Evaluation of disease severity in experimental models of rheumatoid arthritis is inevitably associated with assessment of structural bone damage. A noninvasive imaging technology allowing objective quantification of pathophysiological alterations of bone structure in rodents could substantially extend the methods used to date in preclinical arthritis research for staging of autoimmune disease severity or efficacy of therapeutical intervention. Sodium 18 F-fluoride (18 F-NaF) is a bone-seeking tracer well-suited for molecular imaging. Therefore, we systematically examined the use of 18 F-NaF positron emission tomography/computed tomography (PET/CT) in mice with glucose-6-phosphate isomerase (G6PI)–induced arthritis for quantification of pathological bone metabolism.

2-[F-18]fluoro-2-deoxy-n-glucose ((18)FDG) is a glucose surrogate commonly used in clinical or animal imaging but rarely in plant imaging to trace glucose metabolism. Recently, (18)FDG has been employed in plant imaging for studying photoassimilate translocation and glycoside biosynthesis. There is growing evidence that (18)FDG could be used as a tracer in plant imaging studies to trace sugar dynamics. However, to confirm this hypothesis, it was necessary to show that the observed (18)FDG distribution in an intact plant is an outcome of the chemical nature of the introduced radiotracer and not of the plant vascular architecture or radiotracer introduction method.Methods: In the present work, we fed (18)FDG and [(68)Galgallium-citrate (Ga-68-citrate) solution through mature Arabidopsis thaliana leaf and monitored subsequent radioactivity distribution using positron autoradiography. The possible route of radioactivity translocation was elucidated through stem-girdling experiments. We also employed a bi-functional positron emission tomography/computed tomography (PET/CT) modality to capture (18)FDG radiotracer dynamics in one of the plants in order to assess applicability of PET/CT for 4-D imaging in an intact plant.Results: Autoradiography results showed that [F-18] radioactivity accumulated mostly in roots and young growing parts such as the shoot apex, which are known to act as sinks for photoassimilate. [F-18] radioactivity translocation, in this case, occurred mainly via phloem. PET/CT results corroborated with autoradiography. [Ga-68] radioactivity, on the other hand, was mainly translocated to neighboring leaves and its translocation occurred via both xylem and phloem.Conclusion: The radioactivity distribution pattern and translocation route observed after (18)FDG feeding is markedly different from that of Ga-68-citrate. [F-18] radioactivity distribution pattern in an intact plant is found similar to the typical distribution pattern of photoassimilates. Despite its limitations in quantification and resolution, PET/CT could be a useful tool to elucidate in vivo dynamics of [F-18] radioactivity in intact plants. (C) 2014 Elsevier Inc. All rights reserved.

We synthesized the novel sulfonamide derivative, N,N,N,6-tetramethyl-2-(4- nitrophenylsulfonamido)pyrimidin-4-ammonium chloride ( 7), for use as a precursor in ( 18 F)fluoride chemistry. For this, 4-nitrobenzenesulfonyl guanidine was used as the starting material for condensation with ethyl acetoacetate. The 19 F-compound could be synthesized in four steps with moderate yields. The 18 F-labelled analogue, which has high potential as an intermediate for a positron emission tomography tracer, was produced with a radiochemical yield of 58±12% (n = 4, decay corrected).

Alexander Heidrich, 1 Lydia Würbach, 1 Thomas Opfermann, 1,2 Peter Gebhardt, 1 and Hans Peter Saluz 3 1 Department of Cell and Molecular Biology, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute, Beutenbergstr. 11a, 07745 Jena, Germany 2 Clinic of Nuclear Medicine, Jena University Hospital, Bachstr. 18, 07743 Jena, Germany 3 Friedrich Schiller University of Jena, Jena, Germany

SummaryFungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.

