Experimental Biology and Medicine

This experiment was undertaken for the purpose of comparing the course of syphilitic infection in 2 different breeds of rabbit, one a pure albino and the other a brown variety, inoculated under identical conditions with a virulent strain of Treponema pallidum. The animals were normal young males. The body weight of the albinos varied from 1430 to 1860 gm., that of the brown rabbits from 1340 to 2130 gm. During the experiment they were housed under uniform conditions in a light and well ventilated room, and fed on the same diet.

Experimental. The strain of Treponema pallidum used was that originally isolated by Nichols 1 from the spinal fluid of a patient with syphilitic neurorecurrence. This strain, now 17 years old, has had many passages through rabbits and has attained a high degree of virulence for this animal. The strain was brought to China in September, 1927. The immediate source of virus for this experiment was one of the third group of rabbits through which the strain had passed in this country. This animal was a brown breed and showed clinical evidence of orchitis 28 days after inoculation. When the orchitis was progressing, the right testis was excised under ether anesthesia, finely minced with scissors, and ground up in a sterile mortar with sterile physiologic sodium chloride solution. Of the more fluid portion of this emulsion, 0.3 cc. was inoculated into the right testis of each of 8 albino and 9 brown rabbits. The inoculations were all made on February 24, 1928. The emulsion contained from 2 to 3 actively motile organisms to the microscopic field. One animal in each group of rabbits died of pulmonary disease soon after inoculation.

By applying Ray's “hemolytic” test, 1 Sia 2 found that the blood of Kala-azar patients always hemolizes to a cloudy solution which on standing deposits a flocculent precipitate, whereas in normal subjects and in other patients studied, including severe anemia, it hemolizes to a clear solution which remains clear. Later Sia and Wu 3 showed that the turbidity is due to serum globulin and that Ray's “hemolytic” test is really a globulin precipitation test for Kala-azar. They also found that there is, in the blood of Kala-azar patients, an absolute decrease of serum albumin and an absolute increase of serum globulin. Since the test is performed by adding 20 cmm. of blood to 0.6 cc. of distilled water, 4 and since euglobulin is precipitated simply by dilution with water, it is desirable to know whether the turbidity and precipitate are determined by the amount of euglobulin in the serum. We have, therefore, made quantitative determinations of different fractions of serum protein in normal subjects and in Kala-azar patients. In all cases of Kala-azar the globulin test by Sia's method was positive and stained smears made from splenic pulp showed Leishman-Donovan bodies.

The serum proteins were fractionated with different concentrations of sodium sulphate according to the technic of Howe. 5 Tables I and II give the results of determinations on normal subjects and on patients with Kala-azar respectively. By comparing the figures in these 2 series it will be seen that euglobulin in Kala-azar serum is increased 3 to 13 times and amounts to 30 to 63% of the total serum globulin; the pseudo-globulin I is nearly twice, in some cases more than twice, as much as in normal serum.

Buschke 1 and others believe that thallium acetate acts chiefly on the thyroid gland and the nervous system. Balbi, 2 however, is of the opinion that it does not have such action. Since the Golgi apparatus and the mitochondria are the most vital cellular elements known so far, it is suggested that a study of the action of thallium acetate on these elements in the 2 tissues in question should yield more definite information than ordinary histological examination of the tissue as a whole. The following observations have been made:

Eighteen albino rats of about the same age (3.25 to 3.5 months) and sex (male) were injected with a single injection of 8 mg. of thallium acetate per kilo of body weight and killed at weekly intervals thereafter for the study of the Golgi apparatus, mitochondria and other cellular elements in various organs and tissues. For each experiment, 3 animals were employed.

Normally, the spinal ganglion cells contain a network-like Golgi apparatus and long and short, rod-like mitochondria interspersed with Nissl bodies concentrically around the nuclei. In the thyroid cells the network-like Golgi apparatus lies on the luminal side of the nucleus and the mitochondria run parallel along the long axis of the cells in the basal portions. The colloid substance in the thyroid vesicles exhibits a basophilic staining reaction.

The cellular changes after the administration of thallium acetate are summarized as follows: One week after the injection the Golgi apparatus in some of the thyroid cells is changed from network into droplets, the mitochondria from filaments into segmented granules, and the staining property of the colloid substance from basophilic to an acidophilic reaction. Two weeks after the injection the droplets and granules of the Golgi apparatus and mitochondria became much finer in all the cells and were found dispersed in the distal portions of the cells.

Leigheb 1 and others have observed that the administration of thallium acetate to rats and guinea-pigs produces extensive degenerative changes in the thyroid gland. In view of these observations, the following experiments were undertaken to study the basal metabolism of animals treated with the drug.

Four white rats of the same breed and of approximately the same age were injected subcutaneously with thallium acetate in aqueous solution. Three of the animals received 12 mg. of the thallium salt per kilo of body weight and one, 8 mg. per kilo. Before the administration of the thallium acetate, 4 observations were made on the basal metabolism of each animal at intervals of 3 or 4 days. After the administration of the drug, the basal metabolism was studied at similar intervals, until the basal metabolic rate returned to normal. The technic used for determining the basal metabolism was that described by Wu and Chen. 2 For each experiment there were from 6 to 8 determinations. The average of only those determinations (usually 3) taken at the time the animal was quiet was used as the final result of the experiment.

All of the rats showed diminution of their basal metabolic rates, in parallel with which, defluvium of hair on the back and less briskness of the animals were noticed. In the 3 rats injected with 12 mg. of thallium acetate per kilo of body weight, the basal rates began to diminish from the 8th to the 15th day after the administration of the thallium salt. One of the animals died at the end of the experiment on the 21st day after the injection of the drug, at which time the basal metabolic rate was at its lowest.

Using our modified method of the isolated lung perfusion described by Sollmann and Von Oettingen, 1 we studied the peripheral action of ephedrine and pseudoephedrine on rabbit bronchial muscle. The arrangement of the perfusion was similar to that of Sollmann and Von Oettingen, but the borders of the lung were trimmed with sharp scissors so that the perfusion fluid was allowed to drain freely. The lung was lightly supported with a funnel covered with moist cotton to prevent drying, drops per minute through the funnel being reckoned as the rate of the bronchial outflow. The drugs took from half to one minute to reach the outflow, and 4 to 5 minutes for the concentration of drug of the outflow to be the same as that of the inflow. After 1 to 2 hours of perfusion with Tyrode solution, when the rate of outflow was 50 to 80 drops per minute, and when it had remained nearly constant for over 10 minutes, the perfusion of the ephedrine-Tyrode solution was started, and allowed to continue for 20 to 30 minutes (pH of ephedrine and Tyrode solution was found to be the same). Bronchial constriction was indicated by a decrease of outflow, and dilation by an increase. The bronchial muscle under this condition was found to be exceedingly sensitive to drugs, comparing with the method of Sollmann and Von Oettingen. After 6 to 7 hours of perfusion the bronchioles still responded to drugs. Injection of 0.5 cc. of pilocarpine 1:1,000,000 or atropine 1:1,000,000 into the connecting tube between lung and perfusion apparatus caused respectively a distinct constriction and dilation of the bronchioles.

The response of the bronchial muscle to ephedrine varied with the dosage.

It has already been stated 1 that benzylephedrine, B-phenyl-B-hydroxy-d-methyl-ethyl-methyl-benzyl-amine, has a local anesthetic action about twice as powerful as procaine, as measured by the wheal method.

The principle of adrenaline-procaine synergism was applied to this new compound. Using 0.05% solutions the addition of 0.0005% adrenaline prolonged the time of wheal anesthesia from 8 to 18 minutes. The further addition of adrenaline up to 0.005% prolonged the time to 140 minutes. The latter is a greater potentiation than is obtained with procaine adrenaline mixtures. Increasing the amount of benzylephedrine prolonged the anesthesia, but greater increase obtained with the higher percentage of adrenaline would indicate that vasoconstriction and subsequently delayed absorption is the more important factor.

The known potentiation of the blood pressure action of adrenaline by cocaine with its excessive vasoconstriction is thought to be the factor in the toxicity of this mixture. Comparative experiments upon luminalized dogs with benzylephedrine show that: 1. Benzylephedrine does not potentiate the blood pressor action of adrenaline as is seen with cocaine (see Fig. 1). 2. The pulse did not show such marked changes as are seen with cocaine-adrenaline reactions. 3. After benzyl-ephedrine-adrenaline produced a slower respiration, which effect was greater than that after cocaine.

Fundulus eggs covered for 10 minutes with sea water and 5 cc. of follicular extract, F1, 0.13; F2, 0.0026; F3, 0.00086; F4, 0.00043 units per cc. were removed from the solution and fertilized. In each of the first 3 concentrations 85 eggs were used both in the experimental lots and in the controls with the following results:

Of the 80 eggs exposed to F4 follicular extract 57 were fertilized, while the controls yielded 72 fertilized. Of a total of 335 eggs treated with the follicular extract 180 or 53% were fertilized; of 335 controls, 296 or 88%.

Fertilized eggs were placed for 10 minutes in sea water containing 5 cc. F1 follicular extract and then removed to different salt solutions and the time required for the eggs to sink was noted, with the following results:

On the second day after fertilization 30 eggs were injected for 6 consecutive days with 0.01 cc. F3 follicular extract and the following changes in the embryo were observed: (a) abnormally dark pigment cells, (b) anastomosis of these cells and streaming of the pigment granules, (c) defective lens in one or both eyes, (d) diminutive forebrain. Practically the same abnormalities appeared in the embryos exposed to 5 cc. of F3 follicular extract placed in the sea water.

