Four ACO promoters and four ACO genomic sequences have been isolated and cloned from Trifolium repens L. The promoter sequences were cloned using Gene WalkerTM technology, and are defined as the 5' flanking sequences upstream of the ATG translation start codon, and designated pTR-ACO1 (1006 bp), pTR-ACO2 (1510 bp), pTR-ACO3 (1350 bp), and pTR-ACO4 (1250 bp). To confirm that each 5' flanking sequences represents distinct genes, Southern analysis was undertaken with each of the 5' flanking sequences used as probes. For TR-ACO1 and TR-ACO2, Southern analysis indicated that the genome of white clover contains two copies of each gene, while single copies of TR-ACO3 and TR-ACO4 are evident. However, the pattern of recognition of pTR-ACO3 differs from pTR-ACO4 confirming TR-ACO4 as a newly identified member of the ACO gene family of white clover. The four genomic sequences isolated cover sequences downstream of the ATG codon to the stop codon, and each comprises 4 exons interspersed by 3 introns. In terms of sequence identity, for exon 1, identities over the four genes ranges from 69% to 94%, with 94% identity between exon 1 of TR-ACO3 and TR-ACO4, while for exon 2, identities range from 60% to 99%, with 99% identity between TR-ACO3 and TR-ACO4. For exon 3, sequence identities ranged from 71% to 89%, with 89% identity between TR-ACO3 and TR-ACO4, while for exon 4, identities range from 62% to 100%, with 100% sequence identity between TR-ACO3 and TR-ACO4. For the intron sequences, significantly lower identities are observed, with again, highest identities were observed for TR-ACO3 and TR-ACO4. For intron 1, identities ranged from 40% to 81% with the highest identity of 81% observed between TR-ACO3 and TR-ACO4. For intron 2, an identity range of 32% to 72% was observed with 72% identity between TR-ACO3 and TR-ACO4, while identity values of 13% to 79%, with 79% between TR-ACO3 and TR-ACO4. Analysis, in silico, of the 5' flanking sequences was undertaken to identify putative transcriptional binding domains using the PLACE and Mat-Inspector programmes. The TR-ACO1 5' flanking sequence contains a higher proportion of domains that are associated with young developing tissues, while the TR-ACO2 5' flanking sequence contains domains that are associated with environmental/hormonal cues. In contrast, the TR-ACO3 and TR-ACO4 5' flanking sequences contain a higher proportion of ethylene-response and wound associated domains. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into both tobacco and white clover. In tobacco, the pTR-ACO1 directed expression in the terminal bud and in axillary buds of younger leaves, with expression declining in the older tissues. The pTR-ACO2 directed expression in the petioles and mature-green and senescent leaves, while the TR-ACO3 and TR-ACO4 promoters directed expression in the axillary buds, petioles and leaves of mature-green tissues and those in the early stages of senescence. In white clover, the TR-ACO1 5' flanking sequence directed highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the pTR-ACO2 directed expression in the axillary buds and leaf petiolules in mature-green tissues. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. However, in association with this ontological pattern, all of the 5' flanking sequences directed expression in most cell types examined during leaf ontogeny. In younger tissues, the TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule. In ontological older tissue, the TR-ACO3 and TR-ACO4 5' flanking sequences directed expression in the ground meristem of the axillary buds, the vascular tissue of the stolon and petiolule. However, staining could be observed in the pith and cortex of the stolon, and the cortex of the leaf petiolule, but at a reduced intensity. These expression studies suggest that in leaf development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.