PURPOSE:The chick embryo is a well-known economical in vivo model system and is widely applied in preclinical research, e.g., bone development studies. It is therefore surprising that no studies concerning the application of (18)F-fluoride microPET to bone metabolism have been reported so far. This may be due to motion artifacts or the lack of convenient tracer injection sites.METHODS:We resolved the above problems using a combination of sample preparation, anesthesia, microPET imaging, and computational processing, and describe a convenient way of visualizing three- and four- dimensional features of bone metabolism in living chick embryos.RESULTS:The application of (18)F-fluoride microPET facilitates repeat measurements, highly reproducible and motion-artifact-free skeletal imaging, and provides quantitative measurements of in ovo metabolic activities in the bones of developing chick. During microPET measurement, radio tracer was injected intravascularly using a custom-made catheter system, allowing us to additionally investigate early time points in tracer kinetics and uptake.CONCLUSIONS:Our results show that bone metabolism in living chick embryos can be reproducibly studied and quantified in ovo, even for multiple tracer injections over a longer time period. The use of dynamic (18)F-fluoride microPET imaging made it possible to visualize and analyze even small bone structures with excellent quality. Moreover, as our data are comparable to data from corresponding rodent experiments, the use of embryonated chicken eggs is a convenient and economical alternative to other animal models.

ABSTRACT A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum.

We examined the production, separation, and characterization of Zr-89, including supplementation of a commercial Cyclone (R) 18/9 with a self-made Solid Target System (STS). Obtained [Zr-89]Zr-oxalate was used for the labeling of anti-B cell antibodies with desferrioxamine-p-SCN as a bifunctional chelator. Zr-89-labeled antibodies were injected in DBA/1 mice to examine usability for detection of B cells in vivo. PET measurements showed binding of Zr-89-labeled anti-B cell antibodies in tissues with high frequencies of B cells, i.e. in spleen and lymph nodes. (C) 2011 Elsevier Ltd. All rights reserved.

We examined the production, separation, and characterization of (89)Zr, including supplementation of a commercial Cyclone(®) 18/9 with a self-made Solid Target System (STS). Obtained [(89)Zr]Zr-oxalate was used for the labeling of anti-B cell antibodies with desferrioxamine-p-SCN as a bifunctional chelator. (89)Zr-labeled antibodies were injected in DBA/1 mice to examine usability for detection of B cells in vivo. PET measurements showed binding of (89)Zr-labeled anti-B cell antibodies in tissues with high frequencies of B cells, i.e. in spleen and lymph nodes.

The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation.

ABSTRACT Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from l -tyrosine. Proteome analysis indicated that the l -tyrosine degradation enzymes are synthesized when the fungus is grown with l -tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase ( hmgA ) and 4-hydroxyphenylpyruvate dioxygenase ( hppD ). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by l -tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus . Homogentisic acid is the major intermediate, and the l -tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.

A series of 10,13-disubstituted 16-membered macrolides was synthesized using nitroso Diels-Alder reactions of leucomycin A7. Despite the extensive constituent functionalities in leucomycin, the hetero cycloaddition reactions proceeded in a highly regio- and stereoselective fashion. Subsequent chemical modifications of the nitroso cycloadducts, including N-O bond reduction, were also conducted. Most leucomycin derivatives retained antibiotic profiles similar to leucomycin A7, and, in contrast to leucomycin itself, several exhibited moderate antiproliferative and cytotoxic activity.

Siderophores based on sugar backbones substituted at the 2,3,4- or 2,3,6 positions with hydroxamic or retro-hydroxamic acid chelating units were synthesized and characterized. The alkyl terminus of the iron-coordinating side chain units facilitate lipophilic interactions. Iron coordination properties and complex stability were investigated by ESI-MS and the CAS-Test. The results were correlated to structure activity relationships determined by microbial growth promotion studies under iron limited conditions using wild type strains and iron transport mutants of Mycobacterium smegmatis.

The synthesis and characterization of a novel tripodal mono-hydroxamate, bis catecholate siderophore mimic, Nα,-Nε-bis[2,3-dihydroxybenzoyl]-l-lysyl-(γ-N-methyl-N-hydroxyamido)-l-glutamic acid (H6L), is described. The structure of H6L was established by 2D NMR and mass spectrometry. The chelation chemistry of H6L with respect to iron(III) is characterized in aqueous solution through determination of ligand pKa values and iron(III) binding constants using spectrophotometric and potentiometric titration techniques. Proton dependent iron(III)–ligand equilibrium constants were determined using a model based on the sequential protonation of the iron(III)–siderophore complex. These results were used to calculate the pH dependent speciation, the overall formation constant logβ110 (31.4) and pM value (18.3) for H6L with iron(III). The ability of H6L to deliver the essential nutrient iron to living cells is determined through growth promotion assays using various bacterial strains.