Thirty beating hearts stopped within an hour by 0.5 KCl were placed in sea water and 10 cc. Follicular extract; within 5 hours they recovered and the controls likewise began to beat in the same length of time. Thirty beating hearts placed in sea water and 10 cc. Fl follicular extract were then exposed to the KCl and within 55 minutes they had all stopped beating, while the controls ceased to beat within 58 minutes.

Certain of the free-living and associated protozoa have been selected for a preliminary study of their reaction to the Feulgen thymonucleic acid test. They have been considered as to their status, source, nuclear organization and their reaction to this test. Extra-nuclear inclusions and organelles have received attention, as well.

Thymonucleic acid has been found to be present in some portion of the nuclear apparatus of each protozoan tested, thus far. Whether it can be said that the Feulgen reaction is indicative of the presence of chromatin is somewhat doubtful.

In Paramecium multimicronucleatum and Paramecium aurelia, both the macronucleus and micronuclei of each species gave a decidedly brilliant purple color reaction, while in the “associated” protozoan, Balantidium coli from the guinea pig, the macronucleus was a brilliant purple and the micronucleus a pale violet or negative. Presumably the micronuclei play similar rôles in the life-history of the two ciliates. Division stages of these two forms have not yet been secured for use in this reaction.

The food vacuoles of Paramecium contain bacteria and other ingested food particles. These vacuoles gave a faint violet reaction, which was due doubtless to the presence of the bacteria. The remainder of the protozoan was colorless.

The presence of host intestinal epithelial cells within the endoplasm of Balantidium was definitely established. The nuclei of these host epithelial cells gave the same reaction within the protozoan body as did those nuclei in situ, in the same field of the microscope.

The macronucleus of Balantidium coli showed vesicular areas similar to those previously described by the author 1 for the same species. In the Feulgen preparations these areas appeared as clear colorless vesicles, as they did when stained with alum cochineal, in contrast to their intense dark staining in Heidenbain's Iron Haematoxylin.

Glass cell potentials or other electrode potentials in series with high resistance can be measured conveniently by a modification of Beans and Oaks' 1 condenser technique. Instead of the simple ballistic method employed by them, the cell is compensated by a potentiometer, and a null point reading made, thereby reducing polarization of the cell by reducing the charge on the condenser (Fig. 1).

The Leeds & Northrup type K potentiometer lends itself readily to such an arrangement. Glass cell and condenser are connected in series to the regular E.M.F. binding posts (Fig. 2). From a point between glass cell and condenser a lead passes to the insulated lower contact of the key K and from the upper contact to the galvanometer binding post proximal to the E.M.F. posts. The double point, double throw switch of the potentiometer is short-circuited across its front posts, at S, and the potentiometer may still be checked against the standard cell in the usual manner without complication. The potentiometer tap keys with resistance protection (K₁) are employed as usual in reading the standard cell check, and the extra tap key K only is used in reading the glass cell-potentiometer balance, the condenser acting as a protection to the galvanometer.

A 2 m.f. condenser is sufficient to give a reading to 0.2 millivolt on a Leeds & Northrup high sensitivity galvanometer (10^-10 amp./mm.) critically damped by a shunt. With a glass cell of 150 megohms resistance, the time of charge to 1/2 final voltage of the condenser through this resistance is 3.5 minutes, the time to 90% is 12 minutes. Adjustment to within one millivolt of the null point can be made by readings at intervals of only a few seconds, since the condenser need be charged to only a small fraction of its full capacity to obtain this sensitivity.

Sedimentation of the corpuscles of heparinized blood was determined by noting the extent of fall in glass tubes with an inside diameter of 4 mm. and height of 100 mm. during an interval of 1 hour. The tubes were centrifuged to insure complete sedimentation and the sedimentation-index computed as the ratio between the sedimentation observed in 1 hour and the possible maximum extent of sedimentation.

Precipitability of serum protein was determined by adding various amounts of aluminum sulphate, as contained in a unit volume of 1 cc. to 0.2 cc. of unheated blood serum in small tubes. Serum and reagent were mixed and set aside at room temperature for 1 1/2 hours. A heavy flocculent precipitate that settled out leaving a clear supernatant fluid was recorded as a positive reaction.

Average values for these blood properties as observed in a group of 14 tuberculous and a group of 20 normal individuals are listed in the accompanying table with average values for total protein, fibrin, globulin and albumin as contained in the plasma.

In the presence of tuberculous infection the blood of man exhibits a quantitative shift in plasma protein toward the more labile globulin and fibrinogen fractions, also an increased sedimentability of the corpuscles and an increased precipitability of serum protein. The latter changes are coincident with but not necessarily related to changes in plasma protein. Apparently with the inception of a general biologic reaction in the course of tuberculous disease, a decrease in blood stability (as measured by these properties) becomes apparent and is quantitatively without relationship to the extent of tuberculous involvement.

Clinical and experimental value of the aluminum sulphate flocculation test may be extended by the titration procedure outlined.

It has been found earlier that residue remaining on the membrane after ultrafiltration of bacteriophage through collodion retains a large proportion of the active agent and does not give it off freely into the filtrate on repeated washing with water. But, if broth is substituted for water in washing, the active agent reappears in the filtrate in high concentration. 1

This finding suggested at first that passage of broth through the membrane resulted in coating the filtering bed by the colloids present in the broth, thus rendering it permeable for coarser particles which were held up by the membrane when suspended in water. However, further experiments have shown that preliminary passage of broth through a new membrane similar to that used above failed to increase its permeability to the residual fraction of the phage. On the basis of these findings the results of the earlier experiments were interpreted as indicating that the addition of broth directly to the residue caused detachment (elution) of some of the active agent from the coarser particles and its adsorption on and passage with the finer particles of the broth.

The existence of a method 2 permitting the measurement of the particles which carry phage suggested the means of inquiring into the validity of this tentative conclusion. We determined by this method the size of ordinary broth-phage filtered through Berkefeld filter as being in the average 12 mμ in diameter. 2 Using the same procedure, we now find that particles held back by the membrane after ultrafiltration measure 28mμ in diameter (average). When broth (free from phage) is added to this residue and ultrafiltration repeated, the particles which come through measure 20mμ in diameter (average)—that is, they are smaller than the original particles kept back by the membrane.

Sodium amytal (sodium isoamylethyl barbiturate) was introduced by Page 1 as an anesthetic without influence on blood sugar. He has been supported in this claim by competent observers 2 although Weiss and others 3 disagree with him. It has been stated by some physiologists that unless an animal was under sodium amytal anesthesia, blood sugar figures could not be accepted as being uninfluenced by the emotional state of the animal.

In this connection it is interesting to note the effect of amytal anesthesia on the hyperglycemia induced in dogs by morphine sulphate. In Table I are given blood sugar figures on 7 dogs. Two series of blood sugar determinations, several days apart, were made on each animal; first, following the injection of morphine sulphate alone and secondly, following morphine injection with the animal under amytal anesthesia. The dogs varied in weight from 7 to 18 kilograms. They were on a routine diet and were last fed 14 to 18 hours before the beginning of the experiment. The dose of morphine sulphate was 10 mgm. per kilo body weight given subcutaneously and the dose of sodium amytal was 50 mgm. per kilo intraperitoneally. Blood samples were taken from a vein on the hind leg and sugar determinations were made by the method of Hagedorn. 4

The results indicate that sodium amytal anesthesia in dogs has a marked inhibiting effect on the hyperglycemia due to morphine.

Acetarsone (acetyl-amino-hydroxy-phenyl arsonic acid) was synthesized in 1921 by Tréufouel and Fourneau 1 after having been prepared earlier by Ehrlich. 2 Levaditi 3 noted the drug was toxic for rabbits in doses of 0.66 gm. per kilo on oral administration, and that it was tolerated in doses of 0.3 to 0.4 gm. per kilo. Since then the drug has been recommended for syphilis, amebiasis, and other protozoan infestations. Conflicting reports have appeared on its therapeutic efficiency and toxicity. Pool, 4 Worms, 5 and Kolle 6 found this compound more toxic than Levaditi originally reported, causing death in rabbits when 0.2 to 0.3 gm. per kilo were given by mouth.

Levaditi and Poole worked on material synthesized by Fourneau. We attempted to find the minimal lethal dose on oral administration to cats and rabbits of the commercially available product, and have also studied the toxicity of its calcium and sodium salts. 7

The material was powdered and administered in gelatin capsules washed down with water. It was necessary to anesthetize the cats for this procedure. All animals were kept in individual cages, under identical conditions of diet and hygiene, and were observed for at least 30 days following the administration unless death intervened. Those animals dying within the observation period were subjected to post-mortem examination and sections of the principal organs were taken for histological study. Untreated animals were maintained in the laboratory under similar conditions as controls.

Table I shows the results of this study on 35 cats and 56 rabbits. The determination of the minimal lethal dose on oral administration is difficult because of the uncertainty of absorption. The range of toxicity of these compounds indicated by our figures seems reliable except in the case of calcium acetarsone in rabbits.

When a continuous pressure is maintained upon the bone in the alveolar process of the maxilla the resulting trauma is characterized by several histo-pathological changes.

