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Background Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic efficacy of this novel nanostructured system were assessed. Results Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around − 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin microbiota. Additionally, the stability and sterility of developed NPs were maintained along storage. Conclusion TH-NP showed a promising and efficient alternative for the treatment of skin acne infection, avoiding antibiotic administration, reducing side effects, and preventing microbial drug resistance, without altering the healthy skin microbiota. Additionally, TH-NP enhanced TH antioxidant activity, constituting a natural, preservative-free, approach for acne treatment. Graphical Abstract

There are a large number of remedies in traditional medicine focused on relieving pain and inflammation. Flavanones have been a potential source in the search for leading compounds and biologically active components, and they have been the focus of much research and development in recent years. Eysenhardtia platycarpa is used in traditional medicine for the treatment of kidney diseases, bladder infections, and diabetes mellitus. Many compounds have been isolated from this plant, such as flavones, flavanones, phenolic compounds, triterpenoid acids, chalcones, sugars, and fatty acids, among others. In this paper, natural flavanone 1 (extracted from Eysenhardtia platycarpa) as lead compound and flavanones 1a–1d as its structural analogues were screened for anti-inflammatory activity using Molinspiration® and PASS Online in a computational study. The hydro alcoholic solutions (FS) of flavanones 1, 1a–1d (FS1, FS1a–FS1d) were also assayed to investigate their in vivo anti-inflammatory cutaneous effect using two experimental models, a rat ear edema induced by arachidonic acid (AA) and a mouse ear edema induced by 12-O-tetradecanoylphorbol acetate (TPA). Histological studies and analysis of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were also assessed in AA-inflamed rat ear tissue. The results showed that the flavanone hydro alcoholic solutions (FS) caused edema inhibition in both evaluated models. This study suggests that the evaluated flavanones will be effective when used in the future in skin pathologies with inflammation, with the results showing 1b and 1d to be the best.

Ocular inflammation is one of the most common comorbidities associated to ophthalmic surgeries and disorders. Since conventional topical ophthalmic treatments present disadvantages such as low bioavailability and relevant side effects, natural alternatives constitute an unmet medical need. In this sense, lactoferrin, a high molecular weight protein, is a promising alternative against inflammation. However, lactoferrin aqueous instability and high nasolacrimal duct drainage compromises its potential effectiveness. Moreover, nanotechnology has led to an improvement in the administration of active compounds with compromised biopharmaceutical profiles. Here, we incorporate lactoferrin into biodegradable polymeric nanoparticles and optimized the formulation using the design of experiments approach. A monodisperse nanoparticles population was obtained with an average size around 130 nm and positive surface charge. Pharmacokinetic and pharmacodynamic behaviour were improved by the nanoparticles showing a prolonged lactoferrin release profile. Lactoferrin nanoparticles were non-cytotoxic and non-irritant neither in vitro nor in vivo. Moreover, nanoparticles exhibited significantly increased anti-inflammatory efficacy in cell culture and preclinical assays. In conclusion, lactoferrin loaded nanoparticles constitute a safe and novel nanotechnological tool suitable for the treatment of ocular inflammation., This work was supported by Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/ COMPETE) under the project reference UIDB/04469/2020 (strategic found), co-financed by FEDER, under the Partnership Agreement PT2020: UIDB/04469/2020., info:eu-repo/semantics/publishedVersion

The present work is focused on the development of novel surface-functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with thymol (TH-NPs) for topical administration enhancing thymol anti-inflammatory, antioxidant and wound healing activities against acne. TH-NPs were prepared by solvent evaporation method using different surface functionalization strategies and obtaining suitable physicochemical parameters and a good short-term stability at 4 °C. Moreover, TH-NPs skin penetration and antioxidant activity were assessed in ex vivo pig skin models. Skin penetration of TH-NPs followed the follicular route, independently of the surface charge and they were able to enhance antioxidant capacity. Furthermore, antimicrobial activity against Cutibacterium acnes was evaluated in vitro by the suspension test showing improved antibacterial performance. Using human keratinocyte cells (HaCat), cytotoxicity, cellular uptake, antioxidant, anti-inflammatory and wound healing activities were studied. TH-NPs were non-toxic and efficiently internalized inside the cells. In addition, TH-NPs displayed significant anti-inflammatory, antioxidant and wound healing activities, which were highly influenced by TH-NPs surface modifications. Moreover, a synergic activity between TH-NPs and their surface functionalization was demonstrated. To conclude, surface-modified TH-NPs had proven to be suitable to be used as anti-inflammatory, antioxidant and wound healing agents, constituting a promising therapy for treating acne infection and associated inflammation.

