Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria).

A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.

Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats.

Carbon dioxide (CO2) is the most commonly used agent for euthanasia of laboratory rodents, used on an estimated tens of millions of laboratory rodents per year worldwide, yet there is a growing body of evidence indicating that exposure to CO2 causes more than momentary pain and distress in these and other animals. We reviewed the available literature on the use of CO2 for euthanasia (as well as anaesthesia) and also informally canvassed laboratory animal personnel for their opinions regarding this topic. Our review addresses key issues such as CO2 flow rate and final concentration, presence of oxygen, and prefilled chambers (the animal is added to the chamber once a predetermined concentration and flow rate have been reached) versus gradual induction (the animal is put into an empty chamber and the gas agent(s) is gradually introduced at a fixed rate). Internationally, animal research standards specify that any procedure that would cause pain or distress in humans should be assumed to do so in non-human animals as well (Public Health Service 1986, US Department of Agriculture 1997, Organization for Economic Cooperation and Development 2000). European Union guidelines, however, specify a certain threshold of pain or distress, such as 'skilled insertion of a hypodermic needle', as the starting point at which regulation of the use of animals in experimental or other scientific procedures begins (Biotechnology Regulatory Atlas n.d.). There is clear evidence in the human literature that CO2 exposure is painful and distressful, while the non-human literature is equivocal. However, the fact that a number of studies do conclude that CO2 causes pain and distress in animals indicates a need for careful reconsideration of its use. Finally, this review offers recommendations for alternatives to the use of CO2 as a euthanasia agent.

Formulation of ethical standards for use of animals in medical research.

In the past 200 years, moral theories and attitudes toward both humans and animals have changed considerably. These changes have led to widespread criticism of animal use in some or all research and testing. For the most part, the debate has been marked more by rhetoric than scholarship, but the underlying philosophical theories and their protagonists have been a very important influence on the modern debate over animal use. This paper examines some of the moral philosophy and attempts to present the main arguments concisely and simply and to refute common misconceptions.

Trends in the use of nonhuman primates in biomedical programmes.

Recent surveys of the use of primates in biomedical laboratories indicate that the demand, especially for imported animals, is declining. Reasons for this trend are not clear, although restrictions on export by countries of origin undoubtedly have a significant effect. More precise survey techniques and terminology would aid comparisons and help to identify the factors involved in changes in demand.

Rethinking the morality of animal research.

KIE: A review is provided of major ethical positions concerning the moral status of animals and the use of animals in research. These positions are characterized as ethical skepticism and relativism, absolute dominionism, anthropocentric consequentialism, reverence for life, utilitarianism, and abolitionism. The authors contend that any acceptable ethical discussion of the use of research animals must take into account suffering and well-being, pleasure, thought and reason, purposiveness, and self awareness. Other issues to be addressed are the nature of different animals and the principle of social interaction with humans. Justifications for animal research may be based on claims of benefits to humans, benefits to other animals, or the value of research to science and knowledge.^ieng

The concept of the three R's. An introduction.

The development of the concept of the three R's--Replacement, Reduction and Refinement--is outlined. The concept has changed slightly since the early days and is now known as "alternatives to laboratory animals". Several examples of replacement, reduction and refinement systems are given with special emphasis on biological standardization. Suggestions for action by national and international bodies are included in the conclusion.

The future of animals in research and training. The search for alternatives.

Animal welfare and antivivisection groups have protested against animal research for the last 150 years. However, they have recently (ca. 20 years ago) injected a new element into the debate by pressing for the development and application of alternatives. By this, they mean the development of techniques which could replace or reduce the use of laboratory animals, or the refinement of existing techniques to reduce animal suffering. Animal welfare concerns have focused on toxicology testing in particular. Of course, the state of the art in toxicology had advanced considerably in the last 20 years and some of the changes have resulted in a reduction of the use of animals. Nevertheless, the animal welfare movement seeks a more specific program which will review testing guidelines and support the development of new techniques which will reduce or eliminate animal use. Carcinogen detection, Draize eye irritancy testing, and LD50 testing are three specific areas where the pressure for alternatives development has been strongest. The prospects for alternatives in each of these three areas are briefly discussed together with one or two comments about possible future trends.

Refinement of animal research technique and validity of research data.

Refinement of research techniques using animals will lead to less animal distress and, at the same time, will usually lead to higher quality and more robust data. For example, acclimation of laboratory animals to handling and experimental procedures will produce not only an animal that is easier to handle but also one that reacts to the experimental stimulus rather than to the handler. Even when the animal is acclimated to handling, handling or anesthesia can cause marked changes to commonly measured biological parameters such as serum hormone and tissue metabolite levels. Standard animal housing systems are also stressful to the animals. One research report, for example, found that mice in conventional facilities had plasma corticosterone values of 150-500 ng/ml compared to values below 35 ng/ml from mice kept in special "low-stress" housing. However, assessment of stress in animal research is not easy and few technicians or research scientists are good judges of moderate stress. Investigators in the United Kingdom have recently published a report on a new technique that might allow more objective assessment of discomfort in laboratory rats and mice. The amount of exploratory behavior in treated and control animals can be used for developing a Disturbance Index (DI). The DI is a measure of the extent to which a procedure or treatment causes a departure from normal behavior. The initial data using the DI indicate that it could be a promising approach for assessing animal well-being (or the lack of it).

Changes in the contents of adenine nucleotides and intermediates of glycolysis and the citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle.

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.