EFFECT OF COCAINE ON THE GROWTH OF LUPINUS ALBUS. A CONTRIBUTION TO THE COMPARATIVE PHARMACOLOGY OF ANIMAL AND PLANT PROTOPLASM

1. The effects of cocaine and its decomposition products were studied on the growth of the young roots of Lupinus albus. 2. The results obtained were compared with similar experiments on animal tissues. 3. It was found that, while cocaine is the most toxic of these compounds studied for animal tissues, it was of comparatively low toxicity in respect to its effect on the growth of roots. On the other hand, sodium benzoate, being practically non-toxic for animals, was the most toxic of the compounds studied for the plant roots.     

Preclinical studies of porfiromycin as an adjunct to radiotherapy

The bioreductive alkylating agent porfiromycin (POR) is more toxic to EMT6 cells that are hypoxic at the time of treatment than to aerobic cells. The toxicity of POR to hypoxic EMT6 cells in vitro was similar to that of mitomycin C (MC): the aerobic toxicity of POR was considerably less than that of MC. Treatment of cells in vitro with POR before and during irradiation did not sensitize either hypoxic or aerobic cells to X rays; instead, only additive cytotoxicity was produced. In contrast, treatment of solid EMT6 tumors in vivo with POR plus radiation produced supra-additive cytotoxicity, as assessed by analyses of the complete dose-response curves for the killing of tumor cells by radiation alone or by POR alone. The supra-additivity of the combination regimens appeared to reflect the preferential killing by each agent of those tumor cells which were in an environment conferring resistance to the other agent. In contrast, combinations of POR and X rays produced only additive cytotoxicities to marrow CFU-GM. Supra-additive antineoplastic effects were obtained at doses of POR which produced little hematologic or other host toxicity. The complementary cytotoxicities of radiation and POR to cells in different microenvironments in solid tumors and the absence of a similar effect in normal tissue make optimized regimens combining radiotherapy and POR unusually promising for the treatment of solid tumors.     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : I. THE ACTION OF THE CHLORIDES OF NA,K, CA,AND MG ON THE PROTOPLASM OF AM?BA PROTEUS.

By means of micro-dissection and injection Amœba proteus was treated with the chlorides of Na, K, Ca, and Mg alone, in combination, and with variations of pH. I. The Plasmalemma. 1. NaCl weakens and disrupts the surface membrane of the ameba. Tearing the membrane accelerates the disruption which spreads rapidly from the site of the tear. KCl has no disruptive effect on the membrane but renders it adhesive. 2. MgCl₂ and CaCl₂ have no appreciable effect on the integrity of the surface membrane of the ameba when applied on the outside. No spread of disruption occurs when the membrane is torn in these salts. When these salts are introduced into the ameba they render the pellicle of the involved region rigid. II. The Internal Protoplasm. 3. Injected water either diffuses through the protoplasm or becomes localized in a hyaline blister. Large amounts when rapidly injected produce a "rushing effect". 4. HCl at pH 1.8 solidifies the internal protoplasm and at pH 2.2 causes solidification only after several successive injections. The effect of the subsequent injections may be due to the neutralization of the cell-buffers by the first injection. 5. NaCl and KCl increase the fluidity of the internal protoplasm and induce quiescence. 6. CaCl₂ and MgCl₂ to a lesser extent solidify the internal protoplasm. With CaCl₂ the solidification tends to be localized. With MgCl₂ it tends to spread. The injection of CaCl₂ accelerates movement in the regions not solidified whereas the injection of MgCl₂ induces quiescence. III. Pinching-Off Reaction. 7. A hyaline blister produced by the injection of water may be pinched off. The pinched-off blister is a liquid sphere surrounded by a pellicle. 8. Pinching off always takes place with injections of HCl when the injected region is solidified. 9. The injection of CaCl₂ usually results in the pinching off of the portion solidified. The rate of pinching off varies with the concentration of the salt. The injection of MgCl₂ does not cause pinching off. IV. Reparability of Torn Surfaces. 10. The repair of a torn surface takes place readily in distilled water. In the different salt solutions, reparability varies specifically with each salt, with the concentration of the salt, and with the extent of the tear. In NaCl and in KCl repair occurs less readily than in water. In MgCl₂ repair takes place with great difficulty. In CaCl₂ a proper estimate of the process of repair is complicated by the pinching-off phenomenon. However, CaCl₂ is the only salt found to increase the mobility of the plasmalemma, and this presumably enhances its reparability. 11. The repair of the surface is probably a function of the internal protoplasm and depends upon an interaction of the protoplasm with the surrounding medium. V. Permeability. 12. NaCl and KCl readily penetrate the ameba from the exterior. CaCl₂ and MgCl₂ do not. 13. All four salts when injected into an ameba readily diffuse through the internal protoplasm. In the case of CaCl₂ the diffusion may be arrested by the pinching-off process. VI. Toxicity. 14. NaCl and KCl are more toxic to the exterior of the cell than to the interior, and the reverse is true for CaCl₂ and MgCl₂. 15. The relative non-toxicity of injected NaCl to the interior of the ameba is not necessarily due to its diffusion outward from the cell. 16. HCl is much more toxic to the exterior of a cell than to the interior; at pH 5.5 it is toxic to the surface whereas at pH 2.5 it is not toxic to the interior. NaOH to pH 9.8 is not toxic either to the surface or to the interior. VII. Antagonism. 17. The toxic effects of NaCl and of KCl on the exterior of the cell can be antagonized by CaCl₂ and this antagonism occurs at the surface. Although the lethal effect of NaCl is thus antagonized, NaCl still penetrates but at a slower rate than if the ameba were immersed in a solution of this salt alone. 18. NaCl and HCl are mutually antagonistic in the interior of the ameba. No antagonism between the salts and HCl was found on the exterior of the ameba. No antagonism between the salts and NaOH was found on the interior or exterior of the ameba. 19. The pinching-off phenomenon can be antagonized by NaCl or by KCl, and the rate of the retardation of the pinching-off process varies with the concentration of the antagonizing salt. 20. The prevention of repair of a torn membrane by toxic solutions of NaCl or KCl can be antagonized by CaCl₂. These experiments show directly the marked difference between the interior and the exterior of the cell in their behavior toward the chlorides of Na, K, Ca, and Mg.     

