Mast cell degranulation via MRGPRX2 by isolated human albumin fragments.

BACKGROUND: Mast cells are important modulators of the human immune system via their release of several inflammatory mediators and proteases. The release can be activated by different pathways: the classical immunoglobulin E-dependent pathway and by the non-immunological immunoglobulin E-independent pathway. MAS-related G protein-coupled receptor X2 (MRGPRX2) is expressed in mast cells and it is one of the endogenous receptor responsible for the IgE-independent activation of human mast cell. The MRGPRX2 is classified as orphan receptor and unlike most GPCRs, the MRGPRX2 recognizes a wide range of basic molecules. Thus, there still might be several unknown ligands for the receptor. METHODS: MRGPRX2 activating peptides were isolated from human plasma using consecutive HPLC purification steps. The isolation process was monitored with MRGPRX2 transfected HEK 293 cells. The isolated peptides were sequenced by MS and synthetized. The synthetic peptides were used to determine degranulation of the human LAD 2 mast cell line by measuring ß-hexosaminidase release. RESULTS: Three endogenous MRGPRX2 activating peptides were isolated from human plasma. These peptides are identified as fragments of albumin. The isolated fragments activate MRGPRX2 and degranulate MRGPRX2 expressing LAD 2 cells in dose-dependent manner. CONCLUSIONS: The isolated basic peptides generated from human albumin are able to degranulate mast cells via the MRGPRX2. GENERAL SIGNIFICANCE: These endogenous albumin fragments, cleaved from albumin by mast cell secreted proteases, provide a possible pathway for self-perpetuating mast cell dependent inflammation.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging.

Evolution of flight in maniraptoran dinosaurs is marked by the acquisition of distinct avian characters, such as feathers, as seen in Archaeopteryx from the Solnhofen limestone. These rare fossils were pivotal in confirming the dinosauria-avian lineage. One of the key derived avian characters is the possession of feathers, details of which were remarkably preserved in the Lagerstätte environment. These structures were previously simply assumed to be impressions; however, a detailed chemical analysis has, until now, never been completed on any Archaeopteryx specimen. Here we present chemical imaging via synchrotron rapid scanning X-ray fluorescence (SRS-XRF) of the Thermopolis Archaeopteryx, which shows that portions of the feathers are not impressions but are in fact remnant body fossil structures, maintaining elemental compositions that are completely different from the embedding geological matrix. Our results indicate phosphorous and sulfur retention in soft tissue as well as trace metal (Zn and Cu) retention in bone. Other previously unknown chemical details of Archaeopteryx are also revealed in this study including: bone chemistry, taphonomy (fossilization process), and curation artifacts. SRS-XRF represents a major advancement in the study of the life chemistry and fossilization processes of Archaeopteryx and other extinct organisms because it is now practical to image the chemistry of large specimens rapidly at concentration levels of parts per million. This technique has wider application to the archaeological, forensic, and biological sciences, enabling the mapping of "unseen" compounds critical to understanding biological structures, modes of preservation, and environmental context.

In-office outpatient orthognathic surgery: review of 254 cases where the patients were discharged the same day.

This study was performed to present a single operator's experience of in-office (outside of a hospital setting) outpatient orthognathic surgery over a period of 12 years. A total of 254 surgeries were performed during this period. Average procedure times were comparable with published results from studies of similar material. The mean operating time for bimaxillary surgery (n = 21) was 3 hours and 11 minutes. Regarding single-jaw procedures, the mean operating time for Le Fort I osteotomy (n = 115) was 2 hours and 14 minutes and for bilateral sagittal split osteotomy (n = 118) was 2 hours and 1 minute. All patients were discharged from the office the same day, except one patient who was transported to the hospital after surgery due to an anaesthetic complication. This patient was discharged from the hospital later the same day. In this setting, outpatient orthognathic surgery is both safe and practical when careful attention is given to patient preparation and selection. Emergency phone contact with the surgeon in case of complications is important to avoid unnecessary hospitalization.

Development of spinning-disk solid sample delivery system for high-repetition rate x-ray free electron laser experiments.

X-ray free-electron lasers (XFELs) deliver intense x-ray pulses that destroy the sample in a single shot by a Coulomb explosion. Experiments using XFEL pulse trains or the new generation of high-repetition rate XFELs require rapid sample replacement beyond those provided by the systems now used at low repletion-rate XFELs. We describe the development and characterization of a system based on a spinning disk to continuously deliver a solid sample into an XFEL interaction point at very high speeds. We tested our system at the Linac Coherent Light Source and European XFEL hard x-ray nano-focus instruments, employing it to deliver a 25 µm copper foil sample, which can be used as a gain medium for stimulated x-ray emission for the proposed x-ray laser oscillator.

