Body temperature of free-ranging koalas (Phascolarctos cinereus) in south-east Queensland.

The distribution of the koala (Phascolarctos cinereus) in Queensland is predicted to contract as a result of climate change, driven by the frequency, intensity and duration of heatwaves and drought. However, little is known about the physiological responses of this species to environmental extremes under field conditions. This study aimed to establish the efficacy of surgically implanted thermal radio transmitters and data loggers to measure the body temperature of free-ranging koalas across a range of environmental conditions and ambient temperatures. Five free-ranging koalas in southeast Queensland were implanted with thermal transmitters and data loggers waxed together as a single package. Body temperatures were recorded for variable periods ranging from 3 to 12 months. Diurnal rhythms in body temperature were detected irrespective of season. The long-term diurnal body temperature peak for all koalas occurred between 16:00 and 17:00 h and body temperature was 36.7-36.9 °C, the long-term nadir occurred between 07:00 and 08:00 h and body temperature was 35.4-35.7 °C. Koala body temperatures as low as 34.2 °C and as high as 39.0 °C were recorded. Thermolability became apparent when ambient temperatures were outside the deduced thermal neutral zone for koalas (14.5-24.5 °C): heat was accumulated during the day and dissipated during the cool of the night. While this study is the first to report on body temperature of free-ranging koalas in their normal behavioural context, further investigations are necessary to determine the physiological boundaries of the thermal niche for this species, in order to better equip models that will more accurately predict the impacts of climate change on koalas.

Salt Glands in the Tongue of the Estuarine Crocodile Crocodylus porosus.

The apparent absence of salt glands in marine and estuarine Crocodilia has long been a puzzle. However, we have identified glands in the tongue of Crocodylus porosus which exude a concentrated secretion of sodium chloride. The glands are similar in ultrastructure to other reptilian salt glands and undoubtedly play a major role in electrolyte regulation.

Amplification of the antitumor activity of phleomycins and bleomycins in rats and mice by caffeine.

While having no antitumor effect per se, caffeine substantially enhanced the antitumor effects of the phleomycins PLM-CHP and PLM-PEP, and the bleomycins BLM-CHP and Blenoxane in rats carrying Walker 256 carcinosarcoma and/or mice carrying Ehrlich ascites tumor, even at doses of phleomycin and bleomycin below the minimum effective level. Positive but less conclusive results were also obtained with PLM-A4A4G and PLM-G.

Field test of a paradigm: hysteresis of heart rate in thermoregulation by a free-ranging lizard (Pogona barbata).

The discovery that changes in heart rate and blood flow allow some reptiles to heat faster than they cool has become a central paradigm in our understanding of reptilian thermoregulation. However, this hysteresis in heart rate has been demonstrated only in simplistic laboratory heating and cooling trials, leaving its functional significance in free-ranging animals unproven. To test the validity of this paradigm, we measured heart rate and body temperature (Tb) in undisturbed, free-ranging bearded dragons (Pogona barbata), the species in which this phenomenon was first described. Our field data confirmed the paradigm and we found that heart rate during heating usually exceeded heart rate during cooling at any Tb. Importantly, however, we discovered that heart rate was proportionally faster in cool lizards whose Tb was still well below the 'preferred Tb range' compared to lizards whose Tb was already close to it. Similarly, heart rate during cooling was proportionally slower the warmer the lizard and the greater its cooling potential compared to lizards whose Tb was already near minimum operative temperature. Further, we predicted that, if heart rate hysteresis has functional significance, a 'reverse hysteresis' pattern should be observable when lizards risked overheating. This was indeed the case and, during heating on those occasions when Tb reached very high levels (> 40 degrees C), heart rate was significantly lower than heart rate during the immediately following cooling phase. These results demonstrate that physiological control of thermoregulation in reptiles is more complex than has been previously recognized.

Neuropeptide immunoreactivity and co-existence in cardiovascular nerves and autonomic ganglia of the estuarine crocodile, Crocodylus porosus, and cardiovascular effects of neuropeptides.

