ABSTRACT
Overexpression of the neurotensin receptor type 1 (NTS1R), a peptide receptor located at the plasma membrane, has been reported for a variety of malignant tumors. Thus, targeting the NTS1R with 18F- or 68Ga-labeled ligands is considered a straightforward approach towards in vivo imaging of NTS1R-expressing tumors via positron emission tomography (PET). The development of suitable peptidic NTS1R PET ligands derived from neurotensin is challenging due to proteolytic degradation. In this study, we prepared a series of NTS1R PET ligands based on the C-terminal fragment of neurotensin (NT(8-13), Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) by attachment of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an Nω-carbamoylated arginine side chain. Insertion of Ga3+ in the DOTA chelator gave potential PET ligands that were evaluated concerning NTS1R affinity (range of Ki values: 1.2-21 nM) and plasma stability. Four candidates were labeled with 68Ga3+ and used for biodistribution studies in HT-29 tumor-bearing mice. [68Ga]UR-LS130 ([68Ga]56), containing an N-terminal methyl group and a ß,ß-dimethylated tyrosine instead of Tyr11, showed the highest in vivo stability and afforded a tumor-to-muscle ratio of 16 at 45 min p.i. Likewise, dynamic PET scans enabled a clear tumor visualization. The accumulation of [68Ga]56 in the tumor was NTS1R-mediated, as proven by blocking studies.
ABSTRACT
The hyperphosphorylated protein phosvitin (PV) undergoes a pH-dependent transition between PII- and ß-sheet secondary structures, a process deemed crucial for its role in the promotion of biogenic apatite formation. The transition occurs surprisingly slowly (minutes to hours). This is consistent with a slow aggregation process involving ionic interactions of charged groups on the protein surface. Herein, we determined the associated transition pK values and time constants through matrix least-squares (MLS) global fitting of a series of pH- and time-dependent circular dichroism (CD) spectra recorded in the presence of different mono-, bi- and trivalent cations. Supporting our results with dynamic light scattering data, we clearly identified a close correlation of ß-sheet transition and the formation of small aggregates at low pH. This process is inhibited in the presence of all tested cations with the strongest effects for trivalent cations (Fe3+ and Al3+). In the presence of Ca2+ and Mg2+, larger higher-order particles are formed from PV in the ß-sheet conformation, as identified from the interpretation of differential scattering observed in the CD spectra. Our observations are consistent with the existence of a multi-step equilibrium between aggregated and non-aggregated species of PV. The equilibrium is highly sensitive to the environment pH and salt concentration with exceptional behavior in the presence of divalent cations such as Ca2+ and Mg2+.
Subject(s)
Phosphoproteins , Phosvitin , Cations, Divalent/chemistry , Circular Dichroism , Hydrogen-Ion Concentration , Protein Conformation, beta-Strand , Protein Structure, SecondaryABSTRACT
The recent crystallization of the neuropeptide Y Y1 receptor (Y1R) in complex with the argininamide-type Y1R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design of nonpeptidic Y1R ligands. We designed novel fluorescent probes showing excellent Y1R selectivity and, in contrast to previously described fluorescent Y1R ligands, considerably higher (â¼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y1R binding affinities (pKi values of 9.36-9.95) and proved to be Y1R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry- and fluorescence anisotropy-based Y1R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.