Floral stripes are a common pigmentation pattern in plants. Defining the molecular mechanisms of the striped pattern formation will aid understanding of how a gene can be differentially regulated across a population of similar cells. In the venation phenotype of Antirrhinum majus, the anthocyanin pigment is typically confined to the adaxial epidermal cells overlaying the petal veins. To explore how this pattern forms this study focused on the expression and regulation of Venosa, a Myb regulator of anthocyanin biosynthesis. Pigment complementation experiments demonstrated that the lack of a MYB factor caused the lack of pigment in the cells outside the venation pigmentation domain. An allele of Venosa was isolated and identified. It was a mutant version of functional Venosa due to the central part being replaced by a transposon. Phenotype / genotype analysis indicated that the venation pigmentation patterning was due to the functional Venosa. In situ mRNA hybridisation showed that Venosa was expressed from the xylem to the adaxial epidermis, and was controlled spatially and quantitatively by a signal associated with the petal veins. Venosa expression provided the longitudinal axis for venation pigmentation stripes, and determined the location and intensity of the pigmented cells. Because another factor required for pigmentation, a bHLH factor, is specifically expressed in epidermal cells and it provides the transverse axis. The pigmented stripes are the cross expression domain of these two kinds of factors. The transcriptional controlling property of a 2.4 kb (relative to the ATG) promoter region of the Venosa gene was analysed. The -900 bp fragment was characterised in detail using 5'-end deletion mutagenesis. A heterologous host, tobacco, was used for analysis in stable transgenics. The homologous host, Antirrhinum, was used for transient assays. The efficacy and efficiency of different reporter genes (intron-containing GUS, GFP, Venosa cDNA and genomic Venosa) and enhancement systems (transcriptional enhancer, translational enhancer, inhibitor of post transcriptional gene silencing and a two-step signaling amplification system) for the detection of low-level reporter gene expression were also tested. The strength of expression correlated to the length of the promoter fragment, and expression was detected using deletions down to -500 bp, although only weak expression was found. This expression was flower specific but not vein related in both plant hosts. No expression was detected in petals of either host with fragments shorter than -500 bp. The results suggest that the fragment from -380 bp to -900 bp positively affected Venosa expression at the transcriptional level, but might not be sufficient to define venation. A possibility is that the venation controlling property is negatively controlled at the epigenetic level, such as DNA methylation status and / or chromatin structure. The role of gibberellin and sugar in the pigment and venation patterning formation of Antirrhinum was studied. The results suggest that gibberellin is not required for pigmentation or venation patterning. Convincing evidence on the role of sugar signaling could not be obtained from the experiments, due to the difficulty in separating the impact on pigmentation from other functions of sugars in petal development. In addition, the in situ analysis detected the expression of a gene probably related to aurone biosynthesis that may be a regulatory gene of this biosynthetic pathway.

Clianthus and New Zealand Sophora species are woody legumes endemic to New Zealand, with high ornamental value and biodiversity significance. Research was conducted to address the fact that little is known about the details of their developmental characteristics, genetic structure and relatedness of the wild populations, and their molecular mechanism of flowering. Genetic diversity and relatedness of all remaining wild populations of Clianthus and samples of all New Zealand Sophora species were investigated using ISSR and AFLP markers. Genetic relationships were established for Sophora species, Clianthus wild populations and cultivars, and most individuals in each of the wild Clianthus populations. The molecular evidence did not support the recent separation on morphological grounds of the two Clianthus species, C. maximus and C. puniceus. Postharvest treatments were tested to extend vase life of the short-lived cut Clianthus maximus and Sophora tetraptera flowers. Appropriately treated Clianthus cut flowers lasted 10-12 days in the vase, with over 80% of flowers opening. Similar postharvest treatments did not improve the vase performance of cut Sophora flowers. Detailed calendars of vegetative and reproductive growth, and of floral ontogeny were developed for Clianthus and Sophora. Contrasting behaviours of both vegetative and reproductive growth were observed between these two legumes. A long period of summer-autumn dormancy of vegetative and reproductive growth in Sophora, and mass abortion of initiated Clianthus inflorescences during most of the year were observed. Unusual floral ontogeny processes, with precocious carpel initiation and delayed petal development, were observed in both species. An efficient two-step quantitative real-time RT-PCR protocol for detailed gene expression analysis of large numbers of samples was developed using SYBR Green DNA dye and a LightCycler instrument. The consistency of this protocol was optimised with regards to sample and template preparation, primer design, and determination of appropriate internal controls for gene expression quantification. Differences of gene expression in the range of 5-7 orders were effectively detected. Putative partial homologues of LEAFY, APETALAI, PISTILLATA, and AGAMOUS were isolated from both Clianthus and Sophora. Detailed temporal and spatial expression of each floral identity gene was investigated using quantitative real-time RT-PCR. The expression patterns, together with the sequence similarity, showed that these new isolated gene fragments were most probably LEAFY, APETALAI, PISTILLATA, and AGAMOUS homologues in Clianthus and Sophora, and that the ABC model of floral development is generally applicable to both species. However, there were important variations in temporal expression patterns compared to those of herbaceous species. A bimodal expression pattern of LEAFY and APETALAI homologues was observed in Sophora, but not in Clianthus, coincident with their contrasting patterns of floral initiation and development.