The manner of applying pressure is by means of bands cemented upon the crowns of the teeth. To these spring wire has been previously soldered so that when the appliance is in place the tension may be increased, decreased or changed in direction merely by bending the spring wire. Such appliances have been in place on teeth of monkeys (Macacus rhesus) from one day to 8 months and the results have been so striking that this preliminary report seems appropriate.

When the crowns of teeth are separated distally their roots are forced nearer each other. This pressure produces areas of absorption in the alveolar process as well as on the periphery of the roots. Combined with this absorption is a great amount of fibroblastic activity. These changes commence apparently early in the experiment as shown by the examination of the tissues from an animal on which the appliances had been in place only 24 hours.

In one case, where the greatest possible tension was applied in the effort to produce absorption, the alveolar process between the teeth was fractured.

The force used in producing this result as measured by Jolly balances was found to be one and three-quarters (1 3/4) pounds.

Approximating teeth were subjected to elongation tension and its opposite a depression tension. This double pull stretched the supporting tissues to a great degree and the effect was one in which the bone at the alveolar crest, the overlying connective tissue and mucosa have been pulled or dragged out of their proper position and have followed the movement of the attached tooth.

The present work is a continuation of the studies which have been carried out in this laboratory on the mode of combination of proteins with the inorganic elements. Previous reports have dealt with the compounds of proteins with the alkali metals 1 and with the alkaline earth elements. 2 This work has now been extended to include the compounds which ferric iron forms with certain proteins, amino acids and related substances.

The method employed consists in adding the substances to be tested to a known solution of ferric iron. To this a standard amount of ammonium thiocyanate is added and the reaction is adjusted to a definite pH. The color of this solution is compared with a standard iron thiocyanate solution. If the substance tested forms a compound with iron (e. g., complex ions) which is less dissociated than ferric thiocyanate, the color of the solution will be less intense than the standard. If the ferric compound is dissociated to a greater extent than ferric thiocyanate, no appreciable effect on the color will be noted.

The results showed that at pH 2.5 substances which possess a hydroxyl group alpha to a carboxyl group markedly reduce the color of ferric thiocyanate. The effect is less pronounced when the hydroxyl group is in the beta position. Dicarboxylic acids, which may be considered as hydroxy acids, show some effect. Hydroxy acids which contain a double bond oxygen (=0) and hydroxy groups in an arrangement such as occurs in phosphoric acid, arsenic acid and sulfuric acid markedly reduce the color of ferric thiocyanate. Casein and gelatin likewise exhibit this property. Substances which contain only hydroxyl groups (glycerine, borates, etc.) showed within the limits of concentration tested, no effect.

In a previous study 1 of the circulatory actions of digitalis in dogs we showed that the diminution in cardiac output was dependent upon the lowering of venous pressure, rather than upon cardiac depression, i. e., that it was of vascular and not cardiac origin. It was suggested that this lowering of venous pressure from digitalis was mediated by a hepatic vein constriction with pooling of blood in the portal vessels. In the present study we have amplified the evidence and confirmed the importance of the hepatic mechanism previously suggested.

It was found that intravenous injections of digitalis, in doses corresponding to the full therapeutic for man, and of strophanthus, in 5 dogs caused an increase in arterial pressure, fall of venous (intraauricular) pressure, and increase in portal vein pressure. The increase in portal vein pressure was due to hepatic vein constriction, agreeing with the increased liver volume previously observed by us. The latter actions agree also with similar actions on isolated hepatic veins and on those of perfused livers reported in the literature.

After elimination of the liver (6 dogs) from the circulation, by shunting the portal blood into the vena cava, or of the entire splanchnic circulation by ligation, digitalis and strophanthus did not diminish the venous pressure, but increased it, if anything. Accordingly, the fall of venous pressure, after digitalis and strophanthus in dogs with liver intact, was due to diminished venous flow owing to pooling of blood in the splanchnic (mainly portal) region as a result of block in the liver. This mechanism adequately explains the diminution in cardiac output after digitalis, since a diminished venous return must result in deficient cardiac filling and output.

Comparisons were made with known actions of histamine and epinephrine in the same animals.

The occurrence of a natural salicyluric acid in the body, and its significance as a detoxification product, after administration of salicylates, analogous to hippuric acid after benzoates, have been much discussed but not conclusively settled. Owing to previous negative results by one of us 1 with chemical methods of isolation from urines, it was attempted to obtain indirect evidence on the question by a study of the pharmacological actions of synthetic salicyluric acid. The pure compound was synthesized according to E. Fischer's method 2 by Dr. Robert T. Dillon of the California Institute of Technology, Pasadena, to whom our thanks are due.∗

It was found that synthetic salicyluric acid was comparatively stable in vitro, since it was not hydrolyzed in incubated buffer-salt solutions with hydrion concentrations falling within the range of body fluids, and in these solutions containing ferments, yeast and bile-salts. Some hydrolysis occurred in solutions of pH 2 and 14 and complete hydrolysis in strong acids and alkalies.

On the other hand, the compound suffered marked hydrolysis in its passage through the body, since from one-half to three-fourths of the total excreted salicyl in urines of human subjects (6) consisted of ordinary salicylate. The total salicyl excretion was about 88% of the total administered (from 4 to 28 gm.). The effects on urinary nitrogenous metabolites were variable or insignificant. The symptoms of salicylism in human subjects were weak, even with the highest oral doses, but in white mice, white rats, pigeons and a dog (total, 89 animals) typical symptoms of salicyl poisoning were demonstrated after hypodermic and intravenous administrations. The M.F.D. in the majority or 60% of the mice was 1.13 gm., and in rats 3 gm., per kilo.

It has been shown 1 that oral administrations of certain inorganic salts, alone or in combination, are definitely toxic for ducks. The greatest toxicity in these experiments was found in sodium chloride and sodium sulphate mixtures containing in addition either magnesium or bicarbonate together with nitrate ions. The symptoms exhibited by the birds after feeding with these mixtures resembled in many respects those noted in “duck disease”. The salt composition of soils from disease and non-disease areas was studied to attempt to correlate these experimental findings with the naturally occurring condition.

Soil samples were obtained from the following areas:

A. Disease Areas. 1. San Joaquin Valley (West of Pond, Calif.). 2. Tule Lake (Modoc County, Calif.). 3. Government Sump (South of Klamath Falls, Ore.).

B. Non-disease Areas. 1. San Joaquin Valley (Los Banos area). 2. Isolated ponds south of Klamath Falls, Ore.).

All samples were taken near the surface of the soil and within a few yards of the water's edge. Analyses were made on water extracts. Carbonate, bicarbonate, and chloride were determined by titration. Calcium, magnesium, and sulphate were approximated (after precipitation) by comparative turbidity tests. Nitrate was determined by aluminum reduction followed by distillation and titration. Qualitative tests only were made for nitrite. Sodium was computed by difference in the reaction value of basic and acidic ions. The ions were grouped for study as follows: 1. Chloride, sulphate, and sodium. 2. Carbonate and bicarbonate. 3. Calcium and magnesium. 4. Nitrate.

In Fig. 1 are shown the relative per cents of the different groups of ions in relation to increasing amounts of salts in the soil. This chart includes 50 samples from all areas and is presented for the purpose of showing the general character of the soils studied.

In a previously described series of experiments 1 it was found that the introduction into the venous circulation of an embolus infected with pyogenic organisms may be followed by widely differing pathological changes in the parenchyma of the lung. To determine the exact conditions present within the pulmonary and bronchial circulations following the introduction of such emboli a method for the radiographic demonstration of the two circulations was developed.

Anatomically the bronchial arterial system usually arises as one or more branches from the first portion of the descending aorta anteriorly. Occasionally branches may arise from the first or second intercostal, the internal mammary, or the right subclavian arteries. They enter the lung at the hilum and course along the bronchi and their branches up to the ductus alveolaris. 2

The bronchial artery is best injected with the lung in situ. Under chloroform or ether anaesthesia the chest is opened avascularly by splitting the sternum exactly in the midline. Just before entering the pleural cavity the animal is killed with the anaesthetic to prevent injury to the inflated lung.

The innominate and left subclavian arteries are ligated as they leave the aortic arch. The first intercostal arteries should be clamped laterally to avoid occlusion of the bronchial artery which may arise from one of them. The thoracic aorta is tightly clamped distal to the fifth intercostal arteries so as to limit the injection to the thorax.

The pericardium is opened longitudinally and a large cannula inserted into the aorta through an incision in the left ventricle, all air is excluded from the system by filling the cannula and the attached tube with warm water before insertion into the ventricle. A heavy ligature passed around the base of both the aorta and pulmonary artery holds the cannula firmly with less danger of rupture of the aorta by cutting through of the ligature.

Since water in a layer 10 millimeters or more thick is well known to be inpenetrable by ordinary radiant energy (that is, in the ultraviolet, visible, infra-red, and heat spectrum) of wave lengths longer than 1.4 micron, it has been suggested by Coblenz 1 that living tissue will, in general, be permeable only to wave lengths shorter than these. Sonne 2 found penetration of living tissue in his so-called “visible range”, but his filters (water and 5% ferro-ammonium sulphate in glass chambers) apparently transmitted waves to a length of at least 1.3 micron. 3 On the other hand, Bachem and Reed 4 have recently shown that wave lengths shorter than 0.6 micron practically do not penetrate living skin. Evidence exists to show then, that the wave length range of ordinary radiant energy capable of penetrating living tissue lies between 0.6 and 1.4 microns, i. e., it includes those infra-red rays lying near the red rays of the visible spectrum. Independent experiments initiated by me furnish further evidence in this regard, and indicate the extent to which these rays penetrate living tissue. The required measurements were made by G. H. Cartwright at the California Institute of Technology under the direction of R. C. Burt.

