Accumulation Of Waste Products Research Articles

Microscale variation in Aat (aspartate aminotransferase) is supported by activity differences between upper and lower shore allozymes of Littorina saxatilis

The role of natural selection in maintaining inherited variation in morphological characters is indisputable. In contrast, genetic variation in allozyme loci is mostly assumed neutral or under weak selection, although enzyme functions are critical to central metabolic processes. A locus coding for aspartate aminotransferase (AAT) is polymorphic with two alleles in the rocky shore gastropod Littorina saxatilis. One allele (Aat 120 ) is most frequent (0.8–0.9) in upper shore subpopulations, while the other (Aat 100 ) dominates (0.7–0.8) lower shore subpopulations only meters below. Earlier studies show this variation to be maintained by divergent selection, but the mechanism is unknown. We measured enzyme activities in AAT extracts from individuals of known Aat genotype in populations from two islands and at three different temperatures. In all treatments lower shore homozygotes (Aat 100/100 ) had higher enzyme activity than upper shore homozygotes (Aat 120/120 ), with heterozygotes having intermediate levels of activity. AAT has a central role in anaerobic energy production in marine mollusks during low-tide periods of emergence, and it might appear surprising that upper shore snails, which are most likely to be emerged, have lower enzyme activity. However, upper shore snails are forced to survive long periods of desiccation, and a low metabolic rate will help to conserve limited resources of energy and nutrients and reduce accumulation of waste products.

Insulin-like growth factor-I protects myoblasts from apoptosis but requires other factors to stimulate proliferation.

Insulin-like growth factor-I (IGF-I) has been shown to stimulate myoblast proliferation for a limited time after which serum is required to reactivate IGF-I-stimulated myoblast proliferation. The aim of these studies was to determine whether IGF-I can stimulate myoblast proliferation and/or inhibit apoptosis alone or whether co-factors are necessary. This was achieved by investigating the proliferative response of L6 myoblasts to IGF-I and horse serum (HS) and by examining the status of cells in terms of cell number, substrate adherence, cell viability and DNA laddering following incubation with IGF-I and HS. L6 myoblasts proliferate in response to IGF-I after 36 h is not due to accumulation of waste products or lack of IGF-I. The addition of a low level (1% v/v) of HS restores the ability of myoblasts to proliferate in response to IGF-I and this supports the existence of a mitogenic competence factor. Furthermore, myoblasts failing to proliferate in response to IGF-I after 36 h regain the capacity to respond to IGF-I for a further period of 36 h when exposed to fetal bovine serum. Following the initial (36 h) phase of IGF-I-stimulated proliferation, removal of both IGF-I and HS led to a dramatic (60%) reduction in the number of cells fully attached to the culture vessel, with 60% of the completely detached cells dead. Agarose gel electrophoresis of extracts from these detached cells revealed higher levels of DNA laddering than extracts prepared from attached cells with IGF-I present. This suggests that IGF-I acts as a survival factor by protecting cells from apoptosis. In conclusion these experiments support the presence of a mitogenic competence factor in horse serum, which restores the ability of cells to proliferate in response to IGF-I. Unlike proliferation, protection against apoptosis is achieved by IGF-I or HS independently of each other.

Should protein intake be restricted in predialysis patients?

