Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

al features, including a well-structured a-helical subdomain, a short Trp59--heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur--iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c,a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands. q 2006 Elsevier Ltd. All rights reserved. Keywords: protein folding; denaturation; stability; spectroscopy; NMR *Corresponding author Introduction Small globular proteins often exhibit cooperative unfolding transitions in which only folded

Combined In Vivo Molecular and Anatomic Imaging for Detection of Ovarian Carcinoma-Associated Protease Activity and Integrin Expression in Mice

Most patients with epithelial ovarian cancer (EOC) experience drug-resistant disease recurrence. Identification of new treatments is a high priority, and preclinical studies in mouse models of EOC may expedite this goal. We previously developed methods for magnetic resonance imaging (MRI) for tumor detection and quantification in a transgenic mouse model of EOC. The goal of this study was to determine whether three-dimensional (3D) fluorescence molecular tomography (FMT) and fluorescent molecular imaging probes could be effectively used for in vivo detection of ovarian tumors and response to therapy. Ovarian tumor-bearing TgMISIIR-TAg mice injected with fluorescent probes were subjected to MRI and FMT. Tumor-specific probe retention was identified in vivo by alignment of the 3D data sets, confirmed by ex vivo fluorescent imaging and correlated with histopathologic findings. Mice were treated with standard chemotherapy, and changes in fluorescent probe binding were detected by MRI and FMT. Ovarian tumors were detected using probes specific for cathepsin proteases, matrix metalloproteinases (MMPs), and integrin αvβ3. Cathepsin and integrin αvβ3 probe activation and retention correlated strongly with tumor volume. MMP probe activation was readily detected in tumors but correlated less strongly with tumor volume. Tumor regression associated with response to therapy was detected and quantified by serial MRI and FMT. These results demonstrate the feasibility and sensitivity of FMT for detection and quantification of tumor-associated biologic targets in ovarian tumors and support the translational utility of molecular imaging to assess functional response to therapy in mouse models of EOC

MMP-13 In-Vivo Molecular Imaging Reveals Early Expression in Lung Adenocarcinoma

International audienceSeveral matrix metalloproteinases (MMPs) are overexpressed in lung cancer and may serve as potential targets for the development of bioactivable probes for molecular imaging

Expression of MMP-13 in human lung adenocarcinoma.

<p>Immunohistochemical staining was performed using Clone VIIIA2 diluted 1:20 (Lab Vision Corp., CA). <b>A)</b> Solid predominant adenocarcinoma. MMP-13 index staining (brown) is high in tumor cells (black arrow) and in fibroblasts (yallow arrow) (x100). <b>B)</b> Minimally invasive adenocarcinoma. MMP-13 staining is high in tumor cells (black arrow), and high in fibroblasts (yellow arrow) (x100). <b>C)</b> Adenocarcinoma <i>in-situ</i>. MMP-13 staining is high in tumor cells (black arrow), and high in fibroblasts (yellow arrow) (x100). <b>D)</b> Solid predominant adenocarcinoma and normal lung. Note the absence of MMP-13 staining in normal lung (*). Scale bar: 500 μm. Inserts in the x100 IHCs show x400 magnification.</p

Relative RNA expression of <i>Mmp 2</i>, <i>-3</i>, <i>-9</i>, <i>-13</i> in tumor <i>vs</i>. normal lung tissue in K-ras^LSL-G12D mice.

<p>The expression of <i>Mmp-2</i>, <i>-3</i>, <i>-9</i>, and -<i>13</i> in lung lesions vs. normal lung tissue (n = 12 pairs) was compared using boxplots. The transcript level of each <i>Mmp</i> gene was determined by qRT-PCR and normalized against that of the housekeeping gene <i>Hprt</i>. The fold difference between each sample <i>vs</i>. a mouse universal reference total RNA was calculated using the delta delta Ct method. The middle line is the median, and the bottom & top of the box are the 25th and 75th percentiles (inter-quartile range, IQR). The "whiskers”, extending from the ends of the box, represent the most extreme point (≤ 1.5 times the IQR). More extreme points are considered outliers and are plotted separately. Only the relative expression of <i>Mmp-13</i> was significantly higher in cancerous tissue as compared to normal lung tissue (Wilcoxon signed rank test; * indicates a p value <0.01)</p

The fluorescent signal from MMPs increases in lung tumors.

