L. Joe Berry

Studies of onion root respiration I. Velocity of oxygen consumption in different segments of root at different temperatures as a function of partial pressure of oxygen

Abstract The rate of oxygen consumption and carbon dioxide production in different partial pressures of oxygen at 15°, 20°, 30° and 35°C were measured manometrically in segments of onion root tip 5, 10 and 15 min in length. The rates for the regions 5–10 mm and 10–15 mm above the tip were determined by difference. Curves for the three zones of root tip relating oxygen uptake to pressure of oxygen were hyperbolic at all temperatures but the pressure just supporting the maximum rate (critical pressure) increased with temperature. R.Q.s were essentially unity at pressures equal to or greater than the critical pressure and exceeded unity when the pressures were less than the critical pressure. Rate of carbon dioxide production paralleled that of oxygen consumption but showed a smaller proportionate reduction at low oxygen tensions. Log k vs 1/T curves each root segment at different pressures of oxygen permitted calculations of activation energy. For the two basal zones, μ values of approximately 12000 calories were obtained at all pressures. For the apical 5 mm, a μ value of 4300 calories was found at 5% oxygen and one of about 13000 calories at 100% oxygen. The curves at pressures of 10%, 15% and 21% oxygen for the same segment hadbreaks with the slopes of the two straight-line portions approaching these extremes. From these results, it is concluded that diffusion is the limiting factor in rate of respiration at the lower pressures of oxygen.

Studies of onion root respiration: II. The effect of temperature on the apparent diffusion coefficient in different segments of the root tip☆

Abstract The Gerard-Fenn equation for diffusion into a cylinder was used to calculate the apparent diffusion coefficient oxygen in each of three segments of onion root tip at each of the four temperatures described in the first paper of this series. These coefficients were then used to determine the activation energy of diffusion (log D vs 1/T graphs) with the result that those for the two most apical zones of root agreed closely with the theoretical value. Certain coefficients were also used to calculate the distance to which oxygen penetrates the root when the external pressure of oxygen is limiting. From these values it was shown that the rate of oxygen consumption across the radius of the root is not perfectly linear (as the Gerard-Fenn equation demands) but the departure from linearity is not sufficient to completely invalidate the results.

EFFECTS OF BACTERIAL ENDOTOXIN ON METABOLISM : II. PROTEIN-CARBOHYDRATE BALANCE FOLLOWING CORTISONE. INHIBITION OF INTESTINAL ABSORPTION AND ADRENAL RESPONSE TO ACTH

Urinary nitrogen excretion over a 17 hour period without food and water was determined for mice poisoned with endotoxin and for control mice with and without the subcutaneous injection of 5 mg. cortisone acetate. Endotoxin did not alter the quantity of nitrogen excreted, compared with that of control mice, but in both groups of animals cortisone increased urinary nitrogen by the same amount. A balance between the quantity of protein catabolized (as estimated by increased urinary nitrogen excretion) and the total carbohydrate stored as a result of cortisone administration was found in fasted and in fed mice but not in endotoxin-poisoned mice. Endotoxin inhibited the motility of the gastrointestinal tract and blocked the absorption of food. Injections of ACTH in normal mice increased protein degradation and carbohydrate synthesis. In endotoxin-poisoned animals, ACTH failed to alter urinary nitrogen excretion and carbohydrate reserves. ACTH increased susceptibility to endotoxin. The number of heat-killed cells of S. typhimurium required to block the increase in urinary nitrogen caused by an injection of ACTH was found to be 10(8). A partial block was found with 5 x 10(7) cells but not with 10(5) cells. Mice infected with S. typhimurium excreted less than the normal amount of urinary nitrogen in response to ACTH 6 to 23 hours postinfection while normal amounts of nitrogen were eliminated 54 to 71 hours postinfection. The number of viable cells in the mice during the earlier period was less than the number of dead cells required to alter the nitrogen output, while the reverse was true during the later period.

