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Methicillin-resistant Staphylococcus aureus(MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to β-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

A novel kinetic model for the aqueous phase hydrogen sulfide scavenging reactions using MEA-triazine (HET) is proposed. The assumptions of the model are based on experimental observations obtained by NMR spectroscopy, supporting the existence of 3,5-bis(2-hydroxyethyl)hexahydro-1,3,5-thiadiazine (TDZ) as a quantitative reaction intermediate and showing the protonation behavior of HET and the lack of protonation of 5-(2-hydroxyethyl)hexahydro-1,3,5-dithiazine (DTZ). Experimental kinetic data were obtained with a new in situ Raman spectroscopy setup, which enabled monitoring the time-variation of bisulfide concentrations in a batch stirred reacting system at temperatures of up to 75 °C for HET/HS–initial concentration ratios from 0.5 to 5. The optimal model parameters were regressed from the experimental data using a brute force optimization method. The rate constants of the first and second scavenging reactions were estimated to be 0.435 and 0.004 L mol–1s–1at 25 °C, and the activation energies were 68 and 57 kJ mol–1, respectively.

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (Bothrops atrox). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania(Leishmania) amazonensis. Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSsfrom genome-sequenced producers of chrysogine we identified a candidate NRPScluster comprising five additional genes named chry2–6. Deletion of the two-module NRPS(NRPS14= chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.

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Mixed micelle formation of anionic surfactants sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) have been studied in water and in 5, 10, and 15 mM concentrations of α-cyclodextrin (α-CD) over mole fraction range of αSDSfrom 0 to 1. From the conductivity curves, the critical micellar concentration (CMC) for the pure and binary mixtures were evaluated. The degree of counterion association (χ) or counterion dissociation (δ), the equivalent ionic conductivities of the monomeric species (Λm), the associated species (Λassc), and the micelle (Λmic) were evaluated from the slope of the conductivity vs concentration plots. The CMC values have been used to calculate the thermodynamic parameters such as the standard free energy of micelle formation % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX % garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz % aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaacq % qHuoarieGacaWFhbWaa0baaSqaaiaab2gacaqGPbGaae4yaaqaaiaa % bcdaaaaaaa!3D6B!$$\Delta G^{{\rm{0}}}_{{{\rm{mic}}}}$$and a transfer of standard free energy of micelle % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX % garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz % aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaada % qadaqaaiabfs5aeHqaciaa-DeadaqhaaWcbaGaaeytaiaabYcacaqG % 0bGaaeOCaaqaaiaabcdaaaaakiaawIcacaGLPaaaaaa!3FA7!$${\left( {\Delta G^{{\text{0}}}_{{{\rm{M,tr}}}} } \right)}$$from the aqueous medium to additive medium computed. The apparent CMC of the surfactants varies linearly with α-CD concentrations. From the dependence of CMC of the surfactants on α-CD concentration, we are able to determine the association constant (K) of surfactant-α-CD inclusion complexes assuming 1:1 stoichiometry. Mixed micelle behaves ideally in the pure water as well as at the different concentrations of α-CD, which was evaluated by using the Clint equation, the regular solution approximation, and Motomura’s formulation. Self-diffusion coefficients of the micelle increased upon the induction of SDS into the micelle. 2D-rotating frame Overhauser effect spectroscopy spectra of SDS and SLAS were recorded in the presence of α-CD to investigate the interaction between H-atoms of the alkyl chain of the surfactants and H-atoms of the hydrophobic cavity of α-CD indicating multiple complexation. The fluorescence anisotropy of rhodamine B has been measured to observe the structural behavior of mixed micelle.Mixed micelle formation of anionic surfactants sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) have been studied in water and in 5, 10, and 15 mM concentrations of α-cyclodextrin (α-CD) over mole fraction range of αSDSfrom 0 to 1. From the conductivity curves, the critical micellar concentration (CMC) for the pure and binary mixtures were evaluated. The degree of counterion association (χ) or counterion dissociation (δ), the equivalent ionic conductivities of the monomeric species (Λm), the associated species (Λassc), and the micelle (Λmic) were evaluated from the slope of the conductivity vs concentration plots. The CMC values have been used to calculate the thermodynamic parameters such as the standard free energy of micelle formation % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX % garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz % aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaacq % qHuoarieGacaWFhbWaa0baaSqaaiaab2gacaqGPbGaae4yaaqaaiaa % bcdaaaaaaa!3D6B!$$\Delta G^{{\rm{0}}}_{{{\rm{mic}}}}$$and a transfer of standard free energy of micelle % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbbjxAHX % garmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy0Hgip5wz % aebbnrfifHhDYfgasaacH8qrps0lbbf9q8WrFfeuY-Hhbbf9v8qqaq % Fr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qq % Q8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaeaaakeaada % qadaqaaiabfs5aeHqaciaa-DeadaqhaaWcbaGaaeytaiaabYcacaqG % 0bGaaeOCaaqaaiaabcdaaaaakiaawIcacaGLPaaaaaa!3FA7!$${\left( {\Delta G^{{\text{0}}}_{{{\rm{M,tr}}}} } \right)}$$from the aqueous medium to additive medium computed. The apparent CMC of the surfactants varies linearly with α-CD concentrations. From the dependence of CMC of the surfactants on α-CD concentration, we are able to determine the association constant (K) of surfactant-α-CD inclusion complexes assuming 1:1 stoichiometry. Mixed micelle behaves ideally in the pure water as well as at the different concentrations of α-CD, which was evaluated by using the Clint equation, the regular solution approximation, and Motomura’s formulation. Self-diffusion coefficients of the micelle increased upon the induction of SDS into the micelle. 2D-rotating frame Overhauser effect spectroscopy spectra of SDS and SLAS were recorded in the presence of α-CD to investigate the interaction between H-atoms of the alkyl chain of the surfactants and H-atoms of the hydrophobic cavity of α-CD indicating multiple complexation. The fluorescence anisotropy of rhodamine B has been measured to observe the structural behavior of mixed micelle.