ON THE MECHANISM OF MILK SECRETION : THE INFLUENCE OF INSULIN AND PHLORIDZIN

The four types of experiments on milk secretion herein described really fall into one general class so far as the physiological effects produced are concerned. Starvation lowers the blood sugar and raises the osmotic pressure of the blood. The experiment using parathyroid hormone with or without starvation may have its effects interpreted as simply due to starvation since 1000 units of this hormone produced no visible effects on the blood calcium or milk constituents different from those of starvation. Since insulin produces a marked and rapid drop in blood sugar it too may be looked upon as a rapid starvation effect. It has some other important effects, however. Briggs et al. (21) have shown that potassium and phosphorus of the blood are decreased and Luck, Morrison, and Wilbur (22) indicate a reduction in the amino acids of the blood in insulin treatment. Phloridzin lowers the threshold for sugar retention with the consequence that in time it tends to lower the sugar of the blood to an even greater extent than that noted in starvation. It tends to depress the potassium, to increase the phosphorus content of the blood, and to cause the body to burn protein rather than carbohydrate, thus increasing nitrogen excretion. All of the experiments are characterized by a sharp reduction in the milk yield. Cary and Meigs (23) have studied like reductions in milk yield produced by varying the energy or protein of the diet. They conclude that such decrease in milk production may be interpreted as due to the direct effect of the starvation and the consequent reduction of the energy and protein available to milk secretion. The reduction in milk yield for the experiments herein described can undoubtedly be attributed to the same causes as those cited by Cary and Meigs. The experiment where Cow 47 was given a full ration and at the same time injected with large quantities of insulin is of particular interest in this connection. The ration was adequate and the cow ate well, yet her production declined to a fifth of her normal milk yield. Her chart shows that there was a slight reduction in her blood sugar when insulin was introduced into the blood stream. It seems furthermore likely that this sugar was not as available to milk secretion, since there appears to be more than a corresponding drop in the lactose content of the milk. The work of Luck et al. would seem to indicate that there should be a like drop in the amino acids of the blood. These two conditions would lead, according to the work of Cary and Meigs, to a reduction in the concentration of the nitrogen of the milk. Actually, in the experiment as it was performed, the nitrogen increased to a value about 40 per cent above normal. A somewhat similar conflict is noted in two of the other three insulin experiments where starvation accompanied insulin injection. To this extent it would seem that the factor deserving most emphasis in its immediate effect on milk yield is the energy available, and that the later and more secondary factor is the amino acid concentration of the blood. In the starvation experiments, the butter fat percentage of the milk rises rather uniformly with the duration of starvation. In the insulin experiments, however, the charts appear to show a marked reduction in this butter fat percentage immediately after the introduction of insulin. This is particularly noticed after the second and third injections. Since the dextrose of the blood tends to be reduced and made unavailable to the general physiological processes by the presence of the large excess of insulin, and since this reduction of the butter fat percentage is noted as an accompanying phenomenon, it would appear that the blood dextrose plays a part in the synthesis of milk fat as well as being the source of the milk lactose, possibly as a source of energy in converting body fat to butter fat. In this regard the results for the treatment of Cow 47 with phloridzin are of importance. As noted by others, the introduction of phloridzin causes a marked rise in the fat percentage of the milk. The lactose per cent is also higher than that noted in starvation. Since phloridzin, by lowering the threshold for the blood sugar, causes large quantities of it to be drained from the body through the urine, and therefore reduces the reserve supply, it follows that if the insulin hypotheses are correct we should expect an eventual lowering of the lactose and of the fat below the starvation level. During the last of the experiment this is what was actually observed. The effects of starvation and of insulin furnish concordant proof for the theory that the lactose of milk is derived from the sugar of the blood. The fact that the different constituents of the milk, the fat, the lactose, the nitrogen, and the ash, do not exactly parallel each other in their behavior throughout these experiments indicates that they have in all probability separate origin. This is particularly true of the butter fat percentage, which appears to have a rate of secretion which is more or less independent of the other constituents, and higher in amount. This result would fall in line with the conclusion of the writers in a previous paper in which it was indicated that the fat of the blood was very likely deposited in the udder as fat corresponding to body fat from which source it was metabolized into the fat of milk shortly before it was needed for milk secretion. The wide variation brought about in the constituents of the milk by the treatment all point to the conclusion that in milk secretion a balance is maintained between the osmotic pressure of the milk and of the blood. Thus when the sugar of the milk is reduced either through starvation or by insulin the ash constituents rise to compensate for this reduction and make the osmotic pressure of the milk similar to that of the blood. These results further appear to indicate that the salts and the sugars are more or less independent in their passage and metabolism into milk from the other constituents. These observations are therefore in line with those obtained by Jackson and Rothera (14) and by Davidson (15) in their brilliant experiments where they modified milk secretion by returning milk or milk sugars and salts to the udder. These experiments give direct proof for the conclusion that modifications of the blood of dairy cattle produce direct and predictable modification of the milk secreted.     

