Multimodality multiparametric imaging of early tumor response to a novel antiangiogenic therapy based on anticalins.

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [18F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = -0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.

Limited-projection-angle hybrid fluorescence molecular tomography of multiple molecules.

An advantage of fluorescence methods over other imaging modalities is the ability to concurrently resolve multiple moieties using fluorochromes emitting at different spectral regions. Simultaneous imaging of spectrally separated agents is helpful in interrogating multiple functions or establishing internal controls for accurate measurements. Herein, we investigated multimoiety imaging in the context of a limited-projection-angle hybrid fluorescence molecular tomography (FMT), and x-ray computed tomography implementation and the further registration with positron emission tomography (PET) data. Multichannel FMT systems may image fluorescent probes of varying distribution patterns. Therefore, it is possible that different channels may require different use of priors and regularization parameters. We examined the performance of automatically estimating regularization factors implementing priors, using data-driven regularization specific for limited-projection-angle schemes. We were particularly interested in identifying the implementation variations between hybrid-FMT channels due to probe distribution variation. For this reason, initial validation of the data-driven algorithm on a phantom was followed by imaging different agent distributions in animals, assuming superficial and deep seated activity. We further demonstrate the benefits of combining hybrid FMT with PET to gain multiple readings on the molecular composition of disease.

68Ga-NODAGA-RGD is a suitable substitute for (18)F-Galacto-RGD and can be produced with high specific activity in a cGMP/GRP compliant automated process.

INTRODUCTION: (18)F-Galacto-cyclo(RGDfK) is a well investigated tracer for imaging of ανß3 expression in vivo, but suffers from the drawback of a time consuming multistep synthesis that can hardly be established under GMP conditions. In this study, we present a direct comparison of the pharmacokinetic properties of this tracer with (68)Ga-NODAGA-cyclo(RGDyK), in order to assess its potential as an alternative for (18)F-Galacto-cyclo(RGDfK). METHODS: (68)Ga labeling of NODAGA-cyclo(RGDyK) was done in full automation using HEPES-buffered eluate of an SnO(2) based (68)Ga-generator. Using M21 (human melanoma) xenografted BALB/c nude mice, biodistribution studies and micro-PET scans were performed for both (18)F-Galacto-cyclo(RGDfK) and (68)Ga-NODAGA-cyclo(RGDyK), and for the latter, in vivo stability was assessed. IC(50) was determined in a displacement assay on M21 cells against (125)I-echistatin. RESULTS: (68)Ga-NODAGA-cyclo(RGDyK) was produced with high specific activity (routinely ca. 500 GBq/µmol) within 15 min. IC(50) values are similar for both substances. Tracer uptake was similar in ανß3 positive tumors (1.45%±0.11% ID/g and 1.35%±0.53% ID/g for (68)Ga-NODAGA-RGD and (18)F-Galacto-RGD, respectively) as well as for all other organs and tissues, with the exception of gall bladder and intestines, where (18)F-Galacto-cyclo(RGDfK) uptake was significantly higher, which can be explained by the higher hydrophilicity of (68)Ga-NODAGA-cyclo(RGDyK) (logP=-4.0 vs. -3.2 for (18)F-Galacto-RGD). Only intact tracer was detected 30 min p.i. in organs and tumor; however, minor amounts of metabolites were found in the urine (6% of total urine activity). CONCLUSION: (68)Ga-labeling of NODAGA-RGD can be performed rapidly and efficiently within 15 min in a GMP compliant process. Similar preclinical results were obtained in comparison with (18)F-Galacto-RGD. Therefore, (68)Ga-NODAGA-cyclo(RGDyK) is a suitable replacement for (18)F-Galacto-cyclo(RGDfK).

Calcium regulation in endosymbiotic organelles of plants.

In plant cells calcium-dependent signaling pathways are involved in a large array of biological processes in response to hormones, biotic/abiotic stress signals and a variety of developmental cues. This is generally achieved through binding of calcium to diverse calcium-sensing proteins, which subsequently control downstream events by activating or inhibiting biochemical reactions. Regulation by calcium is considered as a eukaryotic trait and has not been described for prokaryotes. Nevertheless, there is increasing evidence indicating that organelles of prokaryotic origin, such as chloroplasts and mitochondria, are integrated into the calcium-signaling network of the cell. An important transducer of calcium in these organelles appears to be calmodulin. In this review we want to give an overview over present data showing that endosymbiotic organelles harbour calcium-dependent biological processes with a focus on calmodulin-regulation.

Arabidopsis ATPase family gene 1-like protein 1 is a calmodulin-binding AAA+-ATPase with a dual localization in chloroplasts and mitochondria.

Members of the AAA(+)-ATPase superfamily (ATPases associated with various cellular activities) are found in all kingdoms of life and they are involved in very diverse cellular processes, including protein degradation, membrane fusion or cell division. The Arabidopsis genome encodes approximately 140 different proteins that are putative members of this superfamily, although the exact function of most of these proteins remains unknown. Using affinity chromatography on calmodulin-agarose with chloroplast proteins, we purified a 50 kDa protein encoded by AT4G30490 with similarity to the ATPase family gene 1 protein from yeast. Structural analysis showed that the protein possesses a single AAA-domain characteristic for members of the AAA(+)-ATPase superfamily and that this contains all features specific to proteins of the ATPase family gene 1-like subfamily. In vitro pull-down as well as cross-linking assays corroborate calcium-dependent binding of the protein to calmodulin. The calmodulin binding domain could be located to a region of 20 amino acids within the AAA-domain in close proximity to the Walker A motif. Our analysis further showed that the protein is localized in both mitochondria and chloroplasts, further supporting the incorporation of both endosymbiotic organelles into the calcium-signaling network of the cell. Localization of the same calmodulin-binding protein into mitochondria and chloroplasts could be a means to provide a coordinated regulation of processes in both organelles by calcium signals.

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria.

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.

13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA.

The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains LmDeltaprfA and LmDeltaprfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by 13C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-13C6]glucose and glucose with natural 13C abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply 13C-labeled isotopologues were determined quantitatively by signal deconvolution. The 13C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.

The roles of the two zinc binding sites in DnaJ.

All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/GrpE foldase machinery catalytically active.