Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Somatic and Dendritic Encoding of Spatial Variables in Retrosplenial Cortex Differs during 2D Navigation

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Autofluorescence imaging for recurrence detection in skin cancer postoperative scars

This clinical study is a first attempt to use autofluorescence for recurrence diagnosis of skin cancer in postoperative scars. The proposed diagnostic parameter is based on a reduction in scar autofluorescence, evaluated in the green spectral channel. The validity of the method has been tested on 110 postoperative scars from 56 patients suspected of non‐melanoma skin cancer, with eight patients (13 scars) available for the repeated examination. The recurrence diagnosis within a scar has been made after two subsequent autofluorescence check‐ups, representing the temporal difference between the scar autofluorescence amplitudes as a vector. The recognition of recurrence has been discussed to represent the significant deviations from the value of vector angle θ. This new autofluorescence‐based method can be easily integrated into the postoperative monitoring of surgical scars and can help diagnose the recurrence of skin cancer from the early stage of scar development.     

Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a cotranslational protein-folding model

Examination of nascent globin peptides accumulatingin vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of - and -globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.     

Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells

We investigated the relationship between intracellular Ca²⁺ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca²⁺ concentration (Ca _i ²⁺ ). Ca _i ²⁺ and intracellular pH (pH _i ) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca²⁺ influx from the extracellular space into the cell was determined with the Mn²⁺ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca _i ²⁺ oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca _i ²⁺ oscillations. On the contrary, changes of extracellular pH value strongly affected Ca _i ²⁺ oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pH _i changes were less than 0. 1 pH units. However, experiments with the Mn²⁺ quenching technique revealed that extracellular alkalinization significantly reduced Ca²⁺ entry from the extracellular space. Large increases of Ca _i ²⁺ triggered by the blocker of the cytoplasmic Ca²⁺-ATPase, thapsigargin, had no effect on pH _i We conclude: intracellular Ca²⁺ homeostasis in MDCK-F cells is pH dependent. pH controls Ca²⁺ homeostasis mainly by effects on the level of Ca²⁺ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rapid changes of Ca _i ²⁺ .The project was supported by the Deutsche Forschungsgemeinschaft, SFB-176 (A6) and by the Jubilämusstiftung of the University of Würzburg.The authors gratefully acknowledge the valuable discussions with Drs. M.J. Berridge, M. Carew, I. Davidson, G. Law and B. Somasundraman. We are grateful to Applied Imaging for financial and technical support and to the Medical Research Council for financial support.     

Automated measurement of root length with a three-dimensional high-resolution scanner and image analysis

For measuring the length of root samples, the use of a three-dimensional (3D) scanner is proposed to address the problem of a too low resolution. The scanner's high resolution (up to 354 pixels per cm) enables in the resulting grey-value image very thin roots (diameter 100 m) to be segmented from the background by a simple thresholding operation. After skeletonizing, total length of the roots is calculated by multiplying the number of skeleton pixels by a correction factor. A comparison with the modified Newman Line-Intersect Method showed a correlation of r=0.98. Besides its superior resolution, an advantage of this type of scanner is its focusing depth, which allows root samples to be recorded on the scanbed similarly to a camera-oriented system.