CopY of Enterococcus hiraeis a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the copoperon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutanshave similar affinities not only for their native promoters, but also for heterologous coppromoters. CopZ of L. lactisaccelerated the release of CopY from the promoter, suggesting that CopZ of L. lactisacts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureusand related repressors. BlaI and MecI have known structures and belong to the family of ‘winged helix’ proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactisshowed that it contained the same α-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the ‘winged helix’ type.

The purpose of this work was to study the inclusion binding interaction of two structurally related steroids, prednisolone and 6α‐methyl prednisolone with a wide variety of cyclodextrins (CDs), both native (α‐, β‐ and γ‐CD) as well as modified (hydroxypropyl‐, sulfobutyl ether‐, glucosyl‐, and maltosyl‐β‐CD and sulfobutyl ether‐γ‐CD), and to relate the binding constants to structural differences in the steroids. Phase‐solubility diagrams of the steroids with various CDs were obtained. The stability constants (K1:1) revealed that β‐CD formed the strongest complex with prednisolone, followed by γ‐CD. With 6α‐methyl prednisolone, the stability constants of both β‐ and α‐CD were considerably lower compared with prednisolone. In contrast, γ‐CD formed a stronger complex with 6α‐methyl prednisolone compared with prednisolone. Derivatives of β‐CD gave slightly altered stability constants compared with native β‐CD. The structures of the complexes between the two guest molecules and native β‐CD were studied by nuclear magnetic resonance spectroscopy. This clearly showed that the introduction of the methyl group led to a different binding geometry of 6α‐methyl prednisolone compared with prednisolone. Additionally, the nuclear magnetic resonance results indicate the presence of an additional, weaker binding site, which is less populated in prednisolone. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:507–515, 2005

The purpose of this work was to study the inclusion binding interaction of two structurally related steroids, prednisolone and 6α‐methyl prednisolone with a wide variety of cyclodextrins (CDs), both native (α‐, β‐ and γ‐CD) as well as modified (hydroxypropyl‐, sulfobutyl ether‐, glucosyl‐, and maltosyl‐β‐CD and sulfobutyl ether‐γ‐CD), and to relate the binding constants to structural differences in the steroids. Phase‐solubility diagrams of the steroids with various CDs were obtained. The stability constants (K1:1) revealed that β‐CD formed the strongest complex with prednisolone, followed by γ‐CD. With 6α‐methyl prednisolone, the stability constants of both β‐ and α‐CD were considerably lower compared with prednisolone. In contrast, γ‐CD formed a stronger complex with 6α‐methyl prednisolone compared with prednisolone. Derivatives of β‐CD gave slightly altered stability constants compared with native β‐CD. The structures of the complexes between the two guest molecules and native β‐CD were studied by nuclear magnetic resonance spectroscopy. This clearly showed that the introduction of the methyl group led to a different binding geometry of 6α‐methyl prednisolone compared with prednisolone. Additionally, the nuclear magnetic resonance results indicate the presence of an additional, weaker binding site, which is less populated in prednisolone. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:507–515, 2005

