The acanthodians were a common and widespread group of fishes throughout the world during the mid-Palaeozoic. In this study, a full-scale systematic analysis of Silurian to Middle Devonian acanthodian taxa of eastern Australia was undertaken, incorporating a review and updating of historical records and descriptions of taxa. Phylogenetic relationships within the group and with other early gnathostomes are uncertain. The structure, function and modes of growth of acanthodian scales were described and investigated, and these features were used for comparisons between different taxa within the group, and between acanthodians and other early vertebrates. Histological and morphological characters of the scales were incorporated in a cladistic analysis of genera erected for articulated fish. This analysis did not support the traditional ordinal level groups, the Climatiida, Ischnacanthida and Acanthodida. Therefore, the highest taxonomic level used in the study was the family. Rarely were acanthodians preserved as articulated fossils. The only examples known from the Silurian-Middle Devonian of Australia are one specimen of the putative acanthodian Yealepis douglasi Burrow & Young 1999 from the Ludlow of Victoria, five specimens of an indeterminate ?ischnacanthidid from the late Givetian of New South Wales and a rich assemblage from the Givetian lacustrine shales of Mt Howitt, Victoria. The latter fauna (originally dated as Late Devonian) includes six incomplete specimens of the culmacanthidid Culmacanthus stewarti Long 1983 and about 60 specimens of acanthodidid Howittacanthus kentoni Long 1986. In contrast, disassociated remains of the group are ubiquitous in microvertebrate faunas from the mid-Palaeozoic of eastern Australia. Although scales of other fish groups are sometimes more common in particular facies, acanthodian elements are found in all depositional environments, from deep shelf marine to transitional to freshwater. Most of the taxa, particularly those only preserved as isolated scales, had not been systematically described by other workers. This thesis incorporates descriptions of new taxa, and revision and updating of other taxa. Several overseas studies have produced biostratigraphic charts based on acanthodian scales. A similar biostratigraphic overview was undertaken based on a systematic analysis of the Early Devonian acanthodians of eastern Australia, permitting comparisons with acanthodian faunas of other regions. Acanthodian scales and fin spines are the most common elements in the few vertebrate faunas that are known from the Silurian of Australia. Diversity and geographic distribution of the acanthodian faunas peaked during the Early Devonian. This study has been hampered by the low numbers of scales in many samples, and by uncertainty over their dating (particularly for the faunas from non-limestone deposits). Nevertheless, the work shows that changes in the marine assemblages are broadly correlated with the pattern of marine transgressions and regressions. Composition of the acanthodian faunas, and their abundance relative to other vertebrates in the assemblages, depend on the depositional environment. This correlation is best illustrated in Lower Devonian deposits, in which acanthodians are the most prolific and diverse. In transitional and marginal marine deposits, thelodonts are dominant, and acanthodians a minor element of the fauna. In off-shore assemblages, acanthodians and placoderms are dominant, and thelodonts are rare or absent. Vertebrate faunas are poor in the earliest Devonian deposits, but become more common by the late Lochkovian, with near-shore assemblages characterized by Trundlelepis cervicostulata and ANostolepis@ guangxiensis, and deeper shelf assemblages by a new genus, possibly assignable to the Ischnacanthidae. The vertebrate record is sparse through the middle Pragian, though AN.@ guangxiensis is present low in the Coopers Creek Limestone (upper sulcatus-pireneae zones), being replaced by Nostolepoides platymarginata, Gomphonchus? bogongensis, and Radioporacanthodes sp. aff. R. (Gomphonchus) liujingensis by the kindlei Zone. Microvertebrate assemblages are more common by the late Pragian (pireneae Zone), with Radioporacanthodes sp. aff. R. liujingensis in deeper shelf deposits, and N. platymarginata and G.? bogongensis dominating near-shore assemblages. The earliest Emsian (dehiscens Zone) is marked by the incoming Cheiracanthoides wangi. Middle Emsian (perbonus-serotinus zones) assemblages are characterized by two new species, possibly assignable to Gomphonchus.The Middle Devonian cosmopolitan association of Cheiracanthoides comptus and AAcanthoides@ dublinensis, which characterizes early Middle Devonian faunas from North America, Europe and China, appears first in the latest Emsian at the serotinus-patulus zone boundary. As well as showing the value of acanthodians in biostratigraphy and as indicators of environmental settings, their use in biogeography was demonstrated. Although many of the acanthodian taxa are endemic, several are also found in other regions. The Silurian to earliest Devonian faunas of eastern Australia are most closely related to coeval Chinese assemblages. Several latest Silurian-earliest Devonian taxa are also recorded from the circum-Arctic region. The late Lochkovian to early Emsian assemblages, particularly from south-eastern Australia, have many taxa in common with Chinese faunas. The mid-Emsian taxa show highest endemicity; and the latest Emsian-Eifelian assemblages have the most cosmopolitan aspect. Acanthodian faunas become rarer and depauperate in the Middle Devonian, particularly in the south-eastern corner, and are mostly in poorly dated, ?fluviatile/marginal marine deposits. This study of acanthodian faunas has encompassed a full scale systematic review of the group in this region, an appraisal of phylogenetic relationships within the group and with other early vertebrates, their palaeoecology, and their use in biostratigraphy and biogeography.