<p><b>A)</b><i>Ex vivo</i> image of MMP activity in superficial lung lesions of various stages from K-ras^LSL-G12D mice. The nonneoplastic lobe (L1) of freshly isolated lungs imaged immediately after euthanasia did not display any fluorescent signal, while in the tumor-bearing lobes (L2-5), signal increased with the degree of lesion severity. The data confirmed the ability of an epi-illumination fluorescence system (IVIS Spectrum) to image MMP activity at different stages of lung tumor progression. <b>B)</b> Fluorescence intensity of lung adenomas (n = 8), adenocarcinomas (n = 3) and normal lung lobes (n = 25). In this experiment, the lung and tumor fluorescence signal was normalized on liver fluorescence, based on the hepatic activation of the probe. The fluorescence intensity ratio was calculated as a ratio of the median radiant efficiency of the lung lobe / liver. The median values ± interquartile range are shown, and the median differences between cancerous lesions and normal lobes are significant. * p value <0.05 by the Mann-Whitney test.</p

Retrospective analysis of the MMPSense680 fluorescent signal following identification of the tumor on fused FMT-MRI images.

<p>A representative example of one animal, from imaging to histology, is presented. <b>A&B)</b> Axial and sagittal MRI scans, showing a small tumor (white arrows) in the right lower lobe of the lung, behind the liver (L). <b>C)</b> Frontal view of the FMT scan. The tumor is hidden behind the strong fluorescent signal of the liver. <b>D)</b> Overlay of the FMT signal on the MRI image, using the fiducial markers (FM) and the liver (L) for 3D alignment. Note the distinct fluorescent signal localized to the tumor (T). <b>E&F)</b> The position of the tumor was identified on the FMT scan and the corresponding region of interest was circumscribed (E: frontal view of the FMT scan; F: oblique view of the same FMT scan. <b>G)</b> The tumor ROI (ellipse) was reported on the previous FMT scans of the same animal, along with a liver ROI (rectangle) to allow fluorescence ratio quantification, showing an increasing fluorescence with time. The effect of probe activation in the liver on measurements of this tumor—near the liver—cannot be measured. <b>H&I)</b><i>Ex-vivo</i> epifluorescence imaging (H) and histological reviews (I) confirmed the presence of a 2mm adenoma in the right lower lobe (white and black arrows).</p

Imaging of MMP activity in the K-ras^LSL-G12D lung cancer model using MMPSense680.

<p><i>In vivo</i> (A) and <i>ex vivo</i> (B) epi-fluorescent images (IVIS Spectrum) of a large superficial right lung tumor exposed after thoracotomy and laparotomy (B). The fluorescent bioactivatable probe reporting the proteolytic activity of MMP 2, -3, -9 and -13 (MMPSense680, 2 nmol) was injected i.v 24 h prior to imaging. The results confirmed the ability of an epi-illumination fluorescence system to image MMP activity in lung tumors, although the system could not discriminate large lung tumors from the liver (1A). Fluorescence intensity is represented as a pseudocolor image (MATLAB “hot” color map) overlaid on a white-light photographic image. Values of absolute fluorescence efficiency, as measured by the IVIS Spectrum, are shown on the color bar in units of 10⁻⁵ (1A) or 10⁻⁴ (1B). L: Liver; T: Right lung tumor.</p

MMPSense680 retention was detected between week 4 and 8 in developing adenomas and adenocarcinomas and increased over time.

<p>Probe retention was calculated by the TrueQuant software in each ROI at each timepoint. Graph shows the probe retention in adenomas (n = 7) and adenocarcinomas (n = 5) at each time point.</p

Human Immunodeficiency Virus and Leishmaniasis

The Leishmaniases are a group of diseases transmitted to humans by the bite of a sandfly, caused by protozoan parasites of the genus Leishmania. Various Leishmania species infect humans, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1-1.5 million for cutaneous and 500,000 for visceral Leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa, Brazil and the Indian subcontinent. Co-infection with human immunodeficiency virus (HIV) alters the immune response to the disease. Here we review the immune response to Leishmania in the setting of HIV co-infection. Improved understanding of the immunology involved in co-infections may help in designing prophylactic and therapeutic strategies against Leishmaniasis