title: An experimental study of metabolism during anhydrobiosis

authors: Vickers, Murray

abstract: Certain organisms can survive desiccation. Examples are seeds, pollen and spores of plants, lichens, the eggs of many insects, many rotifers and tardigrades and certain nematodes. The term anhydrobiosis has been used to describe the state of such organisms when desiccated. In this thesis anhydrobiosis is defined as ‘the maintenance of the living condition at low water activity’, using the word activity in the thermodynamic sense. The term resting metabolism is proposed to describe any metabolism occurring during anhydrobiosis. The literature on anhydrobiosis and resting metabolism is reviewed. 
The aim of this work has been to study anhydrobiosis. This has required the development of an acceptable technique to study resting metabolism and the evaluation of the technique by the study of a selection of anhydrobiotic organisms. 
The experimental difficulties associated with studying resting metabolism are discussed. Clearly the addition of any solvent to an anhydrobiotic organism will disturb anhydrobiosis, and hence classical biochemical techniques cannot be used. The requirements of a technique that would not disturb the organism are defined. 
A novel technique for studying the resting metabolism of any anhydrobiotic system is described and results from experiments on the resting metabolism of various seeds, pollen and spores are given. It is shown that complex metabolism (involving amino acids, organic acids, sugars and sugar phosphates) occurs above 64% relative humidity. However, these results could be complicated by active fungal growth that may occur above 70% relative humidity. Consequently the experimental work concentrates on metabolism occurring at 64% and four other lower relative humidities to a minimum of 15%. 
This new technique is a development of a method previously used to study the germination metabolism of seeds. The essential principle of the technique is the exposure of the organism to tritiated water vapour. This labels the water and other components containing exchangeable hydrogen in the organism. Since many biochemical reactions proceed with the incorporation of a non-exchangeable hydrogen atom, the detection of a tritium label in a compound indicates that it must have been involved in a chemical or biochemical reaction. If, however, a metabolite is found not to be tritium labelled then this can be evidence that certain metabolic pathways involving this particular metabolic pool are not operating. 
An exposure chamber is described in which biological specimens can be exposed to tritiated water vapour at constant relative humidity. After several days exposure to tritiated water vapour the specimen is extracted and the metabolites that have incorporated tritium are detected and identified. This technique has the advantage of detecting in vivo metabolism and does not alter the resting state of the organism. 
The exposure of plant propagules to tritiated water vapour is shown not to destroy their viability. This was established for samples of Pinus ponderosa pollen, Allium cepa and Sinapis alba seeds, and the spores of Cyathea dealbata. In all cases germination proceeds but at a slower rate than with unexposed propagules. Control experiments show that the tritium activity detected is due to metabolic processes and not due to non-physiological chemical activity. 
The incorporation of tritium into metabolites by the resting propagules is slight. An investigation into the chromatographic and scintillation autographic techniques which would allow maximum sensitivity of detection is described. Thin layer electrophoresis followed by chromatography in a second direction is found to be particularly suited to this work. An attempt to develop low temperature liquid scintillation autography is discussed. It is contended that this shows promise of very great sensitivity. 
Results obtained by the technique show that between 45% and 64% relative humidity the resting metabolism of all propagules studied involves mainly amino acid metabolism with varying amounts of organic acid metabolism. It is suggested that amino acids become labelled by low level transaminations and/or deaminations occurring within the dry propagule. Also the tritium labelling of citric, malic, succinic and possibly fumaric acids may indicate the operation of at least parts of the tricarboxylic acid cycle in the dry propagules at relative humidities of 45% and above.