There are also no data supporting the second assumpIn the last decade, two findings have cast doubt on tion, with the obvious exception of starting dialysis in the wisdom of administering low-protein diets to CRF severely uremic patients. The third assumption is also patients: First, a large body of evidence has documented unsupported by the literature: Protein nutrition of CRF that hypoalbuminemia in dialysis patients is a major risk patients is usually maintained or improved by low-profactor for mortality, whether present at the onset of tein or supplemented very low protein diets. Hence, a dialysis or developed during dialysis [4‐19]. Other indi- well-planned, low-protein diet not only maintains or imces of nutritional status, such as urea nitrogen appear- proves nutritional state (including serum albumin levels) ance and predialysis levels of creatinine and cholesterol, in predialysis patients, but by reducing symptoms, it also are also correlated with mortality [4‐19]. Second, the delays the need for dialysis. Moreover, substantial evihypothesis that protein restriction slows the progression dence suggests that it may also slow the rate of progresof CRF (another rationale for its use) has not been un- sion of renal insufficiency. equivocally confirmed (discussed later here). As a result The benefits of a nutritional regimen include the folof these findings, some workers have questioned the lowing: (a) reduction in (or delayed onset of) symptoms wisdom of restricting protein intake [20‐22]. Further- and signs of uremia; (b) forestalling complications by more, they have suggested that if spontaneous protein lowering the accumulation of waste products at any given intake falls below 0.8 g/kg/day, patients are at increased level of glomerular filtration rate (GFR); and (c) perhaps risk of malnutrition, and the appropriate response is to slowing the rate of decline of GFR. Objections that have start dialysis unless protein intake can be increased. The been raised include cost, poor compliance, and perhaps assumptions underlying these recommendations are that: the increased risk of malnutrition. In this article, we (a) protein nutrition will improve in predialysis patients review the evidence supporting each of these benefits, if protein intake increases; (b) dialysis improves dietary as well as the evidence for each of these objections. intake and nutritional status; and (c) low protein intake causes malnutrition in predialysis patients.

On the relation between size of necrosis and diameter of tumor spheroids

In many previous experimental studies on multicellular tumor spheroids, the spheroid diameter at which central necrosis develops has been determined to be twice the thickness of the viable cell rim measured at a later stage of spheroid growth. This procedure tacitly assumes that there is a linear relation between the diameter of necrosis and that of the whole spheroid over the entire range of emergence and growth of necrosis. However, some experimental investigations have demonstrated that necroses do not grow gradually with spheroid diameter, but show a rapid initial increase, once a few cells have died. The present article offers an explanation for this phenomenon, which is derived from basic diffusion theory. A theoretical relation between sizes of spheroids and of their central necroses is developed, which is based on the assumption that formation of necrosis is caused by depletion of substrates or accumulation of metabolic waste products. In a second part, the theoretical model is fitted to experimental data from the literature, and oxygen consumption rate as a function of spheroid size is determined. It turns out that the model closely mimics the experimentally observed behavior described above. These experimental results, therefore, do not furnish any evidence for assuming other hypotheses of necrosis formation. Resulting O2 consumption rates are well in the range of previously published data. In all cases, approximations to the measured data are better than the corresponding linear squares fits. At least in some tumor cell lines, depletion of substrates or accumulation of waste products can explain formation of necrosis without the assumption of any additional mechanisms. Moreover, the model presented in this article offers an alternative way of determining the turnover rate of a substrate or metabolic waste product provided that depletion/accumulation of this substance represents the cause for necrosis development.

An antistress and antiaging neurometabolic therapy. Accelerated lipofuscinolysis and stimulated anabolic regeneration by the antagonic-stress synergistic formula.

In both stress and aging, an etiopathogenic analysis demonstrates in the brain some common mechanisms and reciprocal accelerating relationships: hypoanabolism (decrease of RNA and protein synthesis), coupled with hypercatabolism (increase of oxidative stress-lipid peroxidation, with waste product accumulation: lipofuscinage pigment and water-insoluble proteins). For therapeutical intervention in these antihomeostatic processes, we successively (1972-1992) developed a neurometabolic antioxidative therapy--finally represented by a specific antistress and antiaging synergistic formula, Antagonic-Stress. Its homeostatic actions have been demonstrated in the stressed aging brains by reestablishing the anabolism/catabolism balance: anabolic regeneration by increasing total RNA, total proteins and water-soluble proteins, coupled with catabolic regulation by accelerated lipofuscinolysis and age pigment elimination (neuronal-->glial-->capillary route) and decreasing of water-insoluble proteins. Specific, synergistic, and superior actions of Antagonic-Stress multiple formula vs. antistress and antiaging monotherapy have been demonstrated by preclinical and clinical studies, confirming our results.