The interconnection between nutrients, metabolites and microbes is a key factor governing the healthy/pathological status of an individual. Thus, microbiota-based research is essential in order to better understand human health and nutrition. Gut bacteria release membrane vesicles (MVs) as an intercellular communication mechanism that allows the direct delivery of factors that prime the host&rsquo, s innate immune system. We have previously shown that MVs from intestinal E. coli activate dendritic cells (DCs) in a strain-specific manner. To gain insights into the regulatory mechanisms involved, here, we have used an RNA deep sequencing approach to identify differentially expressed miRNAs (microRNAs) in DCs which are challenged by the MVs of the probiotic Nissle 1917 (EcN) or the commensal ECOR12. MicroRNAs are post-transcriptional regulatory mediators that permit the fine tuning of signaling pathways. This approach allowed the identification of a common set of miRNAs which are modulated by MVs from both strains and miRNAs which are differentially expressed in response to EcN or ECOR12 MVs. Based on the differential expression of the target genes and subsequent validation experiments, we correlated some of the selected miRNAs with the reported cytokine profile and specific T cell responses. As far as we know, this is the first study to analyze the regulation of miRNAs in DCs by MVs released by gut microbiota.

Pioglitazone (PGZ) is a drug used to treat type 2 diabetes mellitus that has been reported to show additional therapeutic activities on diverse inflammatory parameters. The aim of this study was to optimize a topical PGZ-loaded nanoemulsion (PGZ-NE) in order to evaluate its effectiveness for treating atopic dermatitis (AD). The composition of the nanoformulation was established by pseudo-ternary diagram. Parameters such as physical properties, stability, in vitro release profile, and ex vivo permeation were determined. The efficacy study was carried out using oxazolone-induced AD model in hairless mice. PGZ-NE released the drug following a hyperbolic kinetic. Additionally, its properties provided high retention potential of drug inside the skin. Therapeutic benefits of PGZ-NE were confirmed on diverse events of the inflammatory process, such as reduction of lesions, enhancement of skin barrier function, diminished infiltration of inflammatory cells, and expression of pro-inflammatory cytokines. These results were reinforced by atomic force microscope (AFM), which demonstrated the ability of the formulation to revert the rigidification caused by oxazolone and consequently improve the elasticity of the skin. These results suggest that PGZ-NE may be a promising treatment for inflammatory dermatological conditions such as AD., This work was supported by Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT—Ecuador) (grant number 073-CIBAE-2015).

The synthesis and characterization of two hybrid N-methylated carbazole derivatives containing a thiazolyl or a thienyl ring is reported. The thiazolyl derivative has been also characterised by X-ray diffraction analysis. The study of its reactivity in front of [MCl2(dmso)2] (M = Pd or Pt) or Na2[PdCl4] in methanol has allowed us to isolate and characterize its complexes. However, for the thienyl analogue, the formation of any Pd(II) or Pt(II) complex was not detected, indicating that it is less prone to bind to the M(II) ions than its thiazolyl analogue. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations have also been carried out in order to rationalize the influence of the nature of the thiazolyl or thienyl group on the electronic delocalization. Molecular mechanics calculations show that the free rotation of the thiazolyl in relation to the carbazole requires a greater energy income than for its thienyl analogue. Studies of the cytotoxic activity of the new compounds on colon (HCT116) and breast (MDA-MB231 and MCF7) cancer cell lines show that the thiazolyl carbazole ligand and its Pt(II) complex are the most active agents of the series and in the MCF7 line their potency is higher than that of cisplatin. In the non-tumoral human skin fibroblast BJ cell line, all the compounds were less toxic than cisplatin. Their potential ability to modify the electrophoretic mobility of pBluescript SK+ plasmid DNA and to act as inhibitors of Topoisomerases I and IIα or cathepsin B has also been investigated.

Platinum-based chemotherapy persists to be the only effective therapeutic option against a wide variety of tumours. Nevertheless, the acquisition of platinum resistance is utterly common, ultimately cornering conventional platinum drugs to only palliative in many patients. Thus, encountering alternatives that are both effective and non-cross-resistant is urgent. In this work, we report the synthesis, reduction studies, and luminescent properties of a series of cyclometallated (C,N,N')PtIV compounds derived from amine- imine ligands, and their remarkable efficacy at the high nanomolar range and complete lack of cross57 resistance, as an intrinsic property of the platinacycle, against multiplatinum-resistant colorectal cancer (CRC) and castration-resistant prostate cancer (CRPC) metastatic cell lines generated for this work. We have also determined that the compounds are effective and selective for a broader cancer panel, including breast and lung cancer. Additionally, selected compounds have been further evaluated, finding a shift in their antiproliferative mechanism towards more cytotoxic and less cytostatic than cisplatin against cancer cells, being also able to oxidize cysteine residues and inhibit topoisomerase II, thereby holding great promise as future improved alternatives to conventional platinum drugs.

Gut microbiota plays an essential role in maintaining intestinal homeostasis and human health. Microbiota establishes a complex network of dynamic and reciprocal interactions with the intestinal epithelium and immune system. The mucin layer that covers the epithelium prevents luminal bacteria from accessing host cells. Thus, microbiota–host communication mainly relies on secreted factors and membrane vesicles (MVs), which can cross the inner mucus layer and reach the epithelium. This chapter focuses on the role of microbiota-secreted MVs as key players in signaling processes in the intestinal mucosa. This is an emerging research topic, with the first reports dating from 2012. Microbiota-derived MVs are involved in interspecies communication in the gut, between bacteria and between microbiota and host. Here we present current knowledge on the mechanisms used by microbiota MVs to assist and control the gut microbial community and to modulate host immune and defense responses. Constant stimulation of immune receptors by microbiota MVs results in tightly controlled inflammation that contributes to tolerogenic responses essential to maintaining intestinal homeostasis. Moreover, gut microbiota MVs are emerging as physical vehicles for distribution and delivery of bacterial effectors to distal tissues in human health and disease.

Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were used for elaboration of functionalized NPs, which were characterized physicochemically. Cytotoxicity and ability to modulate the expression of proinflammatory cytokines were evaluated in vitro using human corneal epithelial cells (HCE-2). NPs uptake was assayed in both in vitro and in vivo models. Results: NPs showed physicochemical characteristics suitable for ocular administration without evidence of cytotoxicity. TAT-NPs and G2-NPs were internalized and displayed anti-inflammatory activity in both HCE-2 cells and mouse eye. Conclusion: TAT-NPs and G2-NPs could be considered a novel strategy for the treatment of ocular inflammatory diseases of the anterior and posterior segment.

Extracellular membrane vesicles (MVs) released by gut microbiota are key players in the communication with the host. The aim of this study was to evaluate the immunomodulatory properties of MVs from the probiotic E. coli Nissle 1917 (EcN) in terms of DC-derived adaptive immune responses and to compare the effects with those elicited by commensal E. coli. The effects of MVs were analysed in monocyte-derived DCs by measuring cytokine expression and the ability of activated-DCs to differentiate CD4+ T cells towards specific effector subsets. EcN MVs derived intricate Th1/Th2/Th17/Th22/Treg responses consistent with the beneficial effects of this probiotic. Th2/Th17/Th22 responses were common to commensal E. coli-derived vesicles but specific differences were observed for Th1 and Treg responses. Since MVs activate DCs in a strain-specific manner, probiotic-derived MVs could be explored as a safe (bacteria-free) strategy to develop new functional food ingredients targeting gut microbiota balance or intestinal inflammation.

Background Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. Results Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. Conclusion This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Cisplatin is an efficient anticancer drug, but its effects are often lost after several chemotherapy cycles, showing important secondary effects. For these reasons, new anticancer agents, with different coordination properties and mechanisms of action, are needed. Here we describe the reaction of 2-phenylaniline with cis-[PtCl2(dmso)(2)] and sodium acetate to afford a cycloplatinated compound 2 and the synthesis and some biological studies of 3-6 (two neutral and two ionic compounds): [PtCl(C-N)(L)], C-N cycloplatinated 2-phenylaniline with L = PPh3(3) or P(4-FC6H4)(3) (4) and [Pt(C-N)(L-L)]Cl with L-L = Ph2PCH2CH2Ph2(5) or (C6F5)(2)PCH2-CH2(C6F5)(2) (6). Ionic platinacycles 5 and 6 show a greater antiproliferative activity than that of cisplatin in human lung, breast, and colon cancer cell lines (A-549, MDA-MB-231 and MCF-7, and HCT-116), a remarkable result given the fact that they do not show covalent interaction with DNA. 5 and 6 have also been found able to oxidize NADH by a catalytic process prod- oducing H2O2 as ROS. The activity of these complexes to generate ROS seems to be the key factor to explain their potent anticancer activity; it should be noted that platinum(II) complexes showing biocatalytic activity for hydride transfer from NADH have not been described so far. Ionic complex 6 shows low affinity to some target proteins; the presence of perfluoroaromatic rings seems to hinder its interaction with some biomolecules.

Twelve cyclometallated palladium(II) complexes containing primary aromatic amines [benzylamine (a), (R)-1-(1-naphthyl)ethylamine (b) and 2-phenylaniline (c)] as anionic bidentate (C,N)(-) ligands have been evaluated against a panel of human adenocarcinoma cell lines (A549 lung, MDA-MB231 and MCF7 breast, and the cisplatin resistant HCT116 colon). The results revealed a remarkable antiproliferative activity of the triphenylphosphane mononuclear compounds 3-4 (series a, b, c) and the best inhibition was provided for 3c and 4c with the 2-phenylaniline ligand and a six membered chelate ring. Interestingly, 3c and 4c were 14 and 19 times more potent than cisplatin for the inhibition of the cisplatin resistant HCT116 human adenocarcinoma cell line, respectively. Cyclopalladated complexes 3c and 4c exercise their antiproliferative activity over A549 cells mainly through the induction of apoptosis (38 and 31-fold increase in early apoptotic cells, respectively).