A complete characterization of the aqueous solution Fe(III) and Fe(II) coordination chemistry of a saccharide-based ferrichrome analogue, 1-O-methyl-2,3,6-tris-O-[4-(N-hydroxy-N-ethylcarbamoyl)-n-butyryl]-alpha-D-glucopyranoside (H3LN236), is reported including relevant thermodynamic parameters and growth promotion activity with respect to both Gram-negative and Gram-positive bacterial strains. The saccharide platform is an attractive backbone for the design and synthesis of ferrichrome analogues because of its improved water solubility and hydrogen-bonding capabilities, which can potentially provide favorable receptor recognition and biological activity. The ligand deprotonation constants (pKa values), iron complex (FeIII(LN236) and FeII(LN236)1-) protonation constants (KFeHxL-236-N), overall Fe(III) and Fe(II) chelation constants (beta110), and aqueous solution speciation were determined by spectrophotometric and potentiometric titrations, EDTA competition equilibria, and cyclic voltammetry. Log betaIII110 = 31.16 and pFe = 26.1 for FeIII(LN236) suggests a high affinity for Fe(III), which is comparable to or greater than ferrichrome and other ferrichrome analogues. The E1/2 for the FeIII(LN236)/FeII(LN236)1- couple was determined to be -454 mV (vs NHE) from quasi-reversible cyclic voltammograms at pH 9. Below pH 6.5, the E1/2 shifts to more positive values and the pH-dependent E1/2 profile was used to determine the FeII(LN236)1- protonation constants and overall stability constant log betaII110 = 11.1. A comparative analysis of similar data for an Fe(III) complex of a structural isomer of this exocyclic saccharide chelator (H3LR234), including strain energy calculations, allows us to analyze the relative effects of the pendant arm position and hydroxamate moiety orientation (normal vs retro) on overall complex stability. A correlation between siderophore activity and iron coordination chemistry of these saccharide-hydroxamate chelators is made.

A small library of leucomycin A7 derivatives was prepared by NaCNBH3/ZnCl2-mediated reductive amination of the C18 aldehyde moiety with a variety of lipophilic benzylamines and tested for antibiotic activity.

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SUE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD (similar to 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

New trishydroxamates and triscatecholates based on methyl α-D-glucopyranoside, methyl α-D-galactopyranoside, methyl α-D-ribopyranoside and methyl α-D-xylopyranoside as well as on 1,3,5-tri-O-benzyl-myo-inositol were synthesized. N-Methylsuccinohydroxamate, N-methylglutarohydroxamate and their O-benzoyl derivatives were used as hydroxamate moieties. 2,3-Dihydroxybenzoyl derivatives and acylated compounds as well as 2,3- and 3,4-dihydroxybenzylidenehydrazino derivatives, partly with spacer groups, were utilized as catecholate components. The siderophore activity of the prepared siderophore analogues was examined by a growth promotion assay with various Gram-negative bacteria and mycobacteria and by the CAS-assay. Some trishydroxamates and triscatecholates showed siderophore activity on Gram-negative bacteria and triscatecholates on mycobacteria. Iron complexes of the trishydroxamates act as siderophores for all types of iron transport mutants. The recognition and uptake specificity of these compounds was studied by E. coli siderophore receptor and iron transport mutants. Structure activity correlations are discussed.

New analogues of triscatecholate siderophores based on linear or tripodal triamines with or without spacer groups or lipophilic and hydrophilic substituents were synthesized. The catecholate moieties were prepared in OH-forms, as acetylated compounds or masked as 8-methoxycarbonyloxy-2,4-dioxo-1,3-benzoxazine derivatives. Some of the new compounds were active as siderophores tested by growth promotion assays using various Gram-negative bacteria and mycobacteria under iron limitation and by CAS-assay. Structure-activity-correlations have been studied.