Fatigue development during electrical stimulation in the masseter muscle of rhesus monkeys ( Macaca mulatta)

Fatigue development was investigated in five adult female rhesus monkeys, 9-11 yr old (mean weight, 4.6 kg). After sedation and anaesthesia, silver electrodes were implanted in the anterior and posterior parts of the right masseter; the contralateral muscle was used as a control. The bite force was monitored. Muscle biopsies were obtained from the central part of the masseter and were immediately frozen in liquid nitrogen. After freeze-drying a fluorometric analysis using enzymatic methods for measuring levels of glycogen, glucose, lactate, pyruvate, creatine phosphate, creatine, NADH and NAD was made. The masseters were stimulated for 3 min (100 V, 4 Hz and 2 ms). After a 5-min rest period the stimulation was repeated with the same voltage, frequency and duration. The rhesus monkey masseters were easy to fatigue. After the stimulations 25% of the initial bite force remained. A marked substrate depletion was evident. The precontraction values of glycogen, glucose and phosphocreatine were reduced. The NADH concentration increased and the NAD content decreased. An accumulation of waste products was observed; the pyruvate increased by 92% and the lactate increased by a factor of 3. The substantial substrate depletion in combination with a prominent metabolic waste-product accumulation may induce a decrease in bite-force production.

Production of recombinant proteins in high‐density insect cell cultures

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (beta-galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg beta-galactosidase/10(6) cells and 1.7 microg glucocerebrosidase/10(6) cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 x 10(6) mL(-1). The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L-glutamine, and yeastolate ultrafiltrate.

Production of monoclonal antibodies against human chorionic gonadotropin by hybridoma cultures in calcium alginate capsules.

A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0 x 10(8) cells/cm3 in the capsules led to a high dilution rate of 0.67 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20-30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density.

The Effect of Tension on Perfusion of Axial and Random Pattern Flaps in Foals

Caudally based axial pattern and random pattern skin flaps, 5 cm x 10 cm, were raised on the abdomens of seven foals. Blood flow was measured 7.5 cm from the flap bases during incremental increases in applied tension. At tensions less than 1400 g, cutaneous blood flow was more than four times higher in axial pattern flaps than in random pattern flaps. Blood flow in axial pattern flaps at tensions up to 1500 g was greater than baseline flow in random pattern flaps. After removal of applied tension, perfusion in axial pattern flaps gradually increased but remained below baseline values. A short period of hyperperfusion after removal of tension in random pattern flaps may have been indicative of accumulation of waste products and localized acidosis. All flaps healed in their donor beds without complications.

Interaction between hypometabolism and acid–base balance

This review discusses the changes in acid–base balance that are produced during hypometabolism and the negative feedback role they play in maintaining the hypometabolic state. In prolonged hypometabolism, air-breathing animals consume internal stores of fat, protein, and carbohydrate, while glycogen is the primary fuel supporting anaerobic hypometabolism. Because the excretory processes are greatly reduced, the accumulation of waste products must be dealt with internally. Mitigating strategies to minimise acid–base disturbance are seen in higher buffer capacities, an acid shift in the pH optima of key enzymes, and the use of metabolic pathways that result in a reduction of net H+ production. In some hibernating animals, gut bacteria may play an important role in preventing [Formula: see text] accumulation. However, the compensatory mechanisms are only partially successful, and substantial alterations in acid–base status and related strong ion changes are common. Changes in intracellular pH have wide metabolic effects but the acid–base and ionic status of the cell is dependent on its energy expenditure. The most vulnerable tissue to reduced metabolism is the brain. The turtle brain can greatly lessen its energy requirements by reducing activity; this is achieved by (i) depression of synaptic transmission, (ii) membrane hyperpolarisation through opening of Cl− channels resulting from release of γ-aminobutyric acid, and (iii) slowing transmembrane ion flux by the selective closure of ion channels. CO2 retention is common in hypometabolic animals. Increased levels of CO2 and H+ and decreased [Formula: see text] can directly cause metabolic depression via a variety of mechanisms, as well as a reduction in neural tissue activity. It is concluded that the hypometabolic state represents a very general condition of temporarily reduced energy expenditure which embraces aestivation, hibernation, torpor, and sleep, and that the common phenomena of CO2 accumulation and consequent changes in acid–base balance play a role in the coordinated reductions in energy expenditure and energy cost.