Alcohol and tobacco are drugs of abuse which are legal to sell and consume in most western societies. Addiction to these two substances has major social and health implications worldwide. The brain structure known to mediate addictive behaviour is the dopaminergic mesocorticolimbic system. Dopaminegic neurons arise from the ventral tegmental area, project to the nucleus accumbens and interact with the amygdala and the prefrontal cortex. Chronic alcoholism elicits marked damage in the prefrontal cortex with significant loss of neurons and glia. The key components of addiction, tolerance and dependence, are thought to be the result of semipermanent adaptive changes in gene expression. Gene expression profiling of the mesocorticolimbic system from human alcoholics and alcohol-dependent animals has revealed highly region-specific alterations. How these molecular changes result in the development of alcohol dependence in humans is not fully understood. Complicating factors in human alcoholism include a high comorbidity with smoking, socioeconomic factors and the prevalence of underlying psychological pathologies. Gene expression profiling of the prefrontal cortex of six alcoholics and six controls resulted in the identification of functional gene groups sensitive to alcoholism. Mitochondrial function was found down regulated while mRNA levels of genes involved in stress response and cell protection were elevated. These results correlate with the pathology of the prefrontal cortex in chronic alcoholism. Some of the control cases used for gene expression profiling were later identified as chronic smokers, while all of the alcoholics were heavy smokers. To date the heavy co-morbidity of alcoholism with smoking has not been taken into account. Thus the expression of selected genes were investigated by realtime PCR in an extended case set of non-smoking alcoholics, smoking alcoholics, smoking non-alcoholics and non-smoking, non-alcoholics. This study revealed that alcoholism itself had a significant impact on the expression of midkine, the high affinity glial glutamate transporter, member 1 and the tissue inhibitor of the metalloproteinase 3. Heavy smoking itself led to a small but significant elevation of MDK mRNA levels as well as an increase in variation of excitatory amino acid transporter 1 and metalloproteinase inhibitor, member 3 expression. Apolipoprotein D however was induced by chronic smoking but not by alcohol dependence. These results highlight the need of careful case selection in future studies on gene expression in the human alcoholic brain. Peptide antibodies were produced to midkine and a polyclonal antibody against the excitatory amino acid transporter 1 was obtained from a collaborating laboratory. Western blots utilizing these antibodies revealed a marked increase in midkine and excitatory amino acid transporter 1 protein in alcoholics compared to non-smoking and non-drinking controls. In coronal sections of human prefrontal cortex of alcoholics and non-smoking non-drinking controls, immunofluorescence of midkine was obtained from nuclei throughout the layers of the cortex and from the cell bodies of a distinct set of astrocytes in cortical layer II. Double staining with glial fibrillary acidic protein revealed that a portion of midkine-positive nuclei were localised in glial cells. There was no difference in immunostaining of alcohol and control sections with midkine. In summary these results indicate that midkine protein is induced in the prefrontal cortex of the chronic alcoholic. However, this increase in protein may not be strong enough to be visualised by immunohistochemistry. Midkine induction may be reflective of reparative processes in the prefrontal cortex of the chronic alcoholic. Excitatory amino acid transporter 1 staining in non-alcoholic, non-smoking control cases were obtained as a confluent band in cortical layer II and sparsely in deeper cortical layers. Excitatory amino acid transporter 1 immunoreactivity overlapped partially with glial fibrillary acidic protein labelling. In chronic alcoholics, excitatory amino acid transporter 1 staining in the area between the cortical layer II and VI was significantly increased. At withdrawal, glutamate levels may reach toxic levels in the cortex. The increase in cells expressing excitatory amino acid transporter 1 throughout the cortical layers may indicate a protective measure of this brain region in the chronic alcoholic. Additionally, layer specific expression of midkine and excitatory amino acid transporter 1 in the prefrontal cortex of the healthy individual may implicate a specialised role of these astrocytes.