Subject(s)
Neuropeptide Y , Receptors, Neuropeptide Y , Binding, Competitive , Fluorescent Dyes , Ligands , Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/metabolismABSTRACT
Elastin-like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid-liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least-squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG)20 , (VPGVG)40 , and (VPGVG)60 , we found that coacervation occurs sharply when a certain number of repeat units has acquired ß-turn conformation (in our sequence setting a threshold of approx. 20â repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of ß-turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein-based smart materials.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Peptides/chemistry , Thermodynamics , Circular Dichroism , Humans , Intrinsically Disordered Proteins/metabolism , Models, Molecular , Peptides/metabolism , Protein ConformationABSTRACT
The asymmetric reduction of activated C=C bonds such as enones is well established for non-enzymatic methods as well as in biocatalysis. However, the asymmetric reduction of unfunctionalized C=C bonds is mainly performed with transition metal catalysts whereas biocatalytic approaches are lacking. We have tested two FAD-dependent archaeal geranylgeranyl reductases (GGR) for the asymmetric reduction of isolated C=C bonds. The reduction of up to four double bonds in terpene chains with different chain lengths and head groups was confirmed. Methyl-branched E-alkenes were chemoselectively reduced in the presence of cyclic, terminal or activated alkenes. Using a removable succinate "spacer", farnesol and geraniol could be quantitatively reduced (>99 %). The reduction is strictly (R)-selective (enantiomeric excess >99 %). Hence, GGRs are promising biocatalysts for the asymmetric reduction of unactivated isolated C=C bonds, opening new opportunities for the synthesis of enantiopure branched alkyl chains.
Subject(s)
OxidoreductasesABSTRACT
The structural complexity and bioactivity of natural products often depend on enzymatic redox tailoring steps. This is exemplified by the generation of the bisbenzannulated [5,6]-spiroketal pharmacophore in the bacterial rubromycin family of aromatic polyketides, which exhibit a wide array of bioactivities such as the inhibition of HIV reverse transcriptase or DNA helicase. Here we elucidate the complex flavoenzyme-driven formation of the rubromycin pharmacophore that is markedly distinct from conventional (bio)synthetic strategies for spiroketal formation. Accordingly, a polycyclic aromatic precursor undergoes extensive enzymatic oxidative rearrangement catalyzed by two flavoprotein monooxygenases and a flavoprotein oxidase that ultimately results in a drastic distortion of the carbon skeleton. The one-pot in vitro reconstitution of the key enzymatic steps as well as the comprehensive characterization of reactive intermediates allow to unravel the intricate underlying reactions, during which four carbon-carbon bonds are broken and two CO2 become eliminated. This work provides detailed insight into perplexing redox tailoring enzymology that sets the stage for the (chemo)enzymatic production and bioengineering of bioactive spiroketal-containing polyketides.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Furans/chemical synthesis , Mixed Function Oxygenases/metabolism , Polyketides/chemistry , Spiro Compounds/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biological Products/pharmacology , DNA Helicases/antagonists & inhibitors , Furans/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , Oxidation-Reduction , Polyketides/pharmacology , Spiro Compounds/pharmacologyABSTRACT
Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated ß-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.
Subject(s)
Alcohol Oxidoreductases/chemistry , Bacterial Proteins/chemistry , Ketones/chemistry , Alcohol Oxidoreductases/genetics , Alcohols/chemical synthesis , Alcohols/chemistry , Bacterial Proteins/genetics , Biocatalysis , Mutation , Oxidation-Reduction , Stereoisomerism , Streptomyces/enzymology , Substrate SpecificityABSTRACT
Chiral coordination compounds of Co(II) and other open-shell metal complexes display enhanced vibrational circular dichroism (VCD) spectra associated with the existence of low-lying excited states (LLESs). In addition to the enhancement, a series of Co(II) salicylaldiminato complexes exhibits an almost monosignate pattern of VCD bands, a unique feature if compared with the usual alternation of positive and negative signals. Frequency and excited-state calculations reveal that VCD enhancement and sign reversal selectively affect the normal modes of B symmetry of the C2-symmetric pseudotetrahedral species thanks to their combination with one or more LLES having the same B symmetry. This proves the strict relation between VCD enhancement and monosignate appearance and demonstrates an unprecedented symmetry dependence of the two phenomena.