A model of hypoxic renal failure

Models of ischemic acute renal failure (ARF) must consider the combination of tissue hypoxia, insufficient nutrient flow, and anaerobic waste product accumulation. This study utilized isolated perfused rat kidneys to characterize the renal response to a graded hypoxic insult while maintaining flow. Kidneys were perfused at 37 degrees C with an asanguineous Krebs-buffered saline. After a 40-min baseline period, 10 or 30 min of hypoxia was rapidly achieved by reducing perfusate oxygen tension from approximately 550 to 50 mm Hg. Ten minutes of hypoxia resulted in tubular dysfunction evidenced by a 50% increase in urine flow (UV) and a 10% decrease in percent sodium reabsorption (%Na). Glomerular filtration rate (GFR) decreased by 40% during 10 min of hypoxia and returned to control levels after reoxygenation. Thirty minutes of hypoxia caused an irreversible 85% decrease in GFR accompanied by a 50% decrease in UV. This insult also caused more severe tubular dysfunction evidenced by a 20% decrease in %Na and a 35% decrease in oxygen consumption. These results demonstrate a spectrum of renal dysfunction that corresponds to the clinical spectrum from nonoliguric to oliguric ARF. This model of hypoxic ARF allows more specific investigation into the hypoxic component of postischemic renal dysfunction.

Properties of ectopic proteins synthesis in vitro by human cervical carcinoma cells.

AbstractTwo ectopic proteins, carcinoembryonic antigen (CEA) and ferritin, were synthesized in the culture media from a newly established cervical cancer cell line as measured by radioimmunoassay. The cervical cancer cell line, designated CC‐7T, produced CEA and ferritin ectopically in vitro in increasing quantities as cultures proceeded. In control studies, cultured fibroblasts did not have this secreting phenomenon. The amount of CEA and ferritin production by CC‐7T cells depended upon the cell density, nutrient availability, and the accumulation of waste products. Our findings indicate that CEA and ferritin were directly secreted by cervical cancer cells from cell line CC‐7T. This cell line should be a useful tool for studies of the mechanism of re‐expression of the oncodevelopmental genes which are responsible for the proteins' production. We also presume that there is a parallelism between the ectopic protein production and the advancement of the tumor process in positive cervical cancer patients making them excellent tumor markers for clinical monitoring of patients.

The aging eye. A family physician discusses some inevitable changes and suggests methods for dealing with them.

The aging eye undergoes a variety of structural and physiologic changes that can impair vision and cause functional disability. Among the changes are backward displacement of the eye into the orbit, thickening and yellowing of the lens, decrease in pupil size, increase in laxity of the lids, and accumulation of waste products in the retina. The specific diseases of the eye that occur with aging include macular degeneration, cataracts, glaucoma, diabetic retinopathy, and sudden vision loss. Preventive care both in old age and earlier in life may help forestall ocular changes and the subsequent morbidity and mortality they cause. Environmental changes to accommodate visual change can be important in accident prevention and in improved functioning and quality of life.

Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells

Mammalian cells grown in culture excrete lactic acid and ammonium ions in quantities that may limit growth and reduce product synthesis. Frequent replenishment of the culture medium is often necessary to prevent waste product accumulation which could inhibit cell growth. Since increased medium replenishment results in increased usage of animal serum, the most expensive raw material, excessive production of waste products lowers the cell and product yield on serum, and hence increases production costs. Strategies for reducing the production of lactic acid and ammonium bymammalian cells via controlled addition of glucose and glutamine will be demonstrated. Mathematical relations coupling ammonium and glutamine kinetics will be described. Additionally, a method for automatic on-line estimation of the cell concentration was developed. This method involves calculating the ATP production rate from the oxygen uptake rate and the lactic acid production rate. Automatic online estimation of the cell concentration is critical if nutrient levels in large-scale mammaliancell cultures are to be accurately maintained via process control.

