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51.
Askoxylakis V Garcia-Boy R Rana S Krämer S Hebling U Mier W Altmann A Markert A Debus J Haberkorn U 《PloS one》2010,5(12):e15962
Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.
Methods
Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.Results
In vitro binding experiments of 125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.Conclusions
These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX. 相似文献52.
Emily M. Duncan Annette C. Broderick Wayne J. Fuller Tamara S. Galloway Matthew H. Godfrey Mark Hamann Colin J. Limpus Penelope K. Lindeque Andrew G. Mayes Lucy C. M. Omeyer David Santillo Robin T. E. Snape Brendan J. Godley 《Global Change Biology》2019,25(2):744-752
Despite concerns regarding the environmental impacts of microplastics, knowledge of the incidence and levels of synthetic particles in large marine vertebrates is lacking. Here, we utilize an optimized enzymatic digestion methodology, previously developed for zooplankton, to explore whether synthetic particles could be isolated from marine turtle ingesta. We report the presence of synthetic particles in every turtle subjected to investigation (n = 102) which included individuals from all seven species of marine turtle, sampled from three ocean basins (Atlantic [ATL]: n = 30, four species; Mediterranean (MED): n = 56, two species; Pacific (PAC): n = 16, five species). Most particles (n = 811) were fibres (ATL: 77.1% MED: 85.3% PAC: 64.8%) with blue and black being the dominant colours. In lesser quantities were fragments (ATL: 22.9%: MED: 14.7% PAC: 20.2%) and microbeads (4.8%; PAC only; to our knowledge the first isolation of microbeads from marine megavertebrates). Fourier transform infrared spectroscopy (FT‐IR) of a subsample of particles (n = 169) showed a range of synthetic materials such as elastomers (MED: 61.2%; PAC: 3.4%), thermoplastics (ATL: 36.8%: MED: 20.7% PAC: 27.7%) and synthetic regenerated cellulosic fibres (SRCF; ATL: 63.2%: MED: 5.8% PAC: 68.9%). Synthetic particles being isolated from species occupying different trophic levels suggest the possibility of multiple ingestion pathways. These include exposure from polluted seawater and sediments and/or additional trophic transfer from contaminated prey/forage items. We assess the likelihood that microplastic ingestion presents a significant conservation problem at current levels compared to other anthropogenic threats. 相似文献
53.
Megan L. Steele Stacey Fuller Annette E. Maczurek Cindy Kersaitis Lezanne Ooi Gerald Münch 《Cellular and molecular neurobiology》2013,33(1):19-30
Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD. 相似文献
54.
The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage
is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible PBAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked
immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of
the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility
of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide
expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage
display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides
close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 109 unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the
successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides
shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9. 相似文献
55.
Kurt W. Possinger Annette Staebler Sophia Sgouropoulou Peter J. Langecker Till C. Lorenz Elisabeth Doischer Wolfgang Wilmanns 《Steroids》1987,50(4-6):651-652
In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [3H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [3H]water. The extremely low contamination of the water soluble phase by [1ß-3H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase. 相似文献
56.
Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels 总被引:9,自引:0,他引:9 下载免费PDF全文
Deborah S. Knutzon Thomas R. Hayes Annette Wyrick Hui Xiong H. Maelor Davies Toni A. Voelker 《Plant physiology》1999,120(3):739-746
Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels. 相似文献
57.
A Sørensen BK Ahring M Lübeck W Ubhayasekera KS Bruno DE Culley PS Lübeck 《Canadian journal of microbiology》2012,58(9):1035-1046
The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.?saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.?saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50?°C, and at 60?°C it had a half-life of approximately 6?h. 相似文献
58.
Dorina Zphel Adrian Angenendt Lea Kaschek Keerthana Ravichandran Chantal Hof Sandra Janku Markus Hoth Annette Lis 《Aging cell》2022,21(8)
A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID‐19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time‐resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM) and central memory (TCM) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell‐intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked. 相似文献
59.
Friedrich W. Keppeler Annette S. Engel Linda M. Hooper-Bùi Paola C. López-Duarte Charles W. Martin Jill A. Olin Katelyn J. Lamb Michael J. Polito Nancy N. Rabalais Brian J. Roberts Erick M. Swenson Olaf P. Jensen 《Restoration Ecology》2024,32(3):e14072
Advancing ecological restoration assessments requires a more detailed consideration of species interactions and ecosystem processes. Most restoration projects rely on a few metrics not always directly linked with ecological theory. Here, we used Odum's theory of ecosystem development to assess and compare the ecosystem structure and services of created marshes (4–6 years old) with preexisting, reference marshes in a brackish water region of the Mississippi River Delta. We built ecosystem models for created and reference marshes that integrated large datasets of stomach contents, stable isotopes, and taxa abundances. Despite strong resemblance in community structure, created marshes were at an earlier succession stage compared to the reference marshes, having lower biomass (including exploited species), higher biomass turnover and production, less dependence on detritus, lower material cycling, and less energy flowing through specialist pathways. Although preserving preexisting marshes should be a priority, created marshes may still be an important tool for the restoration of coastal areas and their ecosystem services. In addition, our results show that comparisons of species biodiversity alone may fail to capture essential differences in ecosystem processes between habitats, which reinforces the importance of ecosystem modeling approaches to assess restoration projects. 相似文献
60.
Schmidt-Arras DE Böhmer A Markova B Choudhary C Serve H Böhmer FD 《Molecular and cellular biology》2005,25(9):3690-3703
Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants. 相似文献