ABSTRACT
The initial step in reactions catalyzed by NAD(P)H-dependent alcohol dehydrogenases (ADHs) is the binding of the cofactor to the active site. To study this process, we measured NAD(P)H concentration-dependent circular dichroism (CD) in the presence of purified enzymes (ADH from horse liver, HLADH; ADH-A from Rhodococcus ruber; YGL157w from Saccharomyces cerevisiae) or enzyme-containing whole cell extract (ADH from Lactobacillus brevis, LbADH). We determined the proportions of binding and non-binding NAD(P)H and the associated dissociation constants (Kd) from matrix least-squares global fitting of law of mass action-derived model. Furthermore, the fitting allowed the back calculation of CD spectra corresponding to the cofactor in its bound conformation. With increasing pH and/or increasing ionic strength, we detected an increase in Kd for the NADH·HLADH complex with the shape of the bound cofactor conformation spectrum remaining unaffected. While the bound cofactor spectrum for the ADH-A·NADH complex was similar to that for HLADH, the corresponding spectra obtained for the NADPH-dependent enzymes YGL157w and LbADH exhibited opposite signs of the most prominent band. In comparison to CD spectra calculated on cofactor geometries from the crystal structures at the sTD-DFT level, we found that the sign of the bound cofactor spectrum correlates with the orientation of the nicotinamide ring of the cofactor in the active site. These results demonstrate the usefulness of the global analysis of cofactor titration CD spectra to study the role of cofactor binding and its geometry in ADH catalysis.
Subject(s)
Alcohol Dehydrogenase/chemistry , NADP/chemistry , Alcohol Dehydrogenase/metabolism , Animals , Binding Sites , Circular Dichroism , Horses , NADP/metabolism , Protein Binding , Protein ConformationABSTRACT
Chiroptical broad-range spectral analysis extending from UV to mid-IR was employed to study a family of Co(II) N-(1-(aryl)ethyl)salicylaldiminato Schiff base complexes with pseudotetrahedral geometry associated with chirality-at-metal of the Δ/Λ type. While common chiral organic compounds have well-separated absorption and circular dichroism spectra (CD) in the UV/vis and IR regions, chiral Co(II) complexes feature an almost unique continuum of absorption and CD bands, which cover in sequence the UV, visible, near-IR (NIR), and IR regions of the electromagnetic spectrum. They can be collected in a single (chiro)optical superspectrum ranging from the UV (230 nm, 5.4 eV) to the mid-IR (1000 cm-1, 0.12 eV), which offers a fingerprint of the structure and stereochemistry of the metal complexes. Each region of the superspectrum contributes to one piece of information: the NIR-CD region, in combination with TDDFT calculations, allows a reliable assignment of the metal-centered chirality; the UV-CD region facilitates the analysis of the Δ/Λ diastereomeric equilibrium in solution; and the IR-VCD region contains a combination of low-lying metal-centered electronic states (LLES) and ligand-centered vibrations and displays characteristically enhanced and monosignate VCD bands. Circular dichroism in the NIR and IR regions is crucial to reveal the presence of d-d transitions of the Co(II) core which, due to the electric-dipole forbidden character, would be otherwise overlooked in the corresponding absorption spectra.
