Warburg Vessels Research Articles

The Effect of Some Antihistamines upon the Respiration of Rat Heart Homogenates

Conventional manometric techniques were used to investigate the effects of some antihistamines upon the respiration of rat heart homogenates. The study was undertaken because it has been reported that these drugs exert effects on cardiac arrhythmias but little is known relative to the mechanisms by which these effects are produced. The antihistamines which were studied were promethazine HCl, phenindamine tartrate, methapyrilene HCl, antazoline HCl, dipnenhydramine HCl, tripellenamine HCl, prophenpyridamine maleate, and pyrilamine maleate. Each of these drugs, in 0.007 M concentration, inhibited endogenous respiration as well as respiration in the presence of glucose or pyruvate. Both promethazine HCl and phenindamine tartrate markedly inhibited respiration in the presence of succinate. None of the other antihistamines included in the study produced any significant effect upon respiration in the presence of succinate. The effects elicited by the antihistamines could not be significantly altered by the addition of histamine to the Warburg vessels.

A BIOCHEMICAL AND MORPHOLOGIC STUDY OF MYELINATION AND DEMYELINATION

Samples of normal grey matter, white matter, and peripheral nerves obtained from rats were incubated in Warburg vessels with glucose and a labelled lipide precursor (acetate, phosphate, choline, glycerol, glucose). The total lipides were then extracted and their radioactivity measured. The preparations were compared with respect to dry weight, lipide content, O(2) uptake, and ability to incorporate the various substrates into the lipides. Grey matter was found to be the least damaged by incubation, white matter the most. Damage to the tissue depressed lipogenesis to a greater extent than respiration. Five substrates were compared with respect to their degree of incorporation into the lipides of the various preparations. White matter, which had a greater oxygen uptake than peripheral nerves, showed the lowest degree of incorporation for most of the substrates studied. The results suggest that there are considerable quantitative differences in the metabolism of central and peripheral myelin. In the sciatic preparations, oxygen uptake and lipogenesis from acetate were found to decrease from the proximal to the distal end of the nerve. This finding may be relevant to the pathogenesis of peripheral neuropathies. The growth and metabolic activity of peripheral nerves were studied in rats aged 1 to 500 days, and the biochemical and histological findings were correlated. The results indicated that the lipogenetic activity of the Schwann cell was lowest in the newborn animal, and reached its peak at about 20 days. Comparative data were also obtained from the cerebral cortex. The growth pattern of peripheral nerves was distinctly different from that of the brain. With respect to changes in tissue weight, respiration, and lipogenesis, growing peripheral nerve correlated with body weight, while the brain matured much more rapidly.

Warburg vessel washer

Provides a plan for the construction and use of a container capable of holding 14 ordinary Warburg vessels for washing.

The effect of oxygen on the growth and metabolism of the aerobic thermophilic actinomycete Micromonospora vulgaris.

SUMMARY: In the presence of pure oxygen at one atmosphere pressure Micromonospora vulgaris is unable to form in liquid media a surface pellicle of growth-bearing aerial mycelium. The production of bottom growth is either unaffected or stimulated by oxygen. Vegetative mycelium which develops from spores in Warburg vessels has an oxygen uptake that is similar in the presence of air or oxygen. Aerial mycelium, harvested from growths in air. shows marked differences as regards oxygen uptake in the presence of air or oxygen; oxygen is inhibitory. Some evidence is presented to show that the effect of oxygen may be connected with the inactivation of essential -SH groups of thiol enzymes.

Exposure of human cardiac muscle to radioactive digitoxin in vitro

Slices of human cardiac muscle obtained by biopsy at the time of exploratory thoracotomy were incubated with C 14, labeled digitoxin, in Warburg vessels. The activity of BaCO 3 recovered from CO 2 dissolved in KOH in the center well of the vessels was determined with a thin-window Geiger-Mueller tube and also a flow counter. No radioactivity was detected in the samples even though the slices respired at increased rate in the presence of glycoside. This suggests that digitoxin acts catalytically without conversion to C 14O 2 by the tissue.

The respiration of a thermophilic Actinomycete, Micromonospora vulgaris.

