinations.

The method I wish to describe is a modification of the original method of Noguchi 1 for cultivating spirochaetes, which I think might be of interest to those who are called upon to cultivate these organisms. Noguchi's method, although very efficient, offers one great disadvantage, namely that the rubber stopper connection between the upper and lower parts of the apparatus often becomes a source of contamination of the culture. The method I propose consists in utilizing the principles of anaerobic cultivation in general as well as those special features which were worked out by Noguchi. This new method can be used in two different ways. Firstly, one can use the tube as shown on Fig. 2 which instead of having the rubber connection of Noguchi joining its two parts as shown in Fig. 1, is made entirely out of one glass tube; but otherwise can be used exactly as Noguchi's apparatus, namely the lower part in which the piece of rabbit kidney is put before the tube is drawn out, is filled with the ascitic broth or sheep serum water up to the point where the tube broadens out again; another piece of tissue is placed in the upper portion of the tube and this tube is filled with the ascitic agar into which the spirochaetæ culture is placed. 2 Sterile paraffine oil in a thin layer is placed above the agar and the tube is incubated. Spirochaetes during their growth filter through into the lower portion of the tube exactly as in Noguchi's method. This method is especially convenient when one intends to open the tube many times to examine its contents. The other and better way however of cultivating spirochaetes which does away entirely with the upper part of the tube: is the following: I put a piece of tissue at the bottom of the tube; draw it out as before; introduce by means of a capillary pipette the spirochaetae culture and ascitic broth in the lower tube; connect the tube with the vacuum pump, as shown in Fig. 6, warming the lower part of the tube in a water bath at 37″ to facilitate the exhaustion of the air; cover the ascitic broth, after exhaustion, with sterile paraffine oil by means of a special arrangement taking advantage of the negative pressure in the tube, and finally seal the lower part of the tube at the point of strangulation as shown in Fig. 7.