Factors affecting cell growth and monoclonal antibody production in stirred reactors

Environmental and cultural factors that could affect growth and cell viability of mouse-mouse hybridoma cells in culture were investigated. The aim was to determine conditions that could prolong viability and result in increased yields of monoclonal antibodies in stirred reactors. Factors studied included temperature, level of dissolved oxygen, nutrient depletion, and waste product accumulation. Growing cells at temperatures 3-9 degrees lower than optimum (37 degrees C) increased viability but monoclonal antibody production was lowered. A low level of dissolved oxygen (25% air saturation compared to 60% for optimum growth) prolonged cell viability and increased the monoclonal antibody yield by about 50%. Feeding cultures daily to maintain the glucose level above 1 mg/ml and at the same time feeding cells glutamine (150 micrograms/10(6) cells per day) maintained the level of viable cells at 1.7 X 10(6)/ml for at least 9 days and resulted in an antibody yield of 290 micrograms/ml, about a 70% increase over cultures fed either glucose or glutamine alone. Ammonium ion, added to cell populations at levels produced in cultures, stopped cell growth and decreased antibody production. Another waste product, lactic acid, had no toxic effect when added to media at levels found in cultures. These results agree with our suggestion that monoclonal antibody production is enhanced by maintaining cell viability over a prolonged period and provide a base for investigating modes of hybridoma cell propagation in fermentors.

Dietary problems of the dialysis patient.

The patients on regular dialysis treatment (RDT) usually experience an adaption phase that may sometimes be complicated by intercurrent catabolic episodes before the steady state is reached. In all these clinical settings, and especially during the catabolic episodes, a correct nutritional evaluation is of great importance. The main abnormalities in body composition and metabolism, which may affect the nutritional status in these patients, are reviewed. The caloric intake should provide the ideal amount of 35 kcal/kg b.w., which seems to be critical in maintaining an ideal body weight. A protein intake of 1.0-1.2 g/kg b.w. is adequate to keep a nitrogen balance and to prevent excessive nitrogen-containing waste product accumulation. Carbohydrates should represent 45-50% of total caloric intake and monomeric carbohydrates should be limited. Lipids as 35-40% of total caloric intake are recommended with the polyunsaturated to saturated fatty acid ratio being 1.0. Phosphate intake should not exceed 900-1,200 mg. Calcium supplements are required up to a total intake of 1,500 mg. During the catabolic phases, however, both the caloric and nitrogen intakes should be increased to meet the increased needs of the patients.

The dynamics of life: aging.

Biologic aging arises from various causes, including deterioration of cells by mutations, destruction of the cells in organs through infections, aging by failure to eliminate harmful waste products, poisoning from external sources, and by radiation damage. All of these processes lead to equations of the kind observed experimentally for the aging process, so that a proper assessment of the various factors contributing to aging is especially complicated. Many nonbiological processes in which the products of reaction speed up the aging reaction lead to the familiar equation for the rate of aging. Corrosion of metals is exhibited as a typical example of this; many other examples could be given. The general equation for depth of anesthesia brought about by lipid-soluble materials and how this effect can be mimicked by accumulation of waste products is shown to simulate the typical aging curve; it is well known that such waste products are a frequent cause of death.

The formation and circulation, in Galleria, of hyphal bodies of entomophtoraceous fungi

The hyphal bodies of Entomophthora coronata and Entomophthora apiculata are formed in vivo and in vitro. The number of hyphal bodies produced in stationary culture was less than that produced in shake cultures. The accumulation of waste products and the exhaustion of nutrients apparently did not play a significant role in hyphal-body formation. The hyphal bodies of the two Entomophthora species were not circulated in the blood of the larva of Galleria mellonella , whereas those of Metarrhizium anisopliae and Beauveria bassiana were distributed throughout the larva in the blood of the body cavity. The factors that are apparently involved in the circulation of hyphal bodies in the insect are (1) the size of the hyphal body, (2) the optimal site for the growth of the fungus, and (3) the behavior of the fungus in a specific insect host. Both species of Entomophthora , in general, invaded and multiplied in the solid tissues and organs of the host. On the other hand, M. anisopliae and B. bassiana developed in the hemolymph and did not invade the solid organs and tissues until the host died. M. anisopliae produced chlamydospores in the larvae of Galleria mellonella .

