Experimental Biology and Medicine

The writer has studied the effects of long-continued selection upon several parthenogenetic pure lines (clones) of three species of Cladocera, using their reactiveness to light as a basis for selection. In most of the lines the results, though suggestive, are inconclusive; or there is clearly no effect of selection; or (in two lines) the results even suggest slight differences in the reverse of an effect of selection. But with one line of Simocephalus vetulus, line 757, the result of selection is pronounced and convincing. This line was subjected to selection for a period of 54 months covering 181 generations of descent. In the final ten generations the strain selected for greater reactiveness to light had a reaction time less than one third as large as that for the strain of the same line selected for reduced reactiveness to light.

For the sake of obtaining averages showing less fluctuation than the average reaction times by single broods the data for each strain was averaged by two-month periods of the experiment (each period including the data for all the individuals tested in making the selections for six to eight generations).

For the first two-month period of the experiment the averages for the two strains coincide. But in the next period there is a divergence, the plus showing a greater reactiveness by a small margin. In successive two-month periods this divergence is in general increased. There are considerable fluctuations in the curves as are indicated by the facts,-that for two two-month periods, nineteen months after the experiment was begun, the minus strain was actually the more reactive of the two; and that during three other later two-month periods the mean for the minus strain approached that for the plus strain. But in general the means indicate increasing divergence in mean reaction time throughout the main portion of the experiment until for the last nine months the mean for the plus strain was just half that for the minus strain, and during the last three months it was less than one third that for the minus strain.

A self-colored race of poultry was first produced in China about 1,500 years ago. All buff races of poultry have been derived from this Chinese race, the Buff Cochin. An attempt has been made to create a uniform buff race out of the Jungle Fowl. In 1907 a Jungle Fowl was crossed with a White Leghorn. This gave offspring in which the red of the Jungle Fowl appeared on a white ground, the black of the Jungle Fowl being hypostatic to the white. The birds showing largest amount of red were selected for breeding. During the first two years no progress was made but after that more highly colored red males and more uniformly colored red females were produced. From 1909 to 1918 no evident improvement in the uniformity of the red coloring has appeared. The reason for the marked improvement in 1909 was due to the fact that, unintentionally, blood from the Dark Brahma (also an Asiatic breed) was introduced. This race has no Buff Cochin blood but a type of pattern which is more diffuse than that of the Jungle Fowl and hence permitted the buff pattern to break the limited bounds proscribed by its position on the Jungle Fowl.

Owing to the great amount of inbreeding which the experiment has necessitated and the increasing sterility and weakness of the offspring it will be necessary soon to bring the experiment to a close. The results obtained, however, speak strongly for the impotence of selection in an inbred line, with few genetical factors, unless new mutations afford new foundations upon which to advance. It is quite clear that the original Buff Cochin was not made by slow selection from a bird with the Jungle-fowl type of coloration. The Buff Cochin probably originated as a xanthic sport.

This report presents the training records of the children and grandchildren of a pair of rats heavily alcoholized daily for more than two months before the birth of the young. These alcoholized rats came from the same litter; another pair from this same litter was chosen on the basis of equal weight as the parents of the normal controls. All matings were between sibs in the same litter. Habit formation was tested by training on a Watson maze and the Yerkes multiple choice apparatus. The results of the maze training have been calculated on the basis of the last twelve trials of the training proper as well as twelve memory trials given a month later. The criteria used are: average time per trial, number of “perfect” trials, number of wrong turns, or errors. Problems known as “first door to the left” and “first door to the right,” with tests, were used on the multiple choice apparatus; the results are based upon the number of correct first choices, and the number of wrong choices.

It is obvious from the figures in the following tables that the rats that received alcohol and their unalcoholized descendants were less successful than the controls in meeting the situations presented.

In 1908 R. and M. De W. Pearl 1 published data derived from the vital statistics of the city of Buenos Ayres, concerning the sex ratio in the following types of matings: (1) Italian × Italian, (2) Spanish × Spanish, (3) Argentine × Argentine, (4) Italian × Argentine , (5) Spanish × Argentine .