The acanthodians were a common and widespread group of fishes throughout the world during the mid-Palaeozoic. In this study, a full-scale systematic analysis of Silurian to Middle Devonian acanthodian taxa of eastern Australia was undertaken, incorporating a review and updating of historical records and descriptions of taxa. Phylogenetic relationships within the group and with other early gnathostomes are uncertain. The structure, function and modes of growth of acanthodian scales were described and investigated, and these features were used for comparisons between different taxa within the group, and between acanthodians and other early vertebrates. Histological and morphological characters of the scales were incorporated in a cladistic analysis of genera erected for articulated fish. This analysis did not support the traditional ordinal level groups, the Climatiida, Ischnacanthida and Acanthodida. Therefore, the highest taxonomic level used in the study was the family. Rarely were acanthodians preserved as articulated fossils. The only examples known from the Silurian-Middle Devonian of Australia are one specimen of the putative acanthodian Yealepis douglasi Burrow & Young 1999 from the Ludlow of Victoria, five specimens of an indeterminate ?ischnacanthidid from the late Givetian of New South Wales and a rich assemblage from the Givetian lacustrine shales of Mt Howitt, Victoria. The latter fauna (originally dated as Late Devonian) includes six incomplete specimens of the culmacanthidid Culmacanthus stewarti Long 1983 and about 60 specimens of acanthodidid Howittacanthus kentoni Long 1986. In contrast, disassociated remains of the group are ubiquitous in microvertebrate faunas from the mid-Palaeozoic of eastern Australia. Although scales of other fish groups are sometimes more common in particular facies, acanthodian elements are found in all depositional environments, from deep shelf marine to transitional to freshwater. Most of the taxa, particularly those only preserved as isolated scales, had not been systematically described by other workers. This thesis incorporates descriptions of new taxa, and revision and updating of other taxa. Several overseas studies have produced biostratigraphic charts based on acanthodian scales. A similar biostratigraphic overview was undertaken based on a systematic analysis of the Early Devonian acanthodians of eastern Australia, permitting comparisons with acanthodian faunas of other regions. Acanthodian scales and fin spines are the most common elements in the few vertebrate faunas that are known from the Silurian of Australia. Diversity and geographic distribution of the acanthodian faunas peaked during the Early Devonian. This study has been hampered by the low numbers of scales in many samples, and by uncertainty over their dating (particularly for the faunas from non-limestone deposits). Nevertheless, the work shows that changes in the marine assemblages are broadly correlated with the pattern of marine transgressions and regressions. Composition of the acanthodian faunas, and their abundance relative to other vertebrates in the assemblages, depend on the depositional environment. This correlation is best illustrated in Lower Devonian deposits, in which acanthodians are the most prolific and diverse. In transitional and marginal marine deposits, thelodonts are dominant, and acanthodians a minor element of the fauna. In off-shore assemblages, acanthodians and placoderms are dominant, and thelodonts are rare or absent. Vertebrate faunas are poor in the earliest Devonian deposits, but become more common by the late Lochkovian, with near-shore assemblages characterized by Trundlelepis cervicostulata and ANostolepis@ guangxiensis, and deeper shelf assemblages by a new genus, possibly assignable to the Ischnacanthidae. The vertebrate record is sparse through the middle Pragian, though AN.@ guangxiensis is present low in the Coopers Creek Limestone (upper sulcatus-pireneae zones), being replaced by Nostolepoides platymarginata, Gomphonchus? bogongensis, and Radioporacanthodes sp. aff. R. (Gomphonchus) liujingensis by the kindlei Zone. Microvertebrate assemblages are more common by the late Pragian (pireneae Zone), with Radioporacanthodes sp. aff. R. liujingensis in deeper shelf deposits, and N. platymarginata and G.? bogongensis dominating near-shore assemblages. The earliest Emsian (dehiscens Zone) is marked by the incoming Cheiracanthoides wangi. Middle Emsian (perbonus-serotinus zones) assemblages are characterized by two new species, possibly assignable to Gomphonchus.The Middle Devonian cosmopolitan association of Cheiracanthoides comptus and AAcanthoides@ dublinensis, which characterizes early Middle Devonian faunas from North America, Europe and China, appears first in the latest Emsian at the serotinus-patulus zone boundary. As well as showing the value of acanthodians in biostratigraphy and as indicators of environmental settings, their use in biogeography was demonstrated. Although many of the acanthodian taxa are endemic, several are also found in other regions. The Silurian to earliest Devonian faunas of eastern Australia are most closely related to coeval Chinese assemblages. Several latest Silurian-earliest Devonian taxa are also recorded from the circum-Arctic region. The late Lochkovian to early Emsian assemblages, particularly from south-eastern Australia, have many taxa in common with Chinese faunas. The mid-Emsian taxa show highest endemicity; and the latest Emsian-Eifelian assemblages have the most cosmopolitan aspect. Acanthodian faunas become rarer and depauperate in the Middle Devonian, particularly in the south-eastern corner, and are mostly in poorly dated, ?fluviatile/marginal marine deposits. This study of acanthodian faunas has encompassed a full scale systematic review of the group in this region, an appraisal of phylogenetic relationships within the group and with other early vertebrates, their palaeoecology, and their use in biostratigraphy and biogeography.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.