SUMMARY: The respiration of Micromonospora vulgaris at 60° was studied by means of the Warburg technique. Various cultural methods and heat treatments were used to separate the different growth phases of this structurally complex organism. One- to two-day growths bearing aerial mycelium were most active. Spores were inactive. Vegetative mycelium which developed within 24 hr. in the Warburg vessels was active, but similar 1-day or older growth harvested from cultures proved to be inactive.

THE EFFECT OF FURFURAL UPON THE GERMINATION AND RESPIRATION OF ASCOSPORES OF NEUROSPORA TETRASPERMA

THE WORK of Emerson (1948) showed for the first time that chemical treatment, as well as the application of heat (Goddard 1935, 1939), could activate ascospores of Neurospora. She reported that furfural and furfuryl alcohol in very low concentrations effectively induced germination, but the mechanism of this activating effect was not investigated. Since that time, Sussman (1953) has reported that 11 other heterocyclics also serve as activators of ascospores. The purpose of the following experiments was to follow up these observations with an investigation of the metabolic effects of chemical activators. MATERIALS AND METHODS.-Ascospores were obtained by crossing strains 374 and 377 of Neurospora tetrasperma which were then grown as outlined by Goddard (1935). Since Emerson (personal communication, 1951) has shown that ascospores rapidly lose their sensitivity to chemical activators, these were used within a month after harvesting (fresh spores). In the experiments wherein the ageing effect or loss of sensitivity was studied, old spores were used: these had been harvested at least 6 mo. before use. Both fresh and old ascospores were stored at 40 C. at 65 per cent relative humidity. Respiratory measurements were made in Warburg vessels of 7 ml. capacity with a fluid volume of 1.1 ml. Standard Warburg manometers were used at a shaking rate of 120 oscillations per minute, while the temperature of the bath was maintained at, 260 C. except when otherwise stated. Anaerobic conditions were obtained by flushing the vessels with nitrogen which had been passed through alkaline pyrogallol and then over a heated copper coil to remove contaminant CO2 and 02. Spore suspensions for the respirometric determinations were made up by determining the volume of a weighed sample of spores after centrifuging in a hematocrit tube. The spores were then washed with a 1:4 dilution of sodium hypochlorite (Fishers reagent grade) to kill contaminant conidia, rinsed several times in distilled water, and finally resuspended in 30 parts of distilled water (by volume). Controls of dormant spores suspended in water were always run. The rate of oxygen uptake remained constant; therefore, it was assumed that contamination was controlled effectively. Spores were heat activated by suspending them in distilled water at 600 C. for 30 min. in a constant temperature oil bath. Chemical activation was usu-

THE NATURE OF THE AUXIN‐INDUCED WATER UPTAKE BY POTATO TISSUE. II. THE RELATION BETWEEN RESPIRATION AND WATER ABSORPTION

IT HAS BEEN shown by several workers that the absorption of water by potato discs is an aerobic process, and recently we have demonstrated that it can be inhibited by a variety of metabolic inhibitors (Hackett and Thimann, 1950). This process, furthermore, is promoted by indole-acetic and naphthaleneacetic acids and the resulting increase in water uptake can be blocked by the same inhibitors (Hackett and Thimann, 1952). The auxin-induced water uptake is not accompanied by -any increase, but by a definite decrease, in the osmotic pressure of the cell contents (Hackett, 1952). In order to gain some insight into the mechanisms of these effects, measurements of the respiratory rate (oxygen uptake) were made and the results are reported here. MATERIALS AND METHODS. The method used to prepare and maintain discs of potato tissue throughout the experimental period was described in detail in the previous paper (Hackett and Thimann, 1952). In essence, the discs were supported so as to break the surface of shallow layers of solution in Petri dishes, and the water uptake was calculated from changes in fresh weight. Direct measurements of the oxygen uptake of the discs were made using standard, constant volume Warburg respirometers, according to the technique described by Umbreit et al. (1949). Groups of 10 discs, 9 mm. in diameter, were blotted and weighed fresh, then placed in the respirometer vessels containing 3.0 cc. of water; the center well contained 0.2 cc. of 20 per cent KOH. The vessels were attached to the manometers, placed in 25?C. bath, and shaken at a rate of 140 strokes per minute. After 15-30 min., the manometers were read at 15-min. intervals. The oxygen uptake is given as Qo2, the number of cubic mm. of oxygen absorbed per hour per gram of initial fresh weight. In correcting the flask constant for the volume of tissue, the storage tissue was assumed to have a specific gravity of 1.0. The discs in the Warburg vessels are submerged under water and consequently the conditions are not the same as in the Petri dishes. Since potato discs deteriorate after some hours under anaerobic conditions (Brauner et al., 1940), oxygen uptake was followed for not over 3 hr., and fresh discs were used for each series of measurements. That is, the oxygen (and water) uptake was determined

