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1.
Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems. 相似文献
2.
Susan K. Morton Daniel J. Chaston Brett K. Baillie Caryl E. Hill Klaus I. Matthaei 《PloS one》2014,9(4)
Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacIR, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacIR protein. 相似文献
3.
Frank Baumann Jens Pahnke Ivan Radovanovic Thomas Rülicke Juliane Bremer Markus Tolnay Adriano Aguzzi 《PloS one》2009,4(9)
The prion consists essentially of PrPSc, a misfolded and aggregated conformer of the cellular protein PrPC. Whereas PrPC deficient mice are clinically healthy, expression of PrPC variants lacking its central domain (PrPΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrPC (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrPΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrPΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrPΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants. 相似文献
4.
Xunlong Shi Jianjun Yang Haiyan Zhu Li Ye Meiqing Feng Jiyang Li Hai Huang Qun Tao Dan Ye Lee-Hwei K. Sun Bill N. C. Sun Cecily R. Y. Sun Guizhen Han Yuanyuan Liu Minghui Yao Pei Zhou Dianwen Ju 《PloS one》2013,8(8)
In this study, the in vivo pharmacokinetics and pharmacodynamics
of a novel recombinant human erythropoietin (rhEPO) Fc fusion protein, rhEPO-Fc,
were studied in both rodents and rhesus monkeys. Animal models of anemia induced
by irradiation, cyclophosphamide and partial renal ablation were used to
evaluate therapeutic effects of rhEPO-Fc. We have demonstrated that serum
half-life of rhEPO-Fc was 29.5 to 38.9 h at doses of 8, 25, 80 µg/kg in
rhesus monkeys and 35.5 to 43.5 h at doses of 16, 50, 160 µg/kg in rats.
In anemia animal models, rhEPO-Fc dose-dependently (7.5–30.0 µg/kg
in mice, 5.4–21.4 µg/kg in rats and 5.0–10.0 µg/kg in
rhesus monkeys) increased reticulocyte level, followed by an increase of RBC
count, hemoglobin and hematocrit levels. At reduced intervention frequency of
weekly treatments, rhEPO-Fc showed similar hematopoietic effects as compared
with rhEPO given three times a week. These results indicated that rhEPO-Fc could
potentially be used in treatment of anemia and warrants future clinical
trials. 相似文献
5.
Hye-Rim Lee Han-Jun Kim Ji-Seung Ko Yong-Suk Choi Myun-Whan Ahn Sukyoung Kim Sun Hee Do 《PloS one》2013,8(12)
Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation. 相似文献
6.
7.
Gy?rgy Fejer Lisa Drechsel Jan Liese Ulrike Schleicher Zsolt Ruzsics Nicola Imelli Urs F. Greber Simone Keck Bernd Hildenbrand Anne Krug Christian Bogdan Marina A. Freudenberg 《PLoS pathogens》2008,4(11)
The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease. 相似文献
8.
Peter V. Hauser Jeffrey W. Pippin Cora Kaiser Ronald D. Krofft Paul T. Brinkkoetter Kelly L. Hudkins Dontscho Kerjaschki Jochen Reiser Charles E. Alpers Stuart J. Shankland 《PloS one》2010,5(3)
Podocytes are injured in several glomerular diseases. To alter gene expression specifically in podocytes in vivo, we took advantage of their active endocytotic machinery and developed a method for the targeted delivery of small interfering ribonucleic acids (siRNA). We generated an anti-mouse podocyte antibody that binds to rat and mouse podocytes in vivo. The polyclonal IgG antibody was cleaved into monovalent fragments, while preserving the antigen recognition sites. One Neutravidin molecule was linked to each monovalent IgG via the available sulfohydryl group. Protamine, a polycationic nuclear protein and universal adaptor for anionic siRNA, was linked to the neutravidin via biotin. The delivery system was named shamporter (sheep anti mouse podocyte transporter). Injection of shamporter coupled with either nephrin siRNA or TRPC6 siRNA via tail vein into normal rats substantially reduced the protein levels of nephrin or TRPC6 respectively, measured by western blot analysis and immunostaining. The effect was target specific because other podocyte-specific genes remained unchanged. Shamporter + nephrin siRNA induced transient proteinuria in rats. Control rats injected with shamporter coupled to control-siRNA showed no changes. These results show for the first time that siRNA can be delivered efficiently and specifically to podocytes in vivo using an antibody-delivery system. 相似文献
9.
Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression. 相似文献
10.
Ole S. S?gaard Mette E. Graversen Steffen Leth Rikke Olesen Christel R. Brinkmann Sara K. Nissen Anne Sofie Kjaer Mariane H. Schleimann Paul W. Denton William J. Hey-Cunningham Kersten K. Koelsch Giuseppe Pantaleo Kim Krogsgaard Maja Sommerfelt Remi Fromentin Nicolas Chomont Thomas A. Rasmussen Lars ?stergaard Martin Tolstrup 《PLoS pathogens》2015,11(9)
11.