The extent of tritium labelling at 34% is much reduced. In the cases of Pinus ponderosa pollen and Allium cepa seeds no tritium labelling that can be detected by scintillation autography occurs at all. Other propagules studied at this low relative humidity label only one compound except in the case of Phormium tenax pollen which incorporates traces of tritium into two amino acids. No propagule will incorporate tritium detectable by scintillation autography at 15% relative humidity. 
Scintillation counting of lipids extracted from the propagules shows that small amounts of tritium are incorporated into these compounds by all propagules at all relative humidities. Evidence is adduced to suggest that this incorporation is due to non-metabolic processes.
Tritium labelling of water soluble macromolecules is negligible. This suggests that there is little synthesis of such compounds in resting propagules at 64% relative humidity and below.
The solid residue left after extraction is always found to be tritium labelled. Further examination suggests that the label in the solid residue is acquired by non-metabolic means.
Scintillation counting of extracts containing small molecule water soluble metabolites implies that these contain varying amounts of semi-labile tritium. It is contended that this is lost during chromatography and that chromatography is a very effective means of decontaminating the extracts of semi-labile tritium. Results from further experiments support this contention. 
Sucrose is a carbohydrate often found to be tritium labelled in experiments with germinating propagules in tritiated water. Attempts to detect tritium labelling of sucrose in Pinus ponderosa pollen after exposure to tritiated water vapour are described but only traces of label are found. This does not unequivocally eliminate the possibility of carbohydrate metabolism in the resting pollen but does indicate that it is less comprehensive than in germination. 
The techniques developed by the author have since been used by other workers to study the resting metabolism of Pithomyces chartarum spores and of two examples of lichens in some detail. These results, together with the author’s work on eleven species of pollen, four species of seed, one species of fern spore and one of fungal spore, show a striking similarity in the resting metabolism of diverse anhydrobiotic systems.
It is concluded as a result of this work that there is considerable metabolic activity at surprisingly low relative humidities (that is, low water activities). It is proposed that resting metabolism involves a branch of biochemistry dealing with metabolism at low water activity and consequently solutions of high concentration. Presumably only certain types of metabolic activity are possible in the very concentrated solutions existing in an anhydrobiotic system. It appears that this metabolic activity involves amino acids and organic acids. This hypothesis would predict that the reactions taking place in all anhydrobiotic systems are similar and this proved to be the case for all organisms studied. It will be interesting to determine the nature of resting metabolism of animal anhydrobiotic systems, but time did not permit such studies.
Experiments on extracts from Pinus ponderosa pollen show that tritium labelling does occur at 34% relative humidity but to such a small extent that it could not be detected by scintillation autography. It is suggested that even at this low water activity enzymes in the pollen have not been rendered inoperative but that labelling has been greatly attenuated by the extremely high viscosity of the intracellular fluid. This could mean that diffusion of substrates and products becomes rate limiting. It is suggested that a glass-like state of extremely high viscosity may exist within the cell at 34% relative humidity and below. 
Preliminary experiments indicate that there may be a critical relative humidity between 64% and 75% where the comparatively simple metabolism of intermediate relative humidities become much more complicated. It may be significant that it is in this region of relative humidity that most biochemical substances deliquesce. 
It is suggested that some enzymes will operate in much more concentrated solution than others. As relative humidity increases more enzymes become active. The implications this may have for the nature of enzymes in anhydrobiotic systems is considered. 
Suggestions are made for the application of the newly developed technique to various other biological problems.