THE INSECTICIDAL ACTIVITY OF SUBSTANCES RELATED TO THE PYRETHRINS

The toxicity as contact insecticides of the esters of (±)-3-methyl-2-allyl-cycfopent-3-en-4-ol-i-one with the natural (+ )-trans and with synthetic (±) -cis-trans-chrysaii- themum monocarboxylic acids has been compared with that of the natural pyrethrins. The comparison was carried out on five species of test-insects.
It was found that the figure for both the absolute and the relative toxicities of these compounds varied with the species used as test-subject.
The factors involved in this variation are discussed. The extremes of variation of relative toxicity for the compounds with the natural acid were from about one-eighth as toxic as the pyrethrum standard when the aphid Macrosiphum solanifolii was used as test subject to nearly four times as toxic as the standard with the larvae of the moth Plutella maculipennis. The fully synthetic material varied from about one-sixteenth as toxic as the standard to Macrosiphum solanifolii to nearly twice as toxic to the larvae of Plutella maculipennis. The compound with the natural acid was approximately twice as toxic as the fully synthetic material to three of the insect species, but the fully synthetic material was considerably more than half as toxic as the partially synthetic to the fourth test-species. It is pointed out that while it is widely recognized that large differences in relative toxicity may occur when the effect of chemicals of widely different structure and mechanism of action are compared on a number of different test-species, the fact that these differences may also occur with related chemicals with, presumably, a similar mechanism of action, has not been clearly stated.
Even when the differences in relative toxicity are taken into account, the two synthetic pyrethrin-like esters still show high insecticidal activity.
It seems reasonable to suppose from the results that economic commercial synthesis of pyrethrin-like insecticides is not impossible.     

THE TOXIC ACTION OF COPPER ON NITELLA

1. Using the loss of turgidity of the cells as a criterion it is found that the toxicity curve of copper chloride with Nitella is sigmoid. An empirical equation can be constructed which will approximately fit the curve. 2. When the concentration of the copper chloride is varied the toxic effect varies as a constant, fractional, power of the concentration. This relation holds when the concentration is plotted against either (1) the time necessary to reach a given point on the ordinate of the survivor curve, (2) the maximum speed of toxic action as shown by the tangent to the survivor curve or (3) the first derivative of the equation which fits the survivor curve. 3. When the temperature is varied and the logarithm of the reciprocal of the time necessary to reach a given point on the survivor curves is plotted against the reciprocal of the absolute temperature the resulting figure consists of several intersecting curves. A hypothetical system is described which will give straight lines under normal conditions and curves when acted upon by a toxic agent.     

ON THE RELATION BETWEEN TOXICITY,RESISTANCE, AND TIME OF SURVIVAL,AND ON RELATED PHENOMENA

1. A relation between toxicity, resistance, and time of survival has been derived on the basis of the assumption that the time is a function of a parameter which is the difference between the toxicity and the resistance. Toxicity and resistance act like forces which can maintain an equilibrium-like (or stationary) state. If the equilibrium is upset, the time at which the event (death) occurs is proportional to the logarithm of the difference between toxicity and resistance. 2. It was found that if values proportional to the resistance are calculated with the proposed equation and the percentage mortality plotted against them (instead of against the time as is usual) symmetrical curves are obtained even though the corresponding mortality-time curves are asymmetrical. Assuming that the resistance varies like an error, that is, according to probability rules, theoretical mortality-time curves, similar to the experimental curves, can be constructed from the proposed equations. 3. In the case of a toxic agent acting on a unicellular organism suspended in solution, the toxicity is proportional to the adsorbed amount of the agent, as calculated with the aid of the Langmuir equation. In small concentration ranges the toxicity can be taken as approximately proportional to the concentration. 4. The variation of the temperature affects mainly the constant a which is a function of the temperature similar to that of the velocity constant of a chemical reaction (Arrhenius'' law). 5. The proposed equation has been tested in four different combinations of the variables, concentration, resistance, time, and temperature. The agreement with the experiments is satisfactory. 6. Any noxious agent acting on a unicellular organism may be characterized by three constants: r, the resistance, which is the threshold value at which the agent is still fatally toxic for the organism; a, the reciprocal of the rate constant determining the specific rate (that is, the time corresponding to a difference of 1 between the toxicity and the resistance); and finally the constant γ of the function representing the relation between toxicity and concentration.     

