Disruption of any of these mechanisms may have implications for the function and health of peripheral neurones during the aging process. This review focuses on the impact of advancing age on the overall function of peripheral adrenergic neurones and how these changes in function may be linked to age-related changes in modulation of [Ca2+]i regulation. The data in this review suggest that normal aging in peripheral autonomic neurones is a subtle process and does not always result in dramatic deterioration in their function. We present studies that support the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age-related decline in the function of at least one of the neuronal calcium-buffering systems, smooth endoplasmic reticulum calcium ATPases, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a ‘weak point’ in cellular compensation as this over time may contribute to cell death. In addition, we present more recent studies on [Ca2+]i regulation in the form of the modulation of release of calcium from smooth endoplasmic reticulum calcium stores. These studies suggest that the contribution of the release of calcium from smooth endoplasmic reticulum calcium stores is altered with age through a combination of altered ryanodine receptor levels and modulation of these receptors by neuronal nitric oxide containing neurones.Calcium ions represent universal second messengers within neuronal cells integrating multiple cellular functions, such as release of neurotransmitters, gene expression, proliferation, excitability, and regulation of cell death or apoptotic pathways. The magnitude, duration and shape of stimulation-evoked intracellular calcium ([Ca Calcium is widely recognized as a universal second messenger within neuronal cells and integrates multiple cellular functions. These include release of neurotransmitters, gene expression, proliferation, excitability and regulation of cell death or apoptotic pathways (2+]i) concentration gradient between the extracellular milieu and cytosol. In peripheral neurones, calcium signaling begins with the opening of L and N and some R type calcium channels, allowing calcium to flow from outside of the cell into the cytosol , mitochondrial calcium uptake, plasmalemma calcium ATPases (PMCA) and the sodium–calcium exchanger (Na+/Ca2+), work together to reduce [Ca2+]i from its peak levels, and restore [Ca2+]i back to resting levels  has been suggested to be a peripheral neuroendocrine center because of the plethora of tissues that receive adrenergic input from this source . Thus, tIn the rat cerebrovasculature, adrenergic and sensory innervation is fully developed within the first 30 days of life .In addition to the cardiovascular regulatory effects of the SCG, it also plays a role in protecting the cerebrovasculature from stroke. Hemorrhagic stroke accounts for about 10% of cerebrovascular disease with a peak incidence near the age of 60 years and high probability of morbidity or mortality. Risk factors for stroke include age-related hypertension and changes in the structure of cerebral blood vessels . Given tAging in all creatures is inexorable and has been suggested to be a combination of developmental changes, genetic defects, environmental influences, and an inborn genetic aging process . These s2+]i have been hypothesized to contribute to age-related neuronal dysfunction and degeneration ethane-N,N,N′,N′-tetraacetic acid (BAPTA) decreased stimulation-evoked NA release to a greater extent in old arteries as compared to young, suggesting that age-related changes in NA release is in part due to altered calcium-buffering capacity. Overall, the age-related changes in the function of peripheral adrenergic neurones in terms of NA release appears to be at least due in part to altered [Ca2+]i handling mechanisms. However, altered sensitivity of the NA release mechanisms has not been ruled out.We tested the hypothesis that an age-related alteration in [Ca2+]i in living cells  blocks PMCA function but does not significantly affect SERCA function. Under these conditions, the rate of recovery was diminished to a greater extent in SCG neurones from old as compared to young animals   . Specifi2+]i signaling in SCG neurones, we used molecular techniques of reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay to test the hypothesis that the genetic and protein expression of the predominant RyR isoform(s) in adult rat SCG, along with selective modulators, are altered during late maturation and advancing age in F-344 rats aged 6, 12, and 24 months (As RYRs are integral components in [Ca2+]i (reviewed in Surprisingly, we have found that RyR1 mRNA was undetectable in the rat SCG, in contrast with other studies demonstrating RyR1 mRNA is expressed in excitable cells, including neurones . Thus, iIn addition to measuring the impact of age on RyR gene expression and protein levels, we extended our studies to include possible age-related changes in selected modulators of the RYRs, which include phosphorylation and nNOS levels within the SCG (2+]i regulation, we present the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age-related decline in the function of at least one calcium-buffering system, SERCA, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a ‘weak point’ in cellular compensation as this over time may contribute to cell death (The data suggest that normal aging in peripheral autonomic neurones is a subtle and complex process and does not always result in dramatic deterioration. Moreover, the aging process does not necessarily alter the function of excitable neurones in a uniform manner. In terms of age-related changes in [Ca2+]i signaling and function of peripheral adrenergic neurones represents a complex interplay of mechanisms, including increased sensitivity of the neurotransmitter release mechanism to calcium, a decline in SERCA function that alters calcium buffering and refilling of SER calcium stores, reduced RyR3, and nNOS levels, which in turn modulate cADPr levels and the CICR process. The consequences of these changes are currently being studied in our laboratory by direct measurement of cADPr during aging and the contribution of release of calcium from SER stores to stimulation-evoked increases in [Ca2+]i. Given the advances in molecular techniques future studies may include a comprehensive study on the impact of age on the genetic expression and protein levels of multiple buffering systems, including soluble calcium-buffering proteins, SERCA, mitochondrial calcium pumps, PMCA, and the Na+/Ca2+ exchanger. In addition, another mechanism that modulates calcium influx and SER refilling are store operated calcium channels. Their genetic expression and function with advancing age has not been studied and may provide a fruitful avenue of future research. Finally, SERCA function declines with age thus possibly altering the levels of SER calcium stores and CICR. With calcium indicators available to study SER calcium levels, it is possible to examine how advancing age may impact the calcium levels of the SER. This type of study may provide additional insight on how CICR may be altered with advancing age.This review summarizes our work on the dynamics of intracellular calcium regulation and possible consequences for autonomic nerve function with advancing age. The major findings of all of our studies are summarized in


Drosophila embryos. Many aspects of the signalling pathway are conserved between these lineages, however vertebrates have diverged in at least one key aspect: they have evolved multiple Gli genes encoding functionally-distinct proteins, increasing the complexity of the hedgehog-dependent transcriptional response. Amphioxus is one of the closest living relatives of the vertebrates, having split from the vertebrate lineage prior to the widespread gene duplication prominent in early vertebrate evolution.Hedgehog signalling, interpreted in receiving cells by Gli transcription factors, plays a central role in the development of vertebrate and Gli gene, which is deployed in tissues adjacent to sources of hedgehog signalling derived from the midline and anterior endoderm. This shows the duplication and divergence of the Gli gene family, and hence the origin of vertebrate Gli functional diversity, was specific to the vertebrate lineage. However we also show that the single amphioxus Gli gene produces two distinct transcripts encoding different proteins. We utilise three tests of Gli function to examine the transcription regulatory capacities of these different proteins, demonstrating one has activating activity similar to Gli2, while the other acts as a weak repressor, similar to Gli3.We show that amphioxus has a single These data show that vertebrates and amphioxus have evolved functionally-similar repertoires of Gli proteins using parallel molecular routes; vertebrates via gene duplication and divergence, and amphioxus via alternate splicing of a single gene. Our results demonstrate that similar functional complexity of intercellular signalling can be achieved via different evolutionary pathways. A key challenge faced by embryos with large cell numbers is to regulate the patterning of cell fields. While short-range intercellular signals can play a role in such processes, morphogen gradients provide a conceptually attractive alternative and key examples include nodal signalling in early vertebrate gastrulation and hedgehog signalling in vertebrate limb and neural tube development Drosophila and vertebrates, and significant similarities in genes and mechanisms are observed in these two lineages. There are important