text: An experimental study of metabolism during anhydrobiosis Certain organisms can survive desiccation. Examples are seeds, pollen and spores of plants, lichens, the eggs of many insects, many rotifers and tardigrades and certain nematodes. The term anhydrobiosis has been used to describe the state of such organisms when desiccated. In this thesis anhydrobiosis is defined as ‘the maintenance of the living condition at low water activity’, using the word activity in the thermodynamic sense. The term resting metabolism is proposed to describe any metabolism occurring during anhydrobiosis. The literature on anhydrobiosis and resting metabolism is reviewed. 
The aim of this work has been to study anhydrobiosis. This has required the development of an acceptable technique to study resting metabolism and the evaluation of the technique by the study of a selection of anhydrobiotic organisms. 
The experimental difficulties associated with studying resting metabolism are discussed. Clearly the addition of any solvent to an anhydrobiotic organism will disturb anhydrobiosis, and hence classical biochemical techniques cannot be used. The requirements of a technique that would not disturb the organism are defined. 
A novel technique for studying the resting metabolism of any anhydrobiotic system is described and results from experiments on the resting metabolism of various seeds, pollen and spores are given. It is shown that complex metabolism (involving amino acids, organic acids, sugars and sugar phosphates) occurs above 64% relative humidity. However, these results could be complicated by active fungal growth that may occur above 70% relative humidity. Consequently the experimental work concentrates on metabolism occurring at 64% and four other lower relative humidities to a minimum of 15%. 
This new technique is a development of a method previously used to study the germination metabolism of seeds. The essential principle of the technique is the exposure of the organism to tritiated water vapour. This labels the water and other components containing exchangeable hydrogen in the organism. Since many biochemical reactions proceed with the incorporation of a non-exchangeable hydrogen atom, the detection of a tritium label in a compound indicates that it must have been involved in a chemical or biochemical reaction. If, however, a metabolite is found not to be tritium labelled then this can be evidence that certain metabolic pathways involving this particular metabolic pool are not operating. 
An exposure chamber is described in which biological specimens can be exposed to tritiated water vapour at constant relative humidity. After several days exposure to tritiated water vapour the specimen is extracted and the metabolites that have incorporated tritium are detected and identified. This technique has the advantage of detecting in vivo metabolism and does not alter the resting state of the organism. 
The exposure of plant propagules to tritiated water vapour is shown not to destroy their viability. This was established for samples of Pinus ponderosa pollen, Allium cepa and Sinapis alba seeds, and the spores of Cyathea dealbata. In all cases germination proceeds but at a slower rate than with unexposed propagules. Control experiments show that the tritium activity detected is due to metabolic processes and not due to non-physiological chemical activity. 
The incorporation of tritium into metabolites by the resting propagules is slight. An investigation into the chromatographic and scintillation autographic techniques which would allow maximum sensitivity of detection is described. Thin layer electrophoresis followed by chromatography in a second direction is found to be particularly suited to this work. An attempt to develop low temperature liquid scintillation autography is discussed. It is contended that this shows promise of very great sensitivity. 
Results obtained by the technique show that between 45% and 64% relative humidity the resting metabolism of all propagules studied involves mainly amino acid metabolism with varying amounts of organic acid metabolism. It is suggested that amino acids become labelled by low level transaminations and/or deaminations occurring within the dry propagule. Also the tritium labelling of citric, malic, succinic and possibly fumaric acids may indicate the operation of at least parts of the tricarboxylic acid cycle in the dry propagules at relative humidities of 45% and above.
The extent of tritium labelling at 34% is much reduced. In the cases of Pinus ponderosa pollen and Allium cepa seeds no tritium labelling that can be detected by scintillation autography occurs at all. Other propagules studied at this low relative humidity label only one compound except in the case of Phormium tenax pollen which incorporates traces of tritium into two amino acids. No propagule will incorporate tritium detectable by scintillation autography at 15% relative humidity. 
Scintillation counting of lipids extracted from the propagules shows that small amounts of tritium are incorporated into these compounds by all propagules at all relative humidities. Evidence is adduced to suggest that this incorporation is due to non-metabolic processes.
Tritium labelling of water soluble macromolecules is negligible. This suggests that there is little synthesis of such compounds in resting propagules at 64% relative humidity and below.
The solid residue left after extraction is always found to be tritium labelled. Further examination suggests that the label in the solid residue is acquired by non-metabolic means.
Scintillation counting of extracts containing small molecule water soluble metabolites implies that these contain varying amounts of semi-labile tritium. It is contended that this is lost during chromatography and that chromatography is a very effective means of decontaminating the extracts of semi-labile tritium. Results from further experiments support this contention. 
Sucrose is a carbohydrate often found to be tritium labelled in experiments with germinating propagules in tritiated water. Attempts to detect tritium labelling of sucrose in Pinus ponderosa pollen after exposure to tritiated water vapour are described but only traces of label are found. This does not unequivocally eliminate the possibility of carbohydrate metabolism in the resting pollen but does indicate that it is less comprehensive than in germination. 
The techniques developed by the author have since been used by other workers to study the resting metabolism of Pithomyces chartarum spores and of two examples of lichens in some detail. These results, together with the author’s work on eleven species of pollen, four species of seed, one species of fern spore and one of fungal spore, show a striking similarity in the resting metabolism of diverse anhydrobiotic systems.
It is concluded as a result of this work that there is considerable metabolic activity at surprisingly low relative humidities (that is, low water activities). It is proposed that resting metabolism involves a branch of biochemistry dealing with metabolism at low water activity and consequently solutions of high concentration. Presumably only certain types of metabolic activity are possible in the very concentrated solutions existing in an anhydrobiotic system. It appears that this metabolic activity involves amino acids and organic acids. This hypothesis would predict that the reactions taking place in all anhydrobiotic systems are similar and this proved to be the case for all organisms studied. It will be interesting to determine the nature of resting metabolism of animal anhydrobiotic systems, but time did not permit such studies.
Experiments on extracts from Pinus ponderosa pollen show that tritium labelling does occur at 34% relative humidity but to such a small extent that it could not be detected by scintillation autography. It is suggested that even at this low water activity enzymes in the pollen have not been rendered inoperative but that labelling has been greatly attenuated by the extremely high viscosity of the intracellular fluid. This could mean that diffusion of substrates and products becomes rate limiting. It is suggested that a glass-like state of extremely high viscosity may exist within the cell at 34% relative humidity and below. 
Preliminary experiments indicate that there may be a critical relative humidity between 64% and 75% where the comparatively simple metabolism of intermediate relative humidities become much more complicated. It may be significant that it is in this region of relative humidity that most biochemical substances deliquesce. 
It is suggested that some enzymes will operate in much more concentrated solution than others. As relative humidity increases more enzymes become active. The implications this may have for the nature of enzymes in anhydrobiotic systems is considered. 
Suggestions are made for the application of the newly developed technique to various other biological problems.