ABSTRACT
Xanthocidin and six new derivatives were isolated from the endophytic Streptomyces sp. AcE210. Their planar structures were elucidated by 1D and 2D NMR spectroscopy as well as by HRMS. The absolute configuration of one compound was determined by using vibrational circular dichroism spectroscopy (VCD). The structural similarities of xanthocidin and some of the isolated xanthocidin congeners to the methylenomycins A, B, and C suggested that the biosynthesis of these compounds might follow a similar route. Feeding studies with isotopically labelled [13 C5 ]-l-valine showed that instead of utilizing acetyl-CoA as starter unit, which has been proposed for the methylenomycin biosynthesis, Streptomyces sp. AcE210 employs an isobutyryl-CoA starter unit, resulting in a branched side chain in xanthocidin. Further evidence for a comparable biosynthesis was given by the analysis of the genome sequence of Streptomyces sp. AcE210 that revealed a cluster of homologues to the mmy genes involved in methylenomycin biosynthesis.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Cyclopentanes/metabolism , Acyl Coenzyme A/metabolism , Anti-Bacterial Agents/chemistry , Carbon Isotopes/chemistry , Cyclopentanes/chemistry , Molecular Structure , Multigene Family , Streptomyces/chemistry , Streptomyces/genetics , Streptomyces/metabolism , Valine/chemistry , Valine/metabolismABSTRACT
Birch reductions of aromatic hydrocarbons by means of single-electron-transfer steps depend on alkali metals, ammonia, and cryogenic reaction conditions. In contrast, 2-naphthoyl-coenzymeâ A (2-NCoA) and 5,6-dihydro-2-NCoA (5,6-DHNCoA) reductases catalyze two two-electron reductions of the naphthoyl-ring system to tetrahydronaphthoyl-CoA at ambient temperature. Using a number of substrate analogues, we provide evidence for a Meisenheimer complex-analogous intermediate during 2-NCoA reduction, whereas the subsequent reduction of 5,6-dihydro-2-NCoA is suggested to proceed via an unprecedented cationic transition state. Using vibrational circular dichroism (VCD) spectroscopy, we demonstrate that both enzymatic reductions are highly stereoselective in D2 O, providing an enantioselective pathway to products inaccessible by Birch reduction. Moreover, we demonstrate the power of VCD spectroscopy to determine the absolute configuration of isotopically engendered alicyclic stereocenters.
Subject(s)
Coenzyme A/chemistry , Naphthalenes/chemistry , Oxidoreductases/chemistry , Catalysis , Circular Dichroism/methods , Oxidation-Reduction , Stereoisomerism , Tetrahydronaphthalenes/chemistryABSTRACT
Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.
Subject(s)
Antineoplastic Agents/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/chemistry , Heat-Shock Response/drug effects , Imatinib Mesylate/pharmacology , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Animals , Antineoplastic Agents/chemistry , Binding Sites , Biomarkers, Tumor , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/chemistry , HSP90 Heat-Shock Proteins/metabolism , Humans , Imatinib Mesylate/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Multimerization/drug effects , Spectrum Analysis , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Tranylcypromine/analogs & derivatives , Tranylcypromine/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Cell Line, Tumor , Histone Demethylases/metabolism , Humans , Leukemia/drug therapy , Leukemia/metabolism , Leukemia/pathology , Mice , Models, Molecular , Monoamine Oxidase Inhibitors/chemistry , Monoamine Oxidase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
Studying enzymatic reductions of substrates with more than a single keto group is challenging, as the carbonyl reduction can create a vast array of regio- and stereoisomers. If used as reference compounds, regio- and stereopure hydroxy ketides could facilitate the characterization of reductases with unclear regio- and stereoselectivity. We have combined nonenzymatic and enzymatic reduction and oxidation steps to obtain all four regio- and stereoisomers of tert-butyl hydroxyoxohexanoates in high optical purity (enantiomeric ratio (er) of 99 : 1 for the δ-hydroxy-ß-keto isomers; er of >97 : 3 for the ß-hydroxy-δ-keto isomers). Furthermore, we have prepared seven of the eight possible regioisomers and diastereomers of γ-methylated hydroxyoxohexanoates. These 11 compounds allowed unraveling the complex stereoselectivity of ß,δ-diketo ester reductions catalyzed by carbonyl reductase S1 from Candida magnoliae (CMCR-S1). Our analysis shows that the regio- and stereoselectivity of CMCR-S1-catalyzed reductions is highly sensitive toward modifications at the C-terminus of CMCR-S1: in addition to the expected δ-hydroxy product, the variant with a C-terminal His-tag also led to formation of ß-hydroxy by-products with high optical purity.