Lack of gastric toxicity of nitric oxide-releasing aspirin, NCX-4016, in the stomach of diabetic rats

We compared the gastric toxic effect of aspirin (ASA) in both normal and diabetic rats, with that of NCX-4016, a derivative of ASA with nitric oxide (NO) releasing moiety. Animals were injected with streptozotocin and used after 5 weeks of diabetes with blood glucose levels of >350 mg/dl in the presence of omeprazole. Oral administration of ASA (with 150 mM HCl) did not produce damage at 30 mg/kg in the conscious rat but caused hemorrhagic gastric lesions in STZ-diabetic rats. By contrast, NCX-4016 even at 190 mg/kg (a dose equimolar to 100 mg/kg of ASA) did not cause damage in both normal and STZ-diabetic rat stomachs. Plasma salicylic acid levels were not different between normal and diabetic rats after administration of ASA or NCX-4016, though the latter gave significantly lower levels as compared to ASA. Intragastric application of ASA (80 mM in 50 mM HCl) for 30 min caused a reduction of transmucosal PD and increase of luminal H+ loss with a minimal effect on mucosal blood flow (GMBF) in both normal and diabetic rats, yet resulting in much severe damage in the stomach of the latter group. Mucosal application of NCX-4016, however, did not cause PD reduction and luminal H+ loss, but produced a marked hyperemia, resulting in no damage in the stomach of both normal and STZ-diabetic rats. The increased gastric toxicity of ASA in STZ-diabetic rats was significantly mitigated by co-application of a NO donor FK-409 together with ASA, with an increase of GMBF, despite similar degrees of PD reduction and luminal H+ loss being observed. We conclude that NCX-4016 does not have a toxic effect in either normal or diabetic rat stomachs, although the diabetic rat stomach is more vulnerable to ASA-induced damage. NCX-4016, though absorbed more slowly than ASA, counteracts the injurious effect of aspirin on the gastric mucosa, probably by increasing GMBF mediated by NO.     

THE NERVOUS MECHANISM OF COORDINATION IN THE CRINOID,ANTEDON ROSACEUS

1. Stimulation causes Antedon to swim by means of alternate oral bending and dorsal stroke of the arms. Two arms of a given ray move alternately so that while one is executing the aboral stroke its mate is flexing ventrally. This implies reciprocal inhibition. 2. Recriprocal inhibition between the two arms of an isolated ray can be abolished by the use of either strychnine or nicotine. 3. Coordination between the rays is referable to the conducting properties of the nervous pentagon which connects the five rays. In this system an impulse loses in effectiveness as it passes from the point of origin. 4. When Antedon is made to rest oral face down on the floor of an aquarium, oral flexion of all the rays, swimming movements, and righting result. Antedon is therefore negatively stereotropic with reference to its ventral side. 5. Excitation of the dorsal cirri results in aboral bending of all the rays. Stimulation of the cirri inhibits ventral flexion to the extent of preventing righting movements while on the other hand stimulation of the ventral surface inhibits the grasp reflex of the cirri. Thus oral and aboral sides of Antedon exhibit dynamic symmetry although structurally dissimilar.     

THE EFFECTS OF VARIATIONS IN THE CONCENTRATION OF OXYGEN AND OF GLUCOSE ON DARK ADAPTATION

In this study we have analyzed the effects of variations in the concentrations of oxygen and of blood sugar on light sensitivity; i.e. dark adaptation. The experiments were carried out in an air-conditioned light-proof chamber where the concentrations of oxygen could be changed by dilution with nitrogen or by inhaling oxygen from a cylinder. The blood sugar was lowered by the injection of insulin and raised by the ingestion of glucose. The dark adaptation curves were plotted from data secured with an apparatus built according to specifications outlined by Hecht and Shlaer. During each experiment, observations were first made in normal air with the subject under basal conditions followed by one, and in most instances two, periods under the desired experimental conditions involving either anoxia or hyper- or hypoglycemia or variations in both the oxygen tension and blood sugar at the same time. 1. Dark adaptation curves were plotted (threshold against time) in normal air and compared with those obtained while inhaling lowered concentrations of oxygen. A decrease in sensitivity was observed with lowered oxygen tensions. Both the rod and cone portions of the curves were influenced in a similar way. These effects were counteracted by inhaling oxygen, the final rod thresholds returning to about the level of the normal base line in air or even below it within 2 to 3 minutes. The impairment was greatest for those with a poorer tolerance for low O₂. Both the inter- and intra-individual variability in thresholds increased significantly at the highest altitude. 2. In a second series of tests control curves were obtained in normal air. Then while each subject remained dark adapted, the concentrations of oxygen were gradually decreased. The regeneration of visual purple was apparently complete during the 40 minutes of dark adaptation, yet in each case the thresholds continued to rise in direct proportion to the degree of anoxia. The inhalation of oxygen from a cylinder quickly counteracted the effects for the thresholds returned to the original control level within 2 to 3 minutes. 3. In experiments where the blood sugar was raised by the ingestion of glucose in normal air, no significant changes in the thresholds were observed except when the blood sugar was rapidly falling toward the end of the glucose tolerance tests. However, when glucose was ingested at the end of an experiment in low oxygen, while the subject remained dark adapted, the effects of the anoxia were largely counteracted within 6 to 8 minutes. 4. The influence of low blood sugar on light sensitivity was then studied by injecting insulin. The thresholds were raised as soon as the effects of the insulin produced a fall in the blood sugar. When the subjects inhaled oxygen the thresholds were lowered. Then when the oxygen was withdrawn so that the subject was breathing normal air, the thresholds rose again within 1 to 2 minutes. Finally, if the blood sugar was raised by ingesting glucose, the average threshold fell to the original control level or even below it. 5. The combined effects of low oxygen and low blood sugar on light sensitivity were studied in one subject (W. F.). These effects appeared to be greater than when a similar degree of anoxia or hypoglycemia was brought about separately. 6. In a series of experiments on ten subjects the dark adaptation curves were obtained both in the basal state and after a normal breakfast. In nine of the ten subjects, the food increased the sensitivity of the subjects to light. 7. The experiments reported above lend support to the hypothesis that both anoxia and hypoglycemia produce their effects on light sensitivity in essentially the same way; namely, by slowing the oxidative processes. Consequently the effects of anoxia may be ameliorated by giving glucose and the effects of hypoglycemia by inhaling oxygen. In our opinion, the changes may be attributed directly to the effects on the nervous tissue of the visual mechanism and the brain rather than on the photochemical processes of the retina.     

