16,864 research outputs found
Regulation of breast tumour cell survival by AP-2 transcription factors
No full textPhDAP-2 transcription factors are crucial regulators of embryonic development and also
play important roles in human neoplasia. Over-expression of AP-2α and AP-2γ in
primary breast cancer (BC) correlates with expression of two major breast markers,
ERBB2 and oestrogen receptor. High AP-2γ expression is associated with reduced
survival in BC patients, including those treated with hormone therapy. Our aim is to
define the pathways regulated by AP-2 factors in breast epithelial cells. Data from
AP-2γ depleted MCF-7 cells suggested a role in cell cycle control. Here, regulation
by AP-2α and AP-2γ in additional breast cancer cell lines with differing genetic
background is investigated. Cell cycle analysis of synchronized T47D cells, which
express both AP-2 isoforms but mutant p53, showed a reduction in G1 but increased
S and G2/M-phase populations when AP-2α and AP-2γ were silenced either
independently or together. Despite the lack of growth arrest, p21cip protein levels
increased following AP-2 silencing. ChIP analysis showed AP-2α and AP-2γ binding
at the p21cip/CDKN1A promoter. In addition, cyclin D3 protein levels increased
following AP-2 silencing and ChIP analysis showed AP-2α and AP-2γ binding to its
promoter. Luciferase reporter constructs carrying CCND3 promoter sequences were
repressed when co-transfected with AP-2α or AP-2γ expression constructs. These
findings demonstrate the importance of AP-2 factors in the control of cell cycle
regulation but illustrate cell-type differences in their mode of action. Further work
focused on MCF10A immortalised breast epithelial cells, which express both AP-2
isoforms and wild-type p53. AP-2 silenced MCF10A cells adopted a more rounded
phenotype suggestive of changes in cell adhesion which was also supported by
microarray analysis of their gene expression profile.
KIAA1324, a protein of unknown function with features of a membrane-bound
growth factor receptor, was found to be significantly down-regulated following AP-
2γ silencing in MCF-7 cells. Functional assays using i) inducible knock down of
KIAA1324 in MCF-7 cells, and ii) stable KIAA1324 overexpression in MCF10A
investigated if altering KIAA1324 expression level could affect cell growth.
KIAA1324 did not affect breast epithelial cell proliferation on plastic but may
contribute to the ability of MCF-7 cells to display anchorage-independent growth
A role for amphiphysin in AP-1/clathrin coat formation
No full textTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. Clathrin, in combination with different adaptor proteins, is the major coat protein for vesicle formation at the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized is the formation of clathrin coats for endocytosis at the plasma membrane involving the adaptor protein complex AP-2. Clathrin and AP-2 were shown to be at the centre of a complex interactome of proteins accessory to vesicle formation. Considerably less is known about the formation of clathrin coated carriers at the TGN and endosomes, where the adaptor protein complex AP-1 plays a major role.
In vitro studies showed the minimal requirements for association of AP-1 to liposomal membranes to be activated ARF1, phosphoinositides, and either sorting signals or unknown cytosolic factors. We have used a liposome floatation assay to identify cytosolic proteins collaborating with AP-1 at the membrane. Separation of proteins from bovine brain cytosol with several chromatographic methods yielded an active fraction containing amphiphysin 1, amphiphysin 2, and endophilin A1. All three proteins are expressed in brain and known to be involved in AP-2/clathrin coat formation. They consist of an N-terminal N-BAR (Bin, amphiphysin, Rvs) domain for dimerization and membrane binding and a C-terminal SH3 (Src homology 3) domain for interaction with dynamin and synaptojanin. Amphiphysin 1 and 2 in addition contain a middle domain with binding sites for adaptors and clathrin. It was proposed that amphiphysins and endophilin are targeted to membranes with high curvature, such as the neck of a forming vesicle, where they recruit dynamin and synaptojanin in preparation for vesicle fission and uncoating.
In this thesis, I bacterially expressed and purified all three proteins and tested them in the floatation assay for AP-1 membrane binding activity. Only amphiphysin 2 showed activity, both as a homodimer and as a heterodimer with amphiphysin 1. Activity depended on a motif that was shown to bind to AP-1, AP-2, and clathrin in GST pull-down experiments.