year: 1978

title: An experimental investigation of the self-management therapies: coverant control and covert sensitization

authors: Saunders, Steven Arthur

abstract: The relative efficacies of the SM techniques of Coverant Control (CC) and Covert Sensitization (CS), when applied singly and in combination, are investigated. Rate of cigarette smoking is the dependent variable. 

From an initial sample of 61 volunteers, smokers wishing to quit, 55 subjects (S’s) maintained contact and comprised the study group. Eleven S’s formed a non-treated control condition while the remaining 44 were distributed equally across 4 experimental conditions. These groups included a Placebo condition, CC, CS and CS + CC combined. 

It was hypothesised that CS and CC would prove more effective than a Placebo condition which involved contact only. It was further hypothesised that CS+ CC, administered as a combined package, would prove more effective in maintaining in-treatment gains at follow-up than when either of these treatments was administered singly.

The statistical analyses indicated no significant differences between the experimental groups receiving regular contact. All groups, except the non-treated controls, did reduce cigarette consumption while the programme lasted. However, on follow-up there was an almost uniform return toward baseline rates, with the SM treatment S’s not differing from those in the Placebo group.

These data are discussed in terms of possible implications for the development of SM techniques that are clinically useful with cigarette smokers.

text: An experimental investigation of the self-management therapies: coverant control and covert sensitization The relative efficacies of the SM techniques of Coverant Control (CC) and Covert Sensitization (CS), when applied singly and in combination, are investigated. Rate of cigarette smoking is the dependent variable. 

From an initial sample of 61 volunteers, smokers wishing to quit, 55 subjects (S’s) maintained contact and comprised the study group. Eleven S’s formed a non-treated control condition while the remaining 44 were distributed equally across 4 experimental conditions. These groups included a Placebo condition, CC, CS and CS + CC combined. 

It was hypothesised that CS and CC would prove more effective than a Placebo condition which involved contact only. It was further hypothesised that CS+ CC, administered as a combined package, would prove more effective in maintaining in-treatment gains at follow-up than when either of these treatments was administered singly.

The statistical analyses indicated no significant differences between the experimental groups receiving regular contact. All groups, except the non-treated controls, did reduce cigarette consumption while the programme lasted. However, on follow-up there was an almost uniform return toward baseline rates, with the SM treatment S’s not differing from those in the Placebo group.

These data are discussed in terms of possible implications for the development of SM techniques that are clinically useful with cigarette smokers.

year: 1973

title: Tīaho mai

authors: Maxwell, Te Kahautu

abstract: Waiata ā Ringa (action song). Lyrics composed by Te Kahautu Maxwell. Performed by Ōpōtiki Mai Tawhiti (Kapa Haka group)

text: Tīaho mai Waiata ā Ringa (action song). Lyrics composed by Te Kahautu Maxwell. Performed by Ōpōtiki Mai Tawhiti (Kapa Haka group)

year: 2024