differences too, for example the roles of Drosophila Costal2 (Cos2) and Suppressor of Fused Su(fu) appear to differ from those of their mammalian orthologs Drosophila also differ in the number of genes encoding many pathway components. For example, a single hedgehog gene (hh) has been described from Drosophila, while vertebrates have multiple hedgehog genes falling into three distinct groups, related to Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Dessert hedgehog (Dhh). Studies on the protochordates Branchiostoma floridae (amphioxus) and Ciona intestinalis have shown these extra vertebrate hedgehog genes evolved via gene duplications specific to the vertebrate lineage The hedgehog signalling pathway has been extensively studied in Gli genes in vertebrates and Cubitus interruptus (Ci) in Drosophila (hereafter referred to collectively as the Gli gene family). Evidence from Drosophila suggests all hedgehog signalling is transduced via Ci protein Drosophila cytoplasmic Ci protein is cleaved in the absence of hedgehog signalling to yield an N-terminal form with potent repressor activity. Hedgehog signalling blocks this cleavage, increasing the concentration of full length protein and hence activator activity. Cleavage of Ci requires phosphorylation on specific sites by PKA and additional serine/threonine kinases. These phosphorylation events also appear to result in differently-active protein forms, presenting an additional opportunity for regulation by hedgehog signalling. Thus the single Drosophila Ci gene can produce varying concentrations of activator and repressor protein under the regulation of hedgehog signalling.Receipt of hedgehog signalling by target cells requires the cell membrane proteins patched and smoothened, which then relay the signal intracellularly to a conserved family of transcription factors encoded by the hedgehog genes, in vertebrates there are more Gli genes than in Drosophila. In mammals, three distinct genes, Gli1, Gli2 and Gli3 have been described Gli genes were expressed in Drosophila imaginal discs showed that, at the subcellular level, the combination of activator and repressor activities displayed by all three proteins could be accounted for by Ci alone Gli1, Gli2 and Gli3 genes are differentially expressed during development, for example in the mouse neural tube Gli1 is expressed ventrally, while Gli2 and Gli3 are more dorsally expressed As with the Drosophila and vertebrates, there are also important differences. From an evolutionary perspective, the duplication and divergence of Gli genes in vertebrates has increased the complexity of the mechanism receiving hedgehog signalling. In this context it is intriguing to note that hedgehog signalling is deployed in developing vertebrate embryos in numerous tissues considered to be vertebrate specialisations, for example limb buds Gli gene duplication and divergence occurred, we have characterised a Gli ortholog from the cephalochordate amphioxus. Amphioxus develops a classic chordate body plan, but split from the vertebrate lineage prior to the widespread gene duplications that marked early vertebrate evolution Gli ortholog. Accordingly, we demonstrate the presence of a single Gli ortholog in amphioxus, and show it is expressed in developing embryos and larvae in a pattern consistent with a role in hedgehog signalling. We also find the amphioxus Gli mRNA is differentially spliced to yield two transcripts encoding different open reading frames and with spatially-distinct expression patterns. We utilise tests of Gli protein function to show the proteins encoded by these transcripts differ in function. Together, these results demonstrate that the duplication and divergence of Gli genes is specific to vertebrates, but also show that amphioxus has evolved an increased diversity of Gli proteins through a separate mechanism, that of alternate splicing.Thus, while aspects of Gli family function are similar in C. intestinalis; via the JGI Ciona version 2 website (http://genome.jgi-psf.org/Cioin2/Cioin2.home.html). For mouse (version NCBI m36) and Human (version NCBI 36); via the Ensembl Alternative Splicing Database (.http://www.ebi.ac.uk/asd/). For chicken (version WASHUC2), Xenopus tropicalis (version JGI 4.1) and zebrafish (Zf6); via the Ensembl genome browser (http://www.ensembl.org/index.html).Sequence alignments were constructed using ClustalX AmphiGli-S and AmphiGli-L was examined by in situ hybridisation using a probe derived from the entire ORF of AmphiGli-S. To examine the expression of AmphiGli-L only, the additional exon present in this transcript was amplified by PCR, cloned and verified by sequencing. Probes derived from this fragment, however, generated very high levels of background, with extensive staining appearing on the exterior of the embryos within 15 minutes. Problems with background deriving from 3′UTR sequences have been experienced before , hence, a region of 130 bp derived from the open reading frame was re-amplified, cloned and verified by sequencing. Probes derived from this region did not generate background, though the small size did necessitate extended staining times (up to 5 days). Since extended staining can itself generate background, we also conducted controls using a sense probe derived from this fragment. These did not show any staining when stained for the same period of time.In situ hybridisation and subsequent histology was conducted as previously described Full length cDNAs encoding AmphiGli-S or AmphiGli-L were excised with Xba1 and Xho1, and cloned into the plasmid pCAGGS IRES-NLS-GFP Full length cDNAs encoding AmphiGli-S or AmphiGli-L were excised with EcoR1 and cloned into the EcoR1 sire of pUAST To generate cells expressing these constructs in the fly, the lines were mated to the 30A GAL4 driver line. This line drives expression of the UAS transgene in a ring surrounding the future blade area of the wing imaginal disc To detect effects on the hedgehog signalling pathway, imaginal discs were isolated. For LacZ detection discs were fixed with 0.5% glutaraldehyde. They were washed in PBS and stained for β-Galactosidase activity using standard assays. They were washed in PBS, mounted in 75% glycerol/PBS and imaged using DIC optics on a Zeiss AxioPhot. For immuno-stainings, the tissues were fixed in 4% paraformaldehyde, washed (PBS+0.1% saponin) and blocked using 3% Normal donkey serum. The discs were incubated with primary antibodies in PNT  overnight at 4°C, washed the next day five times for five minutes with PNT and incubated with appropriate secondary antibodies (Jackson ImmunoResearch) for two hours in PNT. Washes were done as before and wing discs were mounted in Vectashield (Vector Laboratories) and imaged using a Zeiss Meta501 Confocal Laser Scanning Microscope.AmphiGli was amplified from an amphioxus embryo cDNA library A 327bp fragment encoding part of the zinc finger region of GLI1 to HOXC and GLI3 to HOXA on human chromosomes 12q13 and 17p13–14 respectively suggests the Gli genes were duplicated at the same time as the Hox clusters, that is before the divergence of jawed vertebrates. We also examined the draft assembly of the Branchiostoma floridae genome (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html) for Gli, Glis and Zic related genes. This identified only one Gli ortholog in the current assembly.Next we sought to assess Gli gene number in amphioxus. Considerable evidence suggests gene duplication via genome duplication marked early vertebrate evolution, as exemplified by the Hox clusters AmphiHhAmphiGli expression was first detected in late gastrulae, in a broad dorsal domain in both cell layers  and p0–p2 interneuron progenitor domain (as detected by Cad7), while it repressed the dorsal marker Pax7 . There are few descriptions of alternate splicing of Gli genes in the literature. An exception is human GLI2, for which alternate splicing in gonad tissue and relating to viral function has been reported GLI2, and hence differs from the splicing in amphioxus, which affects the C terminal. Other vertebrate Gli genes, Ci in Drosophila and Ci-Gli in C. intestinalis have not been reported to be to be alternately spliced. Examination of chordate Gli loci and associated EST data also do not indicate splicing that would alter the C termini (as seen in amphioxus), although there is evidence that other vertebrate Gli genes may also be spliced in a similar way to human GLI2 such that the N terminus is affected . Hence we conclude that, while alternate splicing affecting the N terminus may occur for vertebrate Gli genes, the alternate splicing yielding different C termini observed in amphioxus is specific to the amphioxus lineage.We also show the AmphiGli is dorsally-expressed in both neurectoderm and mesoderm during neurulation. In the neurectoderm, expression is excluded from the floor plate but found throughout the rest of the neural plate. During the same period, AmphiHh is expressed by notochord and floor plate cells Msx, Pax7, Irx3 and Pax6, is repressed by Shh, while expression of ventral genes, such as Nkx6.1, Nkx2.2 and Dbx2, is induced by Shh Mnx, En1, Evx and Lbx1, as well as several Islet and Lim genes. Knowledge of neural DV patterning outside of the amniotes is less advanced, however studies on zebrafish embryos suggest a high degree of similarity within the vertebrates Pax3/7, Msx, Evx, Nkx2.1, Mnx, Islet and Lhx3, have been isolated from amphioxus and shown to be expressed in the developing neural plate and/or specific populations of cells in the neural tube AmphiHh and AmphiGli are expressed at the right