Endogenous amphiphysins in primary neurons, as well as transiently expressed in neuronal or fibroblast cell lines, co-localized with AP-1 at the TGN. In addition, when expressed at high levels in neuronal cells, amphiphysins aggregated and interfered dominantly with the TGN localization of AP-1. Both phenomena depended on the presence of the clathrin and adaptor interaction sequence in the amphiphysins. Furthermore, both amphiphysins could be cross-linked to AP-1 in vivo.
Our results indicate that amphiphysin 1 and 2 function not only in clathrin coated vesicle formation for endocytosis at the plasma membrane, but are also part of the machinery forming AP-1/clathrin coats at the TGN and endosomes. This suggests that the machineries for CCV formation with AP-1 and AP-2 at different locations in the cell share more components than previously anticipated
A cytosolic factor mediating membrane recruitment of AP-1 clathrin adaptors
No full textTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. These vesicles are formed through the recruitment of cytosolic coat proteins to the donor membrane that act as a scaffold to form coated buds and vesicles. At the same time they selectively concentrate cargo proteins by interacting with cytosolic signals. Clathrin, in combination with different adaptor proteins (APs), is the major coat protein for vesicle formation at the plasma membrane, endosomes and the trans-Golgi network. Best characterized is clathrin mediated endocytosis at the plasma membrane which involves AP-2 and a network of associated proteins. Much less is known about AP-1 mediated clathrin coated vesicle formation at the TGN/endosomes. In vitro studies demonstrated that the minimal requirements to recruit AP-1 to liposome membranes are activated Arf1, phosphoinositides, and either sorting signals or an unknown cytosolic factor. In order to identify this factor, we fractionated calf brain cytosol by several chromatographic methods. Fractions were tested for factor dependent AP-1 recruitment activity using an in vitro assay. Purification via ammonium sulfate precipitation, hydrophobic interaction chromatography, anion/cation exchange chromatography or hydroxyapatite chromatography produced a final fraction containing three major proteins: amphiphysin 1, amphiphysinand endophilin A1. All three proteins are known accessory factors in clathrin coated vesicle formation at the plasma membrane. Co-immunodepletion of amphiphysinandresulted in a strong reduction of AP-1 recruitment activity. Therefore we conclude that a heterodimer of amphiphysinandis the long searched for cytosolic factor, required to recruit AP-1 in the absence of sorting signals in vitro. Our results strongly suggest that amphiphysin 1, amphiphysinand endophilin A1 are also involved in AP-1 mediated clathrin coated vesicle formation at the TGN and endosomes in vivo
Characterizing the role of p21-Activated Kinase 3 (PAK3) in AP-1-induced transformation
No full textIncludes bibliographical references.Previous studies identified p21-Activated Kinase 3 (PAK3), a serine/threonine kinase, as a potential AP-1 target gene. PAK3 has been implicated in a variety of pathological disorders and over-expression of other PAK-family members has been linked to cancer. In this study, we investigated AP-1 regulation of PAK3 expression and the role of PAK3 in cJun/AP-1-associated cellular transformation. Our results showed elevated PAK3 expression at both the mRNA and protein level in cJun-over-expressing Rat1a fibroblasts, as well as in transformed human fibroblasts. Elevated PAK3 protein levels were also seen in cervical, ovarian, oesophageal and breast cancer cells lines, while poor survival tracked with high PAK3 expression in ovarian cancer patient material. Elevated PAK3 levels appear to play no role in the proliferation of transformed or cancerous cells, however appears vital for the transformed morphology and actin distribution. These cytoskeletal changes seem to be the underlying force governing cellular migration, as inhibition of PAK3 significantly reduced the motility of both transformed fibroblasts and cancer cell lines. Our data shows that elevated PAK3 expression in response to AP-1 over-expression is regulated through the transcriptional activation of the PAK3 promoter by AP-1 binding directly to a single site in the promoter. We also show that constitutive activation of PAK3 results in changes in cJun phosphorylation and an increase in AP-1 activity, which can be inhibited by a serine/threonine kinase inhibitor. PAK3 and AP-1 proteins were also shown to directly interact with each other. Our study is a first to describe a role for AP-1 in regulating PAK3 expression, and PAK3 in regulating AP-1 activity, identifying a potential feedback loop in which PAK3 is an AP-1 target required for cytoskeletal reorganization and migration observed in transformed cells
Investigation of a molecular mechanism for anesthetic preconditioning