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging.

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its ß-galactosidase activity found 8.3 and 9.8 ng/µg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/µg are obtained for top-down analysis, while 14.5 and 15.9 ng/µg were found in the bottom-up analysis.

Can the new RCP R0/R1 classification predict the clinical outcome in ductal adenocarcinoma of the pancreatic head?

PURPOSE: According to the International Union Against Cancer (UICC), R1 is defined as the microscopic presence of tumor cells at the surface of the resection margin (RM). In contrast, the Royal College of Pathologists (RCP) suggested to declare R1 already when tumor cells are found within 1 mm of the RM. The aim of this study was to determine the significance of the RM concerning the prognosis of pancreatic ductal adenocarcinoma (PDAC). METHODS: From 2007 to 2009, 62 patients underwent a curative operation for PDAC of the pancreatic head. The relevance of R status on cumulative overall survival (OS) was assessed on univariate and multivariate analysis for both the classic R classification (UICC) and the suggestion of the RCP. RESULTS: Following the UICC criteria, a positive RM was detected in 8 %. Along with grading and lymph node ratio, R status revealed a significant impact on OS on univariate and multivariate analysis. Applying the suggestion of the RCP, R1 rate rose to 26 % resulting in no significant impact on OS in univariate analysis. CONCLUSIONS: Our study has shown that the RCP suggestion for R status has no impact on the prognosis of PDAC. In contrast, our data confirmed the UICC R classification of RM as well as N category, grading, and lymph node ratio as significant prognostic factors.

The inhomogeneous structure of water at ambient conditions.

Small-angle X-ray scattering (SAXS) is used to demonstrate the presence of density fluctuations in ambient water on a physical length-scale of approximately 1 nm; this is retained with decreasing temperature while the magnitude is enhanced. In contrast, the magnitude of fluctuations in a normal liquid, such as CCl(4), exhibits no enhancement with decreasing temperature, as is also the case for water from molecular dynamics simulations under ambient conditions. Based on X-ray emission spectroscopy and X-ray Raman scattering data we propose that the density difference contrast in SAXS is due to fluctuations between tetrahedral-like and hydrogen-bond distorted structures related to, respectively, low and high density water. We combine our experimental observations to propose a model of water as a temperature-dependent, fluctuating equilibrium between the two types of local structures driven by incommensurate requirements for minimizing enthalpy (strong near-tetrahedral hydrogen-bonds) and maximizing entropy (nondirectional H-bonds and disorder). The present results provide experimental evidence that the extreme differences anticipated in the hydrogen-bonding environment in the deeply supercooled regime surprisingly remain in bulk water even at conditions ranging from ambient up to close to the boiling point.

Infrared mapping resolves soft tissue preservation in 50 million year-old reptile skin.

Non-destructive Fourier Transform InfraRed (FTIR) mapping of Eocene aged fossil reptile skin shows that biological control on the distribution of endogenous organic components within fossilized soft tissue can be resolved. Mapped organic functional units within this approximately 50 Myr old specimen from the Green River Formation (USA) include amide and sulphur compounds. These compounds are most probably derived from the original beta keratin present in the skin because fossil leaf- and other non-skin-derived organic matter from the same geological formation do not show intense amide or thiol absorption bands. Maps and spectra from the fossil are directly comparable to extant reptile skin. Furthermore, infrared results are corroborated by several additional quantitative methods including Synchrotron Rapid Scanning X-Ray Fluorescence (SRS-XRF) and Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS). All results combine to clearly show that the organic compound inventory of the fossil skin is different from the embedding sedimentary matrix and fossil plant material. A new taphonomic model involving ternary complexation between keratin-derived organic molecules, divalent trace metals and silicate surfaces is presented to explain the survival of the observed compounds. X-ray diffraction shows that suitable minerals for complex formation are present. Previously, this study would only have been possible with major destructive sampling. Non-destructive FTIR imaging methods are thus shown to be a valuable tool for understanding the taphonomy of high-fidelity preservation, and furthermore, may provide insight into the biochemistry of extinct organisms.

Wide-angle X-ray diffraction and molecular dynamics study of medium-range order in ambient and hot water.