Male Drosophila melanogaster double the transcription of most of the genes on their single X chromosome, to equal that from the two female X chromosomes, in a process termed dosage compensation. This process is mediated by the MSL complex, consisting of both protein and non-coding RNA components. This complex is only active in males due to the presence of MSL2, which is not translated in females. The X-linked Lsp1α gene of Drosophila melanogaster appears to escape dosage compensation, and exhibits two-fold higher levels of expression in females compared to males. The apparent lack of dosage compensation of Lsp1α could be due to the promoter being more active in females than in males, or to a lack of regulation by the MSL complex. In this study, the mechanism by which this happens has been addressed. Lsp1α is expressed exclusively in the fat body tissue of third instar larvae, and forms part of a multi-protein complex that acts as a nutrient reservoir during pupariation. In this study it has been shown that transgenes, in which the reporter gene, lacZ, is under the control of the Lsp1α promoter, exhibit variable levels of increased activity in female compared to male third instar larvae. At high levels of transgene expression, activity of the transgene is equal in female and male larvae. When the expression of the transgene is low, the activity of the transgene is much higher in female compared to male larvae. This increased sensitivity of the Lsp1α promoter to position effects in females appears to be mediated by one or more components of the MSL complex. Females ectopically expressing MSL2 exhibit decreased levels of transgene activity. Furthermore, overexpression of MSL1 causes an increase in the activity of transgenes subject to strong position effects. Despite these findings, the sex-specific regulation of the Lsp1α promoter does not account for the non-dosage compensated appearance of Lsp1α. Instead, unlike control dosage compensated X-linked genes, Lsp1α is not enriched for a histone modification, acetylation of lysine 16 of histone H4 that is essential for dosage compensation by the MSL complex. The developmental stage at which the four genes flanking Lsp1α are expressed has been determined using northern RNA hybridization. Expression of the gene immediately 3' of Lsp1α could not be detected at any developmental stage using northern RNA hybridization or in adults by RT-PCR. However, the two genes flanking Lsp1α are expressed in equal levels in male and female Drosophila as determined by quantitative RNase protection analysis. Furthermore, the regions between Lsp1α and these flanking dosage compensated genes do not prevent dosage compensation of an X-linked armlacZ reporter gene. Bioinformatic analysis shows that Lsp1α is present in three species closely related to D. melanogaster but is absent in more distantly related species. It is probable that because of its recent evolutionary origin, the Lsp1α gene lacks the DNA sequences that are required to attract the MSL complex. More generally, a model is proposed in which dosage compensation involves binding of the MSL complex to DNA sequences in actively transcribed regions with possible limited spreading to closely associated active genes.

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.

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.

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.

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.

No description available.