No full textAnesthetic preconditioning (AP) is a potential treatment for patients undergoing procedures that are accompanied by a high risk for ischemic injury (e.g. perioperative stroke) and cannot be treated by anti-coagulants. AP is a phenomenon by which tissues are pretreated with clinical concentrations of a general anesthetic; this minor insult results in the upregulation of cellular protective measures, which defend against greater insults such as ischemia. Our lab has proposed that exposure to isoflurane increases nitric oxide (NO) levels, with microglia being the major producer of NO. This triggers a downstream increase of free Zn2+, which leads to the expression of the Zn2+-binding proteins, metallothioneins (MT). To confirm increases in NO and Zn2+, microglial and neuroblastoma cell lines were exposed to the volatile anesthetic, isoflurane, at clinical concentrations. Cells were shown to produce more NO after isoflurane exposure; however, results for changes in free [Zn2+] were inconclusive. Due to the proposed role in NO production, the function of microglia in AP was tested by depleting microglia from primary neuronal and glial dissociated cultures obtained from murine brain. Microglial depletion had no effect on preconditioning but did result in attenuated cell death by oxygen glucose deprivation (OGD). The exposure of exogenous MT-IIa to cultures prior to OGD also resulted in reduced cell death in wild-type male and female MT-knockout derived cultures. Finally, to understand whether MTs exhibited protection through an extracellular receptor-mediated pathway, cultures were exposed to RAP, a competitive inhibitor of MT on megalin receptors; preliminary results are inconclusive. In summary, experiments showed that isoflurane exposure increases the level of NO in microglia, though microglia are not essential for AP. MTs exhibit protection against in vitro ischemia, but it is unclear if this is through a receptor-mediated pathway. Further understanding of this mechanism could lead to more effective administration in a clinical setting
Increased Action Potential Firing Rates of Layer 2/3 Pyramidal Cells in the Prefrontal Cortex are Significantly Related to Cognitive Performance in Aged Monkeys
No full textThe neurobiological substrates of significant age-related deficits in higher cognitive abilities mediated by the prefrontal cortex (PFC) are unknown. To address this issue, whole-cell current-clamp recordings were used to compare the intrinsic membrane and action potential (AP) firing properties of layer 2/3 pyramidal cells in PFC slices from young and aged behaviorally characterized rhesus monkeys. Most aged subjects demonstrated impaired performance in Delayed Non-Match to Sample (DNMS) task acquisition, DNMS 2 min delay and the Delayed Recognition Span task. Resting membrane potential and membrane time constant did not differ in aged relative to young cells, but input resistance was significantly greater in aged cells. Single APs did not differ in terms of threshold, duration or rise time, but their amplitude and fall time were significantly decreased in aged cells. Repetitive AP firing rates were significantly increased in aged cells. Within the aged group, there was a U-shaped quadratic relationship between firing rate and performance on each behavioral task. Subjects who displayed either low or very high firing rates exhibited poor performance, while those who displayed intermediate firing rates exhibited relatively good performance. These data indicate that an increase in AP firing rate may be responsible, in part, for age-related PFC dysfunction
Indigenous views on the future of public archaeology in Australia. A conversation that did not happen
No full textThis paper was written in response to a request by the editors of the AP: Online Journal of Public Archaeology, Jaime Almansa Sánchez and Elena Papagiannopoulou, for Claire Smith to write on the future of public archaeology in Australia. In Australia, public archaeology focusses on high profile colonial sites such as The Rocks in Sydney (Karskens 1999) and Port Arthur in Tasmania (Steele et al. 2007; Frew 2012), tourism (e.g. Cole and Wallis 2019) or enhancing school curricula (Nichols et al. 2005; Owens and Steele 2005). However, given her decades-long relationships with Jawoyn and Ngadjuri people (Smith 1999; Smith et al. 2016; Smith et al. 2020), Claire Smith decided that a useful way of approaching this topic would be to obtain Indigenous views on the subject. Accordingly, she contacted the Aboriginal co-authors of this article and invited them to co-author the paper. The possibility to write in free form was a boon. The ‘conversation’ format we settled on was designed to facilitate the voices of individuals, to present a range of Indigenous views, to allow people to express their views frankly, and to deal with the constraints of people being located in different parts of Australia as well as occasional lock-downs due to COVID-19. We decided on five topics/questions that would be the basis of the conversation. Each Aboriginal author gave their views either by email or by phone. These views were interwoven into a ‘conversation’. The language has been edited lightly for clarity and to simulate a real-life conversation. The final text was approved by all authors.Full Tex
Magnetic resonance imaging after operative repair of achilles tendon rupture.