Vascular casts of three chondrichthian, one dipnoan, one chondrostean and 14 teleostean species were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of interarterial anastomoses were lacking in the dipnoan (Sarcopterygii) and chondrichthyan species examined, suggesting that a SVS is restricted to actinopterygian fishes. The presence and distribution of a secondary vascular system does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species. Histological sections of primary segmental arteries and associated interarterial anastomoses and secondary vessels from the long-finned eel, Anguilla reinhardtii, were examined by light and transmission electron microscopy. Secondary vessels were found to originate from the primary vasculature as depressions through the tunica intima and media, from where they ran perpendicularly to the adventitial layer, before coiling extensively. From here the anastomoses travelled a relatively linear path in the outer margin of the adventitia to re-anastomose with a secondary vessel running in parallel with the primary counterpart. Secondary vessels had a structure quite similar to that of primary vessels; they were lined by endothelial cells on a continuous basement membrane, surrounded by single layer of smooth muscle cells surrounding the vessel. Smooth muscle cells were also found in the vicinity of interarterial anastomoses in the adventitia, but these were more longitudinally orientated. The presence of smooth muscle cells on all aspects of the secondary circulation suggests that this vascular system is regulated in a similar manner as the primary vascular system. Because interarterial anastomoses are structurally integrated with the primary vessel from which they originate, it was anticipated that flow through secondary vessels would to some extent be affected by an increase in primary vascular tone. Immunohistochemical studies showed that primary segmental arteries displayed moderate immunoreactivity to antibodies against 5-hydroxytryptamine and substance P, while interarterial anastomoses and secondary vessels showed dense immunoreactivity. Secondary vessels were followed to the surface of the animal through consecutive sections, where they eventually give rise to capillary beds overlying the scales. Secondary capillary beds were found to supply chloride cells in the skin, suggesting that this vascular system may be involved in cutaneous ionic exchange. Branchial vascular casts from two species of Tetraodontiformes showed that the vessels previously reported as nutrient vessels are with certainty part of the secondary vascular system. In the three-barred porcupine fish, Dicotylichthys punctulatus, interarterial anastomoses originated at high densities from efferent filamental and the efferent branchial arteries, from where they formed progressively larger secondary vessels. Small branches of this vascular system entered the filament body, where it gave rise to numerous side-vessels along the way. Large secondary vessels running in parallel with the efferent branchial arteries were found to constitute an additional arterio-arterial pathway, in that they exited the branchial basket in company with the afferent mandibular artery, the carotid artery and the efferent branchial arteries, from where they gave rise to vascular beds immediately after exit. The secondary vessels in this species were not found to supply the filament musculature; rather this vascular system was supplied by a single vessel derived from the efferent branchial artery, running in parallel with the afferent branchial artery. Secondary vessels were not found on any branchial component in the banded toadfish, Marylina pleurosticta, but in all other aspects the branchial vascular anatomy was similar to that of D. punctulatus. It is proposed that four independent vascular pathways may be present in the teleostean gill. The blood volume and flow rates of the primary (PVS) and secondary vascular system (SVS) were examined in the catadromous euryhaline teleost Lates calcarifer in order to determine whether any of these parameters were subject to change in individuals acclimated to seawater, compared to a group acclimated to freshwater. There was no significant difference in any measured parameter for the two groups. The volumes of the SVS were 0.67 „b 0.13 and 0.76 „b 0.13 mL 100g-1 body mass for FW and SW acclimated animals respectively. This constituted approximately one-third of the total blood volume in both groups. Turnover times for the SVS ranged from 21.0 to 25.2 minutes, demonstrating in accordance with previous publications, that this system is considerably more dynamic than previously assumed.