For convenience, the experiments on transmission were performed on one subject's cheek which averaged 5 mm. in thickness. Initial experiments indicated that this tissue transmitted about 14% of infra-red rays of a wave length of 0.86 micron. These experiments were made by means of a spectrograph and photographic plates. Opal glass 2 mm. thick was placed in front of the slit of the spectrograph to diffuse the light, the source of which was a 15 Watt electric light bulb.

A number of workers recently have demonstrated that it is possible to produce variants among both plants and animals by exposure to the effect of X-rays We, in turn, have endeavored to demonstrate change by a similar procedure applied to the Bacteriophage and to the organism susceptible to the Bacteriophage, following suggestions made by Dr. Olson.

Seven strains of B. coli bacteriophage were selected and for each of these we cultured the particular strain of B. coli acted upon by its respective strain of bacteriophage. There were selected 7 coli bacteriophages and 6 strains of B. coli for this series of experiments. These bacteriophages were effective as follows upon the bacterial strains: one for all the strains, one for 5, one for 2 and 3 could lyse only one strain each of the organisms. There were thus 42 combinations possible in such a set up of tubes in case any variation were to appear.

The following methods for exposure were followed. The organisms were young cultures in the phase of positive logarithmic growth in beef infusion broth and the phages were recent filtrates in the same medium. These were contained within lead free glass test tubes. Exposure to X-ray was carried out at a distance of approximately fourteen inches from the target of a Roentgen tube actuated by approximately 75,000 volts of electricity. The exposure period was 30 minutes. Following this treatment each baeteriophage was placed in contact with untreated organisms of each culture and the exposed bacterial culture was set up with each of the unexposed bacteriophages. For control purposes a similar series of unexposed bacteriophages and bacterial cultures were used.

The following results were obtained. By this method of treatment 3 of the bacteriophages developed reduced effectiveness when brought into contact with organisms shown previously to be susceptible to them in the control series.

In a previous note 1 we reported that under the influence of canine leucocytic extract horse proteins undergo an initial pseudo-proliferation, presumably due to immunologically symmetrical proteolysis. A similar apparent multiplication of horse proteins takes place on 14-day incubation with an excess (20:1) of normal canine serum. In both cases the apparent multiplication is followed by a marked flattening and distortion of the precipitin graph 2 which we interpret as an index of horse protein denaturization. 3

In contrast with this parenteral type of proteolysis, gastro-intestinal proteolysis has thus far given in our hands no suggestion of pseudo-proliferation and subsequent partial denaturization. If 5% horse serum is added to 0.1% commercial trypsin in Ringer's solution, for example, or to 0.1% commercial pepsin in acidulated (n/100) Ringer's solution and the mixture is incubated at 37.5° C. for several hours, precipitin graphs show from the first a rapid and consistent decrease in horse-protein titer, without flattenings or distortions of the precipitin graph. We interpret this as meaning that in gastro-intestinal proteolysis none of the lytic products retain their original horse protein specificity.

This study represents selected data from 20 titrations of test-tube lytic products. Each titration was accompanied by parallel tests of from 2 to 4 non-lytic controls.

Anti-horse precipitin withdrawn from a rabbit 10 days after injection of the final immunizing dose of horse proteins gives precipitin graphs 1 suggesting a 100% 17th-day retention of intravenously injected horse proteins in the canine circulation, with marked denaturization of these proteins. 2 Parallel tests with precipitin withdrawn 23 days after the final immunizing dose, give graphs suggesting but 1% horse protein retention with no horse protein denaturization.

Our conventional assumption that the specific precipitin is biochemically identical at all stages of sensitization and immunization with the same antigen evidently requires further study.

This report gives the maximum difference observed in 12 parallel titrations of parenteral horse protein derivatives with about 20 different rabbit antisera.

The work here presented was conceived by the observations of Bills 1 on the action of various reagents on the antirachitic vitamin, and was carried to completion in 1927. It occurred to us that an extension of Bills' work to vitamin A might be of value in throwing light on the chemical nature of this factor.

Of the reagents examined by Bills, sulphur dioxide was found to be without action on the antirachitic vitamin. Early in our investigation, however, we found the effect of this substance on vitamin A in cod liver oil to be of such a magnitude as to render desirable an examination of vitamin A from other sources.

In doing so we were guided by two principal considerations: (1) whether vitamin A activity is the property of a single chemical individual or that of a specific atomic grouping possessed in common by several different molecules; (2) assuming that vitamin A of the green plant would be as susceptible to destruction by sulphur dioxide as vitamin A of animal origin, it occurred to us that one important aspect of the damage to vegetation by smelter smoke would be the destruction of vitamin A. With these considerations before us we gave primary attention to experimentation with sulphur dioxide. Cod liver oil, butter, and extracts of alfalfa were used.

Cod liver oil was treated by bubbling with the purified gas at temperatures of 20° to 100° for periods of 15 minutes to 2 hours. The excess of sulphur dioxide remaining at the end of the experimental time was removed by exposure in vacuo.

Butter was melted at 60°, freed of casein by filtration, and treated with sulphur dioxide at 60° for 1 to 24 hours.

Recent work has thrown doubt on the presumption that use of amytal (isoamyl ethyl barbituric acid) as an anesthetic is without interference to blood sugar regulation. 1 In spite of the finding of Page 2 that amytalized rabbits and dogs show normal rise in blood sugar upon splanchnic stimulation, it seemed advisable to determine whether other methods of provoking hyperglycemia would produce normal results. The dosage of amytal employed in our experiments was 50-60 mgm. per kilo intraperitoneally.

The first method tried was intravenous injection of adrenalin. To test our sample of adrenalin 0.5 cc. of a 1:1000 solution was injected into a 16 kilo normal dog, whose post absorptive blood sugar was 0.080. There was no change in 5 minutes, but 30 minutes later the blood sugar had risen to 0.160. In a 21 kilo amytalized dog 1 cc. of 1:1000 adrenalin caused a rise of blood sugar from 0.105 to 0.190 within 5 minutes, 30 minutes later it was 0.185. In an 11 kilo amytalized dog 0.5 cc. 1:1000 adrenalin caused a rise from 0.100 to 0.200 in 5 minutes and 30 minutes later it was 0.192. Injection of adrenalin produces normal hyperglycemia in amytalized dogs.

The second method used was asphyxia. A sheet of rubber dam was wrapped about the dog's muzzle and its air supply entirely cut off for exactly 2 minutes. The dogs' tongues were found to be cyanosed when the bandage was removed. The results are given in the accompanying table.

In none of the amytalized dogs did asphyxia produce the customary rise in blood sugar. Dog 6 was so severely asphyxiated that 5 minutes artificial respiration was necessary before it would breathe on its own, Even this degree of asphyxia produced no significant change in blood sugar.

A method for quantitative estimation of ammonia nitrogen and carbamic acid nitrogen, in a solution containing ammonium salts, urea, and carbamates, has been developed. The method depends on the fact that ammonium carbamate is stable in Nessler solution so that one-half of the nitrogen in the ammonium carbamate does not affect the color of the Nessler solution. However, if the solution is acidified before Nesslerizing, the carbamic acid is almost instantly decomposed and Nesslerization gives the total of ammonia and carbamate nitrogen present. In other particulars analyses are made according to the well-known colorimetric method of determining ammonia. Each solution to be analyzed was Nesslerized under these 2 different conditions. The difference between the two values for nitrogen was equal to the carbamic acid nitrogen. Twice this figure was equal to the ammonium carbamate nitrogen. The method was shown to be quantitative within the limits of accuracy of reading a colorimeter.

A number of experiments have been carried out in which the enzyme was allowed to act on 100 cc. ice-cold 1% urea. The action was stopped after 5 to 7 minutes by adding a trace of potassio-mercuric iodide. During this time about 1 mg. per cc. of total combined nitrogen had been liberated from the urea. This concentration was found to be most convenient for analysis. After poisoning the enzyme, analyses were made immediately for ammonium carbamate and ammonium carbonate and then at intervals until the carbamate had fallen almost to zero. Figure 1 shows the curve for percentage ammonium carbamate nitrogen of the total nitrogen when plotted against time after the enzyme had been stopped.

The percentage of carbamate diminished very rapidly but at a measurable rate. Our method of analysis was not accurate enough to determine the point of equilibrium between the carbonate and the carbamate.

In tissues in differing states of physiological activity the phospholipid percentage was greater when the tissue was more active. In the corpus luteum of the pig the phospholipid content was about 3 times as great during its active state in ovulation and pregnancy as in the resting state. Cholesterol was not greatly different. Malignant tumors had about 3 times the content of phospholipid and twice the content of cholesterol as benign tumors. Mammary glands (rabbit) at the end of pregnancy had twice the phospholipid content of the resting gland, cholesterol remaining the same.

The presence of polynuclear leucocytes in vaginal smears of the rat has been noted by Long and Evans, 1 who observed the presence of such cells during certain phases of the oestrous cycle, no quantitative measurements being made. The present report deals with polynuclear counts made from blood and from vaginal smears taken simultaneously from the same animal. All data were obtained from healthy rats kept under ordinary laboratory conditions.

Blood smears were prepared in the usual manner. The smears, upon drying, were fixed with methyl alcohol for one minute and were stained subsequently with Wright's blood stain. A few smears were stained with “Fadicit”, only Solution I being used. Either stain permits an easy identification of the polymorphonuclear neutrophiles and counts may be made without difficulty.