AN ANALYSIS OF AGGRESSIVE BEHAVIOR, GROWTH, AND COMPETITION FOR FOOD AND SPACE IN MEDAKA (ORYZIAS LATIPES (PISCES, CYPRINODONTIDAE))

The role and consequences of aggressive behavior in competition for food and space were studied among laboratory populations of juvenile medaka. Growth rate, used to measure the success of an individual fish in different competitive situations, was followed for 648 fish in populations of 1, 2, 4, 8, or 16 fish in 1-, 4-, or 8-liter baskets.Aggressive behavior was initiated by internal state of "hunger" and the presence of food stimuli and smaller medaka. Aggression was at a low level when food was supplied in excess and large fish had no competitive advantage over small fish. Reducing the amount of space from 1 to 1/16 liter per fish did not alter the growth consequences of competition as long as the accumulation of waste products was prevented and food was supplied in excess.When food supply was limited, large medaka were socially dominant, chased small fish away from food, and grew faster than small fish. If food was spatially localized the social hierarchical societies changed into territorial societies in which the dominant defended the food area, but aggression only dispersed the subordinates throughout the habitat when food was evenly distributed. The advantage of social dominance was less if population size was large, if food was evenly distributed, and visual isolation between competitors was great. The advantage was high if food was contagiously distributed and visual isolation between competitors was great.

The Oxygen Requirements of Certain Aquatic Animals and Its Bearing Upon the Source of Food Supply

ABSTRACT The original theory of the food supply of aquatic animals put forward by Ptitter, and based upon the results of certain experiments and analyses, claimed that the chief source of food of such animals was organic matter dissolved in the sea, in lakes, rivers, etc., and that this was absorbed directly and indeed often by the gills, if present. The position he took up may be emphasised by his statement regarding fishes : “There is no doubt that a nutrition without dissolved foodstuffs is possible, and it is not impossible that cases of this kind are realised in nature. But the experiments at Naples show that the fish in the Naples aquarium under approximately natural conditions obtain one-half to three-quarters or more of their food requirements by the absorption of dissolved food.” It is quite possible that small quantities of organic matter in solution in water are absorbed by aquatic animals, and in some cases (particularly amongst protozoa living under special conditions) this may be an important, perhaps the most important, source of food. It is also possible that very small quantities of organic matter in solution may eventually be found to exercise a very profound influence (acting like vitamines, for example) on the life of aquatic animals. Evidence for such is not evidence for the main thesis set up by Pütter on the results of his own experiments. The food requirements of many aquatic animals as calculated by Pütter on the basis of oxygen consumption are often remarkably high and need further investigation. It is quite possible that there is an unknown factor at work here. In connection with these calculations we consider that purely theoretical computations based upon the measurement or estimation of the active surface area of the body are not permissible, and that the application of the law of surface area in connection with metabolism must not be allowed to supplant experiment Experiments on goldfish similar to those made by Pütter, show that specimens kept in tap water without any particulate food live for varying periods (which are often of considerable duration) dependent upon the original condition of the fish, and the freedom of the experimental tanks from parasites. The addition of the organic compounds, glycerine and asparagine, makes no difference to the duration of life, and the consumption of oxygen by the fish living in tap water with these compounds does not exceed that of the control fish in tap water only. Gradual starvation takes place, and sections show that the mass of muscle tissue becomes gradually reduced. The cessation of feeding on particulate food makes the fish particularly susceptible to the attacks of parasites (Chilodon cyprini and Gyrodactylus, sp.), if there is any chance of such infection. The consumption of oxygen by plaice eggs during their development agrees fairly well with the amount computed from analyses of the composition of young eggs and eggs shortly before hatching, but the results are only approximate, although they fit in with the assumption that such floating eggs have their own food stores and absorb nothing from the sea water. Aquatic organisms are not to be grouped in one class in so far as nutrition and metabolism are concerned. It had frequently been noted that when the oxygen consumption of aquatic animals is measured, and the determination extends over several hours there is a gradual falling off during the experiment. It is necessary to look for this in every case before estimating the normal oxygen consumption over long periods. The variation may be due to handling the specimens at the beginning of the experiment, to the gradual reduction of the oxygen available, to the accumulation of waste products, or to time of feeding. We have found that goldfish, axolotls, and Anodon used in our experiments are to a certain extent independent of the oxygen pressure, which may fall considerably (until a certain minimum is reached) before the oxygen consumption of the animals per hour is affected.