Actinidia DRM1 : an intrinsically disordered protein whose mRNA expression is inversely correlated with spring budbreak in Kiwifruit

Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

Apple skin patterning is associated with differential expression of MYb10

Background Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.

Characterising transiting exoplanets by way of differential photometry

This paper describes a simple activity for plotting and characterising the light curve from an exoplanet transit event by way of differential photometry analysis. Using free digital imaging software, participants analyse a series of telescope images with the goal of calculating various exoplanet parameters, including its size, orbital radius and habitability. The activity has been designed for a high-school or undergraduate university level and introduces fundamental concepts in astrophysics and an understanding of the basis for exoplanetary science, the transit method and digital photometry.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17β-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17β-estradiol was withdrawn. Stimulation of invasiveness with 17β-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.

On display : Ravensthorpe Wildflower Show and the assembly of place

Each September since 1983 in the rural Shire of Ravensthorpe, Western Australia, volunteers collect samples of up to 700 wildfl ower species which are then displayed in the Ravensthorpe Senior Citizens Centre from 9.00 am to 4.00 pm daily over a two-week period. This chapter offers an ethnographic interpretation of this enduring annual event focusing on the 25th show held in 2007. The study contributes to understanding the complex and nuanced role of local wildflower shows in shaping and supporting rural senses of place and of community. Importantly, this particular type of festival, and more specifically this local instance, foregrounds a less-remarked aspect of festivals, namely the (re)production and celebration of place-specific knowledge through validations of, and interconnections between, scientific flower classification and emotive experience. This feature, encapsulated in Laurel Lamperd’s poem above, invites consideration of the ways in which local place knowledge and the simultaneous (re)production of ‘place’ are constituted by a complex layering of rational, objective ways of knowing and those which emphasize emotions, aesthetics and memories. This rural wildflower show not only mobilises both the rational and the emotional in ‘making sense of the world’ for local residents and for tourists, but also offers insights into the production of place as constituted in and through relations between humans and non-human life forms (Cloke & Jones, 2001; Conradson, 2005; see also Chapter 6).

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP- 2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c- ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT- derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin- positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to he regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA- MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

Hip and knee kinematics display complex and time-varying sagittal kinematics during repetitive stepping: Implications for design of a functional fatigue model of the knee extensors and flexors

The validity of fatigue protocols involving multi-joint movements, such as stepping, has yet to be clearly defined. Although surface electromyography can monitor the fatigue state of individual muscles, the effects of joint angle and velocity variation on signal parameters are well established. Therefore, the aims of this study were to i) describe sagittal hip and knee kinematics during repetitive stepping ii) identify periods of high inter-trial variability and iii) determine within-test reliability of hip and knee kinematic profiles. A group of healthy men (N = 15) ascended and descended from a knee-high platform wearing a weighted vest (10%BW) for 50 consecutive trials. The hip and knee underwent rapid flexion and extension during step ascent and descent. Variability of hip and knee velocity peaked between 20-40% of the ascent phase and 80-100% of the descent. Significant (p<0.05) reductions in joint range of motion and peak velocity during step ascent were observed, while peak flexion velocity increased during descent. Healthy individuals use complex hip and knee motion to negotiate a knee-high step with kinematic patterns varying across multiple repetitions. These findings have important implications for future studies intending to use repetitive stepping as a fatigue model for the knee extensors and flexors.

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.

High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Differential effects of aging on spatial learning through exploratory navigation and map reading

It has been shown that abilities in spatial learning and memory are adversely affected by aging. The present study was conducted to investigate whether increasing age has equal consequences for all types of spatial learning or impacts certain types of spatial learning selectively. Specifically, two major types of spatial learning, exploratory navigation and map reading, were contrasted. By combining a neuroimaging finding that the medial temporal lobe (MTL) is especially important for exploratory navigation and a neurological finding that the MTL is susceptible to age-related atrophy, it was hypothesized that spatial learning through exploratory navigation would exhibit a greater decline in later life than spatial learning through map reading. In an experiment, young and senior participants learned locations of landmarks in virtual environments either by navigating in them in the first-person perspective or by seeing aerial views of the environments. Results showed that senior participants acquired less accurate memories of the layouts of landmarks than young participants when they navigated in the environments, but the two groups did not differ in spatial learning performance when they viewed the environments from the aerial perspective. These results suggest that spatial learning through exploratory navigation is particularly vulnerable to adverse effects of aging, whereas elderly adults may be able to maintain their map reading skills relatively well.