Of these crosses it will be noted that three are not racial crosses and two are. The data obtained by the Pearls stretched over a period of ten years. Although including very large numbers their data has certain minor disadvantages, for the most part frankly recognized by the authors themselves. First in importance is the fact that their data do not take still births into consideration, and second, the Argentine race may properly be considered as itself somewhat of a mixture and therefore less likely to breed as a pure race. If however the first three crosses be recorded as pure racial matings and the last two as racial hybrids, the ratio of males to 100 females in the two types may be compared as follows:

It will be noted that there is a significant excess of males in the hybrids. The Pearls note this fact and state in addition that environmental differences also fail to account for the results. They further agree that experimental investigations are necessary in order to reach adequate explanations for the observed facts.

It is believed that a close approximation to experimental conditions exists at the large lying-in hospitals. Here the patients are observed carefully, the sex of every offspring, including still births recorded, and the environmental conditions are for at least the period of confinement, as nearly equal as possible. The nationality of the parents of every child is recorded and the data thus obtained although not including nearly as large a number of cases as that of the Pearls, may, I believe, properly be considered accurate. During the past few months therefore, a study has been made of the records at the Sloane Lying-In Hospital, New York City.

The Mucorales, represented by the bread molds, and their relatives, are characterized by a sharp sexual differentiation. Certain sexually primitive forms are hermaphroditic with equal gametes. From such forms as a possible starting point sexual differentiation seems to have proceeded in two directions—first toward a constant inequality in the size of the gametes seen in a few hermaphrodites; secondly toward a difference in the individual plants themselves. In these diecious forms, the interaction of two sexual races are necessary for the production of sexual spores.

Not only will the opposite sexes of a single species unite to form sexual spores but a reaction can also be induced between the opposite sexes of different species which shows itself as an “imperfect hybridization”. An imperfect hybridization reaction between the sexual races of diecious species and heterogamic hermaphrodites leads one to believe that the (+) or vegetatively more active race is female and the (-) or less active race is male.

Tests of the sexual reaction of races of a large number of different species have been made and no race of the diecious forms has yet been found by the writer which reacts, if at all, other than as a male or a female. Variations, however, occur in the sexual vigor of different races and some are apparently neutral. Such a neutrality has been induced by environmental factors. Mutations have been observed in an hermaphroditic form producing hermaphroditic races with male, female and neutral tendencies.

1. Mongrel “blood” is eliminated by the pure-sire method not by “quartering out,” but in segments, in degree governed by the relative ontogenetic potency of the particular chromosomes eliminated.

2. The degree of elimination of mongrel “blood” by the puresire method depends upon two factors, (a) the number of chromosomes characteristic of the species, (b) the proportion of mixed dams of each possible pure chromosome-number used in each generation, (c) the relative fecundity of dams of different pure chromosome-number, and (d) the number of generations through which the system is carried.

3. By the pure-sire method, without selection in the F₅ generation in a twelve-chromosome (haploid) species (including man), the probability that a given offspring carries absolutely no mongrel blood, i. e., mongrel-descended chromosomes is 1 :0.205.

4. For mass improvement without selection the pure-sire method in a twelve-chromosome (haploid) species, ceases to be practically effective after the F₅ generation.

In plants there is a rather wide capacity for the development of organs of various kinds from primordia normally destined to produce quite different structures. For example, leaves may replace petals, stamens or carpels; petals may occur in the place of stamens or carpels. The transformation of stamens into carpels is a well-known phenomenon.

Furthermore, the continued development of a growing point the activity of which is usually terminated by the formation of some highly specialized organ, such as the flower or fruit, is quite familiar to those concerned with problems of variation.

Among these morphological abnormalities the continued meristematic activity of the axis which is normally terminated by the formation of the ovary is of very rare occurrence. It is, however, regularly found, although in a small and variable percentage of the cases, in one of the passion flowers, Passiflora gracilis. Here prolification of the fruit consists in the formation of series of carpels, which may or may not be ovuliferous, within the normal fruit. The mass of accessory carpels thus formed may be so large as to rupture the fruit wall.

While the occurrence of prolification may be regarded as a heritable characteristic in P. gracilis the abnormality is of relatively rare occurrence. Physico-chemical factors must, therefore, determine the occurrence of prolification in certain fruits and its absence from others. 1

If the formation of the basal prolification be due to the presence of special formative substances, one might occasionally expect to find the formation of carpellary tissue from other primordia. The only primordia normally developed subsequent to the carpels themselves are the ovules, which are borne on the carpellary margins.

To test this point, and to secure materials for other investigations, a series of dissections was begun in 1908. Those which were made from 1908 to 1915 are summarized in the accompanying table.