We have developed wide-angle X-ray diffraction measurements with high energy-resolution and accuracy to study water structure at three different temperatures (7, 25 and 66 °C) under normal pressure. Using a spherically curved Ge crystal an energy resolution better than 15 eV has been achieved which eliminates influence from Compton scattering. The high quality of the data allows for a reliable Fourier transform of the experimental data resolving shell structure out to ~12 Å, i.e. 5 hydration shells. Large-scale molecular dynamics (MD) simulations using the TIP4P/2005 force-field reproduce excellently the experimental shell-structure in the range 4-12 Å although less agreement is seen for the first peak in the intermolecular pair-correlation function (PCF). The Shiratani-Sasai Local Structure Index [J. Chem. Phys. 104, 7671 (1996)] identifies a tetrahedral minority giving the intermediate-range oscillations in the O-O PCF and a disordered majority providing a more featureless background in this range. The current study supports the proposal that the structure of liquid water, even at high temperatures, can be described in terms of a two-state fluctuation model involving local structures related to the high-density and low-density forms of liquid water postulated in the liquid-liquid phase transition hypothesis.

Complicated sigmoid diverticulitis--Hartmann's procedure or primary anastomosis?

INTRODUCTION: Diverticular disease of the colon is a common condition in developed countries. For perforated diverticulitis Hartmann's procedure is a safe and quick treatment option. But intestinal restoration needs further interventions. This leads to high complication rates and cost. Therefore a critical evaluation of surgical treatment options is necessary. METHODS: During a period of 18 months 88 patients underwent surgical resection for diverticulitis. Forty patients had emergency surgery. Among those a primary anastomosis was performed in 21 patients. The other 19 patients had interval colostomy. Among 21 patients with primary anastomosis major complications occurred in two patients, vs. twelve in patients with Hartmann's operation (p = 0.03). In the Hartmann group eight patients had major general complications, vs. one patient in the group with primary anastomosis (p = 0.06). The mean hospital stay was 38 days after Hartmann's procedure, vs. 13 days for patients with primary anastomosis (p < 0.01). CONCLUSION: In emergency surgery for complicated diverticulitis primary anastomosis is not associated with an increased postoperative morbidity. A primary anastomosis reduces the need for further surgical interventions and complex re-operations. Thus, an overall reduction of morbidity, cost, complication rate and hospital stay is possible. Therefore this technique is advantageous for patients and hospitals.

Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed.

Matrix metalloproteinases (MMPs) catalyze extracellular matrix degradation. Control of their activity is a promising target for therapy of diseases characterized by abnormal connective tissue turnover. MMPs are expressed as latent proenzymes that are activated by proteolytic cleavage that triggers a conformational change in the propeptide (cysteine switch). The structure of proMMP-2 reveals how the propeptide shields the catalytic cleft and that the cysteine switch may operate through cleavage of loops essential for propeptide stability.

Isolation of new ligands for orphan receptor MRGPRX1-hemorphins LVV-H7 and VV-H7.

The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is a member of the GPCR family. The receptor is primate specific and expressed in the sensory neurons of dorsal root ganglion and trigeminal ganglion, where it is considered to be involved in the pain perception. The MRGPRX1 has unusual binding mechanism, as it is activated by several different ligands as well as several different fragments of precursor proteins. Thus, we hypothesize that it is activated by several unknown compounds as well since the receptor is still classified as orphan. Here, we describe the isolation of two novel endogenous ligands for the MRGPRX1 from human platelet preparation. The isolated ligands are hemoglobin ß-chain fragments, known members of the hemorphin family.

Insulin-like growth factor I overexpression in human pancreatic cancer: evidence for autocrine and paracrine roles.

We assessed the potential roles of insulin-like growth factor-I (IGF-I) and the IGF-I receptor (IGF-IR) in human pancreatic cancer. IGF-I enhanced the growth of ASPC-1 and COLO-357 human pancreatic cancer cells, and this effect was significantly inhibited by a highly specific monoclonal anti-IGF-IR antibody (alpha IR3). Both cell lines expressed mRNA transcripts for IGF-IR, and basal cell growth was significantly reduced by an IGF-IR antisense oligodeoxynucleotide. IGF-I mRNA transcripts were not detected in either cell line or in two additional pancreatic cancer cell lines. In contrast, analysis of 12 pancreatic cancers revealed a 32-fold increase (P < 0.01) in IGF-I mRNA levels by comparison with the low levels observed in the normal pancreas. By in situ hybridization, IGF-I mRNA grains were present in both the cancer cells and in the surrounding connective tissue. Six of the cancers exhibited a 4.4-fold increase in IGF-IR mRNA levels. These findings suggest that IGF-I may participate in aberrant autocrine and paracrine activation of IGF-IR in pancreatic cancer in vivo.