Vaginal smears, obtained by means of a wire loop and clean cotton, were stained with Delafield's hemotoxylin. In making a polynuclear count, cells showing fragmentation or obvious distortion were omitted, the count including only cells possessing a distinct cytoplasmic outline.

It will be noted from Table I, a representative cycle, that the blood count is decidedly left-handed as compared to that of normal man, and that the vaginal smear count, while also decidedly left-handed as compared to man, is also somewhat right-handed when compared to the animal's own blood count. The average weighted mean of a group of 35 blood polynuclear counts is 1.09, with a standard error of ±0.0016, while the average weighted mean of a group of 20 vaginal polynuclear counts is 1.69, with a standard error of ±0.039.

The polynuclear count of the rat is similar to the polynuclear counts obtained by Simpson 2 , 3 from the cow, the sheep and the horse.

A study was made of the effects of high voltage cathode rays on the spermiogenic epithelium of the male adult white rat. The animals were about 5 months old, apparently healthy and virile. The shaved scrotal area was exposed in front of the anodal window of the Coolidge cathode ray tube to a current of one milliampere, at voltages of 200,000 and 250,000, about equally divided as to number of animals. All raying of more than one second duration was given in one second periods with about one-half second between each.

Tissues studied one to 50 days after raying.

Protocol of the experiments:

No. of rats Time of exposure Voltage

3 0.5 sec. 200,000

6 1.0 sec. 200,000

8 5.0 sec. 200,000

4 10.0 sec. 250,000

4 15.0 sec. 250,000

3 20.0 sec. 250,000

3 30.0 sec. 250,000

6 normal controls

The changes in the scrotal skin were similar to those in the abdominal skin as described previously by Jacobsen and Waddell, 1 and consisted chiefly of hyaline fusion of the collagen of the corium, acute necrosis of epidermal and hair follicle epithelium, with vascular changes as a much later occurrence.

Definite lesions were produced in the seminiferous tubules in a zone about 0.6 mm. in depth in the animals exposed for from 20 to 30 seconds, with proportionately shallower penetration in the animals exposed for shorter times. Pyknosis, fragmentation and kary-olysis of nuclei, and depression of mitosis were seen in spermatogonia, spermatocytes, and spermatids, the Sertoli cells being least affected. The interstitial cells showed insignificant changes. The tela subcutanea below the upper 0.2 mm. of the skin and the tunica vaginalis testis showed no evidence of injury, indicating very clearly the much greater sensitivity of germinal epithelium to this form of radiation.

Alterations in the Golgi apparatus of the germinal cells were also observed, fragmentation, perinuclear arrangements and fusion with acrosomic material, changes corresponding with findings in the roentgen-rayed testis of cavia cobaya as reported by Gatenby and Wigoder. 2

In a recent note 1 we reported that calcium in the blood could be raised to abnormal levels in dogs and in monkeys by feeding large amounts of irradiated ergosterol, but that hypercalcemia could not be induced by this means after the parathyroids and thyroids had been extirpated. In these experiments, the amount of ergosterol which was given was usually about 40 times that which is prescribed for infants of equivalent weight.

In the course of subsequent experiments of this kind, we have found that if still larger amounts of irradiated ergosterol are given, it is possible to induce hypercalcemia, after the thyroid and parathyroid glands have been excised. For this series of tests, 100 to 400 times the therapeutic dose has been given; in some instances as much as 800 times this dosage has been fed. Under such conditions, the calcium may rise from 6 or 7 mg. to 17 or even 20 mg. per 100 cc. of blood. That the parathyroids had been completely removed from the body, was shown furthermore by the fact that the inorganic phosphorus in the urine became markedly decreased or even was entirely absent following the operation.

As stated previously 2 we are of the opinion that the source of the marked increase in serum calcium induced by excessive amounts of irradiated ergosterol is the tissues, more particularly the bones, which are the great storehouses of calcium in the body. Subsequent investigations have confirmed this point of view. If dogs are fed, for a period of weeks or even months, on a diet which is almost free of calcium, being composed of cracker meal, dried meat and mazola (corn) oil, the calcium of the blood can be raised to abnormal levels by feeding excessive amounts of irradiated ergosterol, in spite of the deficiency of calcium in the ration.

During the course of an experiment with a culture of Hemophilus influenzae, transplants were made on a plate of Levinthal's solid transparent medium. It was observed that the culture contained 2 types of colonies. Well defined small transparent colonies resembling the textbook description of the typical Pfeiffer bacillus colony were found but in addition there were larger, slightly opaque iridescent mucoid smooth colonies. Transplants were made from the transparent colonies on Levinthal agar and all the colonies were found to be identical with the mother colony. The same was true of transplants from the opaque iridescent colonies. It was thus possible to separate 2 cultures each of which produced typical colonies. Since the largest transparent colonies were found to have a rough surface the strain producing these colonies has been called an “R” strain; and since the iridescent colonies from the other culture had a smooth surface this strain has been called an “S” strain.

This culture and 2 others from which “S” and “R” forms were subsequently isolated had been seeded many times on blood or chocolate agar and the variation in the colonies had not been noticed. Yet the difference in the colony appearance was marked when transplants were made on the transparent agar plates. Very striking was the difference when the plate cultures were held before a 100 watt electric light bulb or in the sunshine.

The morphology of the bacteria from the 2 strains has also been found to be dissimilar and characteristic for each type. This is best noticed if the stained preparation is made from a solid medium culture which is not older than 24 hours. The bacteria from the “S” culture appear as small coccobacilli that vary little in size.

This report concerns a correlation of the filterability of certain dyes with the manner of their excretion by the kidney as tested by the method of perfusion. The details of the technique of this method may be found in previous publications 1 and the results of our present experiments briefly summarized as follows:

Filtration of a series of dyes through collodion membranes gave the following average values of filterability. Phenol red, 100%; indigo carmine, 90%; toluidin blue, 60%; and neutral red, 35%. Trypan blue was unfilterable as such, but the pink component of the dye passed through the filter in small amounts, and brilliant red was entirely unfilterable.

By perfusion of either the glomerular or the tubular circulation of frogs with these dyes, it was found that phenol red and indigo carmine were eliminated chiefly through the glomeruli. Toluidin blue and neutral red on the other hand were excreted principally by the tubules. Trypan blue was excreted in the relatively short period that our experiments continued only in traces, and these traces passed through the glomeruli most readily and showed the same separation of the components of the dye as was observed in the filtration process, for the urine was pink in color. Brilliant red did not pass through either tubules or glomeruli.

Anesthesia of the tubules and repression of their function during the course of dye excretion increased the rate of elimination of phenol red and indigo carmine, but decreased the rate of excretion of toluidin blue and neutral red. When the glomeruli were damaged in such a way as to increase their permeability, both components of trypan blue escaped into the urine, which assumed the bluish color of the original dye solution.

It has been shown previously 1 , 2 that, in the collection of gastric juice from dogs with fundus pouches of the Pavlov type, the combined acidity becomes negligibly small if the secretion be collected in such a way as to minimize its content of mucus or serous transudate. With this elimination of combined acid, the free acidity, as determined in 4 dogs, gave an average value of 0.157 N ±0.003 N. During the past summer, several more such fundus-pouch animals were prepared, one of them a pregnant dog of 10 kilos body weight. Five weeks after the operation, this animal dropped a litter of 4 healthy pups which she suckled as long as she was permitted to.

For the 2 weeks immediately preceding parturition, the total volume of juice collected from her pouch never exceeded 15 cc. per day—this amount being secreted wholly within 6 to 8 hours after feeding. Beginning with the day of parturition, however, the flow increased very markedly in amount and duration. In fact, except during certain periods of experimentation, the secretion was practically continuous throughout the 24 hours between single daily feedings. For the first 3 weeks of lactation, the average daily volume of juice collected was 150 cc. A number of times the pouch was so full that the pressure forced evacuation with consequent loss of contents. The increase in daily output, therefore, was well over tenfold. During the sixth week, however, the second week after the pups had been completely weaned, the average daily output had fallen to about 30 cc. and was decreasing further at the time of the accidental death of the dog during the seventh week.

Titration of 66 samples of this hypersecretion juice gave an average value of 0.157 N ±0.007 N for total acidity—titrating with phenol red to pH 7.1.

It is widely believed that freshly shed germ cells are relatively constant. In previous studies 1 , 2 , 3 , 4 an extremely wide variation in many characters and characteristics was noted. In the present study agglutination of sperm by egg water, which agglutination lends itself to fairly exact quantitative determination, was studied under very precise experimental conditions.

The sea urchin (Arbacia punctulata) was used. These were freshly collected, opened immediately upon arrival at the laboratory, the “best” egg lots selected and immediately tested for the duration of agglutination. Duplicate tests and tests of aliquot divisions of the eggs gave a difference of 0 to 4 seconds, averaging 1 second or 4.5%. This was considered the experimental error.

Thirty series of 3 to 4 females each were studied. When eggs from each female in a series were tested separately, under strictly comparable conditions (including the same sperm suspension), the agglutination time varied very extensively, namely, from 2 to 55 seconds, or 9 to 1300%, with an average of 12 seconds or 142%.

Eggs that gave high agglutination values with sperm from one individual gave consistently high, though not the same values, with sperm from other individuals. Similarly eggs that gave intermediate or low values with one male, gave intermediate or low values with other males.