Purification of Clostridium botulinum type a toxin

Abstract The neurotoxin of Clostridium botulinum Type A has been isolated and purified from a liquid culture. The toxin is homogeneuous by anion and cation exchange chromatography, gel filtration, isoelectric focusing and Ouchterlony gel diffusion technique. The specific toxicity of the purified toxin is 10 · 107 minimum lethal doses/1.0 A 278 nm , the molecular weight is 150 000 by gel filtration method and the iso-electric point is pH 6.1. This preparation could not be distinguished from the α fraction isolated from the crystalline toxin. Results presented here failed to confirm the claim of Gerwing et al.5 that a toxin of 12 200 mol. wt. can be isolated from the liquid culture of this organism.

SOME METABOLIC ASPECTS OF HOST-PARASITE INTERACTIONS IN THE MOUSE TYPHOID MODEL*

The influence of host metabolism on susceptibility to bacterial infections, primarily but not exclusively infections with Salmonella lyphimurium, has been studied for several years. I t was first shown that several of the well known inhibitors of specific steps in the tricarboxylic acid cycle, when injected at approximately the same time as the pathogen, reduced dramatically the mean survival time of experimental animals.’-* Also effective in reducing survival time were several intermediates of the same metabolic cycle.3 The extent to which the animals, albino mice, were succumbing to the infection itself, to the toxicity of inhibitor or intermediate, or to a combination of the two was impossible to assess. Until there is some basis for attributing cause of death to specific biochemical lesions in infections with organisms other than exotoxin producers, assessment will continue to be difficult if not impossible. I t was for this reason that the total number of viable pathogens in the mouse was determined.6s6 One of the advantages of using infections with S. typhimurium is the highly selective culture media available for its enumeration. These studies revealed a remarkably constant number of culturable cells of S. typhimurium at the moment of death irrespective of survival time and experimental procedure used to alter it. Approximately lo8 cells were consistently recoverable from the animals a t death. To this extent, it seemed justifiable to assume that altered host metabolism, under the conditions of the experiments, reduced survival time by permitting the “lethal” population of viable pathogens to be reached more rapidly? Within the framework of contemporary knowledge, further pursuit of this type of experimentation seemed to offer promising returns only if developments in related studies would be awaited. As D u ~ o s , * * ~ Pappenheimer,lo and 0ther~ll-I~ have suggested, the disease state in the infected animal may be the result of competition between host and bacterial parasite for an essential metabolite such as a growth factor, a vitamin, or an amino acid, or it may be the consequence of antimetabolite formation that interferes with an essential step in host cell metabolism by one mechanism or another. With the mouse and S. typhimurium, comparatively crude yet potentially revealing techniques yielded only negative evidence for metabolic competition between host and parasite (unpublished observations). It seemed essential, therefore, to paraphrase a 5tatement once made by Racker,” “to search for a thin metabolic trail left by Lhe pathogen in the forest of metabolism.” This search led us to a closer look at some of the effects of bacterial endotoxins in the hope that, a t least in mouse typhoid infections, it might be possible to relate disease symptoms to their presence, despite the important experiment of Hill ef ~ 1 . ’ ~ * The work reported in this paper was supported in part by a grant from the National Science Foundation, Washington, D.C., and by Contract A F 41(-657)-267 between Bryn Mawr College and the School of Aviation Medicine, United States Air Force Aerospace Medical Center (ATC), Brooks Air Force Base, Texas.

Effect of acclimatization to altitude on susceptibility of mice to influenza A virus infection.

Summary 1. Mice acclimatized to a simulated altitude of approximately 20,000 feet for 3 weeks are more resistant to artificially induced infection with the PR8 strain of influenza A virus than (a) normal control mice, (b) mice similarly exposed to altitude but given three weeks of recovery at normal atmospheric pressures prior to infection, and (c) mice made anemic by standardized hemorrhage, the latter 3 groups showing no significant difference in susceptibility. 2. Altitude acclimatized mice maintained at simulated altitude for the postinfection period are significantly more resistant than those maintained at normal atmospheric pressures. 3. There is some indication that these results are due to a metabolic disturbance which accompanies adaptation to altitude as shown by a reduction in the citric acid content of lung tissue of these animals compared to that of normal mice which reduces the animals capacity for virus synthesis.