Enhanced expression of the insulin receptor substrate-2 docking protein in human pancreatic cancer.

Insulin receptor substrate-2 (IRS-2) is a multisite docking protein implicated in mitogenic signaling after activation of the insulin and insulin-like growth factor (IGF)-I receptors. In the present study, we characterized IRS-2 expression and function in human pancreatic cancer. IRS-2 mRNA and protein were expressed in ASPC-1 and COLO-357 human pancreatic cancer cell lines. Insulin, IGF-I, and IGF-II enhanced the growth of both cell lines, stimulated tyrosine phosphorylation of IRS-2, and increased IRS-2-associated phosphatidylinositol (PI) 3-kinase activity. The mitogenic effects of insulin, IGF-I, and IGF-II were markedly attenuated by the PI 3-kinase inhibitor LY 294002. Northern blot analysis of total RNA extracted from normal and cancerous tissues revealed that IRS-2 mRNA levels were increased in the cancer tissues (P = 0.032). In the normal pancreas, IRS-2 immunoreactivity was present at low levels in some ductal and acinar cells and at moderate levels in a heterogeneous pattern in all of the endocrine islets. In the pancreatic cancers, IRS-2 was abundant in the ductal-like cancer cells. These findings indicate that IRS-2 is overexpressed in human pancreatic cancer and suggest that it may contribute to enhanced mitogenic signaling via the PI 3-kinase pathway, thereby leading to excessive growth stimulation in this malignancy.

Evidence for an additional archaebacterial gene cluster in Halobacterium marismortui encoding ribosomal proteins HL46e and HL30.

A small and extremely basic ribosomal protein (HL46e) has been purified from Halobacterium marismortui using reversed-phase high-performance liquid chromatography (HPLC). The amino acid sequence of the protein was determined by automated N-terminal and internal sequence analysis. Comparison of this sequence with other ribosomal protein sequences from eubacteria, archaebacteria and eukaryotes revealed a strong homology to SL46e from Sulfolobus solfataricus, YeaL46 from yeast and RL39 from rat. No significant sequence similarly was found to any eubacterial ribosomal protein so far known. Using a specific oligonucleotide probe the HL46e gene was identified, cloned and the nucleotide sequence including the 5'- and 3'-flanking regions were analysed. The HL46e gene is followed by the gene coding for HL30. A putative halobacterial promoter sequence with the motive 'TTTAAA' has been localized 32 bp upstream of the HL46e gene and a putative terminator sequence localized downstream from the HL30 gene. An equivalent to this HL46e/HL30 operon is apparently not present in Escherichia coli.

HL35e and HLA: primary structure of two very basic and cysteine-rich ribosomal proteins from Haloarcula marismortui.

Two small and very basic ribosomal proteins have been purified from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui by RP-HPLC. The complete primary structures of these two proteins, which we refer to as HL35e and HLA, have been determined by protein chemical methods. Both proteins are characterized by a high content of basic amino acids and the presence of two pairs of cysteines in each polypeptide chain, one of which resembles the C4-zinc-finger motif. Comparison of the protein sequences with those of other ribosomal proteins revealed that HL35e shows significant sequence homology exclusively to eukaryotic ribosomal proteins, namely to yeast L35 and to L37 from rat. For HLA no homologous ribosomal protein so far known could be found. Obviously, HL35e and HLA have no counterparts in eubacterial ribosomes.

Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking.

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution.

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.

X-ray absorption spectroscopy measurements of liquid water.

Recent studies, based on X-ray absorption spectroscopy (XAS) and X-ray Raman scattering (XRS), have shown that the hydrogen bond network in liquid water consists mainly of water molecules with only two strong hydrogen bonds. Since this result is controversial, it is important to demonstrate the reliability of the experimental data, which is the purpose of this paper. Here we compare X-ray absorption spectra of liquid water recorded with five very different techniques sensitive to the local environment of the absorbing molecule. Overall, the spectra obtained with photon detection show a very close similarity and even the observable minor differences can be understood. The comparison demonstrates that XAS and XRS can indeed be applied reliably to study the local bonding of the water molecule and thus to reveal the hydrogen bond situation in bulk water.