Dextrose (30 gms. in 250 c.c. water) given per os with acetyl salicylic acid (1 gm.) in a case of chronic tuberculosis was followed within 2 1/2 hours by a fall in rectal temperature from 38.03° C. to 36.02° C. In numerous observations upon this and other “labile” individuals under like conditions acetyl salicylic acid (1 gm. with 250 c.c.) never has produced a fall of temperature exceeding 1.1° C. in the same length of time.

This observaton has led to an investigation of the action of dextrose upon the heat regulation.

The preceding table shows the effects of dextrose given per os upon the temperature of three individuals, one of whom was in normal health. (Readings were taken at half hourly intervals but the curves are uniform enough for the first and last reading to suffice.)

Calorimetric Observations.-In each of the above experiments also the heat production has been determined by the indirect method, using the Benedict Respiration Chamber at the New Haven Hospital. As would be expected, the CO₂ excretion was constantly above the basal levels for the same individuals; but since the temperature falls, it is evident that dextrose effects a marked increase in heat eliminating processes. The respiratory quotients were also high; but the metabolism data are as yet insufficient to make comparisons between febrile and normal individuals.

Dextrose in Rabbits.-The antipyretic action of dextrose given per os has been confirmed in five rabbits with “peptone fever.” An average temperature fall of 0.8° in one half hour was obtained. The former level was usually regained in about two hours.

The accompanying curve (Fig. I) shows the temperature of an 1,800-gm. “peptone” rabbit given 18 gms. of dextrose by stomach tube in 100 C.C. of water heated to 41° C. The control “peptone” rabbit (1,730 gms.) received only 100 C.C. of water (at 41° C.).'lhe dextrose rabbit exhibited a loss of 0.6° C. in one half hour, the control rabbit no loss of temperature.

Gudernatsch was the first to call attention to the remarkable influence of the feeding of thyroid and thymus glands on frogs'rarvæ. That observer noted that the thyroid causes a dwarfing or shrinkage of the growth and size of tadpoles, and at the same time very rapidly hastens their metamorphosis into frogs, while the thymus, on the other hand, causes giant tadpoles but inhibits their metamorphosis. Gudernatsch and other observers have also studied the effect of feeding of other organs and glands on the development of frogs'rarvæ. As far as the present author has been able to ascertain, however, no experiments concerning the feeding of prostate gland to tadpoles are on record. The present author, in connection with a physiological and pharmacological study of prostatic extracts, conducted a series of experiments in feeding tadpoles with desiccated prostatic substance (Armour). The results are so interesting that it is deemed desirable to make a preliminary announcement on the subject in this place.

Prostate gland was fed to tadpoles of several species of frogs and it was noted that like the thyroid, the feeding of prostate substance tended to hasten the transformation of the larvæ into frogs. Such an effect was occasionally noted after administration of the gland substance for three or four days, and generally was distinctly noticeable after a period of from ten to fourteen days.

Unlike the effects of thyroid feeding, the feeding of prostatic substance while hastening metamorphosis did not produce much shrinkage in the size of the tadpoles. Indeed, it very often seemed to promote the growth of the tadpoles to a greater degree than was noted in the control animals. It was further noted, that prostatic substance was very much less toxic to the larvae than was thyroid substance, so that the tadpoles could be fed on the prostate continuously without being killed.

Sodium citrate was used as anticoagulant. The protein of the plasma was precipitated by acetone—free methyl alcohol and zinc chloride (c.p.). The total sulphur was determined by oxidation with potassium chlorate and precipitation with barium chloride. The total sulphate was estimated by a method similar to the one of Vansteenberghe and Bauzil. The neutral sulphur was computed by subtracting the total sulphate from the total sulphur.

Studies were made on the blood of patients suffering with kidney, liver and malignant disease as well as those suffering with chronic infections.

The blood is treated as in the determination of oxygen by Van Slyke's method. 1 A mixture of gases consisting of oxygen, carbon monoxide and the slight amount of nitrogen gas held in solution in the blood, is obtained. The extraction of the gases must be continued for a somewhat longer time than the one minute which is sufficient when oxygen alone is bound by the hemoglobin, otherwise the technique is the same. After the gases are measured, the oxygen is absorbed by permitting I or 2 C.C. of alkaline pyrogallol solution to flow slowly into the chamber of the apparatus from the cup at the top. The residual gas, corrected for the 0.009 C.C. of nitrogen gas per C.C. of blood known to be present, is the carbon monoxide.