The synthesis and characterization of the new 2-phenylindole derivative: C 8 H 3 N-2-C 6 H 5 -3NOMe-5OMe ( 3c ) and the trans- and cis- isomers of [Pt( 3c )Cl 2 (DMSO)] complexes ( 4c and 5c , respectively) are described. The crystal structures of 4c ·CH 2 Cl 2 and 5c confirm: (a) the existence of a Pt-N indole bond, (b) the relative arrangement of the Cl − ligands [ trans- (in 4c ) or cis- (in 5c )] and (c) the anti- ( E ) configuration of the oxime. The cytotoxic assessment of C 8 H 3 N-2-(C 6 H 4 -4′R 1 )-3NOMe-5R 2 [with R 1 = R 2 = H ( 3a ); R 1 = Cl, R 2 = H ( 3b ) and R 1 = H, R 2 = OMe ( 3c )] and the geometrical isomers of [Pt(L)Cl 2 (DMSO)] with L = 3a – 3c [ trans- ( 4a – 4c ) and cis- ( 5a – 5c ), respectively] against human breast adenocarcinoma cell lines (MDA-MB231 and MCF-7) is also reported and reveals that all the platinum(II) complexes (except 4a ) are more cytotoxic than cisplatin in front of the MCF7 cell line. Electrophoretic DNA migration studies of the synthesized compounds in the absence and in the presence of topoisomerase-I have been performed, in order to get further insights into their mechanism of action.

The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R1–CH N–R2 [R1 = phenyl or ferrocenyl unit and R2 = (CH2)n–CH2–NMe2 where n = 1 or 2) (1 and 2) or C6H4-2-SMe (3)] acting as a bidentate (N,N′) (4–7) or terdentate [C(phenyl or ferrocenyl),N,N′]− (8–10) or [C(ferrocenyl),N,S]− ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116 human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the most relevant complexes have also been performed in order to test: (a) their stability in the aqueous biological medium and/or the formation of biologically active species and (b) their proclivity to react with 9-methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been performed in order to explain the different solution behaviour of the complexes and their proclivity to react with the nucleobase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein–protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of Escherichia coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3′-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.

The synthesis of six novel cyclometallated platinum(IV) iodido complexes is accomplished by intermolecular oxidative addition of methyl iodide (compounds 2a-2c) or iodine (compounds 3a-3c) upon cyclometallated platinum(II) compounds [PtX{(CH3)(2)N(CH2)(3)NCH(4-ClC6H3)}] (1a-1c: X = Cl, CH3 or I). The X-ray molecular structures of platinum(II) compound 1c and platinum(IV) compounds 3b and 3a' (an isomer of 3a) are reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), DNA interaction, topoisomerase I, II alpha, and cathepsin B inhibition, and cell cycle arrest, apoptosis and ROS generation of the investigated complexes are presented. Remarkable antiproliferative activity was observed for most of the synthesized cycloplatinated compounds (series 1-3) in all the selected carcinoma cell lines. The best inhibition was provided for the octahedral platinum(IV) compounds 2a-2c exhibiting a methyl and an iodido axial ligand. Preliminary biological results point to a different mechanism of action for the investigated compounds. Cyclometallated platinum(II) compounds 1a-1c modify the DNA migration as cisplatin. In contrast, cyclometallated platinum(IV) compounds 2a-2c and 3a-3c did not modify the DNA tertiary structure neither in the absence nor in the presence of ascorbic acid, which made them incapable of reducing platinum(IV) compounds 2b and 2c in a buffered aqueous medium (pH 7.40) according to H-1 NMR experiments. Remarkable topoisomerase II alpha inhibitory activity is reported for platinum(IV) complexes 2b and 3a and in addition, for the last one, a moderate cathepsin B inhibition is reported. Cell cycle arrest (decrease in G0/G1 and G2 phases and arrest in the S phase), induction of apoptosis and ROS generation are related to the antiproliferative activity of some representative octahedral cyclometallated platinum(IV) compounds (2b and 2c).

The yiaK–S operon is required for aerobic growth on l-ascorbate in several Enterobacteriaceae. Here we present evidence that the yiaX2 gene belonging to the yiaK–S operon of Klebsiella pneumoniae 13882, which encodes a protein similar to the putative transporters classified as the major facilitator superfamily, is involved in the uptake of l-ascorbate. Concentration kinetic analysis yielded an apparent Km of YiaX2 for l-ascorbate of 161.38 ±8.28 µmol·L–1 and a Vmax of 3.81 ± 0.60 nmol·mg–1·min–1. This carrier uses the energy from electrochemical gradients, since it was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force.

Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.

A series of cyclometallated platinum(IV) compounds (3a, 3a' and 3b') with a meridional [C,N,N'] terdentate ligand, featuring an halido and an aryl group in the axial positions has been evaluated for electrochemical reduction and preliminary biological behavior against a panel of human adenocarcinoma (A-549 lung, HCT-116 colon, and MCF-7 breast) cell lines and the normal bronquial epithelial BEAS-2B cells. Cathodic reduction potentials (shifting from -1.463 to -1.570 V) reveal that the platinum(IV) compounds under study would be highly reluctant to be reduced in a biological environment. Actually ascorbic acid was not able to reduce complex 3a', the most prone to be reduced according its reduction potential, over a period of one week. These results suggest an intrinsic activity for the investigated platinum(IV) complexes (3a, 3a' and 3b'), which exhibit a remarkable cytotoxicity effectiveness (with IC50 values in the low micromolar range), even greater than that of cisplatin. The IC50 for A-549 lung cells and clog P values were found to follow the same trend: 3b' > 3a' > 3a. However, no correlation was observed between reduction potential and in vitro activity. As a representative example, cyclometallated platinum(IV) compound 3a', exercise its antiproliferative activity directly over non-microcytic A-549 lung cancer cells through a mixture of cell cycle arrest (13% arrest at G1 phase and 46% arrest at G2 phase) and apoptosis induction (increase of early apoptosis by 30 times with regard to control). To gain further insights into the mode of action of the investigated platinum(IV) complexes, drug uptake, cathepsin B inhibition and ROS generation were also evaluated. Interestingly an increased ROS generation could be related with the antiproliferative activity of the cyclometallated platinum(IV) series under study in the cisplatin-resistant A-549 lung and HCT-116 cancer cell lines.