Uptake on carbon dioxide, hydrogen and oxygen by Hydrogenomonas facilis.

SUMMARY: The simultaneous uptake of CO2, H2, and O2 by non-proliferating cells of Hydrogenomonas facilis was studied by means of a new method employing ordinary Warburg vessels each provided with one stopper side-arm and one vent side-arm. H. facilis effected the same overall reaction as that carried out by certain anaerobically adapted green algae: 6H2 + 2O2 + CO2 → (CH2O) + 5H2O. In an atmosphere of CO2 and H2, no CO2 fixation accompanied the quantitative reduction of nitrate to nitrite by molecular H2; nor was there any change in concentration of bicarbonate or in total gas pressure when acetone, pyruvate, or α-ketoglutarate were added.

Micro-volumetric respirometry; methods for measuring O2 consumption and CO2 production by cells and enzymic reactions.

When the Arctic Research Laboratory at Point Barrow, Alaska, was ready for use in 1947, it was late autumn. Frost could set in at any time and render unavailable precious material for our projected respiratory studies. This situation brought about an acute need for many more micro than the dozen we had, and prompted the construction of simple, plastic drilled in blocks of lucite. Standard specimen vials attached by rubber stoppers were used as respiration and compensating vessels and tuberculin syringes for the measurements of the oxygen consumption (Scholander, 1950). In a few days our Eskimo friends made enough new to fill our needs. A micro model was similarly constructed and both sizes proved eminently useful on a wide variety of animal and plant material. A model for tissue respiration was designed using standard Warburg vessels. Wennesland (1949) at the same laboratory modified this design to contain an internal rod chamber. The present are a further development of the Barrow respirometers which are all based on the volumetric principle introduced by Winterstein (1912, 1913). In the volumetric system, the pressure is maintained constant, and the volume changes are*read directly. The principle involved can be stated as follows: The pressure of the respiration chamber is balanced against a compensating chamber through a manometer. As gas is absorbed or evolved, it is exactly compensated for by a volumetric device so as to maintain pressure balance with the compensating chamber. The change in gas volume is measured directly on the volumetric device. The volumetric system offers some conspicuous advantages. The volume changes are read directly as such, and are independent of the gas volume of the apparatus. The only calibrated part is the compensating device (piston 375