This wide variation and increase in agglutination time parallels corresponding wide changes in size, color, shape of eggs, loss of jelly, membrane formation, cleavage, etc. 1 , 2 , 3 , 4 The degree of change in any one of these traits measures the extent of overripening of the eggs prior to shedding.

It was also noted that severe storms that delay natural shedding, also higher temperature of the sea water, give rise to overripening of the eggs within the body of the sea urchin, with all the symptoms above enumerated.

A. When freshly shed eggs of Arbacia were physiologically ripe (not overripe) and then aged in sea water at 20° C. there occurred a profound series of changes in agglutination. Three phases may be readily distinguished. In the first phase there was a progressive and marked increase in agglutination, reaching a maximum of about 200% in 3 to 5 hours. This increase is due to a corresponding increase in liberation of agglutinin. In the second phase there was a progressive decrease in agglutination, namely, from about 5 hours to 24 hours. This decrease is associated with a decrease in agglutinin. In the third phase there was continued decrease in agglutination, due to liberation of anti-agglutinin.

When eggs were overripe at the time of shedding, corresponding portions of the first or even second phase did not occur. These phases took place prior to shedding.

Overripening gave rise to the same kind and the same intensity of changes whether within or without the body.

B. When freshly shed sperm from different freshly collected males were aged at 20° C. and tested at each age under strictly comparable conditions, by either the same egg water or by freshly shed ripe eggs, agglutination was widely different. There were 3 phases. In the first there was practically no change, i. e., from 0 to about 3 hours. In the second, agglutination increased progressively and markedly, from the third to the twenty-fourth hour. The average increase was 120%. In the third phase there was a rapid and progressive decrease in agglutination.

When the freshly shed sperm were overripe, or were precociously overripened by high temperature, corresponding portions of the first or second phase were not evidenced.

C. When the freshly shed eggs and sperm were not overripe, and the same germ cells tested at successive ages, under strictly comparable conditions, there occurred the same cyclical increase, then decrease in agglutination.

The previous studies demonstrated that ageing “dry” or concentrated sperm gave rise to a marked increase, then decrease in agglutination, provided, however, the sperm were not overripe when shed. In the present study, the sperm were overripened in a standard 1% suspension at 22° C. and tested at each age by samples of the same egg water. There was the same, but quicker, cyclical change. The first phase began in 5 to 20 minutes after the initial test. The second phase ended in 15 to 125 minutes. The third phase ended in 260 minutes. The increase in agglutination was from 77 to 328%.

The greater the overripeness of the dry sperm when shed, the earlier the maximum values, and the sooner was the cycle ended. When sperm was not overripe when shed, subsequent ageing gave rise to very little or no increased phase, followed by a decreasing phase.

The experiments were so devised as to eliminate such factors as a change in agglutinin, change in jelly content, change in temperature, or H ion concentration of the medium.

To determine whether the increase in agglutination was due to a substance or substances liberated by the sperm, increasing concentrations of sperm were tested with the same egg water solutions. The concentrations of sperm ranged from 1% to 25%. More concentrated suspensions could not be used, on account of the difficulty in distinguishing the agglutinated clusters in the thick medium. Sufficient time was allowed for the substance if present to be liberated. There was no change in agglutination values with the marked increase in concentration of sperm whether ripe or overripe.

The CO₂ liberated by the eggs and the active sperm plays a large role in decreasing the activity of the sperm, in clumping the sperm into aggregations, but plays no role in increasing the agglutination of ageing sperm.

The effects of living for 2 to 5 weeks in an atmosphere containing 40 to 50% oxygen were studied in 5 patients with cardiac insufficiency. The Barach oxygen chamber, with constant temperature and humidity regulation, was used. Studies were made (a) with the patients in the ward, both before and after residence in the oxygen chamber; (b) with the patients in the oxygen chamber with normal (21%) atmospheric oxygen content; and (c) with the patients in the oxygen chamber with 40 to 50% of oxygen in the atmosphere. All other procedures, such as diet, fluid intake and the dosage of drugs, were kept sufficiently constant so that they did not modify the results. The usual clinical observations were recorded and in addition, measurements were made of the basal metabolic rate, pulmonary ventilation, vital capacity, arterial oxygen content, arterial CO₂ content, and the CO₂ dissociation curves of the arterial blood. From the latter were calculated the arterial oxygen saturation, arterial CO₂ tension and the arterial serum pH.

In one patient with mitral stenosis of long standing, great cardiac enlargement and marked chronic passive congestion but no peripheral edema, very little change occurred after a week in 45% oxygen. There was only a slight rise in the level of the CO₂ curve while he was in the chamber. No clinical improvement was noted.

Two patients with chronic valvular heart disease, marked decompensation and edema, together with fever and other evidences of active rheumatic infection, showed moderate subjective improvement. In these individuals, residence for 2 weeks in 45% oxygen was associated with a considerable rise in both the arterial CO₂ content and the level of the CO₂ curve. In one case, there was a rise in arterial oxygen saturation from 84 to 93%.

Several types of microelectrodes have already been devised by different investigators. 1 In all these electrodes the conducting medium, whether of metal or of KCl-agar, is enclosed in a micropipette of glass or quartz. It is possible to make electrodes of similar fineness from metallic wires, but they are too flexible for use.

In the new type of microelectrodes described here, the conducting medium is a continuous film of silver, gold or platinum for stimulating electrodes and silver-silver chloride for non-polarizable electrodes, on the outer surface of glass or quartz micro-needles. The advantages are: (1) they can be easily made and in large numbers at one time, (2) many pure metals can be used, (3) electric contacts of any geometrical shape and fineness can be easily made, (4) the non-polarizable electrode can be made sterile, and (5) their electric resistance is low.

The best way to deposit films of metal on glass and quartz is by spattering, i. e., by placing them in the dark space of high voltage vacuum discharge. But for the deposit of silver films on glass needles, Brashear's 2 method of silvering glass has been found very satisfactory; for bright platinum films Meillere's method 3 is used.

The desired number of micro-needles is drawn in the way described in detail by Chambers. 4 The shank ends of the needles are warmed in a nicroflame and embedded vertically up in a block of paraffin and are then cleaned by immersing the needles vertically down in a beaker of cleaning fluid. After a few hours in the cleaning fluid the needles are thoroughly washed in running water and then kept immersed in a large beaker of distilled water till they are to be placed in the silvering solution.

The following experiments, which are intended as preliminary steps in a study of some of the effects of cathode rays upon cells. provide data for a statistical analysis of the rate of killing of cultures of B. coli, of Staphylococcus aureus and of B. aertryke.

The cathode rays have been obtained from a Coolidge type electron tube operated at a voltage of approximately 200 K.V. Small numbers of the organisms under investigation were evenly spread upon the surfaces of agar plates and known areas were exposed for different lengths of time to the electron stream. After incubation, counts were made of the numbers of colonies growing out in these areas and in similar standard areas shielded from radiation. The ratios of the bacterial colonies in these areas are survival ratios.

A picture of the physical consequences of an electron absorption in a bacterium will be provided by remembering that whenever a fast moving electron is absorbed in matter, some of its energy will be emitted as X-rays but the major part will produce a large number of charged ions within a very small volume. In the present experiments this volume is probably less than 0.001 mm.³ and within it an absorbed electron gives rise to upwards of 10,000 ions. It is natural to attribute the destructive action of cathode rays to the chemical and physical changes resulting from this ionic shower.

By estimating both the number of electrons which strike a bacterium in unit time and the absorption coefficient of these electrons in the bacterium, it is possible to analyse the observed survival ratios by the usual methods of probability theory. Such an analysis will show how many electrons may be stopped by a single bacterium before death results.

Rosenheim's 1 recently announced specific color reaction for ergosterol has been applied to the analysis of tissue from rabbits fed on “Vigantol” (irradiated ergosterol in oil).

The reaction as described by Rosenheim consists in the addition of a saturated aqueous solution of trichloracetic acid to a chloroform solution of the sterol. There is every reason to believe this reaction quite specific for ergosterol or structurally related sterols. We have found after testing many solvents that dichloroethylene (low boiling) is a more satisfactory solvent for the reaction than chloroform. Using this solvent, quantitative estimations of the extracted, partially purified sterols may be made.

Ten adult rabbits were fed “Vigantol” with a stomach tube in doses ranging from 250-870 mg. of irradiated ergosterol. Four were used as controls. Practically all the animals showed the calcification phenomena now well described in the literature. The animals were killed and the organs dried in high vacuum, powdered, extracted with ether (dry), treated with saturated alcoholic barium hydroxide to remove soaps, extracted with petrol ether, the petrol ether evaporated and the residue taken up in dichloroethylene (1 cc.) and treated with one-half cc. of saturated aqueous trichloroacetic acid.

The results show a most striking accumulation of ergosterol in the adrenals and brain with small amounts in the liver and kidneys. Muscular organs appear to be almost free from this substance.

Judging from the color reaction the bone marrow especially seems to contain fairly large amount of a δ^{1, 2} sterin, the nature of which we are now investigating.

The results are all consistent but the amount of ergosterol found by the color test was not in proportion to the amount of ergosterol (Vigantol) fed, e. g., the highest dosed animal did not have a great deal more ergosterol in his body.

