Michael McCabe

The Anomalous Einstein-Stokes Behaviour of Oxygen and Other Low Molecular Weight Diffusants

Almost a century ago, Einstein and Sutherland independently derived equations that describe the relationship between diffusion of solutes and the molecular parameters of those solutes. In that time it has been recognized that, although the equations adequately describe the diffusion of large and medium-sized molecules, there is deviation from this relationship for small molecules. Many authors have attempted to redefine the equations for diffusion, with varying degrees of success, but generally have not attempted to consider the fundamental events that may be occurring at the molecular level during the diffusion of small molecules. In this presentation, we attempt to provide such an explanation, particularly with respect to the diffusion of oxygen through water. We consider the possibility of a random rotational model that complements the (slower) translational process of traditional diffusion and thereby provides accelerated diffusion of small molecules. It is hoped that our description of this model may provide a basis for the development of mathematical modelling of the process.

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the worlds major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)--in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells.

Oxygen as a Regulator of Tissue Perfusion

Tissue perfusion is regulated so that the supply of blood, and in particular the supply of oxygen, can always match the demand of each organ or tissue. Regulation is conducted through the functioning of smooth muscle cells which spiral around the median layers of arteries and arterioles. For cerebral blood vasculature, the bed is innervated by sympathetic (noradrenaline producing) nerves to mediate constriction, and by parasympathetic (acetylcholine producing) nerves to mediate dilation. It is known that cyclic nucleotides also act in the regulation of tissue perfusion, and that cyclic GMP is a mediator of blood vessel relaxation induced by drugs such as nitroglycerine and by agents that function through the stimulation of release of EDRF (now known to be nitric oxide,NO). Thus NO is also a potent vasodilator. Additionally other regulators are involved in the control of perfusion of particular organs or tissues. For example there are a number of peptides present within the nerve fibre network which serves the cerebrovascular bed1 and which are thought to input into cerebral blood flow. All of these regulators are known to be under the direct control of blood pCO2 and blood pH, and so at first sight it might seem unnecessary for oxygen to directly and independently regulate arteriole dilation and contraction. However there are a number of situations where changes in blood pH and pCO2 are not matched by physiologically proportionate changes in blood pO2 (for example during a metabolic acidosis). Additionally, significant changes in pCO2 or pH can be systemic or partly so, suggesting that pH and pCO2 effects can spill from one organ to another. Problems could be expected to arise downstream under such a chain of command, which could have particularly undesirable effects where organs are perfused primarily or significantly by venous blood. It is also known that there are systems in the body which require a different (generally higher) blood pressure for optimum performance, for example the kidneys and lungs. Thus there is a need for input into the regulation of blood perfusion at the level of the individual organ or system.1

Genomic and phenomic correlations in the respiration of basal cell carcinomas

Early last century Warburg1 described differences in metabolism between normal and cancer cells, however subsequent research did not bear out what he considered to be the “primary cause of cancer,” i.e., the replacement of respiration by fermentation 2. Since then attention continues to periodically focus on analysis of the enzymology &/or energetics of oxygen metabolism in cancer cells. Despite such studies, there is still debate as to whether cancers shift towards aerobic metabolism or transform toward a more anaerobic metabolism3. While many theories were advanced to explain those findings, no consistent pattern emerged to correlate the changes observed across all cancers studied. Among many such studies was an investigation we carried out into the enzymology and isoenzymology of human non-melanotic skin cancers (NMSCs). In that research, the levels of three enzymes involved in glucose metabolism, namely lactate dehydrogenase (LDH), aldolase and glucose-6-phospshate dehydrogenase (G-6-PDH), were shown to be depressed in basal cell carcinoma tissue (BCC) compared to normal skin. By contrast, the level of another enzyme, NADP+-dependent isocitrate dehydrogenase was elevated. Those results confirmed the fmdings of Halprin’s group4. In further studies of the same material, the isoenzyme patterns of two of those enzymes were altered in BCC relative to normal skin. The changes seen in the LDH isoenzyme patterns, i.e. increases in the anionic species, were consistent with a shift to a more anaerobic metabolism as were the changes observed in aldolase. To further characterize the oxygen metabolism of BCC, the respiration of small volumes of tissue were directly measured using oxygen electrodes.

Analysis of Sdhd and Mmp12 in an Affected Solar Keratosis and Control Cohort

The incidence of Squamous Cell Carcinoma (SCG) is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 40% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis (SK) lesion and with a small, but significant, chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. More specifically the first aim of this project was to analyse the SDHD and MMP12 genes via Dual-Labelled Probe Real-Time PCR for copy number aberrations in an affected Solar Keratosis and control cohort. It was found that 12 samples had identifiable copy-number aberrations in either the SDHD or MMP12 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The significance of this study is the contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis.