RELATIONS OF RESPIRATION AND GROWTH IN THE AVENA COLEOPTILE

THE AVENA coleoptile is a classical obj ect for the study of the effect of auxin in inducing plant growth. A particularly simple system for the study of the auxin response is constituted by excised sections of the coleoptile which when floated on aqueous solutions containing an active auxin, are capable of extensive and rapid growth (Bonner, 1933). It is known that there is a relation between the auxin induced growth of such sections and their respiration. Thus response to auxin does not occur in an 02 free atmosphere (Bonner, 1933), while HCN, which inhibits respiration of the coleoptile, inhibits response to auxin in a parallel manner (Bonner, 1933, 1936). As a preliminary step in the elucidation of the relation of auxin to respiration, the respiratory systems of the coleoptile have been reviewed and reported in a previous paper (Bonner, 1948). The oxygen uptake of the coleoptile is mediated by the cytochrome-cytochrome oxidase system. Hexose is converted to fructose diphosphate through a series of reactions involving ATP and evidence that pyruvate is the product of initial hexose breakdown has been presented (Bonner, 1948). Pyruvate in turn is metabolized through a system involving succinic dehydrogenase and resembling in general terms the cycle of Krebs. Thus the Avena coleoptile possesses so far as present information goes what might be termed an orthodox respiratory mechanism, one resembling that of animal tissues. The previous paper in this series (Bonner, 1949) has dealt with the influence of several factors including respiratory substrates and inhibitors on the auxin induced growth of A4vena coleoptile sections. This paper deals with experiments which bear on the relation of auxin to respiration. MATERIALS AND METHODS.-A4vena seedlings (var. Siegeshafer) were grown from intact fruits (glumes not removed) in flats in vermiculite and incubated under occasional orange light at 260C. for 5 days before use. At this time the plants were approximately 2 cm. tall and were identical with those used for growth measurements in the preceding paper. For the respiration measurements, tips 2-3 mm. long were removed from the coleoptile and one 5 mm. section cut from each hollow coleoptile cylinder with a double bladed cutting tool. The segments were thoroughly randomized and dispensed into Warburg vessels, twenty per vessel. At the same time one lot of twenty sections was weighed directly in the fresh condition and all respiratory rates are calculated to the basis of 100 mg. fresh wt., twenty sections weighing in general 75-85 mg. Measure1 Received for publication August 28, 1948. Report of work supported in part by the Herman Frasch Foundation. ments of respiration were made in single side-arm conical Warburg vessels of approximately 16 cc. capacity and provided with a central well which contained 5 per cent KOH where oxygen uptake was to be determined. A bath temperature of 30?C. was used throughout. M/100 KH2PO4, pH 4.5,

A technique for the study of oxygen availability to micro-organisms in soil and its possible use as an index of soil aeration

1. In Part I of the paper details are presented of a new technique for the study of oxygen availability to bacteria added to soil. In essentials the technique consists of adding a washed suspension of bacterial cells together with excess of suitable oxidizable substrate to air dried, sieved soil contained in Warburg vessels. The rate of oxygen uptake is looked upon as a function of the aeration conditions within the soil. The results can be expressed by means of a conventional figure called the aeration factor (or A.F.).2. In Part II with the use of the technique it has been shown that two physical properties of soil which influence oxygen availability under the experimental conditions of the technique are (a) moisture content of the soil, (b) the amount of break-up of the soil crumbs. Since the A.F. measures the resultant effect produced by these factors it may be regarded as an index of soil aeration.

Cholinesterase Content of Erythrocytes in Normal and Pathological States. I. Content in Normal Humans.

Several investigators have recently recently reported changes in plasma, or whole blood cholinesterase content in anemias and leukemia. Values reported were expressed in terms of cholinesterase activity per volume of sample. Sabine has calculated the cholinesterase content per cc of red cells but was unable to secure accurate values by direct measurement. The red cells contain only specific cholinesterase and none of the nonspecific type found in plasma or serum. It is unlikely, therefore, that the cholinesterase in the red cells is derived from the circulating plasma, but, more probably, their esterase content is produced by the tissues from which they are derived or with which they are closely associated. Any change of esterase content per cell may be important, therefore, in reflecting the activity of the reticuloendothelial elements of bone-marrow. The production of the esterase by the red cell itself cannot at this time be excluded. Preliminary to some observations of the red cell content in various pathological states, we have determined the cholinesterase content per million cells in 18 normal human subjects. We have been able to secure accurate values by using volumes of washed, resuspended red cells calculated to contain the same number of cells as determined by cell count. The cells were lysed with 10%percnt; NaCl at 2°C. The measurements were then conducted at 37.5° by a modification of the method of Ammon in which the bicarbonate-Ringers solution and the acetylcholine chloride solution were both placed in the large space and the blood sample in the side cup of the Warburg vessels. Values of cholinesterase activity thus secured are expressed as cmm of CO2 produced per hour per million red cells.