Onion (Allium cepa L.) is an example of a species that accumulates very high levels of reduced sulfur (S)-containing compounds, particularly in the bulb as alk(en)yl-L-cysteine-sulfoxides (ACSOs) and it is these compounds, or their derivatives, that confers the distinct odour and pungent flavour. In common with higher plants, the S assimilation pathway in onion begins with the activation of uptaken sulfate (SO4 2-) to 5'-adenylylsulfate (APS), a reaction catalysed by ATP sulfurylase (ATPS; EC 2.7.7.4). Then, APS is reduced to sulfide (S2-) in a two-step process catalysed by the enzymes APS reductase (APSR; EC 1.8.4.9) and sulfite reductase (SiR; EC 1.8.7.1). To complete the reductive assimilation pathway, S2- is incorporated into the amino acid skeleton of O-acetylserine (OAS) to form cysteine, and this reaction is catalyzed by OAS (thiol)-lyase (OAS-TL; EC 4.2.99.8). While the regulation of the pathway is quite well defined in the plant model Arabidopsis, much less is known about its regulation in S accumulating species such as onion. The primary aim of this thesis, therefore, was to characterise the enzymes of the S assimilation pathway in onion, with a particular emphasis on ATPS. As part of this charaterisation two genotypes of onion were compared. These comprised a mild genotype, 'Texas Grano 438' (TG) with a lower level of S-containing compounds in the bulb tissues, and 'W202A' (W), a cultivar with a higher level of S containing compounds in the bulb tissues. As well, comparisons were made between seedlings (typically harvested at 7 weeks) and plants at a designated mature stage (at bulbing; typically after 4 months growth), and for plants grown in S-sufficient (S+) media or S-deficicnt (S-) media, as appropriate. In terms of plant growth, S-deprivation generally had a negative influence for both genotypes, with significant reductions in total biomass (measured as fresh weight) for TG at both the seedlings and mature stages. ATPS activity and accumulation were shown to be present in all tissues examined (leaf, root, bulb) as well as the chloroplasts, with highest activity measured in the roots, particularly in seedlings. ATPS activity and accumulation were also compared between the two genotypes (TG and W) with ATPS activity and accumulation higher in W, particularly at the seedling stage. In terms of the influence of S supply, in general higher ATPS activity was measured in chloroplast, leaf and root extracts from plants of both genotypes grown in the S- media, at the seedling stage. In roots of mature plants of both genotypes, a significant increase in activity was measured in response to S-deprivation, while in chloroplasts isolated from mature plants of both genotypes, highest activity was measure in those grown in the S+ media. Finally diurnal variations were observed in chloroplast, leaf and root extracts of both genotypes with a general trend of an increase in ATPS activity and accumulation a few hours after illumination and upon the onset of the dark period. Although a single gene coding for ATPS is presumed to be present in onion, the enzyme was characterized as two electrophoretic forms using 1D-PAGE during western analyses following fractionation of chloroplasts by anion exchange chromatography and also as an alignment of spots using 2D-PAGE. As protease inhibitors were routinely included in the extraction buffers, these forms suggest the occurrence of ATPS isoforms that may arise as a consequence of post-translational modifications. The regulation of ATPS by one mechanism of post-translational modification, phosphorylation, was therefore investigated using several techniques including the detection of a shift in molecular mass, a change in enzyme activity or pI (as determined by 2D-PAGE) and the capability to bind to 14-3-3 proteins using affinity chromatography. Following treatments of chloroplast extracts to promote either the phosphorylation (P+) or the dephosphorylation (P-) of proteins, no molecular mass change or change in activity was observed. However, after fractionation by 2D-PAGE, differences in the spot alignment of ATPS were visualized, suggesting that ATPS is a phosphoprotein. The enzyme was detected in pull-downs after affinity chromatography, suggesting that ATPS may also interact with 14-3-3 proteins (although this needs to be confirmed unequivocally). A model is advanced, therefore, in which upon phosphorylation, no variation in ATPS activity occurs but a change in the surface charged and possibly a change in conformation of the protein does occur to make the enzyme competent to interact with 14-3-3 proteins.