Unexpected Metastasis of High Grade Serous Ovarian Cancer to Breast: Case Report and Literature Review

Introduction: Metastasis of ovarian serous carcinoma to breast and/or axillary lymph nodes represents an unusual event. Nevertheless, their detection and distinction from mammary carcinoma are of huge clinical importance because the treatment and prognosis diverge significantly. Case presentation: We report a case of a 47 year-old Caucasian female patient with unforeseen metastasis to the breast and to axillary lymph nodes due to ovarian serous carcinoma. Conclusion: In patients with history of OSC who present with axillary or breast mass, an accurate histological diagnosis should be obtained since this has a great impact on treatment outcomes

Conformational Changes of Calpain from Human Erythrocytes in the Presence of Ca2

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution

Saccharose solid matrix enbedded proteins: a new method fro XAS sample preparation

i.f. 1,950; biophysic

Saccharose solid matrix embedded proteins:a new method for sample preparation for X-ray absorption spectroscopy

In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy

Conformational changes of calpain revealed by small angle X-ray scattering

INTRODUCTION: Calpains (EC 3.4.22.17) are a family of cytosolic calcium-dependent cysteine endopeptidases widely distributed in all mammalian cells and consisting of several genetically distinct isozymes structurally organized as heterodimers (1). The physiological role of calpains is related to the transduction of extra cellular signals mediated by changes in the permeability of membranes to Ca2+ or by the mobilization of this ion from internal stores. These proteins are also involved in other physiological and pathophysiological conditions such as cell cycle regulation, apoptosis, cytoskeletal remodeling, Alzheimer’s or Parkinson’s diseases and muscular dystrophies (2). The crystal structure of calcium-free recombinant human and rat m-calpain have recently been determined at 2.3 Å and 2.6 Å resolution respectively (3;4).Synchrotron radiation X-ray small angle scattering measurements were carried out on calpain isolated from human erythrocytes. Solutions of the proteinase were analyzed in the absence and in the presence of Ca 2+ and the structural effects of two different inhibitors, the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane (E64) and the neutral serine- and thiol-protease inhibitor leupeptin, were investigated.MATERIALS AND METHODS: Calpain was purified from erythrocytes according to Michetti et al. (5) and dissolved in Sodium Borate buffer pH 7.5 containing 0.1 mM EDTA in order to obtain a monodisperse protein solution. SAXS measurements were performed at the synchrotron radiation beam line D24 in the DCI storage ring of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The homogeneity of each sample was checked immediately before SAXS measurements by SE-HPLC chromatography (Waters 486 System with a Shodex Protein KW-802.5 column ). Eight frames of 100s each were recorded using a position sensitive proportional detector placed 1819 mm downstream from the sample so as to cover the range of momentum transfer Q from 0.01 to 0.15 Å-1 (Q = 4 sin/ where 2 is the scattering angle, * is the radiation wavelength, *=1.488 Å). The distance distribution function p(r), corresponding to the distribution of distances between any two volume elements within the protein particle, has been determined using the indirect transform method as implemented in the program GNOM. The scattering intensities were computed from the atomic coordinates of the crystal structure of the human m-calpain (1kfu.pdb) by using the program CRYSOL. The ab initio shape determination was performed with the dummy atom model (DAM) method (6) using the program DAMMIN running on a Silicon Graphics O2 workstation.RESULTS: The X-ray scattering model of the unligated enzyme in solution does not display any significant difference with that calculated from the crystal structure. The value of the radius of gyration calculated from the Guinier analysis of the scattering intensity results to be Rg = 35.8 0.4 Å. The molecular weight of the native protein calculated from the zero-angle intensity, calibrated by means of a reference sample has a value of 110 10 kDa, in close agreement with the values typical for both - and m- isoforms of calpains. Calculation of the p(r) function of the unliganded enzyme yields a value for the maximal diameter of 120 Å with a value of the radius of gyration of 36.3 0.4 Å, very close to that derived from the Guinier analysis. The SAXS pattern obtained for the native calpain in the presence of 100 M Ca2+ displays conspicuous differences with that of the unliganded protease indicating the formation of large aggregates in solution. In contrast, Ca2+ addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change[...

Conformational changes of calpain from human erythrocytes in the presence of Ca2+.

Small angle x-ray scattering has been used to monitor calpain structural transitions during the activation process triggered by Ca(2+) binding. The scattering pattern of the unliganded enzyme in solution does not display any significant difference with that calculated from the crystal structure. The addition of Ca(2+) promotes the formation of large aggregates, indicating the exposure of hydrophobic patches on the surface of the protease. In contrast, Ca(2+) addition in the presence of the thiol proteinase inhibitor E64 or of the inhibitor leupeptin causes a small conformational change with no dissociation of the heterodimer. The resulting conformation appears to be slightly more extended than the unliganded form. From the comparison between ab initio models derived from our data with the crystal structure, the major observable conformational change appears to be localized at level of the L-subunit and in particular seems to confirm the mutual movement already observed by the crystallographic analysis of the dII (dIIb) and the dI (dIIa) domains creating a functional active site. This work not only provides another piece of supporting evidence for the calpain conformational change in the presence of Ca(2+), but actually constitutes the first experimental observation of this change for intact heterodimeric calpain in solution.[...

Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins. The first shell analysis of the EXAFS modulation is consistent with the presence of n = 3N∈2 imidazole nitrogens at an average distance of 1.92±0.03 Å from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n = 5 with average distances of 1.93 Å. Alternatively, by separating the contribution of N∈2 imidazole nitrogens and of peroxide O-atoms, n = 3 ligands at 1.98 ± 0.03 Å and n = 2 ligands at 1.87 ± 0.03 Å are found

Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropod hemocyanins

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins. The first shell analysis of the EXAFS modulation is consistent with the presence of n = 3 N\uf0652 imidazole nitrogens at an average distance of 1.92\ub10.03 \uc5 from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n = 5 with average distances of 1.93 \uc5. Alternatively, by separating the contribution of N\uf0652 imidazole nitrogens and of peroxide O-atoms, n = 3 ligands at 1.98\ub10.03 \uc5 and n = 2 ligands at 1.87\ub10.03 \uc5 are found