The anaerobic utilization of L-ascorbate by gene products of the ula regulon in Escherichia coli has been widely documented. Under aerobic conditions, we have shown that this metabolism is only functional in the presence of casein acid hydrolysate. Transcriptional fusions and proteomic analysis indicated that both the ula regulon and the yiaK-S operon are required for the aerobic utilization of this compound. The aerobic dissimilation of l-ascorbate shares the function of three paralogous proteins, UlaD/YiaQ, UlaE/YiaR and UlaF/YiaS, which encode a decarboxylase, a 3-epimerase and a 4-epimerase, respectively. In contrast, l-ascorbate enters the cells through the ula-encoded phosphotransferase transport system, but it is not carried by the yiaMNO-encoded ABC transporter. Proteomic analysis also indicated enhanced expression of the alkyl hydroperoxide reductase encoded by the ahpC gene, suggesting a response to oxidative stress generated during the aerobic metabolism of l-ascorbate. Control of ahpC expression by the OxyR global regulator in response to l-ascorbate concentration is consistent with the formation of hydrogen peroxide under our experimental conditions. The presence of certain amino acids such as proline, threonine or glutamine in the culture medium allowed aerobic l-ascorbate utilization by Escherichia coli cells. This effect could be explained by the ability of these amino acids to allow yiaK-S operon induction by l-ascorbate, thus increasing the metabolic flux of l-ascorbate dissimilation. Alternatively, these amino acids may slow the rate of L-ascorbate oxidation.

We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound. This suppressing phenotype was assigned to yjcG , a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate. On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family. Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs , encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate. The fortuitous presence of an IS 2 element in acs , which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS 2 in the suppressed strain. The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yjcG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP ) could be acetate transport and allowed us to define an operon involved in acetate metabolism. The time course of [1,2- 14 C]acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate.

The enzyme 2-phosphoglycolate phosphatase fromEscherichia coli, encoded by thegphgene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (kcat/Km), which enabled the conversion of this substrate even at low intracellular concentrations. A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism. Western blot analysis revealed constitutive expression of thegphgene, regardless of the carbon source used, growth stage, or oxidative stress conditions. We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3′-phosphoglycolate ends. DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin. The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism. We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery.

The synthesis and preliminary biological evaluation of neutral and cationic platinum derivatives of chiral 1-(1-naphthyl)ethylamine are reported, namely cycloplatinated neutral complexes [PtCl{(R or S)-NH(2)CH(CH(3))C(10)H(6)}(L)] [L = SOMe(2) ( 1-R or 1-S ), L = PPh(3) (2-R or 2-S), L = P(4-FC(6)H(4))(3) (3-R), L = P(CH(2))(3)N(3)(CH(2))(3) (4-R)], cycloplatinated cationic complexes [Pt{(R)-NH(2)CH(CH(3))C(10)H(6)}{L}]Cl [L = Ph(2)PCH(2)CH(2)PPh(2) (5-R), L = (C(6)F(5))(2)PCH(2)CH(2)P(C(6)F(5))(2) (6-R)] and the Pt(ii) coordination compound trans-[PtCl(2){(R)-NH(2)CH(CH(3))C(10)H(6)}(2)] (7-R). The X-ray molecular structure of 7-R is reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), cell cycle arrest and apoptosis, DNA interaction, topoisomerase I and cathepsin B inhibition, and Pt cell uptake of the studied compounds are presented. Remarkable cytotoxicity was observed for most of the synthesized Pt(ii) compounds regardless of (i) the absolute configuration R or S, and (ii) the coordinated/cyclometallated (neutral or cationic) nature of the complexes. The most potent compound 2-R (IC(50) = 270 nM) showed a 148-fold increase in potency with regard to cisplatin in HCT-116 colon cancer cells. Preliminary biological results point out to different biomolecular targets for the investigated compounds. Neutral cyclometallated complexes 1-R and 2-R, modify the DNA migration as cisplatin, cationic platinacycle 5-R was able to inhibit topoisomerase I-promoted DNA supercoiling, and Pt(ii) coordination compound 7-R turned out to be the most potent inhibitor of cathepsin B. Induction of G-1 phase ( 2-R and 5-R ), and S and G-2 phases (6-R) arrests are related to the antiproliferative activity of some representative compounds upon A-549 cells. Induction of apoptosis is also observed for 2-R and 6-R.