The work of MacCallum and Oppenheimer 1 and of Craciun and Oppenheimer 2 offered at least the hope that the “washed granules” prepared from commercial calf vaccine might represent purified vaccinia virus. We have accordingly investigated further the nature of the washed granular material obtained by the method of Craciun and Oppenheimer. 2

In confirmation of the earlier work we have found that the granular centrifugalization sediments after repeated washings in Ringer's solution were infectious even in high dilution; characteristic lesions of vaccinia resulted from intradermal injection into rabbits; the supernatant fluids were infectious only in low dilution. When mixed with the sera of rabbits recently recovered from vaccinia the granules were rendered non-infectious; the infectivity of the granules was not neutralized by normal rabbit sera. Consideration of the results of previous work 1 , 2 , 3 and of this work leaves no doubt in our minds that the property of infectivity is borne by some of the particles in the washed granular material.

The washed granules have been found to become non-infectious at hydrogen ion concentrations more acid than pH = 5.2 or 5.3 (See Table I). This is in sufficient agreement with the results of Douglas and Smith, 4 who found that “diffusates” of vaccine virus from infected rabbits' testes were rapidly inactivated at reactions more acid than pH = 5.5.

We have found that infectivity could be restored to non-infectious mixtures of granules and immune serum by washing the serum-treated granules in saline or Ringer's solution. 5 , 6 , 7 Yet recovery of infectivity on washing was not complete; the infecting power of the rewashed granules treated with immune sera was considerably inferior to that of granules similarly treated with normal sera.

The phenomenon here reported was first observed in the course of an unsuccessful attempt to discover among a number of specimens of B. coli bacteriophage one that would be active upon B. pyocyaneus. Of 25 filtrates showing marked lysis of one or more strains of B. coli none could be demonstrated to have any lytic power for any of the 7 strains of B. pyocyaneus tested. The bacteriophage specimens were obtained from the following sources: sewage 4; urine 7; feces 15; pus 2; unknown source 1. The phages were tested on the pyocyaneus cultures both as fresh filtrates and after their lytic power for B. coli had been materially enhanced by repeated passage. Neither clearing of young broth cultures nor plaque formation could be demonstrated with any of the strains of B. pyocyaneus.

An attempt was then made with one bacteriophage specimen to bring about pyocyaneus lysis by serial passages upon cultures of the latter organism. A filtrate having marked lytic activity on several strains of B. coli was chosen for this test and its activity was brought to a maximum by a number of passages upon a strain of B. coli, S 1. This coli strain was isolated from a case of pyelitis, belonged to the slow lactose-fermenting group, gave gas on saccharose and marked hemolysis on blood agar plates. One one-hundred-millionth (10^-8) cc. of the lytic filtrate produced complete lysis of a young broth culture in less than 18 hours and, spread upon agar, gave numerous plaques.

Three pyocyaneus strains were used in the test. These all gave the typical abundant growth on agar with the characteristic blue-green pigments and funnel shaped gelatin liquefaction. There was a striking difference in the fermentation reactions of the 3 strains.

Sufficient clinical evidence has accumulated in recent years to show that certain persistent infections in degenerated mucous membranes lining the paranasal sinuses require surgical removal. In the usual operative procedure in the antrum the diseased tissue is scraped out with a curette; or the entire membrane is removed in one piece by sub-periosteal dissection through an opening in the canine fossa (Dr. Kistner). Clinically it is observed that repair occurs with variable results. In some cases the new lining is thick and in others thin. The exact nature of the new tissue has never been determined.

In our investigations human material is employed, inasmuch as there is no available experimental animal with the surgical and pathological characteristics of sinusitis in man. At the reoperation the specimens are immediately mounted on thick paper supports and immersed in Zenker's fluid. The tissues are dehydrated in alcohol, cleared in cedar oil, imbedded in paraffin and stained with hematoxylin-eosin.

Repair After Part of the Mucous Membrane is Removed:

1. Repair in a Small Area—Approximately one square centimeter was cut from the nasal wall of the antrum in the usual gland-bearing portion of the mucous membrane. Four months later the healed area with some adjacent membrane was removed. Microscopic sections show a layer of ciliated columnar pseudo-stratified epithelium resting on white fibrous scar tissue. No submucous glands are found.

2. Repair of a Large Area—About one-third of the mucous membrane was removed from 2 cases. Two years later the sinuses were reoperated and the entire antrum lining removed. Microscopic sections show the original portion of the membrane containing the normal histologic structures as follows: (1) A layer of pseudo-stratified ciliated columnar epithelium; (2) a superficial spongy layer of areolar connective tissue with large tissue spaces; (3) tubo-alveolar mucous and serous glands in the layer of loose connective tissue; (4) an orderly arrangement of branching arteries and arterioles in definite layers; (5) a definite periosteal layer of compact connective tissue.

Using a modified heart-lung preparation of Starling, with the arterial cannula inserted in the brachial cephalic artery in the first experiments, and in the later experiments in the arch of the aorta, the following experiments were done.

In drawing conclusions as to the external work of the heart, we consider that our experiments establish the diastolic volume of the heart as an index of the energy consumption of the heart and therefore a measure of the work of the heart, provided the heart muscle function remains as constant throughout the period under consideration. Only one experiment is given here.

Calibration of elastic coefficient in terms of percentage volume increase of arterial system to mm. Hg. increase pressure or mm. Hg. increase in pressure/% increase in volume. This is the “effective” elastic coefficient or rigidity of arterial system.

Without Air Chamber or Arteriosclerosis. Content of “effective” arterial system = 210 cc. 2 cc. blood injected into system gives a rise of 56 mm. Hg. in pressure.

2/210 = 0.95% increase in volume for 56 mm. Hg., or 1% increase in volume for 59 mm. Hg. A. V. Hills normal artery between 80-120 mm. Hg. showed 6.6% increase in volume for 40 mm. Hg., or 1% increase in volume for 6 mm. Hg. Ergo in this system without air chambers and representing extremely severe arteriosclerosis, there is a rigidity 9.8 × normal “mean” artery of A. V. Hill at normal pressure.

With Air Chamber or Normal. Content of arterial system = 240 cc. 20 cc. blood injected into system gives a rise of 27 mm. Hg. 20/240 = 8.3% for 27 mm. Hg., or 1% increase for 3.3 mm. Hg. Ergo rigidity is 1/2 that of A. V. Hill “mean” normal artery.

In previous communications from this clinic it has been stated that the urinary alkaline tide is primarily due to the secretion of hydrochloric acid by the stomach, for the tide was absent in most cases of achlorhydria. 1 This observation has been confirmed by Ackman 2 and Davies. 3 Further study showed that it was necessary to distinguish between alkalinity developing during the day-time and that shown immediately after awakening, 4 for this latter change is frequently seen in patients where hydrochloric acid is not found in the stomach, and is probably due to the respiratory adjustment of the subject to waking conditions. 5 Recently we have studied a patient with achlorhydria who showed a tide exactly resembling those found in normal subjects. It seems desirable for us to report this case because the absence of hydrochloric acid was found after histamine had been injected—a method giving as complete proof of functional disability of the stomach as can be obtained. In many cases where a urinary tide has been present, but hydrochloric acid apparently was absent from the gastric juice, further gastric studies have failed to confirm the abnormal finding of the first one, 6 and it has been claimed by some that the presence of a satisfactory alkaline tide furnishes definite proof that the stomach secretes hydrochloric acid.

The patient was a man 46 years old who was a mechanical engineer by profession. He had occasional periods of mild diarrhoea, and symptoms which were somewhat suggestive of the presence of a peptic ulcer. The stomach emptied rapidly, as frequently is the condition in cases of achlorhydria. Gastric analyses were carried out, and the reaction of successive samples of morning urine determined.

Recent work by Hart, Steenbock and others 1 and Myers and Beard 2 has given rise to opposing results as to the value of metals other than copper in the regeneration of hemoglobin in the anaemic white rat. The suggestion has been made 3 that these differing results were due to the fact that the work was carried out in 2 rather widely separated localities. It has seemed to us that the publication of our results obtained in a third and entirely different section of the country might be of value as bearing on this point.

The rats used were from a local laboratory strain, originally a Wistar Institute stock. The milk used came entirely from one cow, was milked in an enamelled vessel and brought to the laboratory in glass bottles, with the result that there was no contact with metal previous to feeding. This precaution was found to be necessary since neither raw nor pasteurized milk bought on the open market sufficed to produce an anaemia in the white rat, although it comprised the sole diet of the animals for a period of 6 months.

Several attempts were made to obtain iron, free from traces of other metals, but electrolytic deposition was the only method producing a sample of iron which does not allow a regeneration of hemoglobin in anaemic white rats. Whether or not it may prolong the life of the animal, 4 we are not at this time prepared to state. The method used for the electrolytic deposition of iron was that of Classen and v. Reis. 5 Copper was prepared electrolytically by the method of H. Sand, 6 , 7 and cobalt by that of Denso.

The interrelationships between the hypophysis and the ovary have been studied extensively. Changes in the ovary following the administration of anterior lobe substance and extracts have been described by Goetsch, 1 Evans, 2 Zondex and Ascheim, 3 and others. Erdheim and Stumme 4 called attention to changes in the hypophysis during pregnancy. Zondex and Ascheim have demonstrated, in the urine of pregnant women, the presence of a hormone which has all the ovarian growth properties of the anterior hypophysis, and have utilized this in their test for pregnancy. Fels 5 found the same hormone in the blood of pregnant women. Recently Engle and Smith 6 have demonstrated that the amount of ovarian stimulating substance in the hypophysis of the guinea pig varied during the sexual cycle, and was much diminished during the time the vagina was open. Evans and Simpson 7 further showed that this substance was increased by castration, and was present in larger amounts in males than in females.

