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Purpose: Helicobacter pylori (H. pylori) is a significant risk factor for development of gastric cancer (GC), one of the deadliest malignancies in the world. However, the mechanism by which H. pylori induces gastric oncogenesis remains unclear. Here, we investigated the function of IL-6 in gastric oncogenesis and macrophage-epithelial cell interactions., Methods: We analyzed publicly available datasets to investigate the expression of IL-6 and infiltration of M2 macrophages in GC tissues, and determine the inter-cellular communication in the context of IL-6. Human gastric epithelial and macrophage cell lines (GES-1 and THP-1-derived macrophages, respectively) were used in mono- and co-culture experiments to investigate autocrine-and paracrine induction of IL-6 expression in response to H. pylori or IL-6 stimulation., Results: We found that IL-6 is highly expressed in GC and modulates survival. M2 macrophage infiltration is predominant in GC and drives an IL-6 mediated communication with gastric epithelium cells. In vitro, IL-6 triggers its own expression in GES-1 and THP-1-derived macrophages cells. In addition, these cell lines are able to upregulate each other's IL-6 levels in an autocrine fashion, which is enhanced by H. pylori stimulation., Conclusion: This study indicates that IL-6 in the tumor microenvironment is essential for intercellular communication. We show that H. pylori enhances an IL-6-driven autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells, which may contribute to gastric carcinogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Nuclear magnetic resonance spectroscopy combined with modeling represents a powerful tool for the structural analysis of heterogeneous materials. In this contribution we describe an upgraded method, particularly suited for the structural analysis of mixed-linker metal-organic framework materials, which is based on the measurement and modeling of proton spin diffusion among constituents. We tested the method on a UiO-66-type metal-organic material, in which the organic building units were 1,4-benzenedicarboxylate and trans-1,4-cyclohexanedicarboxylate anions distributed within the framework in an unknown manner. We showed that resolution of the signals of different building units could be significantly enhanced by the carbon-detected version of the proton spin-diffusion measurement. Because this kind of measurement is much more time consuming than the proton-detected measurement and because one has to carry out several two-dimensional measurements to extract spin-diffusion curves, we inspected the possibility of reducing the number of such measurements. This could be done by limiting the analysis to short mixing times, for which, as shown in this contribution, linear approximation is valid. When working in the linear regime, only a few experimental points are needed to determine the slope of spin-diffusion curves. Usage of short spin-diffusion mixing times significantly shortened the total measurement time and also markedly simplified the modeling of spin-diffusion curves., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections., (© 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.)