time and place to be involved in regulating the expression of these genes in amphioxus, while the functional analysis of AmphiGli (discussed below) demonstrates that AmphiGli is able to act as an effector of hedgehog signalling. These data suggest conservation of neural DV patterning between amphioxus and vertebrates, with an ancient role for Gli proteins in transducing midline-derived hedgehog signals and hence in generating the bilateral organisation of the central nervous system.Orthologs of many of these vertebrate genes, including AmphiGli mesodermal expression is excluded from the midline (i.e. the notochord), but found through the majority of each mesodermal segment. Amphioxus mesodermal segments are often referred to as somites. However these differ from vertebrate somites, as in vertebrates the ventral mesoderm remains unsegmented, while in amphioxus all mesoderm (other than the notochord) is segmented. Hence each amphioxus somite includes cells fated to form the myotome , plus cells fated to form the mesoderm that surrounds the gut  and an additional population of dorsolateral cells of distinct but uncertain fate (with no clear counterpart in vertebrates) Msx and Zic in the dorsal somite FoxF and Vent in ventral, gut-associated mesoderm AmphiGli through the majority of the amphioxus somite indicates the same mechanism is operating in amphioxus. Therefore, as for neural DV patterning, mesodermal DV patterning regulated by midline hedgehog/Gli signalling is an ancient mechanism.Similar to its expression in the neurectoderm, AmphiGli includes long and short forms, or just the long form. However as the long form is definitively expressed and acts as an activator of hedgehog targets in response to hedgehog signalling (see below), we can conclude that midline-derived hedgehog signalling in amphioxus involves activation of ventrally-expressed target genes in ventral cells, and may also involve repression of these genes in dorsal cells, or indeed of other genes in ventral cells.We were not able to determine if neural and mesodermal expression of C. intestinalis, hedgehog expression is present in the ventral cells of the neural tube, but not the notochord, while Gli is expressed in the neural tube but not by mesodermal cells C. intestinalis, mesodermal patterning has been lost.Recent revision of chordate phylogeny has placed vertebrates and urochordates as sister groups, with amphioxus more distantly related to this clade AmphiGli transcripts in the developing left head cavity towards the end of neurulation, and in the developing cerebral vesicle, pre-oral pit (which forms in part from the left head cavity) and endoderm associated with the developing club-shaped gland and gill slits. These sites correlate with expression of AmphiHh in adjacent tissues, specifically the anterior notochord, central pre-oral pit and pharynx AmphiHh and AmphiGli in the developing pre-oral pit may reflect a conserved role. Similarly, Shh is expressed by vertebrate anterior endoderm cells and plays a role in patterning the developing pharynx AmphiHh and AmphiGli in the amphioxus pharynx may indicate that the regulation of pharyngeal development by hedgehog/Gli signalling is a primitive chordate character.We also detected AmphiGli-L and AmphiGli-S are functionally distinct. All three tests show AmphiGli-L is an activator of the hedgehog pathway, and in the chick neural tube assay it has similar activating ability as full length vertebrate Gli2Drosophila imaginal disc assays it functioned as a repressor of the hedgehog pathway. This is similar to the activity of Gli3 in these assays. A similar effect on the transcriptional activity of vertebrate Gli2 was achieved by deleting the last 93 amino acids of the protein Drosophila reveals a high level of conservation in the C terminal region (Three different tests of Gli protein activity demonstrate that the proteins encoded by l region . This raGli gene demonstrates that two different chordate lineages have used different genetic routes to evolve an increased complexity of transcription factor capability downstream of hedgehog signalling. In vertebrates, Gli genes have duplicated then diverged both in the function of the encoded proteins and in spatiotemporal expression. The amphioxus lineage originated before these duplications, and amphioxus has not independently duplicated the Gli gene. However, diversification of protein function and regulation of expression has evolved via alternate splicing. Perhaps most interestingly, in vertebrates Gli3 usually acts as a strong repressor, with a major role of hedgehog signalling being to alleviate this repression. This is illustrated by −/− Gli3−/−Shh mouse embryos, which show partial rescue of the phenotype seen in −/−Shh embryos Our data suggest a model for the diversification of hedgehog signalling in the chordates . The cha


Drosophila, neural precursors (neuroblasts) divide in a stem cell-like manner generating a larger apical neuroblast and a smaller basal ganglion mother cell. The cell fate determinant Prospero and its adapter protein Miranda are asymmetrically localized to the basal cortex of the dividing neuroblast and segregated into the GMC upon cytokinesis. Previous screens to identify components of the asymmetric division machinery have concentrated on embryonic phenotypes. However, such screens are reaching saturation and are limited in that the maternal contribution of many genes can mask the effects of zygotic loss of function, and other approaches will be necessary to identify further genes involved in neuroblast asymmetric division.The asymmetric segregation of determinants during cell division is a fundamental mechanism for generating cell fate diversity during development. In Mosaic Analysis with a Repressible Cell Marker) system to generate postembryonic clones of mutations with an early lethal phase. We have screened a total of 2,300 mutagenized chromosomes and isolated alleles affecting cell fate, the localization of basal determinants or the orientation of the mitotic spindle. We have also identified a number of complementation groups exhibiting defects in cell cycle progression and cytokinesis, including both novel genes and new alleles of known components of these processes.We have performed a genetic screen in the third instar larval brain using the basal localization of Miranda as a marker for neuroblast asymmetry. In addition to the examination of pupal lethal mutations, we have employed the MARCM  in the targeting of determinants to the basal cortex. The identification and analysis of the remaining mutations will further advance our understanding of the process of asymmetric cell division. We have also isolated a number of mutations affecting cell division which will complement the functional genomics approaches to this process being employed by other laboratories. Taken together, these results demonstrate the value of mosaic screens in the identification of genes involved in neuroblast division.We have identified four mutations which affect the process of neuroblast asymmetric division. One of these, mapping to the  Drosophila neural progenitors, have served as one of the major models for studying asymmetric division . Neurobiewed in ,3, to geDrosophila homologue of Par-3), Par-6 and atypical protein kinase C (DaPKC) – and the heterotrimeric G protein subunit Gαi together with the guanine nucleotide dissociation inhibitors Partner of Inscuteable (Pins) and Locomotion defects (Loco). The apical complex has several important functions during neuroblast asymmetric division including the correct orientation of the mitotic spindle along the apical-basal axis of the cell, the displacement of the spindle towards the basal cortex [The initial step in defining the asymmetry of neuroblast divisions is the establishment at the apical cortex of a multi-protein complex  contal cortex  and the l cortex . This gil cortex -11. Myosl cortex , althougbicoid mRNA [pros mRNA basally [Miranda functions as an adapter protein, localizing Staufen and Prospero (Pros) to the basal cortex -15. Stauoid mRNA , is empl basally -19. The  basally -22. In t basally -25.Gαi, Pins or Loco – three components of the apical complex identified biochemically or by a candidate gene approach – are viable, albeit with locomotion defects, and fertile, indicating that alleles of these genes would not have been found in a zygotic loss of function genetic screen [Several molecules known to be involved in asymmetric neuroblast division have been identified in zygotic loss of function screens looking for embryonic phenotypes but the major limitation of this method is that maternal contribution of mRNA will mask the effects of the loss of many genes during embryogenesis. In support of this idea, animals lacking zygotic c screen -29.A systematic germline clone screen might be an effective way to identify new components of the asymmetric cell division machinery. However, components such as Myosin II and Jaguar are required during oogenesis and do not give rise to fertilized eggs in germline clones ,31, and pins mutants has been examined in this way [Drosophila is to generate postembryonic clones of cells homozygous for the mutation of interest, and we have used a variation of such a strategy in which clones are positively marked by the expression of GFP: the MARCM system , which  Marker; . Core toand Cdc2 , as welland Cdc2 ,38 – havIn this study, we describe the results of a MARCM screen on chromosome arm 3L, together with a screen of pupal lethal and semi-lethal mutations on chromosome 3. We identified 78 mutations