From the foregoing work the following facts are known: (1) the hypophysis has a powerful action on the ovary; (2) the ovary affects the hypophysis; (3) there is a change in the hypophysis during pregnancy. With these conclusions in view, it seemed reasonable to believe that the ovarian growth stimulating properties of the anterior hypophysis could be increased by the injections of placental extracts which contain a high concentration of oestrin.

Our experiments tend to support this belief. Adult female rats, of approximately the same weight, were castrated, in order to exclude any cyclical variation, allowed to recover for 2 weeks, and then given a series of injections of a commercial alcoholic extract of human placenta which had been carefully standardized.

In 1922 Evans and Bishop 1 found that a diet poor in salts had a definite effect on the oestrous cycle of the white rat. They found that on such a diet the experimental animals reached maturity later than their litter-mate controls and that only 15% of the oestrous cycles of these animals were of the 4 or 5 day length as opposed to 48% of those of the control animals.

We have carried out similar experiments on the white mouse. The animals used were about 60 days of age. Vaginal smears were made daily for 3 weeks, and all animals not showing cycles of 4 to 5 day length were discarded. They were then divided into experimental and control groups.

The following experimental diet was used: Casein 31%, dextrin 41%, Crisco 20%, cod liver oil 3%, yeast 5%.

The control animals received the same diet, with the exception that salts were fed at a 7% level and the dextrin reduced to 35%.

There was a very definite change in the oestrous cycles of the experimental animals, only 25% of the cycles being of the 4 on 5 day length, while many of the long cycles were 12 to 20 days in length. In the control group, 70% of the cycles were 4 or 5 days in length and in the remaining cycles, only a few were over 7 days in length.

The experimental animals grew at a slightly lower rate than the control animals. The difference at the peak of the experiment was 13.75%. In all cases, however, the latter exhibited a rather sleek coat, as contrasted with the coarse, roughened fur of the experimental mice.

It is evident from these experiments that the oestrous cycle was affected much more than the growth ability, indicating that the oestrous cycle is a much more delicate indicator of the well-being of the animal than is the growth curve.

According to Mendez 1 ergotamine produced depression of motility in the isolated intestine of the rabbit. Later Issekutz and Leinzinger 2 reported that it was without effect on the intestine of these animals. More recently Rothlin 3 stated that ergotamine produced no effect in some rabbits and caused decreased motility in others. He also reported that in experiments with ergotamine on the intestine in situ of dogs his results were negative. Planneles 4 observed in the guinea pig that the isolated intestine of these animals was depressed by ergotamine. Thienes 5 recently carried out experiments on the isolated intestine of the guinea pig, rabbit and cat with adrenalin and low concentrations of ergotamine (1:250,000 and 1:500,000). He made no statement, however, of the action of ergotamine but a tracing of the intestine of a rabbit shows that no change in motility occurred as a result of the treatment with ergotamine. In the course of studies on the effect of intestinal motility 6 in which ergotamine was occasionally employed, it was noticed that the intravenous injection of the substance stimulated peristalsis in cats under urethane anesthesia. More extensive observations carried out since fully corroborated our earlier findings, and we were able also to show that the increase in the motor functions was in some cases very striking. These studies, which were carried out by the Trendelenburg method, on the intestine in situ were extended to experiments on the isolated intestine of the cat, suspended in oxygenated Locke's solution and kept at body temperature. Ergotamine in concentration of about 1:200,000 to 1:500,000 produced depression, the tonus being slightly decreased and the rhythmic movements were abolished or greatly diminished in force and frequency.

As stated in the preceding communication, 1 the response of the intestine to ergotamine has been studied in different animals. No observations have been made, however, to determine whether changes in ionic content of the surrounding medium modified the action of ergotamine. We, therefore, undertook the investigation of this problem and selected the intestine of the rat for the test object. As far as we know, no studies with ergotamine on the intestine of this animal have been reported. The experiments of Rosenmann, 2 however, are of interest in this connection. He observed that ergotoxin produced stimulation in the isolated intestine of the rat.

Our experiments were carried out by means of the Magnus method, segments of different parts of the intestine being suspended in oxygenated Locke solution maintained at almost uniform temperature, usually 37.5° C. The variations which sometimes occurred seldom exceeded 0.2° C. We found that concentrations of 1:100,000 and sometimes 1:1,000,000 ergotamine in Locke solution with a pH of about 7.2 or 7.3 and containing 0.014% calcium chloride, produced a considerable decrease of tonus, the relaxation being more marked in the ileum and colon than in the duodenum. The rhythmic movements, however, were usually stimulated, especially the amplitude. When the amount of calcium in the solution was increased to 0.028 or 0.056% a striking change in the action of ergotamine occurred when the H-ion concentration was decreased below neutrality, especially when it was around pH of 6.5. The same amounts of ergotamine now caused a very marked increase of tonus which occurred within a few seconds after it was added to the solution. The duration of the effect varied in different experiments, but it usually lasted several minutes or longer.

The Smith and Engle phenomenon, first demonstrated in the rat, has been extended to other laboratory mammals. E. Allen induced sudden stimulation of the ovaries and genital tract in an immature rhesus female by means of implants of anterior lobe from 3 spayed females of the same monkey species. He also found that dog hypophysis had no effect. 1 It is, therefore, of interest to report a striking effect of pig anterior lobe upon a non-ovulating and amenorheic monkey.

A preliminary experiment was done with the glands implanted whole and intact. Female No. 22 of the Carnegie Colony of rhesus monkeys had been running irregular cycles as follows: 82, 98, 98, 25, 54, 91 days. These were her only menstrual periods; but minor cycles were reflected in the curve of vaginal desquamation. She was, moreover, blind, with complete optic atrophy, the sequel of a disease a year before the experiment. Laparotomy June 4, 1929, 8 days after the last bleeding of 5 days' duration, showed an infantile uterus and tiny ovaries absolutely devoid of visible graafian follicles, corpora lutea, or corpora albicantia. Two whole interior lobes of castrated male pigs were implanted retroperitoneally at the time of the laparotomy; 2 on June 5, intramuscularly, and again 2 on June 6. The animal was killed June 10 and embalmed with Regaud's fluid: uterus of middle finger size (15×10 mm.); ovaries 9×6.8×6 and 9×6.2×6 mm., with follicles of pin-head and mustard-seed size. The effect of the implants was noticeable but not striking.

An experiment with female No. 31 gave unequivocal results. This rather vigorous female of about 4000 gm. weight passed through the following menstrual cycles: 28, 28, 22, 19, 65, 31, 35, 21, 57, 45 days.

The condition of pseudopregnancy in the rat was first described by Long and Evans. 1 These authors demonstrated that pseudopregnancy could be produced artificially by the introduction of a small glass rod into the cervical canals at the time when the animal was in stages 1, 2 or 3 of the oestrous cycle, as specified by them. Wang 2 corroborated the findings of Long and Evans and recently Slonaker 3 has made additional contributions to the subject.

Long and Evans showed that there was no direct nervous connection between cervix and ovary. Slonaker suggests that a substance, most likely to be found in the vaginal or uterine mucosa, is responsible for the continued action of the corpora lutea in pseudopregnancy and possibly in pregnancy. He further suggests that this substance could act either directly on the corpora lutea or through some intermediary agency, possibly the anterior part of the pituitary gland. It has been shown by Smith, 4 Smith and Engle, 5 Evans and Simpson 6 and others that there are one or more substances secreted by the anterior portion of the pituitary body which influence the growth of follicles and the formation of corpora lutea of the ovary.

We wish to report results obtained when rats in the first 2 stages of the oestrous cycle are anesthetized for a short time with ether, nitrous oxide, or ethylene, and the cervical canals then stimulated by a small glass rod, as compared with controls which were not anesthetized. The rats used in this study were all healthy and sexually mature. A number of the animals were selected from the stock cages at the time when they were in oestrum, without first determining the regularity of their cycles.

In a communication now in press we have described a case of asthma due to a saprophytic fungus. This patient's attacks occurred in damp houses or other places where molds and similar fungi flourish. On testing his skin for its reaction to extracts of fungi isolated from places where he had had severe attacks, we found a consistently positive reaction to a species of Altenaria (mali?). His serum also gave a positive Kustner-Prausnitz reaction to Altenaria in the skin of 3 out of 4 individuals. Inhalation of a spray of extract of this fungus produced characteristic paroxysms of asthma. Other saprophytes produced milder reactions. We concluded that his spontaneous attacks were caused by the spores of these fungi which abound in the air.

This patient also has a chronic eczema involving the hands and feet and to a lesser extent the forearms and legs. The lesions begin as groups of minute papules and vesicles on red patches of skin. The areas slowly enlarge and become infiltrated and desquamating. Their appearance resembles that of the eczematous lesions which frequently follow dermatophytosis of the feet—lesions which Jadassohn and Peck have recently shown to be due to sensitization to trichophyta. In our case cultures from the lesions have never yielded pathogenic fungi, though a common saprophyte, Aspergillus nidulans, has been repeatedly isolated from a lesion on the leg.

Some years ago Hilgermann and Niethe reported that certain patients with eczema were sensitized to saprophytic fungi which grew on their lesions. In this patient we could obtain immediate intradermal reactions to extracts of the altenaria which produced asthma, of the aspergillus which we obtained from his skin, and of a number of other fungi obtained from house dust.