SOME EFFECTS OF RADIUM RADIATIONS ON WHITE MICE

It has been estimated that 92 per cent of the total radiation emitted by radium in equilibrium with its subsequent products is given off in the form of α-rays. This, however, cannot be utilized when the source is enclosed in an ordinary container, because the α-rays are absorbed completely by even a small thickness of glass. About 3.2 per cent of the total radiation is emitted in the form of β-rays, and 4.8 per cent as gamma radiation. The effects produced on the radiated mice of these experiments were due mainly to the β-rays, which are easily absorbed by tissue. The γ-rays, being only slightly absorbed by organic matter, probably contributed very little to the observed effects. It is interesting to correlate the different effects produced by the same dose of radiation. The mice which received a dose of 1.9 millicurie hours showed no local effects on the skin or hair. Neither females nor males were sterilized, and the time at which they opened their eyes or reached sexual maturity was not affected, as far as we could tell. The only difference noted between the radiated animals and the controls was in the body weight. This dose accelerated the growth of the young mice, that is, while initially of the same weight, soon after irradiation they became distinctly bigger than the controls, but finally the animals of each group had substantially the same average weight. That this variation in body weight should be accidental is unlikely, since it was observed also in the animals treated by a slightly larger dose (2.4 millicurie hours). The number of animals (seven) which showed this effect is too small to prove conclusively the accelerating effect of small doses of radiation on the body growth of mice. But considering that similar results have been. obtained by radiating plants and beetles, it is reasonable that the observed increase in weight might be attributed, at least in part, to the effects of radiation. Since this paper was first written Russ, Chambers, and Scott have shown that small doses of x-rays accelerate the body growth of rats. In view of this additional evidence there can be little doubt that the increase in weight observed in our experiments was due to the radiation. A dose of 2.4 millicurie hours applied over the backs of the animals produced no local skin effects, but it accelerated the growth of the mice as in the previous case. In addition it caused permanent sterilization of all the females. A similar result was obtained with 4.9 millicurie hours, except that the effect on the rate of growth was uncertain. A dose of 6.8 millicurie hours produced a definite but mild skin erythema and retarded the development of lanugo hair. But since in this instance the emanation was applied over the heads of the animals, the dose reaching the ovaries was not sufficient to cause sterilization, as already explained. No other definite effect was noted. In connection with the sterilization of the females it should be noted that a dose of radiation which produced no visible skin changes was sufficient to cause permanent sterility. On account of the greater distance of the ovaries from the source of radiation as compared with that of the skin directly below the tube, and the depth of tissue which the rays had to traverse to reach the ovaries, the amount of radiation acting on the latter was much smaller than the amount falling on the skin. The radiation emitted by the emanation tube is reduced to about 50 per cent of its initial value after traversing 1 mm. of tissue. Still, while the skin was not visibly affected, the mice were sterilized. This shows that the ovaries are influenced very easily by radiation of this type. We can estimate the amount of radiation reaching the ovaries which is sufficient to cause sterility to be less than 25 per cent of the amount necessary to produce visible skin changes in the mice. It should be noted also that whenever sterility of the female mice was induced, it was permanent. Furthermore, those mice which were not rendered sterile by radiation were, as far as the experiments enable us to say, as prolific as the controls. Remembering that a dose of 1.9 millicurie hours had no apparent effect on the ovaries, while a slightly larger dose, 2.4 millicurie hours, caused permanent sterility, it might be concluded that it is not possible to produce temporary sterility by radiation. We know, however, that temporary sterility can be produced, at least when the animals are radiated at a later stage in their development. The mice in our experiments were radiated for the first time soon after birth, and it is not improbable that under these conditions temporary sterility cannot be obtained. Large sublethal doses produced severe skin burns, retarded the body growth of the animals, but failed to sterilize the males. About one-third of the total skin area of the mice showed marked effects from the radiation. The animals were very sick for a time, and their growth was temporarily stunted. But nevertheless they recovered and finally became apparently normal except for the narrow hairless strip of skin which had been closest to the emanation tube. Only the females were rendered permanently sterile. The males did not show even temporary sterility when the doses of radiation were close to the lethal dose. While the testes of mammals are known to be very easily affected by radiation, still they are more resistant than the ovaries. In addition, in these experiments they were at a greater distance from the source of radiation than the ovaries, and they were better protected by the thicker layer of tissue in the path of the rays. The fact that no sublethal dose in these experiments sterilized the males shows that under the conditions of irradiation adopted the amount of radiation reaching the testes was not sufficient to affect them noticeably. If the source of radiation had been applied closer to the reproductive organs of the males, they would have been sterilized by millicurie hour doses much smaller than the lethal dose. Some of the radiated animals were killed with ether, and macroscopic and microscopic examinations of the reproductive organs were made. The ovaries of the sterile females were generally atrophied and colored yellow. The normal histological structure was altered. The characteristic findings were the destruction of the Graafian follicles, with absence of ovum cells. The testes and the epididymis of the radiated mice of the present experiment appeared macroscopically and histologically normal, with the presence of abundant spermatozoa. Owing to the method adopted for the irradiation of the mice, the testes were too far from the source of radiation, and too well protected by the intervening tissue to be definitely affected by the rays.     