Carbonates and phosphates are removed by adding 2 grams of calcium hydroxide to 100 c.c. of urine, and filtering after 10 minutes. 25 c.c. of the filtrate is brought to a pH of approximately 8 by adding 0.2 _N HCl with phenolphthalein as indicator, till the pink color disappears. Then 5 c.c. of 0.02 per cent. Tropeolin 00 solution are added, and the solution is titrated to a pH of 2.7 with 0.2 _N HCl, the volume being brought to approximately 50 c.c. by addition of water towards the end of the titration. The color is compared with that of 50 c.c. of a control solution with the same pH and indicator. The amount of 0.2 _N HCl required to give the endpoint with a control in which water replaces the urine is subtracted. Of the organic acids known to be present in urine in quantitatively significant amounts, the titration measures from 93 to 100 per cent. of each. It also includes very weak bases, but apparently of this class of substances only creatine and creatinine are significant; they are titrated to nearly 100 per cent. The titration figure, corrected for the amounts of these two bases, represents the organic acids.

With the view of ascertaining the systemic effect of malignant neoplasms upon the organism, a series of sixty cases of various types of malignancies have been studied and contrasted with benign tumors. The data accumulated comprise determinations of the uric acid, urea, creatinine, sugar, diastatic activity and carbon dioxide combining power of the blood; the phthalein excretion; the occurrence of proteinuria and casts; and the blood pressure.

Two thirds of our cases of malignancies present evidence of an impairment of kidney function. The appearance and progress of this renal insufficiency follows the order characteristic of interstitial nephritis, described by Myers and his co-workers. The nitrogenous waste product first to be retained is uric acid, later urea and finally creatinine, and paralleling the accumulation of these nitrogenous substances there was noted a drop in the carbon dioxide combining power of the blood. A hyperglycemia and an increased diastatic activity pointing to a lowered carbohydrate tolerance, were encountered only in those cases manifesting a nitrogen retention, and as this retention became more marked, the hyperglycemia and increased diastatic activity kept pace with it. Apparently, then, the hyperglycemia and lowered carbohydrate tolerance, as gauged by the diastatic activity of the blood, must be attributed not specifically to the malignant newgrowth but rather to the impairment of renal function. A further evidence of this impairment of kidney function was furnished by the decreased phthalein excretion, and the presence of protein and casts in the urine. Hypertension was noted in only a few instances.

This renal involvement was found to be associated invariably with general carcinomatosis; in 90 per cent. of carcinomata of the bladder, prostate, uterus and rectum and in about 50 per cent. of gastric carcinomata. On the other hand, carcinomata of the breast and epitheliomata produced no such changes. Of the small number of sarcomata coming under our observation, but one, a sarcoma of the kidney, disclosed a renal impairment.

The investigations recorded in these two papers are founded upon numerous series of animal experimentations and also clinical observations of human mustard gassing.

1. Local Action: Skin.—Dichlorethylsulphide (“mustard gas”), in liquid or in vapor form, even in very low concentrations, is an escharotic poison for the animal tissues (skin, conjunctivæ, cornea, mucous membranes of respiratory and gastrointestinal tracts) with which it comes in direct contact. The degree of the injury is proportionate to the concentration of the gas, the time of exposure, individual susceptibility, and local physical conditions, such as moisture, sweating, warmth, pressure and friction. The escharotic action is, for the greater part, painless, the anesthetic effect being especially notable upon the skin; while upon the mucous membranes its action may be more irritant, probably chiefly reflex in character. The cutaneous surfaces most susceptible are those with thinner, more delicate skin, well supplied with sweat glands and hair follicles, where sweat may collect, and which are exposed to friction or pressure, such as the axillæ, flexor surfaces, genitals, inner surface of arms and corresponding surface of trunk, inner surfaces of thighs, between the fingers, etc. There is a penetration of the gas into the sweat and sebaceous glands, and a re-solution of mustard gas vapor into the sweat or sebum occurs. The injuries are particularly striking in their insidious, slowly progressive development, becoming first apparent only some hours after the exposure. Upon human skin the lesion appears as a hyperemia, followed by vesication, eschar formation, sloughing and slow healing, with more or less pigmentation. Depilation may occur; in severe cases the eschar may extend entirely through the corium into the subcutaneous tissues. Secondary infection and gangrene of the eschars occur invariably in cases not properly treated. Milder lesions may show only the earlier stages of hyperemia, vesication or pigmentation. In general the injuries may be classed as burns of first, second or third degree.