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS·Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.

A computer program (‘BBReader’) was developed which performs an inverse search in theBioMagResBank database. Given (cross) peak positions of a protein, the program searchesfor atoms with matching chemical shifts and suggests possible assignments for user-specifiedhomo- and heteronuclear one- to three-dimensional COSY- and NOESY-type experiments.It can handle 1H, 13C and 15N spectra. Distance information from PDB files can be utilizedfor filtering possible NOESY cross peak assignments.

Photocyclodehydrogenation of methylated azobenzene derivatives to the corresponding benzo[c]cinnolines was utilized for the synthesis of a number of compounds featuring sterically hindered methyl groups with different degrees of sterical hindrance including close mutual methyl-methyl interaction. Such compounds are of special theoretical interest in the context of intramolecular dynamics, spectroscopy, and quantum (tunneling) effects. Factors determining synthetic accessibility and product distribution are discussed. Photocyclodehydrogenierung von methylsubstituierten Azobenzolderivatven zu den entsprechenden Benzo[c]cinnolinen wurde zur Synthese einer Reihe von Verbindungen benützt, die sich durch Methylgruppen unterschiedlicher sterischer Hinderung – einschließlich gegenseitiger Methyl-Methyl-Wechselwirkung – auszeichnen. Derartige Verbindungen sind aus theoretischer Sicht in Bezug auf intramolekular Dynamik, Spektroskopie und Quanten-(Tunnel-)Effekte von großem Interesse. Faktoren, die synthetische Zugänglichkeit und Produktverteilungen bestimmen, werden diskutiert.

The overall and intramolecular rotational diffusion behavior of 1,3,7,10-tetramethylbenzo[c]cinnoline was determined from longitudinal13C NMR relaxation and {1H}–13C NOE measurements in dilute chloroform solution. The four methyl groups in this compound represent three different situations of sterical hindrance. One pair of methyl groups is in close mutual sterical contact, forming the ends of an open six-membered ring. Assuming completely anisotropic overall molecular tumbling combined with a 120° jump model for the internal methyl rotations the jump rates of methyl groups were evaluated and compared to earlier results on different sterically hindered compounds, in particular with respect to a potential cogwheel-like intermethyl interaction. To characterize intermethyl interactions in different sterical situations, a new gauge—the “methyl interaction volume”—is introduced. Implications for correlated rotational diffusion of methyl groups are discussed.

The plant pathogenic fungus Fusarium graminearumis known to produce a wide array of secondary metabolites during plant infection. This includes several nonribosomal peptides. Recently, the fusaoctaxin (NRPS5/9) and gramilin (NRPS8) gene clusters were shown to be induced by host interactions. To widen our understanding of this important pathogen, we investigated the involvement of the NRPS4gene cluster during infection and oxidative and osmotic stress. Overexpression of NRPS4led to the discovery of a new cyclic hexapeptide, fusahexin (1), with the amino acid sequence cyclo-(d-Ala-l-Leu-d-allo-Thr-l-Pro-d-Leu-l-Leu). The structural analyses revealed an unusual ether bond between a proline Cδto Cβof the preceding threonine resulting in an oxazine ring system. The comparative genomic analyses showed that the small gene cluster only encodes an ABC transporter in addition to the five-module nonribosomal peptide synthetase (NRPS). Based on the structure of fusahexin and the domain architecture of NRPS4, we propose a biosynthetic model in which the terminal module is used to incorporate two leucine units. So far, iterative use of NRPS modules has primarily been described for siderophore synthetases, which makes NRPS4a rare example of a fungal nonsiderophore NRPS with distinct iterative module usage.

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