Liver transplants have their highest technical failure rate in the first two weeks following surgery. Currently, there are limited devices for continuous, real-time monitoring of the graft. In this work, a three wavelengths system is presented that combines near-infrared spectroscopy and photoplethysmography with a processing method that can uniquely measure and separate the venous and arterial oxygen contributions. This strategy allows for the quantification of tissue oxygen consumption used to study hepatic metabolic activity and to relate it to tissue stress. The sensor is battery operated and communicates wirelessly with a data acquisition computer which provides the possibility of implantation provided sufficient miniaturization. In two in vivo porcine studies, the sensor tracked perfusion changes in hepatic tissue during vascular occlusions with a root mean square error (RMSE) of 0.135 mL/min/g of tissue. We show the possibility of using the pulsatile wave to measure the arterial oxygen saturation similar to pulse oximetry. The signal is also used to extract the venous oxygen saturation from the direct current (DC) levels. Arterial and venous oxygen saturation changes were measured with an RMSE of 2.19% and 1.39% respectively when no vascular occlusions were induced. This error increased to 2.82% and 3.83% when vascular occlusions were induced during hypoxia. These errors are similar to the resolution of a commercial oximetry catheter used as a reference. This work is the first realization of a wireless optical sensor for continuous monitoring of hepatic hemodynamics. 相似文献
12.
Background
Although APP and its proteolytic metabolites have been well examined in the central nervous system, there remains limited information of their functions outside of the brain. For example, amyloid precursor protein (APP) and amyloid beta (Aβ) immunoreactivity have both been demonstrated in intestinal epithelial cells. Based upon the critical role of these cells in absorption and secretion, we sought to determine whether APP or its metabolite amyloid β (Aβ), had a definable function in these cells.Methodology/Principal Findings
The human colonic epithelial cell line, Caco-2 cells, were cultured to examine APP expression and Aβ secretion, uptake, and stimulation. Similar to human colonic epithelium stains, Caco-2 cells expressed APP. They also secreted Aβ 1-40 and Aβ 1-42, with LPS stimulating higher concentrations of Aβ 1-40 secretion. The cells also responded to Aβ 1-40 stimulation by increasing IL-6 cytokine secretion and decreasing cholesterol uptake. Conversely, stimulation with a sAPP-derived peptide increased cholesterol uptake. APP was associated with CD36 but not FATP4 in co-IP pull down experiments from the Caco-2 cells. Moreover, stimulation of APP with an agonist antibody acutely decreased CD36-mediated cholesterol uptake.Conclusions/Significance
APP exists as part of a multi-protein complex with CD36 in human colonic epithelial cells where its proteolytic fragments have complex, reciprocal roles in regulating cholesterol uptake. A biologically active peptide fragment from the N-terminal derived, sAPP, potentiated cholesterol uptake while the β secretase generated product, Aβ1-40, attenuated it. These data suggest that APP is important in regulating intestinal cholesterol uptake in a fashion dependent upon specific proteolytic pathways. Moreover, this biology may be applicable to cells beyond the gastrointestinal tract. 相似文献13.
Pascale Hermant Céline Demarez Tanel Mahlak?iv Peter Staeheli Philip Meuleman Thomas Michiels 《PloS one》2014,9(1)
The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-λ) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-λ. In contrast, the IFN-λ response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-λ response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-λ. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-λ response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-λ. The lack of mouse hepatocyte response to IFN-λ was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-λ. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-λ in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-λ in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment. 相似文献
14.
Jeehee Kim Aurora Badaloni Torsten Willert Ursula Zimber-Strobl Ralf Kühn Wolfgang Wurst Matthias Kieslinger 《PloS one》2013,8(11)
Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo. 相似文献
15.
The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate. During multi-fork replication severe under-initiation occurred in cells lacking the Fis protein; the cells initiated at an elevated mass, had fewer origins per cell and the origins were not initiated in synchrony. These results suggest a positive role for the Fis protein in the initiation of replication. 相似文献
16.
Henk Koning Antoon J. M. van Oosterhout Uilke Brouwer Lisette E. den Boef Renée Gras Marjan Reinders-Luinge Corry-Anke Brandsma Marco van der Toorn Machteld N. Hylkema Brigitte W. M. Willemse Ian Sayers Gerard H. Koppelman Martijn C. Nawijn 《PloS one》2014,9(7)
Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo. 相似文献
17.
Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo. 相似文献
18.
Matthias Gralle Michelle Gralle Botelho Fred S. Wouters 《The Journal of biological chemistry》2009,284(22):15016-15025
The amyloid precursor protein (APP) is implied both in cell growth and
differentiation and in neurodegenerative processes in Alzheimer disease.