The magnitudes of MIC for the antibacterial activity and of the IC50 for the antioxidant activity reported in μM are really mM. Thus, in page 84: 0.18-0.34 μM is 0.18-0.34 mM; in Tables 2 and 3: μM is mM; in page 85: 0.12 and 0.14 μM is 0.12 and 0.14 mM; and in page 87: 0.12-0.14 μM is 0.12-0.14 mM. The authors apologize for any inconvenience caused.

The antitumor, antibacterial and antioxidant activity, DNA interaction and cathepsin B inhibition of cyclo- ortho -palladated and -platinated compounds [Pd(C,N)] 2 (μ-X) 2 [X = OAc ( 1 ), X = Cl ( 2 )] and trans - N , P -[M(C,N)X(PPh 3 )] [M = Pd, X = OAc ( 3 ), M = Pd, X = Cl ( 4 ), M = Pt, X = Cl ( 5 )] are discussed [(C,N) = cyclo- ortho -metallated benzophenone imine]. The cytotoxicity of compound 5 has been evaluated towards human breast (MDA-MB-231 and MCF-7) and colon (HCT-116) cancer cell lines and that of compounds 1 – 4 towards the HCT-116 human colon cancer cell line. These cytotoxicities have been compared with those previously reported for compounds 1 – 4 towards MDA-MB-231 and MCF-7 cancer cell lines. Compound 3 and 4 were approximately four times more active than cisplatin against the MDA-MB-231 and MCF-7 cancer cell lines, and compound 5 , was approximately four times more potent than cisplatin against the HCT-116 cancer cell line. The antibacterial activity of compounds 1 – 5 was in between the ranges of activity of the commercial antibiotic compounds cefixime and roxithromycin. Complexes 1 – 2 and 4 – 5 presented also antioxidant activity. Compounds 1 – 5 alter the DNA tertiary structure in a similar way to cisplatin , but at higher concentration, and do not present a high efficiency as cathepsin B inhibitors. Compound 5 has not been previously described, and its preparation, characterization, and X-ray crystal structure are reported.

The synthesis of the endo five-membered cyclo-ortho-palladated benzophenone imines [Pd{C6H4(Ph)C═NR}]2(μ-X)2 [1 (X = OAc), 2 (X = Cl), a (R = phenyl), b (R = 1-naphthyl), c (R = benzyl), d (R = α-methylbenzyl)], and trans-N,P-[Pd{C6H4(Ph)C═NR}X(PPh3)] [3 (X = OAc), 4 (X = Cl), a (R = phenyl), b (R = 1-naphthyl), c (R = benzyl), d (R = α-methylbenzyl)] and the X-ray molecular structure of 1a, 1c, 1d, 4a, 4b, and 4c are reported. The antitumor activity, DNA interaction, and cathepsin B inhibition of palladium compounds a-d were studied and compared with those previously reported for palladium compounds e with R = H and compound 4f analogous to 4e but with a platinum(II) center. The IC50 values against a panel of human cancer cell lines allowed the establishment of a qualitative relationship between their structure and antitumor activity. Compounds 3e, 4e, and 4f were the most active ones in relation to their in vitro anticancer activity. Compounds 3e and 4e were about 4 times more active than cisplatin against the MDA-MB-231 and MCF-7 breast human cancer lines, and compound 4f was about 4 times more active than cisplatin against the cisplatin-resistant HCT-116 colon human cancer cell line. In addition, compound 3e was 3 times less cytotoxic than cisplatin toward the quiescent HUVEC cells. Accumulation of palladium compounds e and b in the MDA-MB-231 cell line was considerably greater than that of cisplatin in the same cell line, but palladium compounds b were noncytotoxic. Some of these complexes altered the DNA tertiary structure in a similar way to cisplatin but at higher concentration, and most cytotoxic ones did not present a high efficiency as cathepsin B inhibitors.

The new compound [Pt2(4-FC6H4)4(μ-SEt2)2] (A) was prepared and fully characterized. The reactions of compound A with ligands ArCH=NCH2CH2NMe2 (Ar = 2-BrC6H4, 1a; 2,6-Cl2C6H3, 1b; 4-ClC6H4, 1c; 2-Cl,6-FC6H3, 1d) were studied under different conditions and produced platinum(II) compounds [Pt(4-FC6H4)2(ArCH=NCH2CH2NMe2)] (2b−2d), containing a bidentate [N,N′] ligand, as well as cyclometalated platinum(IV) or platinum(II) compounds such as [PtBr(4-FC6H4)2(C6H4CH=NCH2CH2NMe2)] (4a) or [PtCl{(3-FC6H3)(2-XC6H3)CH=NCH2CH2NMe2}] (5b: X = Cl; 5d: X = F), containing a tridentate [C,N,N′] ligand and either a five (4a) or a seven (5b, 5d) membered metallacycle. These compounds exhibit a great antiproliferative activity against non-small lung cancer cells (A549), and the best result was obtained for compound 2c (IC50 = 0.3 ± 0.1 μM). While compounds 5 alter the mobility of plasmid DNA in a similar way to cisplatin, compound 4 was less efficient in removing the supercoils from DNA. In spite of the very low IC50 value obtained for compound 2c, this compound does not interact with DNA, and it is neither an intercalator nor a topoisomerase I inhibitor.