Subject(s)
Alcohol Oxidoreductases/metabolism , Candida/enzymology , Histidine/metabolism , Biocatalysis , Histidine/chemistry , Molecular Structure , StereoisomerismABSTRACT
NADP(H)-dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d-glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized 1 Hâ NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.
Subject(s)
Glucose 1-Dehydrogenase/metabolism , Imines/metabolism , Bacillus subtilis/enzymology , Biocatalysis , Chromatography, Gas , Glucose/metabolism , Imines/chemistry , Magnetic Resonance Spectroscopy , NADP/metabolism , Oxidation-Reduction , Stereoisomerism , Substrate SpecificityABSTRACT
The interplay between noncollagenous proteins and biomineralization is widely accepted, yet the contribution of their secondary structure in mineral formation remains to be clarified. This study demonstrates a role for phosvitin, an intrinsically disordered phosphoprotein, in chick embryo skeletal development, and using circular dichroism and matrix least-squares Henderson-Hasselbalch global fitting, unravels three distinct pH-dependent secondary structures in phosvitin. By sequestering phosvitin on a biomimetic 3D insoluble cationic framework at defined pHs, access is gained to phosvitin in various conformational states. Induction of biomimetic mineralization at near physiological conditions reveals that a disordered secondary structure with a low content of PII helix is remarkably efficient at promoting calcium adsorption, and results in the formation of biomimetic hydroxyapatite through an amorphous calcium phosphate precursor. By extending this finding to phosphorylated full-length human recombinant dentin matrix protein-1 (17-513 AA), this bioinspired approach provides compelling evidence for the role of a disordered secondary structure in phosphoproteins in bone-like apatite formation.
Subject(s)
Phosphoproteins/chemistry , Adsorption , Animals , Apatites , Biomimetics , Chick Embryo , Chickens , Durapatite , HumansABSTRACT
Understanding the composition of the adsorbed protein layer on a biomaterial surface is of an extreme importance as it directs the primary biological response. Direct detection using labeled proteins and indirect detection based on enzymatic assays or changes to mass, refractive index or density of a surface have been so far established. Nevertheless, using current methodologies, detection of multiple proteins simultaneously and particularly in a three-dimensional (3D) substrates is challenging, with the exception of radiolabeling. Here using fluorescence molecular tomography (FMT), we present a non-destructive and versatile approach to quantify adsorption of multiple proteins within 3D environments and reveal the dynamics of adsorption of human serum albumin (HSA) and fibrinogen (Fib) on 3D polymeric scaffold. Furthermore, we show that serum starved human articular chondrocytes in 3D environment preferentially uptake HSA over Fib and to our knowledge this represents the first example of direct visualization and quantification of protein adsorption in a 3D cell culture system. STATEMENT OF SIGNIFICANCE: The biomaterial surface upon exposure to biological fluids is covered by a layer of proteins, which is modified over a period of time and dictates the fate of the biomaterial. In this study, we present and validate a new methodology for quantification of protein adsorption on to a three-dimensional polymer scaffold from unitary and binary systems, using fluorescence molecular tomography, an optical trans-illumination technique with picomolar sensitivity. In additional to being able to follow behavior of two proteins simultaneously, this methodology is also suitable for studying protein uptake in cells situated in a polymer environment. The ability to follow protein adsorption/uptake in a continuous manner opens up new possibilities to study the role of serum proteins in biomaterial compatibility.
Subject(s)
Chondrocytes/metabolism , Fibrinogen , Molecular Imaging , Optical Imaging , Serum Albumin, Human , Adult , Chondrocytes/cytology , Female , Fibrinogen/chemistry , Fibrinogen/pharmacokinetics , Fibrinogen/pharmacology , Humans , Male , Serum Albumin, Human/chemistry , Serum Albumin, Human/pharmacokinetics , Serum Albumin, Human/pharmacologyABSTRACT
Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A-D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.
ABSTRACT
The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired ß-strands.