Glutathione is an intracellular redox-active tripeptide thiol with a central role in cellular physiology across all kingdoms of life. Glutathione biosynthesis has been traditionally viewed as a conserved process relying on the sequential activity of two separate ligases, but recently, an enzyme (GshF) that unifies both necessary reactions in one platform has been identified and characterized in a number of pathogenic and free-living bacteria. Here, we report crystal structures of two prototypic GshF enzymes from Streptococcus agalactiae and Pasteurella multocida in an effort to shed light onto the structural determinants underlying their bifunctionality and to provide a structural framework for the plethora of biochemical and mutagenesis studies available for these enzymes. Our structures reveal how a canonical bacterial GshA module that catalyzes the condensation of L-glutamate and L-cysteine to γ-glutamylcysteine is linked to a novel ATP-grasp-like module responsible for the ensuing formation of glutathione from γ-glutamylcysteine and glycine. Notably, we identify an unprecedented subdomain in the ATP-grasp module of GshF at the interface of the GshF dimer, which is poised to mediate intersubunit communication and allosteric regulation of enzymatic activity. Comparison of the two GshF structures and mapping of structure-function relationships reveal that the bifunctional GshF structural platform operates as a dynamic dimeric assembly., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Four new triphenyltin(IV) complexes of composition Ph(3)SnLH (where LH=2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1-4) were synthesized and characterized by spectroscopic (((1))H, ((13))C and ((119))Sn NMR, IR, ((119))Sn Mössbauer) techniques in combination with elemental analysis. The ((119))Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph(3)SnL((1))H (1), Ph(3)SnL((3))H (3), Ph(3)SnL((4))H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ((119))Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1-4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6-7) and tributyltin(IV) complexes (8-11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1-3 are also comparable to those of its o-analogs i.e. 4-7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID(50) values in the range 41-53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1-3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8-11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1-7 and tributyltin(IV) complexes 8-11 is also discussed against a panel of human tumor cell lines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A series of tributyltin(IV) complexes based on 2/4-[(E)-2-(aryl)-1-diazenyl]benzoate ligands was synthesized, wherein the position of the carboxylate and aryl substituents (methyl, tert-butyl and hydroxyl) varies. The complexes, Bu(3)SnL(1-4)H (1-4), have been structurally characterized by elemental analysis and IR, NMR ((1)H, (13)C, and (119)Sn) and (119)Sn Mössbauer spectroscopy. All have a tetrahedral geometry in solution and a trigonal bipyramidal geometry in the solid-state, except for Bu(3)SnL(4)H (4) that was ascertained to have tetrahedral coordination by X-ray crystallography. Cytotoxicity studies were carried out on human tumor cell lines A498 (renal cancer), EVSA-T (mammary cancer), H226 (non-small-cell lung cancer), IGROV (ovarian cancer), M19 MEL (melanoma), MCF-7 (mammary cancer) and WIDR (colon cancer). Compared to cisplatin, test compounds 1-4 had remarkably good activity, despite the presence of substantial steric bulk due to Sn-Bu ligands. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of organotin(IV) benzoates, along with some reference drug molecules, is also discussed against a panel of human tumor cell lines. Molecular structures of the tributyltin(IV) complexes (1-4) were fully optimized using the PM6 semi-empirical method and docking studies performed with key enzymes associated with the propagation of cancer, namely ribonucleotide reductase, thymidylate synthase, thymidylate phosphorylase and topoisomerase II. The theoretical results are discussed in relation to the mechanistic role of the cytotoxic active test compounds (1-4)., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

A series of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes has been synthesized and characterized by (1)H-, (13)C-, (119)Sn NMR, ESI-MS (electrospray ionization mass spectrometry), IR and (119m)Sn Mössbauer spectroscopic techniques in combination with elemental analyses. The structures of four di-n-butyltin(IV) complexes, viz., (n)Bu(2)Sn(L(3))(2) (3), (n)Bu(2)Sn(L(4))(2) (4), (n)Bu(2)Sn(L(5))(2) (5) and (n)Bu(2)Sn(L(7))(2).0.5C(6)H(6) (7) (LH=5-[(E)-2-(aryl)-1-diazenyl)quinolin-8-ol) were determined by single crystal X-ray diffraction. In general, the complexes were found to adopt a distorted cis-octahedral arrangement around the tin atom. These complexes retain their solid-state structure in non-coordinating solvent as evidenced by (119)Sn and (13)C NMR spectroscopic results. The in vitro cytotoxicity of di-n-butyltin(IV) complexes (3-8) is reported against seven well characterized human tumour cell lines. The basicity of the two quinolinolato donor N and O atoms of the ligands are discussed in relation to the cytotoxicity data.

Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

Glutathione reductase (GR) plays a vital role in maintaining the antioxidant levels of the cytoplasm by catalyzing the reduction of glutathione disulfide to reduced glutathione, thereby using NADPH and flavin adenine dinucleotide as cofactors. Chromatiaceae have evolved an unusual homolog that prefers both a modified substrate (glutathione amide disulfide [GASSAG]) and a different cofactor (NADH). Herein, we present the crystal structure of the Chromatium gracile glutathione amide reductase (GAR) both alone and in complex with NAD(+). An altered charge distribution in the GASSAG binding pocket explains the difference in substrate specificity. The NADH binding pocket of GAR differs from that of wild-type GR as well as that of a low active GR that was engineered to mimic NADH binding. Based on the GAR structure, we propose two attractive rationales for producing an efficient GR enzyme with NADH specificity.