No full textWe performed a magnetic resonance imaging (MRI) study in 16 consecutive patients who had undergone open repair of a unilateral Achilles tendon rupture (ATR) at an average of 32.5 (SD 3.2) (range 29-36) months from the operation. We measured the widest antero-posterior diameter of the tendon, the longest distance between the insertion of the Achilles tendon on the calcaneum and the musculo-tendinous junction of the soleus muscle on the Achilles tendon, the distance between the insertion of the Achilles tendon on the calcaneum and the point of maximal width of the tendon. We also ascertained whether areas of altered signal were present in and around the tendon. The operated tendons were always significantly thicker than the non-operated ones. There was a non-significant trend for the other measurements to be greater in the operated tendons. In five patients, areas of dishomogeneous signal were present in the operated tendon. These areas were less than 25% of the antero-posterior diameter of the tendon, and were clinically silent. These findings probably represent normal features of long-term tendon healing following open repair of an ATR
Base Excision Repair of Oxidative Guanine Lesions in Nucleosomes Initiated by Formamidopyrimidine DNA Glycosylase
No full textDeoxyribonucleic acid (DNA) is constantly under attack from reactive oxygen species (ROS) from endogenous and exogenous sources, which can cause various forms of damage, including mutagenic damage that threatens genomic integrity. Oxidative DNA damage has been implicated in inflammation, carcinogenesis, neurodegenerative diseases, and aging. Of the four canonical nucleobases, guanine (G) has the lowest reduction potential and is most easily oxidized to the lesion 8oxoguanine (8oxoG), which has an even lower reduction potential and can be further oxidized to hydantoin lesions. One of these lesions is spiroiminodihydantoin (Sp), a chiral propeller-shaped lesion that can destabilize the DNA duplex and transversion mutations. Oxidative lesions and other non-bulky lesions can be repaired by the base excision repair (BER) pathway, which occurs in five core steps. Bifunctional glycosylases can perform the first two of these steps: extrusion and excision of the damaged nucleobase, leaving an abasic (AP) site, which is then incised by AP lyase activity to form a single-strand break (SSB). The bacterial bifunctional glycosylase formamidopyrimidine (Fpg) can excise both 8oxoG and Sp at comparable rates in free duplex (leippold, krishnamurthy). However, most DNA in cells is packaged in nucleosomes: 145-147 base pairs of DNA wrapped around an octameric histone protein core. Nucleosomes form the polymer chromatin, which condenses into chromosomes and compacts the genome. Nucleosome core particles (NCPs) pose a steric hindrances to glycosylases and decrease the efficiency of excision. It is therefore necessary to study the kinetics of glycosylase activity in nucleosomes to better understand lesion excision in physiological conditions. This project’s eventual goal is to determine the rate of excision of Sp by Fpg in nucleosomes using an Fpg glycosylase assay. The extent of reaction can be visualized and quantified using polyacrylamide gel electrophoresis (PAGE). But in order to form nucleosomes, it is necessary to synthesize and purify strands of DNA long enough to wrap around a histone core
The Treatment of Ties in AP Correlation
No full textThe Kendall tau and AP correlation coefficients are very commonly use to compare two rankings over the same set of items. Even though Kendall tau was originally defined assuming that there are no ties in the rankings, two alternative versions were soon developed to account for ties in two different scenarios: measure the accuracy of an observer with respect to a true and objective ranking, and measure the agreement between two observers in the absence of a true ranking. These two variants prove useful in cases where ties are possible in either ranking, and may indeed result in very different scores. AP correlation was devised to incorporate a top-heaviness component into Kendall tau, penalizing more heavily if differences occur between items at the top of the rankings, making it a very compelling coefficient in Information Retrieval settings. However, the treatment of ties in AP correlation remains an open problem. In this paper we fill this gap, providing closed analytical formulations of AP correlation under the two scenarios of ties contemplated in Kendall tau. In addition,we developed an R package that implements these coefficients.Best Short Paper Accepted author manuscriptMultimedia ComputingWeb Information System
- …