The two aortas of the crocodile are in open connection at two sites, the foramen of Panizzae immediately outside the ventricles, and the arterial anastomosis at the level of the gut. The present study was performed to elucidate the innervation of the cardiovascular structures of the crocodile, in part to provide a further basis for the assumption that the apertures of the foramen and the anastomosis may be altered, possibly leading to changes in the flow profiles of the central vessels. The presence of smooth muscle arranged at the circumference of the foramen and in the walls of the anastomosis was demonstrated. The cardiovascular structures were innervated by nerves containing co-existing tyrosine hydroxylase, NPY and somatostatin immunoreactivities, which also occurred in neurons of the sympathetic ganglia. CGRP and substance P immunoreactive material co-existed in cardiovascular nerves, and in the nodose ganglion. In addition, bombesin, VIP and galanin immunoreactive nerves were found. Effects of neuropeptides on blood flows and blood pressures were studied in vivo. Substance P increased all blood flows measured, NPY increased the flow through the arterial anastomosis while neurotensin caused an initial decrease in the flow through the arterial anastomosis. In conclusion, there is a rich innervation of the heart and major vessels of the estuarine crocodile, including the foramen of Panizza and the arterial anastomosis. These nerves possibly regulate the distribution of blood in the cardiovascular system, which is further suggested by the results of the injection of neuropeptides.

The sub-pulmonary conus and the arterial anastomosis as important sites of cardiovascular regulation in the crocodile Crocodylus porosus

We present evidence to support the hypothesis that the arterial anastomosis and the cogteeth-like valves located in the sub-pulmonary conus in the right ventricle are important sites of cardiovascular regulation in the crocodile Crocodylus porosus. The influence of the arterial anastomosis on the development of the 'foramen spike' in the left aortic pressure trace, which occurs at the onset of diastole when the pressures in the right and left aortas become equal, and on gastrointestinal blood flow was examined in unanaesthetised C. porosus using blood vessel occluders. Measurements of blood flow in the arterial anastomosis showed that, during non-shunting conditions, there was a substantial systolic blood flow from the right aorta into the coeliac artery. The total coeliac artery blood flow was the sum of the anastomosis flow from the right aorta plus the left aortic flow originating from the right aorta via the foramen of Panizza during diastole. During mechanically induced pulmonary-to-systemic shunting, the anastomosis blood flow was reversed, with blood flowing from the left to the right aorta. The magnitude of the 'foramen spike' was directly related to the vascular resistance in the anastomosis. When vascular resistance in the anastomosis was high, such as during mechanical occlusion, there was an increase in the right aortic to left aortic pressure gradient during systole which resulted in an increase the foramen spike amplitude. Recordings of right intraventricular pressure in unanaesthetised C. porosus showed spontaneous changes in right intracardiac systolic pressure. The pressure recordings were biphasic, with the second contraction (isometric) being highly variable in size, indicating the control of pulmonary outflow resistance, possibly via the 'cogteeth valves' located in the sub-pulmonary conus in the right ventricle.

Dynamic anatomical study of cardiac shunting in crocodiles using high-resolution angioscopy

Prolonged submergence imposes special demands on the cardiovascular system. Unlike the situation in diving birds and mammals, crocodilians have the ability to shunt blood away from the lungs, despite having an anatomically divided ventricle. This remarkable cardiovascular flexibility is due in part to three anatomical peculiarities: (1) an 'extra' aorta (the left aorta) that leaves the right ventricle and allows the blood from the right ventricle to take an alternative route into the systemic circulation instead of going to the lungs; (2) the foramen of Panizza, an aperture that connects the right and left aortas at their base immediately outside the ventricle; and (3) a set of connective tissue outpushings in the pulmonary outflow tract in the right ventricle. Using high-resolution angioscopy, we have studied these structures in the beating crocodile heart and correlated their movements with in vivo pressure and flow recordings. The connective tissue outpushings in the pulmonary outflow tract represent an active mechanism used to restrict blood flow into the lungs, thus creating one of the conditions required for a right-to-left shunt. We observed that the foramen of Panizza was obstructed by the medial cusp of the right aortic valve during most of systole, effectively differentiating the left and right aortic blood pressure. During diastole, however, the foramen remained open, allowing pressure equilibration between the two aortas. Contrary to current theories, we found that the left aortic valves were unable to cover the foramen of Panizza during any part of the cardiac cycle, supporting the reversed foramen flow hypothesis. This would ensure a supply of blood to the coronary and cephalic circulation during a complete shut-down of the left side of the heart, such as might occur during prolonged submergence.