affecting neuroblast division that fall into 48 complementation groups. The majority of these represent genes required for cell division, 12 of which correspond to previously described loci, and several of which also appear to have effects on asymmetric cell division. Although the bulk of the cell division mutants isolated in our screen do not have clear polarity phenotypes, we have deficiency mapped many of them to small genomic regions, reasoning that this would complement several RNAi-based screens currently being conducted to look for genes involved in cell division (for example ). In addelavGAL4C155, which directs expression in neuroblasts as well as neuroblast progeny  were mutagenized with ethyl methanesulfonate (EMS) and stocks established carrying mutations balanced over TM6B, Tb. In total we generated 1923 stocks carrying mutations causing lethality before the wandering third instar larval stage, as assayed by the absence of Tb+ larvae, and approximately 350 pupal-lethal and semi-lethal chromosomes.To generate mutant lines, male flies isogenic for a chromosome carrying an Tb larvae. The crossing scheme for generating somatic clones of the remaining 1923 mutant lines is given in figure tub-GAL80 on the FRT2A chromosome together with elavGAL4C155, hsp70Flp and UAS-CD8::GFP. Induction of mitotic recombination by 37° heatshocks at first and second instar stages resulted in the generation of clones within neural lineages that were homozygous for mutations on 3L and positively marked by the expression of CD8::GFP. Clones were readily identifiable by examination of intact larvae under UV light revealing that the great majority of Tb+ female third instar larvae contained mutant clones in the brain. Nervous systems from these larvae were dissected and screened by antibody staining against Miranda and GFP and confocal microscopy. At least three metaphase or anaphase neuroblasts in at least two brains were examined looking for alteration or absence of the basal Miranda crescent or any misorientation of the metaphase spindle.The 350 pupal- and semi-lethal lines giving rise to third instar larvae homozygous for the mutant chromosome were screened by antibody staining of non-We initially screened our collection of approximately 350 third chromosome pupal- and semi-lethal mutations for those that showed a neuroblast phenotype in the larval brain, scoring at least five dividing neuroblasts in at least two brains. We identified two lines with neuroblast asymmetry defects , and we have been unable to map the PL26 phenotype further.In our pupal lethal screen, we identified a single mutation with a neuroblast polarity phenotype. In animals homozygous for the e Figure , althougs Figure . Clones PL17, was initially identified in our pupal lethal screen as having a high mitotic index but overall reduction in the size of homozygous larval brains, suggesting a defect in cell cycle progression (described below). In addition to these defects we observed a frequent mislocalization of Miranda to discrete cytoplasmic regions, although localization of the apical marker Inscuteable appears unaffected , and we interpret this as a consequence of the perdurance of wild-type protein within clones. Deficiency mapping and complementation testing revealed that PL17 is allelic to imaginal discs arrested (ida), which encodes the Drosophila APC5 homologue, and a more detailed characterization of the defects observed in this line will be presented elsewhere .One mutant, d Figure . Co-staiJ16, which appears to have defects in the size asymmetry of neuroblast divisions within mutant clones. Wild type clones invariably contain a single large neuroblast accompanied by a number of much smaller GMCs and neurons. Miranda expression is found in neuroblasts, and occasionally in GMCs, but never in post-mitotic neurons Exel9636) survive to the third larval instar, and brains of these animals contain appear to have a high rate of metaphase arrest in neuroblasts . Although neuroblasts form correctly in J16, by stage 9 we observe a loss of expression of Worniu, a marker for all embryonic neuroblasts [J16 embryos . The low penetrance of neuroblast defects in the embryo suggests the perdurance of maternal protein may be masking the embryonic phenotype. As we do not obtain fertilized eggs in J16 germline clones, even with the restoration of polo function, we have not explored this further.To investigate the consequence of these defects in neuroblast division we examined the expression of neuroblast and cell fate markers in homozygous roblasts ,43,44, as Figure . In wildJ16 mutation appears to lie outside the region uncovered by the Bloomington deficiency kit, and further investigation will be required to identify the genomic region responsible for these defects.The D76, in which dividing neuroblasts are almost never seen. On examining large numbers of clones we were able to determine that the rare neuroblasts which enter metaphase do not form a basal Miranda crescent, although Miranda is expressed and can be detected at low levels in the cytosol and Inscuteable is localized appropriately to the apical cortex . However, we have been unable to identify additional lethal deficiencies within this interval, and so have not mapped the D76 mutation further.We isolated a single allele, x Figure . In somex Figure , the ideIn addition to the four lines with phenotypes during neuroblast asymmetric division, we identified a total of 76 mutations, in 46 complementation groups, which exhibit defects in cell division (Table Proliferation defects). Two of these mutations – PL13 and LVC73 – give similar phenotypes. The optic lobes of the brain are generally small, with fewer neuroblasts than wild type, and we rarely observe mitotic cells , compared to 45% in PL13/FRT2A heterozygotes (n = 60)), although when we do see neuroblasts in metaphase the localization of Miranda and orientation of the mitotic spindle appear normal. Despite their phenotypic similarities, these mutant lines do not show any lethality as transheterozygotes and their lethal regions map to distinct regions of the chromosome.In our pupal lethal screen we identified three lines in which the brains of homozygous larvae appear reduced in size ), suggesting a delay at meta- or anaphase HR119 and Df(3L)GN34, which fail to complement the lethality of PL17, and complementation testing revealed that PL17 is allelic to imaginal discs arrested.An additional line, e Figure . We idenIn our clonal screen, we isolated nine mutations in which we see clones of only one or two cells but in which the neuroblasts do not show any gross morphological defects. Because of the small numbers of mutant clone neuroblasts generated in our screening regimen we have not attempted to accurately measure the mitotic index in these mutations, but in eight out of nine cases the lack of mitotic neuroblasts observed suggests that the mitotic index is greatly reduced Figure .Proliferation defects). Testing known mutants in these regions revealed that the two mutations that fall into complementation group 7 are allelic to small-minded (smid). Furthermore, the phenotype observed in clones of these mutations reflects the described phenotype of smid homozygous larvae in which proliferation of postembryonic neuroblasts is reduced [D76, also gives rise defects in Miranda localization in the rare metaphase neuroblasts observed.These eight mutations fall into five complemention groups which we have mapped to separate regions of 3L . These three complementation groups give rise to clone neuroblasts which appear to have an abnormally high DNA content but which do not contain multiple nuclei , previously known to be required for chromosome separation, and sequence analysis indicated that the C93 chromosome carries a G→A transversion at the exon 6 splice acceptor of sse, presumably leading to the formation of a truncated protein. Transheterozygotes of C93 with sseM13 or hemizygotes with Df(3L)Exel6106, which uncovers the sse locus, survive until late third instar stages and show phenotypes similar to those observed in C93 clones. These brains contain large cells which have an increased DNA content Exel6106 hemizygotes frequently contained several cortical crescents of Miranda instead of a single basal crescent. Interestingly, when we examined the localization of Inscuteable in these larvae, we found that neuroblasts with several Miranda crescents also had several Inscuteable crescents, and that these never overlapped  and γ-tubulin . One of these mutations, C33, shows a severe cell division phenotype in neuroblasts and was mapped to the pebble locus, previously shown to be required for cytokinesis ([TBP-associated factor 4 (Taf4), and the multinuclear phenotype is presumably a downstream consequence of defects at the transcriptional level. The remaining complementation groups mapped to a number of chromosomal regions with the exception of groups 30 and 31 which complement the entire Bloomington deficiency kit and were not mapped further.In the clonal screen we identified a further allele of kinesis . Whereas in wild type neuroblast clones we observe the CD8::GFP outlining the cell and nucleus, and surrounding the mitotic spindle, in a number of these lines we frequently see multiple brightly labelled punctae within the cell, the identity of which is unclear . Several of the other mutations isolated in the screen are also likely to have a direct influence on the asymmetry machinery. In particular, we have identified a mutation affecting metaphase spindle orientation in neuroblasts, a process which