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367).

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme. No effect on glycolate metabolism was detected by insertional mutation in glcG. Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript.

A series of seven-membered cyclometallated Pt(II) complexes containing a terdentate [C,N,N'] ligand (1a-1c and 2a-2c) have been developed as potential monofunctional DNA binding agents. By reactions of cis-[Pt(4-C 6H 4Me) 2(μ-SEt 2)] 2 or cis-[Pt(C 6H 5) 2(SMe 2) 2] with imines 2-ClC 6H 4CHNCH 2CH 2NMe 2 (b) or 2-F,6-ClC 6H 3CHNCH 2CH 2NMe 2 (c) the new compounds 1b, 1c and 2c were synthesized and characterized. Complex 1b and 1c were further characterized by X-ray crystallography. The cytotoxicity assessment of the seven-membered platinacycles 1 (1a-1c) and 2 (2a-2c) against a panel of human cancer cell lines (A549 lung, HCT116 colon, and MDA MB231 breast adenocarcinomas) revealed that the six cycloplatinated complexes exhibit a remarkable antiproliferative activity, even greater than cisplatin in the three human cancer cell lines. From a pharmacological point of view, platinacycles 1 (1a-1c) and 2 (2a-2c) may represent compounds for a new class of antitumor drugs. Electrophoretic DNA migration studies showed that all of them modify the DNA tertiary structure. Induction of S-G2/M arrest and apoptosis were also observed for one of the representative compounds (1c) of the series. © 2012 Elsevier Masson SAS. All rights reserved., This work was supported by the Ministerio de Ciencia y Tecnología (projects CTQ2009-11501 and CTQ2009-07021/BQU) and the AGAUR, Generalitat de Catalunya (Grants 2009-SGR-1111, 2009SGR01308, 2006ITT-10007 and 2009CTP-00026). This study was also supported by the project SAF2011-25726 and by RD06/0020/0046 from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, both funded by the Ministerio de Ciencia e Innovación-Spanish government and European Regional Development Funds (ERDF) “Una manera de hacer Europa”. Marta Cascante acknowledges the support received through the prize “ICREA Academia”, funded by ICREA foundation-Generalitat de Catalunya.

Genes for d -galactarate ( gar ) and d -glucarate ( gud ) metabolism in Escherichia coli are organized in three transcriptional units: garD , garPLRK , and gudPD . Two observations suggested a common regulator for the three operons. (i) Their expression was triggered by d -galactarate, d -glucarate, and d -glycerate. (ii) Metabolism of the three compounds was impaired by a single Tn 5 insertion mapped in the yaeG gene (proposed name, sdaR ), outside the d -galactarate and d -glucarate systems. Expression of the sdaR gene is autogenously regulated.

3 páginas, 3 figuras, 1 tabla -- PGS nros. 36-38, UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å3 Da−1 and a solvent content of 43%, This work was supported by grants from the Spanish Ministry of Science and Education (MEC): BFU 2006-15573 to MCV, BFU 2004-03586/BMC to JB and GEN2003-20642 to MC. FG was the recipient of a predoctoral fellowship from the Generalitat de Catalunya. FJF was supported by an I3P postdoctoral contract from MEC

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) is an anchorless, multifunctional protein displayed on the surface of several fungi and Gram-positive pathogens, which contributes to their adhesion and virulence. To date a role for extracellular GAPDH in the pathogenesis of Gram-negative bacteria has not been described. The aim of this study was to analyze the extracellular localization of GAPDH in enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains and to examine its interaction with host components that could be related to the infection mechanism. Recombinant E. coli GAPDH was purified and polyclonal antibodies were obtained. Western blotting and immunoelectron microscopy showed that GAPDH is located on the bacterial surface and released to the culture medium of EHEC and EPEC strains. GAPDH export in these Gram-negative pathogens depends on the external medium, is not mediated by vesicles and leads to an extracellular active enzyme. Non-pathogenic E. coli strains do not secrete GAPDH. Two-dimensional electrophoresis analysis showed that in E. coli GAPDH is present at least in two major forms with different isoelectric points. Of these forms, the more basic is secreted. Purified GAPDH was found to bind human plasminogen and fibrinogen in Far-Western blot and ELISA-based assays. In addition, GAPDH remained associated with colonic Caco-2 epithelial cells after adhesion of EHEC or EPEC. These observations indicate that exported GAPDH may act as a virulence factor which could contribute to EHEC and EPEC pathogenesis. This is the first description of an extracellular localization for this enzyme, with a function other than its glycolytic role in Gram-negative pathogens.