Bismuth dithiocarbamate complexes of general formula Bi(S(2)CNR(2))(3) demonstrate potent in vitro cytotoxicity against a panel of seven human cancer cell lines; a structure-activity relationship has been established. Potency exhibited by the R=Et (2) derivative, for example, is unrivalled by standard cancer drugs with the exception of paclitaxel. In vivo studies indicate a significant anti-tumor effect exerted by (2) against both OVCAR-3, an ovarian cancer cell line, and HT-29, a colon carcinoma cell line.

Aspartate carbamoyltransferase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allosteric regulation. The structure of the Escherichia coli enzyme has been thoroughly analyzed by X-ray crystallography, and recently the crystal structures of two hyperthermophilic ATCases of the same structural class have been characterized. We here report the detailed functional and structural investigation of the ATCase from the psychrophilic deep sea bacterium Moritella profunda. Our analysis indicates that the enzyme conforms to the E. coli model in that two allosteric states exist that are influenced by similar homotropic interactions. The heterotropic properties differ in that CTP and UTP inhibit the holoenzyme, but ATP seems to exhibit a dual regulatory pattern, activating the enzyme at low concentrations and inhibiting it in the mM range. The crystal structure of the unliganded M. profunda ATCase shows resemblance to a more extreme T state reported previously for an E. coli ATCase mutant. A detailed molecular analysis reveals potential features of adaptation to cold activity and cold regulation. Moreover, M. profunda ATCase presents similarities with certain mutants of E. coli ATCase altered in their kinetic properties or temperature relationships. Finally, structural and functional comparison of ATCases across the full physiological temperature range agrees with an important, but fundamentally different role for electrostatics in protein adaptation at both extremes, i.e. an increased stability through the formation of ion pairs and ion pair networks at high physiological temperatures, and an increased flexibility through enhanced protein solvation at low temperatures.

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.

Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.

Current methods for quantitative determination of chlormequat residues in food crops are characterized by rather low recoveries and the need for derivatization (in case of gas chromatography, GC), or by high capital investment (in case of liquid chromatography-mass spectrometry, LC-MS). We propose a cation-exchange chromatography method for the analysis of chlormequat in pears. The method is based on extraction of the target compound with 40 mM HCl, followed by centrifugation and filtration. The filtrate is directly injected into an ion chromatograph equipped with a commercially available cation-exchange column and a suppressed conductivity detection system. While the limit of detection (LOD) (0.5 mg/kg) may not be small enough to allow dietary analysis, the method meets all validation requirements and is an alternative for the existing GC and LC-MS methods in quality control.

An overview of the development of anti-tumor organotin derivatives, sometimes as active in vitro as doxorubicin, is presented and discussed. Solubility in water is an important issue, dominating the in vivo testing of compounds with promising in vitro properties. Several water-soluble organotin compounds gave the best in vitro activities. Novel, useful organotin anti-tumor compounds should be designed toward improved water solubility.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

A gas-liquid chromatographic method for the determination of the gamma-aminobutyric acid-like drug 1-hydroxy-3-aminopyrrolidone-2 (HA-966) in plasma is described. HA-966 was converted into its diacetyl derivative AC2HA-966 with acetic anhydride. This compound could be suitable eluted from a capillary OV-17 support-coated open tubular column. A sensitive detection method was achieved by making use of nitrogen-phosphorus-selective flame ionization.

The gas--liquid chromatography of cyclophosphamide has been extensively investigated. Several methods for the assay of cyclophosphamide in plasma are reported, those with the nitrogen--phosphorus-specific detection being most sensitive and selective.