7,901 research outputs found
Oral History Interview with Billy Jackson, June 17, 2005
No full textThe National Museum of the Pacific War presents on oral interview with Billy Jackson. Jackson joined the Navy in August, 1944 and was assigned aboard USS Fergus (AP-82). Jackson recalls hauling troops to various destinations in the Pacific and returning to the US with a load of former prisoners of war
Oral History Interview with Billy Jackson, June 17, 2005
No full textThe National Museum of the Pacific War presents on oral interview with Billy Jackson. Jackson joined the Navy in August, 1944 and was assigned aboard USS Fergus (AP-82). Jackson recalls hauling troops to various destinations in the Pacific and returning to the US with a load of former prisoners of war
Transcription Factor AP-2 Regulatory Signatures in Breast Cancer
No full textPhDAP-2 transcription factors are highly conserved basic helix-span-helix proteins whose
members ((x, ß, y, S and c) are crucial regulators of bryonic development. They also
play an important role in human neoplasia. uohis ochemical studies have detected
high levels of AP-2y expression in primary tumo of breast cancer patients. This high
expression has been correlated with reduced survival in all patients and reduced survival
in an ERa positive subset treated with hormone therapy. In breast cancer cell lines, AP-
2 factors have been implicated in the regulation of the ERBB2 proto-oncogene and ERa.
In an effort to further understand the role of AP-2y in breast carcinoma, this study has
sought to identify additional AP-2 activated cellular pathways and ultimately novel
transcriptional targets for AP-2 through the use of gene expression profiling.
RNAi using three independent AP-2y targeting sequences, has been used to deplete AP-
2y levels in the ERa positive MCF-7 breast carcinoma cell line, chosen as it exclusively
expresses the AP-2y family member. Microarrays were then utilised to create an AP-2y
dependent transcription profile. Statistical comparisons between non-silencing control
siRNA and AP-2y targeting siRNA groups identified a total of 162 gene expression
changes (p<0.01). These changes implicate AP-2y in the control of cell cycle
progression and developmental signalling. Indeed a role for AP-2y in the control of cell
cycle, in particular at the GUS transition, has been verified using flow cytometry.
Several of these gene expression changes, including IGFBP3, Transgelin and
KIAA1324, have been confirmed using qPCR and immunoblotting.
Finally, elevated levels of p21 mRNA and protein have been observed following AP-2y
silencing in MCF-7 cells. Additionally, the activity of a p21 promoter reporter is
repressed following transfection with an AP-2y expression construct in HepG2 cells.
These results coupled with ChIP experiments showing AP-2y occupancy at the proximal
promoter region of p21 in cycling MCF-7 cells, implicate AP-2y in the repression of
p21 transcription and suggest a role for AP2y in- the, control of cell cycle in breast
carcinoma in part through the transcriptional repression of p21
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
Recruitment of AP-1 clathrin adaptors to liposomal membranes
No full textProtein and membrane traffic between organelles within the endocytic and exocytic pathway is mediated most prominently by coated vesicles. These vesicles are formed by the assembly of cytosolic coat proteins onto the donor membrane, which deform it into a bud so that vesicles can pinch off. Clathrin with its associated adaptors, COPI and COPII are the three major coats. Various in vitro studies allowed insight into the mechanism of coat formation. COPI and COPII vesicle budding from chemically defined liposomes has been reconstituted in vitro, using pure coat compounds. Further, it has been demonstrated that cargo is sorted into these vesicles. The mechanism of clathrin-coated vesicle formation appears to be more complicated. The AP-1 clathrin adaptor is involved in vesicle formation at the transGolgi network and endosomes. This work presents an in vitro assay where AP-1 is recruited to peptidoliposomes, presenting covalently linked peptides corresponding to sorting signals. In this system, AP-1 recruitment depends on myristyolated ADP-ribosylation factor(ARF1), GTP or GMP-PNP, tyrosine signals and a small amount of phosphoinositides, most prominently phosphatidyl inositol 4,5bisphosphate. In such a minimal system AP-1 is recruited as a highmolecular weight complex indicating the formation of a precoat in the absence of clathrin. GTP hydrolysis, induced by ARF GTPase-activating protein(ARFGAP1), disassembled this complex. Further, AP-1 is able to enhance the GAP activity of ARFGAP1 on myristoylated ARF1, suggesting a regulatory function of GTP hydrolysis in early steps of coat recruitment. This work provides insights into the mechanism of AP-1 clathrin coat formation which might also be used to investigate the recruitment of other coats
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
Blurry terminal
No full textBlurry photograph of people waiting in line at a service desk in a travel terminal, like an airport. About twenty-one people are shown waiting with their luggage and coats under florescent lights. Handwritten caption by J.B. Jackson reads: "AP L.V."
Donkey in a Lion's Cage
No full textAndrew O'Keefe (Producer); Siobhan Jackson (Writer / Director); Tuuli Forward (Producer
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
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