Survival and growth of hatchling Crocodylus porosus in saltwater without access to fresh drinking water.

It has been suggested that C. porosus select nest sites which provide a source of freshwater for hatchlings during the dry season. From a mark-recapture study, we conclude that hatchling C. porosus can survive and grow in hyperosmotic saltwater without drinking freshwater. Hence, the siting of nests is unlikely to be the consequence of a requirement by hatchlings for freshwater. Considered along with other information, our observations imply that hatchling C. porosus have functional salt glands.

The origins of the back-mutation assay method: a personal recollection.

The back-mutation assay method for determining the mutagenicity of various treatments was first developed a little over 50 years ago and has been in continuous use ever since. Shortly after the method was first used it became evident that certain factors of cell density, composition of media, etc., had to be carefully controlled to preserve an acceptable reliability of the method. A factor of particular importance was the suppression of growth of back-mutant prototrophic cells by the large number of auxotrophic cells present, a phenomenon which later became known as the "Grigg Effect." This review describes the origins of the back-mutation method and of the confounding competitive suppression phenomenon, the cause of competitive suppression, methods of diagnosing whether it is likely to bias the interpretation of a particular back-mutation experiment, and an experimental design which removes it entirely as a possible source of error. A number of other phenomena, such as phenotypic lag and coincident mutation associated with back-mutation, are also discussed as possible sources of error.

A general method for selecting strains of Escherichia coli having altered spontaneous or induced mutabilities.

A simple method is described for selecting mutant strains of E. coli differing from their parent in the ability to spontaneously mutate via any mechanism. The method detects hypo- and hyper-mutators. It relies on the detection as papillae on thy colonies of second mutations either at the dra or drm locus.

Sulphydryl-mediated DNA breakage by phlemomycin in Escherichia coli.

Sulphydryl-mediated DNA breakage, which is induced by the antibiotic phleomycin in vitro, has been found to contribute significantly to the DNA damage produced by phleomycin in Escherichia coli. The effect of pleomycin was inhibited in vivo, as in vitro, by chelating agents, sulphydryl blocking agents and antioxidants. An increase in the intracellular concentration of free sulphydryl resulted in an increased response to phleomycin, while mutants containing very low levels of free sulphydryl due to a defect in glutathione synthesis showed greatly reduced DNA breakage, particularly at low phleomycin concentrations. In spheroplasts of these gshA mutants, restoration of the response to phleomycin of dithiothreitol. Sulphydryl-mediated breakage appears to be the principal mechanism for DNA damage in E. coli at libly enzymic, operates at higher phleomycin concentrations.

DNA methylation and mutation.

5-Methylcytosine (5mC) in DNA is produced by post-synthetic modification of cytosine residues, and it occurs primarily in CpG doublets in the mammalian genome. 5mC is a mutable site, because it can undergo spontaneous deamination to thymine. There is a repair mechanism which specifically recognises G.T mispairs, and replaces thymine with cytosine. However, this repair is not fully efficient, because the 5mC-->T transition mutation occurs about 10 times as frequently as other transitions. Such mutations are frequently seen in inherited diseases, and mutations in the p53 gene in tumours are also very commonly in 5mCpG doublets. As well as mutations, there can also be heritable changes in DNA methylation, known as epimutations, which may be of particular significance in somatic cells. Whereas the pattern of DNA methylation is very constant for any one cell type, the pattern becomes very variable in tumour cells. The breakdown of the normal controls of DNA methylation in tumorigenesis can lead to increased gene expression or to gene silencing. DNA damage increases not only mutation, but also heritable changes in methylation. At present, little is known about the ability of DNA repair to preserve the normal pattern of methylation in somatic cells.