Biochemical Nitrogen Fixation Studies. IV. Experiments with Excised Legume Nodules

Experiments with excised nodules of sweet clover, cowpea, crown vetch, soybean, and hairy vetch attempted to determine whether such nodules possess the ability to fix atmospheric nitrogen when maintained in culture solutions in the laboratory. Nitrogen fixation was determined by a standard gas analysis method, as well as directly in Warburg respiration vessels and also by Kjeldahl analysis for total nitrogen. The results were as follows: 1. No nitrogen fixation was observed with whole or crushed nodules kept in Warburg vessels at 28⚬C. in various gas mixtures and in a mineral-culture solution containing sucrose, glucose, or mannite. 2. A 1-3 per cent increase in nitrogen, which is scarcely significant, was obtained in a few cases where nodules were kept in thin layers of a mannite-glucose medium at 4⚬-12⚬ C. for 1-3 days. 3. Pure cultures of rhizobia did not fix nitrogen when supplied with oxalacetic acid. A few similar experiments with excised nodules gave negative or inconclusive results. 4. Fifteen ser...

Temperature Characteristics for Respiration in the Newt Under Chloretone and Nembutal Anesthesia

It is well known that various narcotics and anesthetics have a depressing effect upon respiration but this has not been systematically investigated as a function of temperature. Thus, while something is known of the influence of this class of drugs on the absolute respiratory rate, little information is available for changes in the relative respiratory rate.The newt, Triturus viridescens. was used in the present experiments both because it is a poikilothermous animal and therefore useful for temperature analyses and because its respiration has already been investigated by means of a Warburg apparatus suitably modified for this purpose.The minimum dose required to maintain newts immobile for 24 hours under the conditions of temperature variations was determined experimentally for two suitable drugs. After preliminary anesthesia for 15 minutes in a 1-10 dilution of a saturated aqueous solution of chloretone, animals were transferred to modified Warburg vessels containing 1 cc of a 1-20 dilution of a saturat...

Utilization of Serine by Clostridium botulinum.

A number of amino acids are utilized by washed suspensions of Clostridium botulinum, for the most part in coupled reactions between pairs of different amino acids. For example, alanine is deaminatively oxidized to CO2 and acetic acid by glycine or proline which are reduced to acetic acid or δamino-n-valeric acid, respectively. However, amino acids such as leucine and particularly serine appeared to be deaminated when present singly in washed suspensions of this organism. The utilization of serine has been studied in detail by the technic already described, ammonia being determined by the method of Parnas as described by Niederl and Niederl. Serine is decarboxylated in the presence of washed suspensions of Types A or B Cl. botulinum at a rate (Qco2 of 7-10) approximately one-third of that observed with pyruvic acid (Qco2 of 25-30) as the substrate at optimal pH values for both reactions. Typical results of CO2 production are presented in Fig. 1. It is apparent that the pH optimum for the utilization of serine by Cl. botulinum lies on the alkaline side of neutrality, probably near pH 7.5. This is in marked contrast with the pH optimum of 6.0 observed with pyruvic acid as the substrate. In further studies it was observed that apparently only one optical form of serine is attacked at an appreciable rate, deamination and decarboxylation of dl-serine proceeding only approximately 50% to completion. In semimacro experiments in Warburg vessels of 40 ml capacity with Clerici fluid in the manometers the fermentation was studied in more detail. Ammonia, CO2, ethyl alcohol and acetic acid were identified as the chief products of degradation of serine. In these experiments the reaction was not allowed to go to completion due to the slow rate observed by the time approximately one-half of the serine had been utilized.

Action of a Quaternary Ammonium Type of Wetting Agent on Metabolism of Microorganisms Associated with Dental Caries

In the search for a compound which can effectively inhibit the metabolism of microörganisms found in lesions of dental caries, or in plaques associated with such lesions, we have studied the action of alkyl dimethyl benzyl ammonium chloride (Zephiran) on these bacteria. The germicidal action of this compound was described by G. Domagh. Pure cultures of 5 microörganisms, which are found constantly or very frequently in association with human dental caries, were studied. These were a lactobacillus, 2 strains of M. tetragenus, Staphylococcus albus, an unidentified, aerobic, acid-producing, Gram-positive diplococcus and a yeast of the genus Monilia. Washed suspensions of the organisms were placed in appropriate buffers containing 0.02M glucose, and their rate of respiration or glycolysis measured in Warburg vessels. The alkyl compound exerts a pronounced inhibitory effect on both respiration and glycolysis at extremely low dilutions (concentrations estimated at M/10,000 to M/50,000). At a concentration of M/10,000 the compound exerts its full effect on one billion cells within 5 to 10 minutes. That the inhibition is complete and irreversible is demonstrated by the following experiment: organisms which were exposed to the compound for several minutes, and then centrifuged off and washed with saline, did not regain their metabolic activity when resuspended in fresh glucose-buffer solution. Table I shows the extremely small quantities of the compound required to inhibit one billion cells of the particular organisms studied. It can be seen that the alkyl dimethyl benzyl ammonium chloride has an activity comparable to that of the unusually potent compounds recently obtained by Dubos from soil bacteria. The marked inhibition of the metabolism of dental bacteria by the alkyl dimethyl benzyl ammonium chloride makes this compound of unusual interest.