is critical to the correct segregation of cell fate determinants, and another which appears to perturb the size asymmetry of the neuroblast division and the correct establishment of different identities in daughter cells.We found a total of five complementation groups that affect the distribution of Miranda in dividing neuroblasts or the size asymmetry of the neuroblast division. At the time of writing, two have been mapped to a gene. One of these is miranda, prospero and scribbled, a tumour suppressor in the Lgl/Dlg pathway . This suggests that our screen methodology is effective at finding genes involved in neuroblast asymmetric division, and that it can in the future be applied to the whole genome to find further novel components of this machinery. The major limitation with the approach at present is that it is rather labour intensive, which necessarily leads to a low throughput compared to other screening methods. The use of live imaging methods, coupled with the GFP and RFP fusion reagents widely in use, would most likely relieve this bottleneck and allow a higher throughput. Similarly, improvements to the crossing schemes could be made. If chromosomes could be screened without the need to establish stocks of each one, perhaps with mutations being recovered from the siblings of the larvae examined, an F1 screen could be conducted which would enable a genome-wide saturating screen to be carried out much more rapidly.We did not find as many asymmetric division mutations as we might have expected given the scale of the screen: we estimate, using a Poisson approximation, that we have achieved approximately 80% saturation of 3L. Although it is possible that the design of our screen has prevented the detection of some genes, we believe that this is a reflection of the rather small number of genes involved exclusively in asymmetric cell division. Indeed, a similar screen of 3R has detected new alleles of several known players of neuroblast asymmetric division including taf4, involved in transcriptional initiation, appear to cause a failure in cytokinesis. Nonetheless, we have identified alleles of a number of previously reported cell division genes and we anticipate that a substantial number of the mutations described here will be directly involved in the processes of cell division.In addition to finding several new polarity genes, we have isolated large numbers of mutations affecting cell division, and this seems to be a particular strength of such a clonal approach. Looking only at complementation groups which give rise to multinucleate neuroblasts we have found mutations in 13 regions not previously known to be involved in cytokinesis, and similar screens may prove beneficial to laboratories with a specific interest in aspects of cell division. As with other screening methods, in several cases mapping of the gene responsible for a cell division phenotype has yielded unexpected results: for example, mutations in lethal giant larvae (lgl) and discs large (dlg), involved in the localization of basal components in neuroblasts, are both tumour suppressors, and in their absence larval brains and discs show a dramatic overgrowth . Similaiewed in ,12. This unclear ,53.elavGal4 directing expression of GFP, the presence of an EP insertion [FRT site, would lead to transcription of genes downstream of this element specifically in labelled clones, and circumvent any early lethality caused by ectopic expression, a frequent limitation of such screens.Aside from the clear utility of the MARCM system in screening for mutations affecting aspects of cell division, we consider that it could easily be adapted for use in a mis-expression screen. As clones are positively labelled by nsertion , or one Previous screens for components of the asymmetric division machinery have focused on embryonic phenotypes, and are now reaching saturation. Here we have used a clonal approach to screen neuroblasts in the third instar larval brain, and have identified several novel mutations, the identification and further study of which will advance our understanding of the process of neuroblast asymmetric division.Drosophila stocks were reared and maintained on standard yeast-cornmeal-agar medium [w flies carrying an FRT element inserted at polytene segment 79D-F (FRT 2A) were first isogenized for the third chromosome. Three to five day old males were then mutagenized by feeding with 1% sucrose solution containing 25 or 38 mM EMS as described previously [en masse to virgin females of the genotype TM8/TM6B, Tb, Ubi-GFP. Single male progeny were crossed to the balancer stock to establish stocks in which the mutagenized third chromosomes were balanced over TM6B, Tb, Ubi-GFP. Lines in which the mutagenized chromosome was homozygous viable were discarded.All r medium  and all eviously . MutagenFRT2A chromosome were viable at the wandering third larval instar stage (350 lines), as assayed by the absence of the Tb marker, were screened by antibody staining of mutant brains. Lines lethal before the third instar (1923 lines) were screened using the MARCM system [elavGAL4C155,hsp70Flp/Y; UAS-CD8::GFP, UAS-LacZ; tub-GAL80, FRT2A (a gift from A. Gould and B. Bello). Crosses set up using females from the MARCM driver line and males from a mutant stock were significantly less productive, and these crosses were avoided except in cases in which few females of a mutant stock could be obtained.Stocks in which animals homozygous for the mutagenized M system . FemalesTb wandering third instar larvae were dissected and screened by antibody staining.After 24 hour periods of egg laying, progeny were heat-shocked twice for 2 hours in a 37° water bath at first instar and second instar stages to induce mitotic recombination. Brains of female non-pebble, encore, nuclear fallout, four wheel drive, pavarotti, fumble, polo, sticky and kinesin-like protein at 61F (Klp61F). This allowed us to identify alleles of pebble, polo, sticky and Klp61F. The remaining complementation groups were mapped initially by crossing to the third chromosome deficiency kit, provided by the Bloomington Drosophila stock centre. We performed further fine scale mapping with smaller deficiencies obtained from the Drosdel and Exelixis collections [small-minded, TBP-associated factor 4 and -6 (Taf4 and -6), reptin, Isoleucyl-tRNA synthetase (Aats-ile), separase, makos, Int6 and neurexin. A number of complementation groups containing only a single allele failed to complement more than one region of 3L. In several of these cases we were able to place the phenotype in a single region by examination of hemizygous phenotypes or by meiotic recombination with appropriate markers from a ru h th st cu sr es ca chromosome followed by clonal analysis. Several groups complemented the entire Bloomington deficiency kit for 3L and were not mapped further.Following screening, lines were placed in complementation groups by pairwise complementation testing and tested against alleles of the following candidate genes on 3L: lections ,58, as wlections . Testingmks1 [P{GFP-polo} [sseM13 [Stocks, other than those used to test candidate genes and available from the Bloomington or Szeged stock centres, were mks1 , P{GFP-pFP-polo}  and sseM [sseM13 .2HPO4/NaH2PO4 (PBS) and fixed in 4% formaldehyde (Polysciences) in PBS for 20 min at room temperature. Brains were washed in PBT (PBS + 0.1% Triton X-100) for 2 × 30 min, blocked in 5% normal goat serum in PBT for 30 min, incubated with primary antibody at 4° overnight, washed 3 × 20 min in PBT, incubated with secondary antibody for 2 hours at room temperature, washed 3 × 20 min in PBT, further dissected and mounted in Vectashield (Vector Laboratories). Antibodies used were mouse anti-Miranda [Brains of wandering third instar larvae were dissected in 100 mM Na-Miranda , rabbit -Miranda ), rabbit-Miranda ), rabbit-Miranda ), mouse -Miranda ) and mou-Miranda , anti-Ac-Miranda  and antiSecondary antibodies were obtained from Jackson labs (Cy3/HRP) or Molecular Probes (Alexa-488) and used at a concentration of 1:1000 (fluorescence) or 1:500 (HRP). DNA was visualized by the addition of ToPro-3-iodide  to one of the wash steps. Antibody staining of embryos was performed essentially as previously described . SamplesCS, WGS, WC and PMO designed the screen, which was carried out by CS, WGS and PMO with help from RS-N; CS and PMO performed deficiency mapping and further analysis of mutations; PMO wrote the manuscript with help from CS; all authors read and approved the manuscript.


Thus, the proportion of positively reacting neoplasms increased as the stage progressed, tumours which had invaded regional lymph-nodes overexpressed p53 more frequently than localised carcinomas and an elevated level of p53 was associated with early relapse and death. In colorectal carcinoma p53 positivity was also linked with site and macroscopic configuration of the primary tumour and was most frequently expressed in carcinomas from the rectum and in ulcerative tumours. p53 overexpression was irrespective of tumour grade. Uniform negative reactivity with anti-p53 antibody was seen in normal epithelium adjacent to carcinoma, intestinal metaplasia, atrophic gastritis and in colonic adenomas. There was a good correlation between immunohistochemical staining on paraffin and frozen sections. These studies suggest that in gastric and colorectal carcinoma, immunohistochemical detection of p53 protein in routinely fixed tissue can be used along with other established parameters to assess prognostic outcome, especially to identify patients with poor short-term prognosis.


