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1.
Haas PJ de Haas CJ Kleibeuker W Poppelier MJ van Kessel KP Kruijtzer JA Liskamp RM van Strijp JA 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(9):5704-5711
Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd=35.4 +/- 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes. 相似文献
2.
Tempels FW Wiese G Underberg WJ Somsen GW de Jong GJ 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,839(1-2):30-35
An on-line size exclusion chromatography (SEC)-solid-phase extraction (SPE)-capillary electrophoresis (CE) system using a Tee-split interface has been developed for the analysis of peptides in biological fluids. The SEC column fractionates the sample by molecular size and the low-molecular-weight fraction, which contains the peptides, is directed to a C(18) SPE microcolumn, where the peptides are trapped and concentrated. The SPE column is desorbed with 425 nL acetonitrile and the effluent is sent to the Tee-split interface, which hydrodynamically splits (1:40) the flow and, thus, allows appropriate injection of analytes into the CE system. The performance of the system is investigated by the analysis of enkephalins in cerebrospinal fluid (CSF). It is demonstrated that the SEC step efficiently removes potentially interfering proteins, permitting reproducible SPE and CE. The total system provides efficient separations of the enkephalins with plate numbers up to 100,000. Concentration limits of detection (S/N = 3) for the peptides are about 100 ng/mL for injection of 20 microL spiked CSF samples. Plots of enkephalin peak areas versus concentration showed good linearity over the 0.25-10 microg/mL range (R2 > or = 0.985). Repeatability of migration time and peak area was within 2% and 10% R.S.D., respectively. 相似文献
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4.
Johannes H. Ippel Carla J. C. de Haas Anton Bunschoten Jos A. G. van Strijp John A. W. Kruijtzer Rob M. J. Liskamp Johan Kemmink 《The Journal of biological chemistry》2009,284(18):12363-12372
Complement component C5a is a potent pro-inflammatory agent inducing
chemotaxis of leukocytes toward sites of infection and injury. C5a mediates
its effects via its G protein-coupled C5a receptor (C5aR). Although under
normal conditions highly beneficial, excessive levels of C5a can be
deleterious to the host and have been related to numerous inflammatory
diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of
Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein
excreted by S. aureus. It binds the N terminus of the C5aR (residues
1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated
responses in human leukocytes. Therefore, CHIPS provides a starting point for
the development of new anti-inflammatory agents. Two O-sulfated
tyrosine residues located at positions 11 and 14 within the C5aR N terminus
play a critical role in recognition of C5a, but their role in CHIPS binding
has not been established so far. By isothermal titration calorimetry, using
synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal
peptides, we demonstrate that the sulfate groups are essential for tight
binding between the C5aR and CHIPS. In addition, the NMR structure of the
complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise
binding motif as well as the distinct roles of sulfated tyrosine residues sY11
and sY14. These results provide a molecular framework for the design of novel
CHIPS-based C5aR inhibitors.The human complement system is a key component of the innate host defense
directed against invading pathogens. Complement component C5a is a 74-residue
glycoprotein generated via complement activation by cleavage of the
α-chain of its precursor C5. C5a is a strong chemoattractant involved in
the recruitment of neutrophils and monocytes, activation of phagocytes,
release of granule-based enzymes, and in the generation of oxidants
(1,
2). C5a exerts its effect by
activating the C5a receptor
(C5aR).3
Although this is a highly efficient process, excessive or erroneous activation
of the C5aR can have deleterious effects on host tissues. C5a has been
implicated in the pathogenesis of many inflammatory and immunological
diseases, including rheumatoid arthritis, inflammatory bowel disease, immune
complex disease, and reperfusion injury
(3,
4). Consequently, there is an
active ongoing search for compounds that suppress C5a-mediated inflammatory
responses.Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is
a 121-residue protein excreted by S. aureus, which efficiently
inhibits the activation of neutrophils and monocytes by formylated peptides
and C5a (5,
6). CHIPS specifically binds to
the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity
(Kd = 35.4 ± 7.7 nm and 1.1 ± 0.2
nm, respectively)
(7), thereby suppressing the
inflammatory response of the host. A CHIPS fragment lacking residues 1-30
(designated CHIPS31-121) has the same activity in blocking the C5aR
compared with wild-type CHIPS
(8). CHIPS31-121 is
a compact protein comprising an α-helix packed onto a four-stranded
anti-parallel β-sheet (8).
C5a has an entirely different fold (PDB ID code 1KJS) and is comprised of an
anti-parallel bundle of four α-helices stabilized by three disulfide
bonds (9,
10). Preliminary experiments
indicated that CHIPS binds exclusively to the extracellular N-terminal portion
of the C5aR (7). In contrast,
the binding of C5a by its receptor involves two separate binding sites: C5a
residues located in the region between 12-46
(11,
12) bind to a primary binding
site partly coinciding with the binding site of CHIPS, while the C terminus of
C5a (residues 69-74) binds to the activation domain of the C5aR located in the
receptor core (13). Because of
their dissimilarity in sequence and structure, the binding sites of CHIPS and
C5a are not identical (11).
The present working model is that CHIPS interferes with the primary binding
site of C5a located at the N terminus of the C5aR, thereby preventing the
C-terminal tail of C5a from contacting the activation domain of the C5aR and
blocking downstream signaling. Currently, the development of C5aR inhibitors
has been focused primarily on mimicking C5a in order to directly interrupt
C5a-mediated C5aR signaling (3,
4,
14). Understanding the
interactions between CHIPS and the C5aR may provide valuable insights toward
the development of new C5aR antagonists.Postma et al. (15)
proposed that residues involved in CHIPS binding are located between residues
10-18 of the C5aR. Specifically, the acidic residues Asp-10, Asp-15, and
Asp-18 and residue Gly-12 appear to be critical for binding. High affinity
binding was observed between 125I-labeled CHIPS and the N-terminal
portion of the C5aR (residues 1-38) expressed on the cell surface of HEK293
cells (Kd = 29.7 ± 4.4 nm). In contrast,
very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide
(residues 1-37; Kd = 40 ± 19 μm),
measured by isothermal titration calorimetry (ITC), was recently reported by
Wright et al. (16).