Effects of Temperature on Oxygen Transport in Sheets and Spheres of Respiring Tissues

The effect of temperature upon the oxygen partial pressure profiles (and hence upon flux) of oxygen through respiring tissues of differing architecture is examined. We have considered the two situations of respiring sheets of tissue and of respiring spheres. Sheets of respiring tissue can model to some extent the behaviour of skin (which abandons its own temperature stasis in response to its obligations in the control of overall body temperature). The oxygen profiles of spheres of respiring tissues subject to temperature shifts is investigated since it is a model for solid tumour oxygen kinetics where a spherical tumour, inadequately supplied with a capillary network, is being treated by one or another form of hyperthermia during cancer therapy.

The Flux Of Oxygen Within Tissues

Diffusive flux of oxygen through tissues which are essentially connective and have few cells, display reduced diffusion coefficients when compared to that through an equivalent lamina of water. In general even significant reductions can be explained in terms of the exclusions imposed on small molecular weight diffusates by the large hydrodynamic domains of the connective tissue components. An alternative way of explaining this large exclusion is to point to the very large microscopic viscosities which large interacting polymers impose upon the solvent (water). By contrast, the diffusive flux of oxygen through tissues composed of contiguously packed and actively respiring cells, shows an increased diffusive flux for oxygen when compared to that through an equivalent water lamina. This increase can be explained in terms of the substantial solubility of oxygen within the membrane phase of the cells. This high oxygen partition coefficient into cell lipids has several consequences. Firstly oxygen diffusion will be directed and two dimensional rather than random and three dimensional. Secondly this diffusion will be directed towards the oxygen-consuming sites which are located at lipid surfaces. Thirdly the aqueous oxygen partial pressure will be kept low (since re-supply is constrained while consumption is continuous). This low aqueous environment permits all of the cell soluble redox systems to be maintained efficiently at low metabolic cost, as well as minimising the risk of unscheduled oxidations. Viewed from this perspective, the high value found for oxygen partition coefficient into the erythrocyte membrane suggests that evolution of membrane structure and components may have been driven in part by the selective advantages of high oxygen solubility.

Wyman's Equation and Oxygen Flux through the Red Cell

Wymans equation of 1966 describes the facilitation of flux of a reversibly bound substrate such as oxygen, consequent on the translational diffusion of the binding protein (the carrier). While Wymans equation, or some modification of it such as that by Murray 2, may provide a realistic description of the flux of oxygen through a dilute solution of haemoglobin (see also Wittenburg), it is unlikely to be the complete explanation, nor even the basis, for oxygen transport through the intact red cell. The mature erythrocyte contains approximately 350 g/l haemoglobin, and while this suggests that only 35% of the available water volume is actually occupied by the protein, the remaining 65% is unavailable for protein translational diffusion due to the mutual exclusion of the haemoglobin molecules. For this reason we have examined other possible mechanisms whereby haemoglobin may facilitate the translational diffusion of oxygen within the erythrocyte. Possible alternatives include rotational diffusion by the haemoglobins, intracellular shuffling of haemoglobins due to shape changes by the erythrocyte, and haemoglobin rotations and oxygen exchange consequent on the charge change which accompanies substration and desubstration of the haemoglobin molecule. Finally the dipole interactions are shown to generate significant intermolecular attractions between adjacent haemoglobins.

Pseudogenes and The Electron Transport Chain

With the advent of easy access to the human genome sequence, molecular biology techniques to target respirome-specific genes have begun to be exploited in the study of human disorders and in particular human cancers. In some recent publications it would appear that some investigators have inappropriately targeted pseudogenes rather than functional genes. The high transcription level and generally small size of many of the genes in the respirome make them prone to duplications in the form of processed pseudogenes within the human genome. Such genes can be challenging to analyse using standard molecular genetics approaches. In this presentation, we offer an analysis of pseudogenes that have been identified to have significant homology with some elements of the respirome. Other sequence elements such as Alu repeats, which present similar research obstacles, are also discussed.

Alu sequences in the human respirome.

The concept of the ‘ome’ has dominated some scientific literature in recent years since the introduction of the various ‘genome’ projects began in the late eighties. In a presentation at a recent conference on inborn errors of metabolism (ISIEM 2003), almost fifty such examples were cited. The concept is a useful one in so far as it serves to amalgamate and expedite access to a body of specific information within an area of scientific interest. The application of the concept is perhaps best illustrated by the published outcomes of the human genome projects in both the printed versions and by the easily accessible web-based version. The latter is particularly convenient in terms of ease of revision of information and also in terms of ability to link to related sites. It is the purpose of the current paper to highlight the need to develop and maintain a web-site that encompasses the information of interest to the members of the International Society on Oxygen Transport to Tissues (ISOTT). The structure of the ‘respirome’ and the specific components of this concept is presented in broad terms. As an example of the facility of such a resource, an example is given that encompasses the search for particular DNA sequence motifs (Alu sequences) within the human respirome. Analysis of the results of such searches can often provide insights into fundamental concepts. In this instance the search highlights the relative paucity of Alu repeats and an almost complete lack of inverted Alu repeats in exonic regions of some components of the human respirome elements investigated.