Several histological classification systems have been developed, but assessing the histological type/grade of DCIS lesions is still challenging, making treatment decisions based on these features difficult. To obtain insight in the molecular basis of the development of different types of DCIS and its progression to invasive breast cancer, we have studied differences in gene expression between different types of DCIS and between DCIS and invasive breast carcinomas.Ductal carcinoma in situ and 40 invasive breast cancer cases.Gene expression profiling using microarray analysis has been performed on 40 n = 6), intermediately (n = 18), and poorly (n = 14) differentiated type. Of the 40 invasive breast cancer samples, five samples were grade I, 11 samples were grade II, and 24 samples were grade III. Using two-dimensional hierarchical clustering, the basal-like type, ERB-B2 type, and the luminal-type tumours originally described for invasive breast cancer could also be identified in DCIS.DCIS cases were classified as well- (Using supervised classification, we identified a gene expression classifier of 35 genes, which differed between DCIS and invasive breast cancer; a classifier of 43 genes could be identified separating between well- and poorly differentiated DCIS samples. DCIS is characterised by malignant epithelial cells accumulating in the ducts of the breast without invading through the basement membrane into the surrounding tissue. DCIS accounts for approximately 3% of symptomatic breast malignancies and for approximately 20% of breast malignancies in patients from population-based screening programs [Ductal carcinoma programs .Different histological types of DCIS can be recognised, and a variety of classification systems have been developed . Due to in situ lesions to invasive disease and developing diagnostic tests would be of great clinical benefit.It is assumed that most cases of DCIS will progress to invasive breast cancer. Because this progression may take many years and may not occur within the lifetime of a patient, elucidating the mechanisms of progression from Several models of the evolution of DCIS to invasive cancer have been suggested. One model suggests the linear progression from low-nuclear-grade DCIS to high-nuclear DCIS and the subsequent development of invasive cancer . Based oin situ (LCIS) [Several specific genetic alterations have been found in DCIS. HER2 gene amplification and protein overexpression are detected in up to 70% of poorly differentiated DCIS cases  in approu (LCIS) . Severalu (LCIS)  and Allru (LCIS) .in situ carcinomas of the breast and also lead to a more reproducible classification of DCIS lesions.Gene expression profiling has been shown to be a powerful tool for identifying profiles of tumour subtypes -15 and fTo date, only a few studies of gene expression profiling of DCIS and a comparison with the gene expression pattern of invasive samples have been published and these are based on a small number of samples ,20.in situ and 40 invasive breast carcinomas were studied.In the study presented here, gene expression profiling was performed on one LCIS and 39 DCIS samples to identify differentially expressed genes between well-, intermediately, and poorly differentiated DCIS. In addition, differences in gene expression between these cases of carcinoma n = 16) or mastectomy (n = 24). All samples were reviewed by two pathologists independently to determine the histological classification of the samples according to Holland and colleagues [Cases of DCIS were selected from the tissue bank of the Netherlands Cancer Institute . These samples were obtained within 1 hour after surgery from patients who underwent wide local excision  measuring between 1 and 5 cm and were graded as grade 1, 2, or 3 according to the method described by Elston and Ellis . The stuRNA isolation and amplification were performed essentially as described by Weigelt and colleagues . Thirty The procedures applied are described in the supplementary information provided online .Labeling of the amplified cRNA and microarray hybridisations were performed as previously described . Equal aMicroarrays were scanned with the DNA Microarray Scanner G2565B . Self-self hybridisations were used to validate the quality of the hybridisations and as a negative control in the error model.Information on data processing is provided in the supplementary information .Two-dimensional unsupervised hierarchical clustering using Pearson correlation as distance function and complete linkage was performed using Genesis software  ,28.We performed supervised classification applying methods described previously ,29,30. PThe microarray data, additional information on the methods, and the filtering results are provided as supplementary information .This study was performed to identify differences in gene expression (a) between DCIS and invasive breast cancer and (b) between different histological types of DCIS.n = 6), intermediately differentiated (n = 18), and poorly differentiated (n = 14). For analysis purposes, the group of intermediately differentiated cases was further subdivided in well-intermediately (n = 10), true intermediately (n = 2), and intermediately-poorly (n = 6) differentiated type. One sample contains a mixture of well- and poorly differentiated DCIS components in the same tissue specimen. In addition, one case of LCIS was included.Thirty-nine cases of DCIS of the breast were included in the analyses. By histological examination, they were assigned to the following groups: well differentiated  14 poorly differentiated DCIS cases versus 38 invasive grade 3 tumours, and (c) six cases of well-versus 14 cases of poorly differentiated DCIS.We performed supervised classification on different data sets to identify the genes differentially expressed between the groups of interest. These groups are (a) 40 in situ and invasive breast carcinoma samples. We therefore used the whole data set and assigned all 40 in situ samples to one group and all 40 invasive samples to a second group . To obtain a profile taking into account the expression sets of both tumour types, significantly regulated genes were identified independently for both groups. The 1,706 overlapping genes were used for analysis. Monte Carlo randomisation revealed approximately 300 genes differentially expressed between in situ and invasive samples.We investigated the differences in gene expression between After cross-validation, classifier consisting of 35 genes resulted in a stable prediction of the differences between DCIS and invasive breast carcinomas, with an average performance of 91%. The gene list is provided in Table n = 14) and grade 3 invasive tumours (n = 24) . Again, the filtering procedure was applied to both groups independently. The overlapping fraction of this gene list contains 1,119 genes that were used to perform the analyses. Monte Carlo randomisation showed that 80 genes are differentially expressed between poorly differentiated DCIS and grade 3 invasive breast carcinoma samples. After cross-validation in 14 steps, the best performance of 93% is reached, when at least 50 genes are used to build the classifier. This performance remains stable with increasing numbers of genes. This means that 50 to 80 genes are able to discriminate between poorly differentiated DCIS and invasive grade 3 breast tumours  resulted in a set of 43 genes separating these groups with a performance of 90%  and a second group containing poorly/intermediately-poorly differentiated samples (n = 20). Supervised classification of these data revealed a set of 78 genes separating these two groups with an average performance of 89%  are negative for ER expression by IHC. There was a slight chance that our classifier would detect mainly the differences of ER-associated genes. We identified only one gene (LIV-1), beside the ER itself, directly ER-regulated in the classifier of 43 genes. Additionally, we compared the 43 genes with 2,460 ER-associated genes identified by van 't Veer and colleagues [Twenty-one genes are overlapping between the 43 genes of analysis set 3 and the 78 genes of analysis set 4. It is known that many poorly differentiated lleagues . Thirteein situ samples, on one hand, and DCIS and invasive samples on the other hand. These findings may indicate that gene regulation involved in progression from in situ to invasive breast cancer affects molecular mechanisms other than the mechanisms responsible for the development of the different types of DCIS.Remarkably, completely different gene lists are found describing the differences in gene expression between different Although studies to identify gene expression signatures in DCIS are limited by difficulties in obtaining frozen material from DCIS, we were able to collect a relatively large series of DCIS cases for this purpose. It should be kept in mind that we did not have a sufficient number of cases to validate the gene expression signatures that we identified.n = 6) and poorly (n = 14) differentiated DCIS show different gene expression profiles and can be distinguished by a classifier of 43 genes. Most of the genes differentially expressed between well- and poorly differentiated DCIS are involved in metabolism  and cell communication .We were able to show that well-  can be found within the 15 downregulated, but not in the 28 upregulated, genes.Twenty-eight of the 43 genes are upregulated and 15 genes are downregulated in the well-differentiated samples Figure . Whereasin situ samples, we found a positive log2-ratio for HER mRNA expression in six of 14 poorly differentiated DCIS cases (43%) and in one case of intermediately-poorly differentiated DCIS. In all the other in situ samples, the log2-ratios of HER2 are negative. These results are in agreement with the hypothesis that HER2 overexpression is an early event in the development of poorly differentiated in situ breast carcinomas.It is known that HER2 is overexpressed in poorly differentiated DCIS in approximately 42% of the cases , and it Supervised classification of well-, well-intermediately, intermediately-poorly, and poorly differentiated DCIS samples  showed a separation of these samples in three groups: a 'good' group, a 'poor' group, and an 'intermediate' group containing mostly samples that were identified as intermediately-poorly differentiated samples by pathologists. This group also contains some samples pathologically classified as well-intermediately differentiated, whereas most of these samples fall in the 'good' group. These results indicate that well- and well-intermediately differentiated DCIS are more similar to each other than poorly and intermediately-poorly differentiated DCIS are. Following this idea, well- and well-intermediately differentiated samples may be considered to be one group, whereas poorly and intermediately-poorly differentiated samples seem to be two distinct groups of DCIS. If these results can be validated in additional studies, this classification could help to decrease controversial classification of DCIS due to interobserver variability and to recognise well-differentiated DCIS with more accuracy.Bcl-2, Ack1, CCL19, and CELSR2, among others.Within the gene lists describing the differences between well- and poorly differentiated DCIS, a number of genes refer to proteins for which antibodies are available. There is no single gene discriminating between the different types of DCIS, but it has to be investigated whether a combination of protein stainings in a patient's tissue can assist in better classification of DCIS. From the study presented here, potential candidates for such an approach are in situ and invasive breast tumour samples. This classifier contains many genes involved in signal transduction  and cell growth and maintenance .Thirty-five genes are able to describe the global differences in gene expression between in situ to invasive breast cancer lesions is the existence of distinct pathways for the evolution of well- and poorly differentiated DCIS. Following this idea, well-differentiated in situ lesions develop into grade 1 IDC, whereas poorly differentiated samples develop into grade 3 IDC [n = 14) and grade 3 invasive breast cancer (n = 24).The most likely model describing the progression from de 3 IDC ,6. We thin situ from grade 3 invasive breast carcinomas. Thirteen of these 80 genes are upregulated and 67 genes are downregulated in poorly differentiated DCIS samples. The genes in this classifier are involved mostly in cell growth and protein metabolism. Many of them have a function in protein binding  and nucleic acid binding .Approximately 80 genes discriminate poorly differentiated in situ and invasive breast cancer by using 18 paired samples and combining laser capture microdissection and gene expression profiling on oligonucleotide microarrays. They showed that 546 probes were differentially expressed between DCIS and IDC. From the 18 genes they validated by real-time polymerase chain reaction, four  are also present in our filtered data sets of significantly regulated probe sets comparing DCIS and invasive samples. They all show the same expression pattern as described by Schuetz and colleagues and are expressed at higher levels in the groups of invasive tumours. One of these genes (MMP11) is also part of the 35-gene and 80-gene classifiers. MMP11 and PLAU have already been correlated to invasion and poor prognosis [FAP (seprase) is a membrane-bound protease that has been suggested to reduce the dependence of breast cancer cells on exogenous growth factors in vitro and thereby to facilitate tumour growth and metastasis [CXCR4 does not appear in the set of significantly regulated genes, indicating that it does not play a crucial role in our series of tumours, which reflects the data of a mixed population of cells enriched for tumour cells, whereas Allinen and colleagues performed gene expression profiling on microdissected cell populations.A limited number of previous studies have addressed gene expression profiles in DCIS, and most of these studies have analysed a small number of samples. One study by Seth and colleagues  comparedrognosis . FAP  are upregulated in the invasive cancer cell line in the data set of Nagaraja and colleagues, whereas we see a downregulation in the invasive grade 3 tumours compared with the group of poorly differentiated samples. This may be due to the fact that Nagaraja and colleagues generated in vitro data, which we compared with our human breast cancer data set.A recent study by Nagaraja and colleagues  describeXBP1 is present in one of our classifiers (78 genes). Porter and colleagues describe this transcript as tumour-specific, meaning upregulated in in situ and invasive tumours compared with their normal samples. We find that XBP1 is significantly more highly expressed in well- and well-intermediately differentiated DCIS samples than in poorly/intermediately-poorly differentiated ones.Porter and colleagues  identifiWulfkuhle and colleagues  performein situ breast cancer samples of well-versus poorly differentiated type. There appear to be a group of poorly differentiated samples, a group of well- and well-intermediately differentiated samples, and a third group containing mainly intermediately-poorly differentiated in situ cases. The quantitative differences in gene expression between these groups are mainly between twofold and fourfold. These differences are difficult to detect by classical IHC, because this technique is not very accurate in the quantification of small differences in protein expression. So far, there are no single markers that distinguish between the different types of DCIS, but the possibility of identifying a manageable panel of markers to distinguish the different types of DCIS lesions has to be further investigated.We demonstrate here that gene expression profiling can distinguish between in situ; ER = oestrogen receptor; IDC = invasive ductal carcinoma; IHC = immunohistochemistry; LCIS = lobular carcinoma in situ; SNR = signal-to-noise ratio.DCIS = ductal carcinoma The authors declare that they have no competing interests.JH performed data analyses, participated in the study design, and drafted the manuscript. AV participated in data analyses. JBGH and BK carried out microarray hybridisations. JP and MV reviewed the histological specimens. MV participated in designing the study and drafting the manuscript. All authors read and approved the final manuscript.


Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species. The maintenance of genetic integrity in the male germ line has major repercussions for conception, the progress of pregnancy and, ultimately, the health and well-being of the progeny . The humThese observations raise important questions about the aetiology of genetic damage in the male germ line and the causal links that exist between the induction of such damage and the inheritance of many childhood diseases. As early as 1912, Wilhelm Weinberg  as opposed to an ovulated egg (approximately 22 divisions regardless of age). If this is the case, then the mutations would have to be generated and retained in mitotically active spermatogonia. In the case of Apert syndrome, for example, there is good reason to believe that the causative mutation  arises in spermatogonia and is selectively retained in the germinal epithelium because the mutant germ cells enjoy an unspecified selective advantage [However, the replication-error hypothesis does not hold for all dominant genetic mutations; achondroplasia being a particular case-in-point. By taking sperm DNA from donors of different ages, Tiemann-Boege and colleagues  have exaIn order for the premutational lesion hypothesis to account for a mutation that is present in every cell of the body, rather than a mosaic, this putative aberrant repair would have to precede the S phase of the first mitotic division. The oocyte is well endowed with poorly characterized enzymes for effecting DNA repair ,8, incluOf course, aberrant repair of DNA damage in the oocyte could account for a wide variety of genetic aberrations in embryos generated from DNA damaged spermatozoa, not just point mutations. Thus, exposure of spermatozoa to xenobiotics or X-irradiation is known to induce dominant lethal effects (post implantation pregnancy loss) and heritable translocations in the embryos of mated females as a consequence of chromosome mutations (breaks and rearrangements) as well as specific locus mutations ,11 that As indicated above, the mutation seen in a vast majority of achondroplasia patients is a CG-AT transition in the FGFR3 gene. Since this is the most common base substitution observed following oxidative damage to DNA ,14, it i2O2, has been confirmed in a number of independent studies [2O2 to suppress tyrosine phosphatase activity, and partly on the ability of these molecules to stimulate cAMP generation by the soluble form of adenylyl cyclase (sAC) [Oxidative stress, and its role in the origins of male infertility was first appreciated in 1943, when the Scottish andrologist John MacLeod demonstrated that catalase could support the motility of human spermatozoa incubated under aerobic conditions . His expse (sAC) ,17. The se (sAC) , mouse [se (sAC)  and stalse (sAC) .This redox drive to capacitation involves a low, steady state level of ROS production. However, if, for any reason, this physiological rate of ROS generation should increase, or the spermatozoa should become exposed to exogenous ROS generated by, for example, infiltrating leukocytes, then a state of oxidative stress can be readily induced. Spermatozoa are particularly susceptible to such stress as a consequence of their high unsaturated fatty acid content and their limited store of antioxidant enzymes such as superoxide dismutase or glutathione peroxidase . In keepFollowing the initial report from Macleod , Aitken 2-•) [2-• production in phagocytic cells and in cell-free ROS-generating systems, such as xanthine plus xanthine oxidase [2-• in rat sperm suspensions isolated from the cauda epididymides [To further understand the biochemical basis of ROS generation in spermatozoa, attention soon focused on the use of other ROS-detecting probes apart from luminol. Owing to its sensitive nature, a popular reagent to use in this context was lucigenin. Lucigenin has been frequently deployed for the detection of ROS and is certainly capable of detecting superoxide anion (O2-•) ,30. For  oxidase . In the idymides . This siidymides . Althougidymides .Notwithstanding the fact that mitochondrial production of ROS in spermatozoa remains largely unexplored, Vernet et al.  generate2-• that then dismutates to H2O2 under the influence of intracellular superoxide dismutase , (ii) a reaction between the latter and ground state oxygen to produce O2-• and recycle the lucigenin back to its native state (L), (iii) and finally a reaction between LH+• and O2-•, generated as a result of the redox cycling of lucigenin, to produce a dioxetane that, in turn, decomposes with the generation of chemiluminescence  used to detect this activity. In a similar fashion, NADH-induced lucigenin-dependent chemiluminescence has been shown to be associated with another intracellular reductase, cytochrome b5-reductase (CYB5R) in spermatozoa [As a consequence of this chemistry, Ormatozoa . Thus, Crmatozoa ) or alte2-• production, it has also been argued that the reaction between LH+• and O2 is thermodynamically unlikely [2-• production.Although this general redox cycling concept Fig.  seems tounlikely  and thatunlikely  reflect 2-• generation, the diagnostic significance of this activity may still reside in its ability to reflect oxidative stress in human sperm populations [Even if NAD(P)H-induced lucigenin-dependent chemiluminescence does simply reflect the presence of oxidoreductases capable of initiating the redox cycling of the probe, rather than primary Oulations ,42,43. Tulations . These oulations ,28.An explanation for this association between excess cytoplasmic retention and ROS production has been put forward, based on the correlation between the latter and the cellular content of glucose-6-phosphoate dehydrogenase in human sperm suspensions . This en2-• and produce 2OH ethidium we have recently secured incontrovertible evidence that human spermatozoa do in fact generate this free radical species and that the cellular production of O2-• is significantly elevated in populations of defective sperm populations. Furthermore, the data we have generated to date suggests that this is not a consequence of defective mitochondrial function since a variety of mitochondrial inhibitors fail to disrupt the 2OH ethidium signal.Although this explanation is consistent with the data generated in numerous independent studies, it still has the status of an unproven hypothesis rather than an experimentally proven fact. Definitive evidence for the presence of NOX 5 in human spermatozoa has still not been obtained and we still await independent confirmation of the ability of NOX 5 to generate ROS in spermatozoa. It has even been questioned whether human spermatozoa produce ROS from any source. Using the ability of dihydroethidium to react with O2-• in human spermatozoa that is extra-mitochondrial. A number of possibilities exist in this context. First of all, the above-mentioned NADPH-oxidase, NOX5, originally identified by Banfi et al. in human testes [Such results suggest there must be a source of On testes ,45, has n testes . Althougn testes  employedIn addition to NADPH oxidase activity, it is also clear that spermatozoa will generate ROS when placed in contact with certain xenobiotics. Examples of such compounds include endocrine disruptors with estrogenic properties that are capable of inducing ROS production by male germ cells and initiating free radical-mediated DNA damage . Similar+/NADH couple. Further characterization of this putative electron transport chain is clearly warranted. However, this task will not be easy given that, by definition, the redox activity ascribed to such systems depends on the close interaction of several independent constituents, not on a single identifiable entity.Another potential source of ROS in human spermatozoa is a trans-plasma membrane oxidoreductase system that removes electrons from NAD(P)H on the cytoplasmic surface of the cell and transfers them to oxygen on the outer leaf of the plasma membrane via intermediate carriers such as ubiquinone. Evidence to support the existence of such systems in somatic cells, arises from the ability of the latter to reduce artificial membrane-impermeant electron acceptors such as potassium ferricyanide, in concert with the concomitant oxidation of cytosolic NADH . Interes2-•. Exogenous NAD(P)H will certainly reduce extracellular electron acceptors such as WST-1 in the presence of human spermatozoa [Another form of oxidase activity detected by Berridge and Tan ,55 is a rmatozoa . Moreovermatozoa ,55. Howermatozoa ,54,55.2O2-mediated attack on arginine [Nitric oxide (NO) is another oxygen free radical which is apparently generated by defective populations of human spermatozoa. NO is normally generated from L-arginine by three isoforms of nitric oxide synthase (NOS). Recent mouse knock out experiments indicate that the selective deletion of these NOS isoforms has no impact on the ability of the spermatozoa to achieve fertilization. In other words, NO does not appear to have