THE INFLUENCE OF ELECTROLYTES ON POLLEN TUBE DEVELOPMENT

These experiments serve to show that neutral salts in amounts considerably below those commonly employed in culture solutions may be very injurious to pollen. It has been found, for example, that NaCl, one of the least toxic salts tried, excepting CaCl₂, added to a sucrose solution in a concentration of 0.0002 M, or about 11 parts per million, reduces the growth of sweet pea pollen tubes 15 per cent. When it is considered that MgCl₂ and BaCl₂ are about fifteen times as toxic as NaCl it becomes evident that the susceptibility of pollen tubes to injury by these substances amounts virtually to hypersensitiveness. On the other hand calcium salts in concentrations ranging from 0.02 to 0.002 M markedly enhance the growth of sweet pea pollen tubes. MgCl₂ has a similar action in the case of Nicotiana. Calcium, moreover, exerts a strong protective action in the presence of the injurious monovalent cations Na and K. So far as can be determined by microchemical means these salts do not alter the wall of the pollen tube; presumably, their effect is on the protoplast itself. In the light of recent experimentation (Osterhout) with other forms better adapted to precise investigation of these phenomena it seems probable that the explanation of the facts presented here lies in changes brought about in the permeability of the cells. Since several gaps exist in our evidence, however, conclusions drawn at this time must necessarily be provisional. The highly injurious action manifested by the cations of several of the salts used indicates that they penetrate the protoplast very rapidly. Possibly in pure sucrose cultures, exosmosis is a limiting factor in pollen tube growth. The addition of salts of calcium or magnesium may favor development by retarding or preventing this outward diffusion. The protective effect of calcium in the presence of the toxic cations K and Na is best interpreted on the assumption that the entry of these latter into the protoplast is retarded by the calcium. The mode by which hydrogen ion concentration affects pollen tube growth is largely a matter of speculation. It has previously been been shown by Brink that the time relations of the growth process simulate those of an autocatalytic reaction. It has been demonstrated also that elongation of the tubes in artificial media is related to the digestion of the reserve food materials contributed by the pollen grain. In the case of the sweet pea these stored substances are largely fats and their hydrolysis may constitute the most important chemical reaction in growth. If, as seems not improbable, the other reactions involved wait upon this one, it is the "master reaction" according to Robertson''s hypothesis. If this conception really applies to the case in hand as outlined, the effect of the concentration of hydrogen ions on growth may be a direct one. It is known that the action of the fat-splitting enzyme lipase is favored by a certain amount of free acid. The maximum rate of germination of the pollen and the greatest amount of growth of the pollen tubes occur at pH 6.0. This may be due in large part to the immediate effect of this concentration of hydrogen ions upon the digestion of the reserve food.     

EFFECTS OF ANDROSTENEDIONE, 19-NORETHYNYLTESTOSTERONE, PROGESTERONE, AND 17α-HYDROXYPROGESTERONE UPON THE MANIFESTATION OF SECONDARY SEXUAL CHARACTERS IN THE MEDAKA, ORYZIAS LATIPES.

Effects of androstenedione, 19-norethynyltestosterone, progesterone, and 17α-hydroxyprogesterone upon the manifestation of secondary sexual characters were studied in the orange-red variety of the medaka. The steroids were consecutively administered per os for 15 days. Androstenedione and 19-norethynyltestosterone induced formation of new segments in the fin rays and produced papillar processes on the anal fin of the female. 19-norethynyltestosterone proved to have a strong potency, estimated to be more than 300 times that of androstenedione. Progesterone and 17α-hydroxyprogesterone had neither androgenic nor estrogenic effect on the secondary sexual characters. This is quite in harmony with the fact that they have neither androtermonic nor gynotermonic potency. The administration of these steroids did not impair the fertility of females. The administered females produced offspring by mating with administered males.     

Morphological differences elicited by two weak acids, retinoic and valproic, in rat embryos grown in vitro

We compared in rat whole-embryo culture the morphological changes elicited by valproic acid (VPA) with those elicited by trans-retinoic acid (RA). Rat embryos explanted on day 9.5 of gestation were treated on day 10 with RA or VPA at concentrations producing equivalent reductions in embryonic protein. The concentrations selected for morphological assessment by scanning and transmission electron microscopy, 2.3 and 800 microM, respectively, for RA and VPA, produced approximately a 50% incidence of abnormally open anterior neuropores in initial range-finding experiments in the culture system. Protein and DNA analyses were also performed on corresponding groups of embryos at three different doses. With concurrent control groups used as reference standards, the two treatment groups were compared for differences in external and internal morphology, protein and DNA contents, and growth indices. While certain variables responded similarly in the two treatment groups, e.g., the growth variables, protein and DNA contents, each drug produced selective morphological effects. Whereas treatment with RA produced underdeveloped branchial arches, symmetrically cleft cranial defects resulting in openings in rhombencephalic and prosencephalic regions, and exteriorized neural tissue in the caudal neuropore region, VPA produced irregular clefts with wavy margins along the entire length of the neural tube, and an open caudal neuropore without eversion of the neuroepithelium, while producing no detectable effect on the branchial arches. The similar effects of these two drugs on protein and DNA contents suggest comparable degrees of overall toxicity; however, the dissimilar effects on neural tube and branchial arches, coupled with the large difference in concentration of the drug required to produce the effects, add to the evidence that their mechanisms for elicitation of abnormal development are qualitatively different.     

Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

Summary Cell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.     