The discrepancy in the magnitude of these dissociation constants may be
explained by the presence of two sulfate groups on tyrosine 11 and 14 of the
C5aR N terminus expressed on the cell surface of HEK293 cells, which are
absent in the synthetic C5aR peptide utilized by Wright et al.
(16). Farzan et al.
(17) stressed the critical
role of these sulfate groups in activation of the C5aR by C5a. Previous
mutational studies employing FITC-labeled CHIPS, however, suggested that the
sulfate groups had only a limited effect on the binding affinity
(15).To resolve these discrepancies, we set out to chemically synthesize several
sulfated and unsulfated peptides representing the N terminus of the human
C5aR. We have measured the binding affinities of these peptides to
CHIPS31-121 by ITC and used the C5aR peptide with the highest
affinity to determine the structure of the complex between
CHIPS31-121 and the C5aR N terminus by NMR spectroscopy. 相似文献
5.
6.
Julius Mulindwa Joyce Namulondo Anna Kitibwa Jacent Nassuuna Oscar Asanya Nyangiri Magambo Phillip Kimuda Alex Boobo Barbara Nerima Fred Busingye Rowel Candia Annet Namukuta Ronald Ssenyonga Noah Ukumu Paul Ajal Moses Adriko Harry Noyes Claudia J. de Dood Paul L. A. M. Corstjens Govert J. van Dam Alison M. Elliott Enock Matovu TrypanoGEN+ Research group 《PLoS neglected tropical diseases》2022,16(7)
BackgroundKnowing the prevalence of schistosomiasis is key to informing programmes to control and eliminate the disease as a public health problem. It is also important to understand the impact of infection on child growth and development in order to allocate appropriate resources and effort to the control of the disease.MethodsWe conducted a survey to estimate the prevalence of schistosomiasis among school aged children in villages along the Albert-Nile shore line in the district of Pakwach, North Western Uganda. A total of 914 children aged between 10–15 years were screened for Schistosoma mansoni using the POC-CCA and Kato Katz (KK) techniques. The infection intensities were assessed by POC-CCA and KK as well as CAA tests. The KK intensities were also correlated with POC-CCA and with CAA intensity. Anthropometric measurements were also taken and multivariate analysis was carried out to investigate their association with infection status.ResultsThe prevalence of schistosomiasis using the POC-CCA diagnostic test was estimated at 85% (95% CI: 83–87), being highest amongst children living closer to the Albert-Nile shoreline. Visual scoring of the POC-CCA results was more sensitive than the Kato Katz test and was positively correlated with the quantified infection intensities by the CAA test. The majority of the children were underweight (BMI<18.5), and most notably, boys had significantly lower height for age (stunting) than girls in the same age range (p < 0.0001), but this was not directly associated with S. mansoni infection.ConclusionHigh prevalence of S. mansoni infection in the region calls for more frequent mass drug administration with praziquantel. We observed high levels of stunting which was not associated with schistosomiasis. There is a need for improved nutrition among the children in the area. 相似文献
7.
Michael E. T. I. Boerrigter Erik Mullaart Govert P. Van Der Schans Jan Vijg 《Experimental cell research》1989,180(2):569-573
Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair. 相似文献
8.
Hogewoning SW Wientjes E Douwstra P Trouwborst G van Ieperen W Croce R Harbinson J 《The Plant cell》2012,24(5):1921-1935
The mechanisms underlying the wavelength dependence of the quantum yield for CO(2) fixation (α) and its acclimation to the growth-light spectrum are quantitatively addressed, combining in vivo physiological and in vitro molecular methods. Cucumber (Cucumis sativus) was grown under an artificial sunlight spectrum, shade light spectrum, and blue light, and the quantum yield for photosystem I (PSI) and photosystem II (PSII) electron transport and α were simultaneously measured in vivo at 20 different wavelengths. The wavelength dependence of the photosystem excitation balance was calculated from both these in vivo data and in vitro from the photosystem composition and spectroscopic properties. Measuring wavelengths overexciting PSI produced a higher α for leaves grown under the shade light spectrum (i.e., PSI light), whereas wavelengths overexciting PSII produced a higher α for the sun and blue leaves. The shade spectrum produced the lowest PSI:PSII ratio. The photosystem excitation balance calculated from both in vivo and in vitro data was substantially similar and was shown to determine α at those wavelengths where absorption by carotenoids and nonphotosynthetic pigments is insignificant (i.e., >580 nm). We show quantitatively that leaves acclimate their photosystem composition to their growth light spectrum and how this changes the wavelength dependence of the photosystem excitation balance and quantum yield for CO(2) fixation. This also proves that combining different wavelengths can enhance quantum yields substantially. 相似文献
9.
Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry
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Wang X Permentier HP Rink R Kruijtzer JA Liskamp RM Wösten HA Poolman B Robillard GT 《Biophysical journal》2004,87(3):1919-1928
The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly. 相似文献
10.
Stefanie Knopp Paul L. A. M. Corstjens Artemis Koukounari Colin I. Cercamondi Shaali M. Ame Said M. Ali Claudia J. de Dood Khalfan A. Mohammed Jürg Utzinger David Rollinson Govert J. van Dam 《PLoS neglected tropical diseases》2015,9(5)