In Escherichia coli the glc operon involved in glycolate utilization is located at 67.3 min and formed by genes encoding the enzymes glycolate oxidase (glcDEF) and malate synthase G (glcB). Their expression from a single promoter upstream of glcD is induced by growth on glycolate and regulated by the activator encoded by the divergently transcribed gene glcC. Gene yghK, located 350 bp downstream of glcB, encodes a hydrophobic protein highly similar to the L-lactate permease encoded by lldP. Expression studies have shown that the yghK gene (proposed name glcA) is transcribed from the same promoter as the other glc structural genes and thus belongs to the glc operon. Characterization of a glcA::cat mutant showed that GlcA acts as glycolate permease and that glycolate can also enter the cell through another transport system. Evidence is presented of the involvement of L-lactate permease in glycolate uptake. Growth on this compound was abolished in a double mutant of the paralogous genes glcA and lldP, and restored with plasmids expressing either GlcA or LldP. Characterization of the putative substrates for these two related permeases showed, in both cases, specificity for the 2-hydroxymonocarboxylates glycolate, L-lactate and D-lactate. Although both GlcA and LldP recognize D-lactate, mutant analysis proved that L-lactate permease is mainly responsible for its uptake.

Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pathway, or alternatively it can be reduced to glycolate by constitutive glyoxylate reductase activity. At present no information is available on this latter enzyme in Escherichia coli, although similar enzymes, classified as 2-hydroxyacid dehydrogenases, have been characterized in other organisms. A BLAST search using as the query sequence the hydroxypyruvate/glyoxylate reductase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subsequent BLAST search yielded the ycdW gene as a good candidate to encode glyoxylate reductase in this bacterium. Cloning and overexpression of the ycdW gene showed that its product displayed a high NADPH-linked glyoxylate reductase activity, and also catalysed the reduction of hydroxypyruvate with a lower efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransferase (‘CAT’) cassette did not totally abolish the glyoxylate reductase activity, indicating that another enzyme accomplished this function. The similarity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE proteins permitted their kinetic characterization and comparison. Analysis of the catalytic power (kcat/Km) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate.

Genes yiaP and yiaR of the yiaKLMNOPQRS cluster of Escherichia coli are required for the metabolism of the endogenously formed l -xylulose, whereas yiaS is required for this metabolism only in araD mutants. Like AraD, YiaS was shown to have l -ribulose-5-phosphate 4-epimerase activity. Similarity of YiaR to several 3-epimerases suggested that this protein could catalyze the conversion of l -xylulose-5-phosphate into l -ribulose-5-phosphate, thus completing the pathway between l -xylulose and the general metabolism.

Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.

1,2-Propanediol oxidoreductase, which reduces the L-lactaldehyde formed in the fermentation of L-fucose or L-rhamnose to L-1,2-propanediol in E. coli, was inactivated by a component of E. coli cell extracts in the presence of oxygen. Pure propanediol oxidoreductase preparations were shown to be inactivated in vitro by aerobic incubations in the presence of Fe3+ and ascorbate. The Fe3+ ascorbate-mediated inactivation reaction was inhibited by catalase, although not by superoxide dismutase. Under anaerobic conditions, the presence of H2O2 strongly inactivated the enzyme. Propanediol oxidoreductase was rapidly degraded in the presence of oxygen, while the native enzyme displayed high stability as long as no oxygen was present.

Regardless of the respiratory conditions of the culture, Escherichia coli synthesizes an active propanediol oxidoreductase. Under anaerobic conditions, the enzyme remained fully active and accomplished its physiological role, while under aerobic conditions, it was inactivated in a process that did not depend on protein synthesis or on the presence of a carbon source.

The three structural genes rhaA, rhaB and rhaD, that specify the enzymes rhamnose isomerase, rhamnulose kinase and rhamnulose 1-phosphate aldolase respectively, have been cloned from Escherichia coli K-12. The precise location of the genes has been determined by gene complementation analysis and by enzymatic assays of strains transformed with recombinant plasmids containing different parts of the cloned region. The corresponding gene products have been studied by their expression in maxicells. Protein products of 47 kDa, 52-54 kDa and 32 kDa have been assigned to rhamnose isomerase, rhamnulose kinase and rhamnulose 1-phosphate aldolase respectively.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response. KEYWORDS: DNA repair; Endonuclease IV; Glyceraldehyde-3-phosphte dehydrogenase; Single-stranded DNA binding protein; Uracil DNA glycosylase

An equimolar amount of 1,2-propanediol was detected in the medium when Salmonella typhimurium or Klebsiella pneumoniae fermented L-fucose or L-rhamnose. These metabolic conditions induced a propanediol oxidoreductase that converted the lactaldehyde formed in the dissimilation of either sugar into the diol. The enzyme was further identified by cross-reaction with antibodies against Escherichia coli propanediol oxidoreductase. This indicates that L-fucose and L-rhamnose fermentation takes place in these species by 1,2-propanediol production and excretion.

Salmonella typhimurium excreted the rhamnose fermentation product 1,2-propanediol not only under anaerobic conditions, but also under aerobic conditions. The absence of an aldehyde dehydrogenase enzymatic activity that oxidizes to lactate the lactaldehyde formed in the dissimilation of rhamnose raised the intracellular concentration of the aldehyde which was alternatively reduced to the excretable 1,2-propanediol by a residual propanediol oxidoreductase activity.