OBSERVATIONS ON THE TRANSPORT OF CARBON DIOXIDE IN THE BLOOD OF SOME MARINE INVERTEBRATES

Although the results we have recorded merely serve to indicate the possibilities of this interesting field of investigation, we have sufficient data to enable us to draw certain general conclusions. In the first place it is evident that the bloods of the more highly developed marine invertebrates, such as the active Crustacia and the Cephalopods, are specially adapted for the carriage of carbon dioxide. The quantity of carbon dioxide taken up by the blood of Maia, Palinurus, or Octopus at any given tension of the gas is, in general, about twice or three times as great as that which is taken up by sea water under the same conditions. On the other hand, the blood of a slow, creeping form, such as Aplysia, or of a sessile animal such as the ascidian Phallusia shows no more adaptation for the carriage of carbon dioxide than does sea water. But our estimations of the CO₂ content of the blood as it circulates in the bodies of these more active invertebrates show that the conditions of transport of this gas differ considerably in some respects from those which obtain in mammals. For the invertebrate blood in the body contains only a relatively small quantity of carbon dioxide, averaging in the forms we examined from 3 to 10 cc. per 100 cc. of blood. This forms a marked contrast with the condition found in mammals where even the arterial blood contains about 50 cc. of CO₂ per 100 cc. of blood. The invertebrate, therefore, works at a very low CO₂ tension. There is a twofold significance in this circumstance. In the first place, it means that only the first portion of the carbon dioxide dissociation curve is in use in the respiratory mechanism. Now an inspection of our curves will show that at these low carbon dioxide tensions the dissociation curves tend to be steeper than at higher tensions. As we intend to show in a later paper it can be proved mathematically that, other things being equal, a blood with a carbon dissociation curve of moderate steepness, i.e. one in which the carbon dioxide content of the blood increases fairly rapidly with increase of carbon dioxide tension, is a more efficient carrier of the gas from the tissues to a respiratory surface than a blood in which the dissociation curve is either steeper or flatter. It would seem as if the active invertebrates avoid the use of too flat a part of their CO₂ dissociation curves by working over the initial steeper portion. Furthermore, it is seen that over the range of this initial steep portion of the curves the changes of reaction produced by the uptake of carbon dioxide are much smaller than at higher tensions of the gas; for these initial portions of the curves are more nearly parallel to the lines of constant reaction calculated for a temperature of 15°C. according to Hasselbalch''s method (10) on the assumption that the whole of the combined CO₂ is in the form of sodium bicarbonate. It is evident also that on this assumption the hydrogen ion concentration of the blood of invertebrates (with the exception of the tunicates) would appear to be practically the same as that of the warm-blooded vertebrates—a conclusion confirmed by the direct measurements of Quagliariello (9). On the other hand, our measurements do not lend support to the idea put forward by Collip (4) that in order to maintain an appropriate faintly alkaline reaction an invertebrate needs to retain carbon dioxide in its blood at a comparatively high tension. This idea was based on the observation that at comparatively high CO₂ tensions the blood of invertebrates contains considerably more sodium bicarbonate than does sea water. But our curves show that this is no longer true at the lower values of carbon dioxide tension, the amount of sodium bicarbonate falling off more rapidly in the blood than in the sea water with diminution of the carbon dioxide tension so that in order to maintain an appropriate reaction in the blood only a comparatively small tension of CO₂ is required. The largest amount of carbon dioxide that we found present in the circulating blood of any of the types examined was 9.7 cc. per 100 cc. of blood in the case of Maia, and in most cases the amount was considerably less. But even this lowest value corresponds to a tension of CO₂ of only about 3 mm., so that the tension gradient across the gill membrane must be even less than this. We would emphasize rather the circumstances that as the portion of the dissociation curve over which the reaction is approximately constant is of but small extent, it is necessary that in an active form like Octopus the carbon dioxide produced should be removed rapidly lest an accumulation of it should cause the limits of normal reaction to be exceeded; and this need is correlated with the extreme efficiency of the respiratory apparatus in this animal. It is interesting to notice that the mammal which, in order to obtain an appropriate reaction in the blood, has to work at relatively high carbon dioxide tensions where the dissociation curve is comparatively flat, secures a steeper physiological CO₂ dissociation curve in the body, and with it a more efficient carriage of carbon dioxide and a more constant reaction in the circulating fluid, in virtue of the effect of oxygenation on the carbon dioxide-combining power of its blood (3, 6). Returning now to the consideration of the actual form of the dissociation curves we have obtained—it is a significant fact that it is in those forms such as Maia, Palinurus, and Octopus whose bloods are rich in proteins—particularly hemocyanine—that the initial steep portion of the curve is observed. This suggests that in these forms the blood proteins act as weak acids and expel carbon dioxide from the blood at the low tensions which include the physiological range, just as in vertebrates the hemoglobin similarly displaces carbonic acid from its combination with alkali metal. On the other hand the cœlomic fluid of Aplysia contains no pigment and only 0.00672 per cent of protein nitrogen (Bottazzi (11)) and shows no initial rapidly ascending portion of the CO₂ dissociation curve. This is supported by the observation of Quagliariello (9) that the acid-neutralising power of the blood of an invertebrate is roughly proportional to its protein content. It seems as if the proteins of invertebrate blood like the blood proteins of vertebrates, exist in the form of sodium salts which are capable of giving up sodium for the transport of carbon dioxide as sodium bicarbonate. That this is so in the case of hemocyanine follows from the fact that the isoelectric point of this pigment occurs at a hydrogen ion concentration of 2.12 x 10^–5 N, i.e. at a pH of 4.67 (Quagliariello (12)) so that in the alkaline blood of the invertebrates possessing it, hemocyanine will act as a weak acid. It is probable that the initial steep portion of the carbon dioxide dissociation curves which we have found to be of such importance in the respiration physiology of Octopus, Palinurus, and Maia is produced by the competition of this acid with carbonic acid for the available sodium of the blood.     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : II. THE ACTION OF THE CHLORIDES OF LEAD,MERCURY, COPPER,IRON, AND ALUMINUM ON THE PROTOPLASM OF AM?BA PROTEUS.