Reticuloendothelial response and liver enzyme induction in relation to endotoxicosis in mice

Abstract The influence, at a metabolic level, of inert, nonmetabolizable, RE-active, colloidal materials was investigated in relation to endotoxin poisoning in mice. Repeated injections of glucan or zymosan increased RE activity and sensitized mice to endotoxin lethality. The liver TO activity was normal after treatment with either colloid but liver pyridine nucleotide (PN+) levels were depleted. Cortisone induced liver TO, and increased liver PN+ levels, in zymosan or glucan pretreated mice, and it protected such animals against the lethal effects of endotoxin. Such observations are consistent with the view that a cause and effect relationship may exist between hormone induction of selected hepatic enzymes and survival against stress. Since zymosan and glucan share many properties in common with endotoxin the possibility was considered that the colloids are contaminated with the toxin. This was not substantiated when it was shown that purposeful contamination of zymosan with 0.01 LD50 of endotoxin yielded results distinct from those obtained with the colloidal agent alone. These and other observations suggest a metabolic relationship between RE response and the behaviour of selected inducible liver enzymes.

Effect of malonate on selected body defenses.

Summary Our data fail to implicate an alteration in capacity of blood neutrophiles to ingest bacteria or in the bactericidal property of blood serum as a possible explanation for the mechanism by means of which malonate injections reduce survival time of experimentally infected rats.

Altitude stress: its effect on tissue citrate and salmonellosis in mice.

Summary 1. Citric acid content (γ/g wet wt) of blood, diaphragm, duodenum, heart, kidney, liver, lung, and spleen was determined colorimetrically in the following groups of mice: (a) normal mice, (b) mice exposed to simulated altitude of 20,000 ft for 3 to 4 months, (c) mice given daily intraperitoneal injections of 1 mg cortisone acetate for 2 weeks, and (d) mice starved for 17 hours. 2. Mice of group (b) had 20–30% less tissue citrate than control mice. In group (c) there was no change in tissue citrate except in kidney, which increased, and in spleen, which decreased. Tissues from starved animals showed a significant increase in citrate in all tissues. Thus the change observed in group (b) cannot be correlated with cortisone injections nor with a reduced blood sugar level induced by inanition. 3. Altitude exposed mice are more susceptible to Sal. typhimurium infection than control mice.

EFFECT OF COMPONENTS OF THE TRICARBOXYLIC‐ACID CYCLE ON BACTERIAL INFECTIONS

In order to establish the kind of tactical problem with which investigators in the area of natural resistance to infections are often confronted, assume for the moment that the survival time of an infected susceptible host, previously subjected to a set of experimental manipulations, is significantly shorter than the survival time of a control group of the same host. How is one to proceed in the laboratory to gain some insight into the mechanisms responsible for this change in resistance to infection? For the purposes of the report to follow, the notion that greater susceptibility is the result of a reduced resistance will be implied even though the potential fallacy of the reasoning, as emphasized so clearly by Schneider (1951), is recognized. When confronted with a situation of this type it is possible to postulate a number of potential explanations, all based on confirmed experimental evidence. For example, the shorter survival time could be the result of an impaired cellular defense, a depressed humoral defense, both natural and induced, or it could be due to various combinations of these factors. The final analysis inevitabry becomes complicated and exceedingly difficult to circumscribe with any assurance. The investigator is faced with a bewildering array of uncertainties because most of the important body defenses against bacterial infections cannot be quantitatively evaluated. Determinations of phagocytic indices yield data of some reliability in experienced hands of 1 step, and 1 step only, in 1 group of cells of importance in the cellular defense. In cases in which this 1 step is normal, may it be concluded that the whole cellular defense is normal? Obviously not. Similarly, antibody titers may be semiquantitatively evaluated to give some idea of the ability of an animal to respond to antigenic stimulation, but this by no means yields the complete picture of the humoral defense status of the animal. Even if all such possible tests were made, the picture of an animals defense against bacterial infection would not be complete. To compound the picture further, it is self-evident that any viable infectious agent, such as pathogenic bacteria, must grow and reproduce in and at the expense of the host organism. Regardless of whether the pathogen is intracellular or extracellular, the host cells and the pathogen must require a t least some, if not most, of their nutriments in common. A priori, it would seem that competition might exist between the cells of host and parasite for those molecules they both need for growth and maintenance. Is it not theoretically conceivable that, in this sphere of interaction between the 2 species of cells, events may occur to alter the course of a disease, even in the absence of a specific defense change that makes itself manifest in profoundly altered survival times