The specific microscopic pathology of the local lesions of dichlorethylsulphide poisoning consists in degeneration and necrosis of the cells with which it comes in contact. The earliest microscopic change is pyknosis of the nucleus and cell body, followed by hydropic degeneration, liquefaction or coagulation necrosis. In the skin, hyperemia, with regeneration of the damaged cells, pigmentation, vesicle formation, desquamation of the dead epidermis or eschar formation mark varying stages of severity of the lesion. The degenerative changes extend deepest in the hair follicles and sweat glands. In mild burns without vesication the papillary layer of the corium may show a greater degree of necrosis than the epidermis itself, thus explaining the frequent occurrence of Nikolsky's sign. Large pigmented chromatophores may be the only living cells left in the papillary layer. In severe burns the necrosis may extend entirely through the corium. In the cornea, pyknosis and simple or coagulation necrosis of the corneal epithelium and interstitial substance, even to the endothelial layer, in extent varying with the degree of exposure, constitute the microscopic features. On the conjunctivæ the epithelium shows pyknosis, hydropic degeneration, liquefaction necrosis, or there may be a deeper necrosis extending into the subconjunctival tissues. The conjunctival surface suffers to a less degree proportionately than the epidermis. On the mucous membranes the epithelium shows pyknosis, hydropic or mucoid degeneration, desquamation, liquefaction or coagulation necrosis. The necrosis may extend into the submucosa, but the depth of the lesions on the conjunctivæ and the mucous membranes of the respiratory tract is never so great from identical exposures, as it is in the skin. Following the necrosis there is marked hyperemia, and the development of an edema, more marked in the subcutaneous and subconjunctival tissues in animals, but less marked in man. Human skin, however, shows a much greater tendency to vesication.

1. B. typhosus has a range of growth from P_H+5.0 — P_H+8.6, with an optimum growth at P_H+6.8 — P_H+7.0.

2. Stock cultures isolated from stools, blood, and urine have a more decided optimum than recently isolated cultures. In such cultures, the plateau is much more pronounced and extends over a wider range than in stock cultures. The latter is suggestive of microbic adaptation to changes in H-ion concentration in body fluids, particularly urine and bile.

3. The growth curve is influenced by changes in H-ion concentration.

At P_H+7.0, the minimum generation time at 36.0° C. — 37.0° C., is 31 minutes. Maximum growth begins on the average after five hours incubation.

At P_H+7.8, the minimum generation time is 33.4 minutes. The maximum rate of growth is reached by the eighth or ninth hour.

At P_H+5.4 the minimum generation time is 41.3 minutes. The maximum rate of growth is reached after 9¾ hours.

The very close relationship of B. abortus (Bang) to B. melitensis demonstrated by A. C. Evans 1 has been confirmed by our observations. Experimental studies, to be published in the near future, have furnished two additional characteristics of similarity.

1. The guinea pigs infected with B. abortus developed striking reactions of cutaneous hypersensitiveness with melitensis-protein, and vice versa in animals successfully infected with B. melitensis skin reactions are obtained with aborto-protein (see Table 1.).

2. In a series of attempts to infect guinea pigs with various known strains of B. melitensis obtained from the Hygienic Laboratory, U. S. Public Health Service (same strains as studied by Miss Evans), we finally succeeded in producing by intratesticular injection of 1/10 agar slant (48 hours'rrowth on peptic digest agar) a disease with pathological changes which could not be distinguished from those seen in about one hundred guinea pigs suffering from abortion disease. The lesions of two guinea pigs, which consisted of a very large spleen, general lymphadenopathy, liver and lung lesions with infiltrations of epithelioid cells and lymphocytes were only definitely diagnosed to be the result of a B. melitensis infection by cross agglutination and absorption

tests with the recovered bacteria. The observations thus far completed are summarized in Table 1. For comparison, the records of two guinea pigs successfully infected with the B. abortus, and four animals unsuccessfully inoculated intraperitoneally with B. melitensis are included.

Experiments with B. melitensis strains obtained from Algiers and with the B. paramelitensis (Nègre and Raynaud) are in progress.