I. Plasmalemma. 1. The order of toxicity of the salts used in these experiments on the surface membrane of a cell, taking as a criterion viability of amebæ immersed in solutions for 1 day, is HgCl₂, FeCl₃> AlCl₃> CuCl₂> PbCl₂> FeCl₂. Using viability for 5 days as a criterion, the order of toxicity is PbCl₂> CuCl₂> HgCl₂> AlCl₃> FeCl₃> FeCl₂. 2. The rate of toxicity is in the order FeCl₃> HgCl₂> AlCl₃> FeCl₂> CuCl₂> PbCl₂. 3. The ability of amebæ to recover from a marked tear of the plasmalemma in the solutions of the salts occurred in the following order: AlCl₃> PbCl₂> FeCl₂> CuCl₂> FeCl₃> HgCl₂. II. Internal Protoplasm. 4. The relative toxicity of the salts on the internal protoplasm, judged by the recovery of the amebæ from large injections and the range over which these salts can cause coagulation of the internal protoplasm, is in the following order: PbCl₂> CuCl₂> FeCl₃> HgCl₂> FeCl₂> AlCl₃. 5. AlCl₃ in concentrations between M/32 and M/250 causes a marked temporary enlargement of the contractile vacuole. FeCl₂, FeCl₃, and CuCl₃ produce a slight enlargement of the vacuole. 6. PbCl₂, in concentrations used in these experiments, appears to form a different type of combination with the internal protoplasm than do the other salts. III. Permeability. 7. Using the similarity in appearance of the internal protoplasm after injection and after immersion to indicate that the surface is permeable to a substance in which the ameba is immersed, it is concluded that AlCl₃ can easily penetrate the intact plasmalemma. CuCl₂ also seems to have some penetrating power. None of the other salts studied give visible internal evidence of penetrability into the ameba. IV. Toxicity. 8. The toxic action of the chlorides of the heavy metals used in these experiments, and of aluminum, is exerted principally upon the surface of the cell and is due not only to the action of the metal cation but also to acid which is produced by hydrolysis.     

Effects of beauvericin to mammalian tissue and its production by Austrian isolates ofFusarium proliferatum and Fusarium subglutinans

Austrian isolates ofFusarium subglutinans andFusarium proliferatum were studied for their ability to produce beauvericin, moniliformin and fumonisin B₁ and B₂ under laboratory conditions. Analytical methodology for beauvericin was specially adapted for this task. Our analyses showed that the strains produced beauvericin up to 687 mg /kg maize and moniliformin up to 70 mg/kg. The culture ofF. proliferatum in addition produced fumonisin B₁ and B₂ at levels of 106 and 61 mg/kg,respectively. The preliminary toxicity experiments performed in this study clearly indicated a toxic effect of beauvericin on the contractility of mammalian smooth muscle and thus on mammalian cells.     

Influence of quercetin, a bioflavonoid, on the toxicity of copper to Fusarium culmorum

The effect of quercetin on copper toxicity to the mycelial growth of Fusarium culmorum was investigated. Increasing concentrations of copper produced dose-dependent inhibition in yeast extract and malt extract agar. However, the toxic level of copper against fungal growth was significantly affected by the concentration of yeast extract in the medium, compared to that of malt extract. Apart from the difference in toxic level of copper, the addition of quercetin antagonized copper toxicity to hyphae morphology and resulted in the reversal of fungal growth inhibition. Quercetin showed a protective ability similar to citrate, and was more effective than acetate and proline.     

Effect of proximate environment on drug susceptibility of mice (author's transl)

The present study was performed in order to elucidate the effect of proximate environment on drug susceptibility of mice. Three experiments were carried out independently. In the first experiment, mongrel and ddS mice produced under an unsatisfactory control of proximate environment were purchased, and acute toxicity tests of thiamine hydrochloride (B1HCl) and isonicotinic acid hydrazide (INAH) were practiced at two different conditioned rooms. In the second experiment, ddY mice produced under the conventional environment controlled to a certain extent were purchased, and the toxic effect of B1HCl was examined under the similar environment. In the third experiment, the sensitivity to B1HCl of RFVL mice produced under the strict barrier system was tested at the severe air-conditioned room. LD50 and their fLD50 values were calculated by Litchfield-Wilcoxon's method, and the variance analyses were carried out. The severer the environmental control after the purchase of mice turned to the higher the drug sensitivity. This respect was more remarkable in INAH of which the toxic response is appeared slowly compared with B1HCl. Furthermore, seasonal variation was found in LD50 values. However, seasonal effect differed from rearing and experimental conditions. In the third experiment which these proximate environments were controlled severely, seasonal variation was very small. From the results of these experiments, it was defined that the use of animals produced under the satisfactory rearing condition and severe environmental control are necessary for animal experiments.