a positive role to play in the generation of functional gametes . Howeverarginine .Clearly there are many potential sources of ROS in the male germ line. The task that now confronts us is to determine which of these multifarious sources are responsible for the oxidative stress observed in the spermatozoa of male patients.2-• and H2O2. This activity is of fundamental biological importance in regulating the signal transduction pathways that control sperm capacitation. However, excess exposure to ROS can lead to pathological damage to human spermatozoa curtailing their competence for fertilization and disrupting their genetic integrity. DNA damage in these cells appears to be largely oxidative and is associated with a wide variety of adverse outcomes including impaired conception rates, increased incidences of abortion and defects in the offspring, including childhood cancer and dominant genetic diseases such as achondroplasia. It is hypothesized that such effects in the F1 generation involve the aberrant repair of oxidative DNA damage in the newly fertilized zygote. The etiology of oxidative stress in the male germ line is being actively researched at the present time. While spermatozoa certainly generate ROS, the biochemical basis of this activity is uncertain and may be multifactorial. Errors of spermiogenesis associated with the retention of excess residual cytoplasm appear to be associated with oxidative stress as a consequence of enhanced ROS production by uncharacterised 'oxidases', plasma membrane electron transport chains or oxidoreductases capable of activating redox-cycling xenobiotics. Electron leakage from defective sperm mitochondria represent yet another potential source of oxygen radicals. Given the clinical significance of oxidative stress in human spermatozoa, resolving the biochemical basis of this condition is a high priority task for the future.Human spermatozoa are redox active cells that are capable of generating O


Electrical storm occurring in a patient with the Brugada syndrome is an exceptional but malignant and potentially lethal event. Efficient therapeutic solutions should be known and urgently applied because of the inability of usual antiarrhythmic means in preventing multiple recurrences of ventricular arrhythmias. Isoproterenol should be immediately infused while oral quinidine should be further administrated when isoproterenol is not effective. In case of failure of these therapeutic options, ablation of the triggering ventricular ectopies should be attempted. The Brugada syndrome is an inherited cardiac electrical disorder occurring in the absence of obvious structural heart disease, initially defined by the association of right bundle branch block, ST elevation in right precordial leads and sudden cardiac death related to polymorphic ventricular tachycardias .In Brugada syndrome, the prevalence of malignant ventricular arrhythmias varies from 5 % at two years follow-up in asymptomatic patients to 45 % in patients with previous cardiac arrest . While iTrue prevalence of electrical storm in Brugada syndrome has not been evaluated, such events having only been seldom reported in isolated case reports. To our knowledge, to date, only twelve such cases have been published -16..Although exceptional, electrical storm in Brugada syndrome is a major event with dramatic consequences ,10, leadCausal factors should be searched and immediately corrected, such as fever ,15,16, iApart from class 1A agents (see further) no antiarrhythmic drug has been shown to be effective in preventing recurrence of arrhythmia in Brugada syndrome  21], th, th21], Isoproterenol infusion at a sufficiant dosing, ranging usually from 0.1 to 1-3 μg/mn ,10,18,19Both Brugada electrocardiographic pattern and ventricular excitability or vulnerability are believed to be dependent on the sympathetic imbalance and on cardiac rate. Worsening of the electrophysiological conditions have been described when vagal tonus increases and/or heart rate decreases ,25. MajoCaL and restores the dome of epicardial action potentials, reducing the degree of local and transmural heterogeneicity  has has49] hAll patients were studied within 2 weeks of their arrhythmic storm and had been documented to have frequent VPB’s. The triggering role of VPB’s in the initiation of VF was observed by ambulatory monitoring or stored electrograms of the defibrillator. Premature beats in the Brugada syndrome were monomorphic in all, coming from the RVOT in three patients  and with left bundle branch block-superior axis morphology in one . The monomorphic VPB’s were first observed at the time of VF in 2 patients, whereas in 2 patients they had been documented together with a normal ECG 14 and 11 years before they triggered VF, preceding the apparition of ECG abnormalities. Exercise testing and isoproterenol infusion eliminated all premature beats, excluding catecholaminergic polymorphic ventricular tachycardia.Mapping and ablation was performed as previously described in patients with idiopathic VF . In the While catheter ablation techniques are emerging, the initial experiences with idiopathic VF, and latterly with VF related to repolarization disorders have provided important insights into the role of focal triggers from RVOT and the Purkinje system in the initiation of VF associated with a number of clinical substrates in humans. Reducing the incidence of VF with localized ablation may reduce defibrillation requirement and replacement and improve the patients’ quality of life. The excellent long-term results after successful ablation of these triggers has been confirmed utilizing the data-logging capabilities of defibrillators in many of these patients and is being achieved in several centers.


If breast cancer cells escape from the initial tumor site, they often travel to axillary lymph nodes (ALNs) in the armpit. Surgical removal and biopsy of axillary lymph nodes are routinely used to assess the stage of a patient's cancer, and prevent cancers that have spread into the lymph nodes from further metastasis. Because ALN dissections can cause uncomfortable chronic arm swelling, they are often preceded by a biopsy of the sentinel lymph node, the first node into which the tumor tissue drains. If sentinel lymph node biopsy reveals evidence of metastasis, surgeons then remove and dissect the ALNs. Lymph nodes are primarily examined for the presence of metastatic tumor cells, and, more recently, researchers are even searching for the presence of isolated tumor cells and breast cancer–associated gene expression patterns in local nodes.In light of increasing evidence that the immune system is perturbed both locally at the tumor site and systemically as the cancer progresses, Peter Lee and colleagues set out to study the state of the immune system in draining lymph nodes. They determined profiles of CD4 and CD8 lymphocytes and dendritic cells from sentinel node biopsies and (nonsentinel) axillary lymph node dissections. Their results suggest that these immune profiles harbor independent information about the likelihood of tumor recurrence.The researchers used automated high-resolution imaging to determine the numbers of CD4 T cells, CD8 T cells, and CD1a-positive dendritic cells in 47 sentinel and 104 axillary nodes from 77 patients with breast cancer. Five-year follow-up data were available for all patients, and 33 patients had disease recurrence within that time. The researchers found that sentinel and axillary nodes from cancer patients (whether the nodes contained tumor cells or were tumor free) contained fewer CD4 and CD8 T lymphocytes than nodes from cancer-free control patients. Dendritic cells were also reduced in tumor-positive nodes, but increased in tumor-free axillary nodes. By dividing the patients into a “training set” of 29 individuals and a “test set” of 48 individuals, the researchers could test whether the correlations found in the test set could predict disease-free survival. They found that axillary node CD4 T cell and dendritic cell numbers, regardless of tumor status, were correlated with disease-free survival, but that this was not the case for immune parameters in the sentinel nodes . Moreover, in their dataset, the predictive power of the immune parameters in the axillary nodes was better than that of any other characteristics of the patients, including pathological parameters such as tumor size and extent or size of nodal metastases.These results suggest that, in patients with tumor-positive sentinel nodes, immune profile data from axillary nodes hold additional information on the probability of disease recurrence. As the authors suggest, these results warrant larger prospective studies to test these relationships and explore them in more detail. Another important open question is whether immune profile information from lymph nodes can predict risk of recurrence even in women whose cancers are caught at a stage where they have not yet spread to any lymph nodes.