Regulated proteolysis of APP generates biologically active fragments such as
the neuroprotective secreted ectodomain sAPPα and the neurotoxic
β-amyloid peptide. Furthermore, it has been suggested that the intact
transmembrane APP plays a signaling role, which might be important for both
normal synaptic plasticity and neuronal dysfunction in dementia. To understand
APP signaling, we tracked single molecules of APP using quantum dots and
quantitated APP homodimerization using fluorescence lifetime imaging
microscopy for the detection of Förster resonance energy transfer in
living neuroblastoma cells. Using selective labeling with synthetic
fluorophores, we show that the dimerization of APP is considerably higher at
the plasma membrane than in intracellular membranes. Heparan sulfate
significantly contributes to the almost complete dimerization of APP at the
plasma membrane. Importantly, this technique for the first time structurally
defines the initiation of APP signaling by binding of a relevant physiological
extracellular ligand; our results indicate APP as receptor for neuroprotective
sAPPα, as sAPPα binding disrupts APP dimers, and this disruption
of APP dimers by sAPPα is necessary for the protection of neuroblastoma
cells against starvation-induced cell death. Only cells expressing reversibly
dimerized wild-type, but not covalently dimerized mutant APP are protected by
sAPPα. These findings suggest a potentially beneficial effect of
increasing sAPPα production or disrupting APP dimers for neuronal
survival.The amyloid precursor protein
(APP)4 is known both
for its important role in the development and plasticity of the nervous system
(1–6)
and for its involvement in Alzheimer disease (AD)
(7,
8). Despite intensive research
efforts, the initial events that lead to the prevalent sporadic, i.e.
non-familial, forms of AD are still unclear. Furthermore, although a higher
gene dose of APP (9) or the
presence of pathological APP mutations is sufficient to induce familial AD
(for review, see Ref. 10), the
exact pathological mechanism that is triggered by APP is still under
debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are
thought to contribute to synaptic dysfunction and neurotoxicity
(11,
12). On the other hand, the
α-secretase-derived extracellular fragment of APP (sAPPα), which
is present at lower levels in AD patients than in controls
(13), has been shown to be
beneficial for memory function, to possess neuroprotective properties, and to
counteract the effects of Aβ
(14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration
in AD
(19–24);
however, the signal transduction pathway for transmembrane APP remains
unknown, although several potential regulatory proteins, glycosaminoglycans,
and metal ions are known to bind with high affinity to APP and sAPPα
(25,
26). The most common form of
signal transduction for single-pass transmembrane proteins is the
ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the
dimerization of transmembrane APP has been implied several times in the past.
Several studies have investigated the effects of presumed dimer-breaking
perturbations on biological read-outs, such as the production of Aβ
(27,
28), but without directly
measuring the APP aggregation state, or have investigated the aggregation
state of APP subdomains, often reconstituted in cell-free systems
(27–32).
Dimerization interfaces in both the extracellular and the transmembrane domain
have been suggested.In the studies investigating the aggregation state of full-length APP, most
of the employed methods, such as chemical cross-linking and
co-immunoprecipitation, do not lend themselves readily to a rigorous
quantitative analysis of the abundance of potentially instable dimers
(31,
33), whereas in other cases
the use of chimeras may have influenced the dimerization potential or
precluded the search for a natural stimulus
(23,
34). The only previously
reported direct observation of APP dimerization by Förster resonance
energy transfer (FRET) microscopy uses an assay in which the FRET efficiency
varies with the level of overexpression
(35). Therefore, a
concentration-dependent FRET component due to nonspecific stochastic
encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the
selective detection of APP dimers on the surface of live cells, where they
would encounter ligands, they could not differentiate between subpopulations
of APP. This may be one reason why no natural ligand of APP has ever been
shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective
sAPPα
(36–39).
The ligand-dependent dimerization of sAPPα in solution
(40) and its origination from
transmembrane APP suggest that APP might serve as receptor for sAPPα,
but this binding has never been experimentally shown. 相似文献
19.
Nagarjun V. Konduru Yulia Y. Tyurina Weihong Feng Liana V. Basova Natalia A. Belikova Hülya Bayir Katherine Clark Marc Rubin Donna Stolz Helen Vallhov Annika Scheynius Erika Witasp Bengt Fadeel Padmakar D. Kichambare Alexander Star Elena R. Kisin Ashley R. Murray Anna A. Shvedova Valerian E. Kagan 《PloS one》2009,4(2)
Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells. 相似文献
20.
John A. Gaynes Hideo Otsuna Douglas S. Campbell John P. Manfredi Edward M. Levine Chi-Bin Chien 《PloS one》2015,10(9)
Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3’UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development. 相似文献