Effect of 1-2-4 Dinitrophenol on Oxygen Uptake of Rat Tissue

Since Cutting and Tainter1 and Magne, Mayer and Plantefol2 have observed that small quantities of dinitrophenol cause a marked acceleration of the metabolism of animals, it seemed desirable to determine whether this drug accelerates the oxygen uptake of excised tissue.The experiments here reported are a continuation of those given in a previous preliminary report.3 The preparation of the rat tissue slices and the measurement of the oxygen uptake was done as previously described,4 the tissue being suspended in glucose phosphate (pH 7.4) Ringer's solution. Four Warburg vessels were employed in each instance, 2 serving for the control observations. The following experiments were performed. The oxygen uptake of rat liver, kidney and muscle tissue obtained from rats previously injected with dinitrophenol was determined when the tissue was suspended in normal Ringer's solution. Three or more animals were employed in each case. The oxygen uptake of rat liver and kidney tissue obtained from normal animals was als...

Effect of Dinitrophenol on Oxidation of Tissues

The marked acceleration of metabolism of animals injected with small quantities of dinitro α napthol reported by Heymans and Bouchaert,1 Van Uytvanck,2 and Euler,3 and a similar action of dinitrophenol observed by Magne, Mayer and Plantefol4 and by Tainter and Cutting,5 make it of interest to learn whether these substances affect the rate of respiration of isolated tissues and cell suspensions in vitro. If that be the case, it may be hoped that a study of the action of these substances will aid in elucidating the mechanism of cellular oxidation and metabolism. We have studied the effect of 2-4 dinitrophenol on the respiration and anaerobic fermentation of yeast and of frog tissues, using Warburg vessels. With muscle, lactic acid was also determined chemically.Dinitrophenol added to the solutions in which the tissues or yeast are suspended, in concentrations of the same order as perhaps exists in the tissue fluids after administration of toxic or even large therapeutic doses to man or animals, causes a mar...

Effect of Sodium Fluoride on Lactic Acid Production by Strepto-bacterium Casei

Fluorides, like the halogenated acetic acids, inhibit the formation of lactic acid from hexose or glycogen in biological systems by causing the formation of stable hexose esters. We have reported observations on the course of iodoacetate poisoning in Streptobacterium casei, and can now add to these the course of events in fluoride poisoning. The experimental procedure was the same as previously reported except that various concentrations of sodium fluoride instead of iodoacetate, made up in the suspension medium, were placed in the sidearms of the Warburg vessels. As in iodoacetate poisoning there was a brief latent period on addition of fluoride. However, the rest of the picture was quite different. Whereas inhibition was observed in all cases with iodoacetate, in concentrations ranging from 0.0004% to 4.0%, with fluoride we found that concentrations up to 0.08% actually stimulated lactic acid production. At a concentration of 0.10%, the lactic acid-time curve was approximately the same as that of the control. Increasing the fluoride concentration up to 0.18% caused small decreases in the rate of lactic acid production, then there was a sharp decrease in this rate when the fluoride concentration reached 0.20%. Little more inhibition resulted from a fluoride concentration up to 0.5%. Unlike the smooth continuous decrease in rates observed with iodoacetate, fluoride poisoning causes a rather sharp break in the rate of lactic acid production, after which the rate remains constant for several hours, at a characteristic value for each fluoride concentration. As in previous experiments, lactic acid production in the control vessels was carried out at a constant rate for some 7 hours, the duration of the experiments.