Amplification of the antitumor activity of phleomycins in rats and mice by heterocyclic analogues of purines.

Significant and often substantial enhancement of the antitumor properties of several individual phleomycins , by co- administration via intraperitoneal injection of a number of purine analogues, is demonstrated in rats and mice having three diverse tumors. It is evident that the dose levels of both the phleomycin and the amplifier are very significant and that optimal levels vary widely with the actual agents used. Constant serum levels of amplifier can be maintained for several days by administration via silastic-pellet implantation rather than injection, and this route of administration is an effective alternative for amplifiers of low solubility.

Amplification of the antibiotic effects of the bleomycins, phleomycins and tallysomycins: its dependence on the nature of the variable basic groups.

The bleomycins, phleomycins and tallysomycins are structurally similar glycopeptide antibiotics. Within each class, individual members differ only in the structure of a basic group. The antibiotic effect of phleomycin (Bristol batch A9331-648) against Escherichia coli is amplified substantially by a number of simple heterocyclic and aromatic compounds. In this paper a sample of 26 such compounds were tested for this property with 25 different phleomycins, bleomycins and tallysomycins. The nature of the variable basic group of the phleomycins, bleomycins and tallysomycins determined the response obtained with all amplifiers, although variation of response was much less marked with caffeine which potentiated the cytotoxic effects of all the phleomycins, bleomycins and tallysomycins tested. Phleomycins and bleomycins having two or three guanidino groups in the variable basic group, or phleomycins having a secondary amino group within a methylene chain and a terminal 2-phenylethyl substituent, were amplified by most compounds, whereas the cytotoxicity of others was enhanced little or not at all. Similar phleomycins, having a secondary amino and a terminal guanidino group and no 2-phenylethyl substituent showed little enhancement, and in these cases the inclusion of a 2-phenylethyl substituent had a major influence in determining amplifiability. Bleomycins and phleomycins having identical basic groups were amplified to similar extents by the sample of 26 amplifying agents used.

Amplifications of phleomycin and bleomycin-induced antibiotic activity in Escherichia coli by aromatic cationic compounds.

A wide range of aromatic compounds has been shown to amplify phleomycin-induced cell killing in Escherichia coli. They include acridines, acridinium chlorides, dihydroanthracenes, anthracenes, dianthracenes, phenanthridinium salts, phenazinium chlorides, phenoxazones, triphenyl methane dyes, benzoquinolizinium chloride, diphenylmethane derivatives, stilbene and diphenyl derivatives. Low concentrations of these amplifiers also amplified the DNA breakage and degradation effects of phleomycin. The minimum structural specification for activity as an amplifying agent is suggested. A representative sample of compounds effective as amplifiers of phleomycin also amplified the antibiotic effects of bleomycins B4 and B6. The amplifiers described are known to vary in their ability to penetrate and accumulate in different organisms or tissues. This suggests the possibility of developing a series of antibiotic regimes using these amplifiers (or the large number of derivative compounds also likely to be active) where the therapeutic index is determined by the properties of the amplifier chosen rather than of the phleomycin or the bleomycin.

Comparative erythrocyte metabolism in marsupials and monotremes.

Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

Comparative study of the antioxidant defence systems in the erythrocytes of Australian marsupials and monotremes.

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes "detoxify" H2O2, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC.

Sequencing 5-methylcytosine residues by the bisulphite method.

Measuring patterns of cytosine methylation in genomic DNA is most efficiently accomplished by use of the bisulphite method. This method depends on the large difference in reactivity of cytosines relative to 5-Methyl cytosines in genomic DNA to bisulphite. The chemistry and history of the method and recent developments which greatly increase its sensitivity, simplicity and reliability are described. An updated protocol to guide users is appended.