Novel nanostructured lipid carriers loading Apigenin for anterior segment ocular pathologies.

Dry eye disease (DED) is a chronic multifactorial disorder of the ocular surface caused by tear film dysfunction and constitutes one of the most common ocular conditions worldwide. However, its treatment remains unsatisfactory. While artificial tears are commonly used to moisturize the ocular surface, they do not address the underlying causes of DED. Apigenin (APG) is a natural product with anti-inflammatory properties, but its low solubility and bioavailability limit its efficacy. Therefore, a novel formulation of APG loaded into biodegradable and biocompatible nanoparticles (APG-NLC) was developed to overcome the restricted APG stability, improve its therapeutic efficacy, and prolong its retention time on the ocular surface by extending its release. APG-NLC optimization, characterization, biopharmaceutical properties and therapeutic efficacy were evaluated. The optimized APG-NLC exhibited an average particle size below 200 nm, a positive surface charge, and an encapsulation efficiency over 99 %. APG-NLC exhibited sustained release of APG, and stability studies demonstrated that the formulation retained its integrity for over 25 months. In vitro and in vivo ocular tolerance studies indicated that APG-NLC did not cause any irritation, rendering them suitable for ocular topical administration. Furthermore, APG-NLC showed non-toxicity in an epithelial corneal cell line and exhibited fast cell internalization. Therapeutic benefits were demonstrated using an in vivo model of DED, where APG-NLC effectively reversed DED by reducing ocular surface cellular damage and increasing tear volume. Anti-inflammatory assays in vivo also showcased its potential to treat and prevent ocular inflammation, particularly relevant in DED patients. Hence, APG-NLC represent a promising system for the treatment and prevention of DED and its associated inflammation.

Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen.

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.

Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli.

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 (k(cat)/K(m)) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate.

Regulation of expression of the yiaKLMNOPQRS operon for carbohydrate utilization in Escherichia coli: involvement of the main transcriptional factors.

The yiaKLMNOPQRS (yiaK-S) gene cluster of Escherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate L-xylulose. Transcription of yiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5' end has been located at 60 bp upstream from the ATG. Expression of the yiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of the yiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. The lacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from -50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. The lacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at -106, is essential for yiaK-S expression. The 5' end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-S promoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of the yiaK-S promoter.

Role of the yiaR and yiaS genes of Escherichia coli in metabolism of endogenously formed L-xylulose.

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.

A common regulator for the operons encoding the enzymes involved in D-galactarate, D-glucarate, and D-glycerate utilization in Escherichia coli.

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 Tn5 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.

Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli.

Growth experiments with Escherichia coli have shown that this organism is able to use allantoin as a sole nitrogen source but not as a sole carbon source. Nitrogen assimilation from this compound was possible only under anaerobic conditions, in which all the enzyme activities involved in allantoin metabolism were detected. Of the nine genes encoding proteins required for allantoin degradation, only the one encoding glyoxylate carboligase (gcl), the first enzyme of the pathway leading to glycerate, had been identified and mapped at centisome 12 on the chromosome map. Phenotypic complementation of mutations in the other two genes of the glycerate pathway, encoding tartronic semialdehyde reductase (glxR) and glycerate kinase (glxK), allowed us to clone and map them closely linked to gcl. Complete sequencing of a 15.8-kb fragment encompassing these genes defined a regulon with 12 open reading frames (ORFs). Due to the high similarity of the products of two of these ORFs with yeast allantoinase and yeast allantoate amidohydrolase, a systematic analysis of the gene cluster was undertaken to identify genes involved in allantoin utilization. A BLASTP search predicted four of the genes that we sequenced to encode allantoinase (allB), allantoate amidohydrolase (allC), ureidoglycolate hydrolase (allA), and ureidoglycolate dehydrogenase (allD). The products of these genes were overexpressed and shown to have the predicted corresponding enzyme activities. Transcriptional fusions to lacZ permitted the identification of three functional promoters corresponding to three transcriptional units for the structural genes and another promoter for the regulatory gene allR. Deletion of this regulatory gene led to constitutive expression of the regulon, indicating a negatively acting function.

A rare 920-kilobase chromosomal inversion mediated by IS1 transposition causes constitutive expression of the yiaK-S operon for carbohydrate utilization in Escherichia coli.

The regulator of the yiaK-S operon, currently assigned a carbohydrate utilization function in Escherichia coli, is inactivated by a genome rearrangement that leads to the constitutive expression of the operon. The yiaK-S constitutive cells acquire the ability to utilize the rare pentose L-lyxose. Restriction analysis and sequencing of the regulator gene indicate that it is disrupted by foreign DNA. The insert consists of a large inverted fragment of DNA of 920 kilobases flanked by two IS1 elements with opposite polarity. One corresponds to that found naturally at min 0.4 of the bacterial chromosome and the other to a new copy transposed into the regulator gene located at min 80.6. This insertion-inversion could be the result of the intramolecular transposition mechanism itself, a gene rearrangement rarely originated by IS1. Alternatively, it could be attributed to the homologous recombination between the IS1 at min 0.4 and the IS1 transposed intermolecularly into the yiaK-S regulator gene. The participation of a rare IS1-mediated inversion in the evolution of a stable phenotype is thus identified.

Transcriptional regulation of the Escherichia coli rhaT gene.

Transcriptional regulation of the rhaT gene, one of the operons forming the rhamnose regulon in Escherichia coli, was studied by fusing its complete or deleted promoter to the reporter gene lacZ. Analysis of beta-galactosidase activities induced in these constructions grown under different conditions predicted the presence of two putative control elements: one for the RhaS regulatory protein and activating the gene not only by L-rhamnose but also by L-lyxose or L-mannose, the other for cAMP-catabolite repression protein and activating this gene in the absence of glucose. Anaerobiosis increased the promoter function two- to threefold with respect to the aerobic condition. Experiments involving complementation of strains containing the rhaT-promoter fusion and carrying a deletion in the rhaS and/or rhaR genes with plasmids bearing the rhamnose regulatory genes showed that rhaT is controlled by a regulatory cascade, in which RhaR induces rhaSR and the accumulated RhaS directly activates rhaT.

glc locus of Escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein.

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.

Activation of a cryptic gene encoding a kinase for L-xylulose opens a new pathway for the utilization of L-lyxose by Escherichia coli.

A silent gene encoding a kinase that specifically phosphorylates L-xylulose was activated and rendered constitutive in mutant cells of Escherichia coli. L-Xylulose kinase was purified to homogeneity and found to be a dimer of two subunits of 55 kDa, highly specific for L-xylulose with a Km of 0.8 mM, a Vmax of 33 mumol/min/mg, and an optimum pH of 8.4. Physical (thin layer chromatography) and spectroscopic (nuclear magnetic resonance and optical rotation) characterization of the product of L-xylulose kinase indicated that the enzyme phosphorylated the sugar at position 5. The gene encoding L-xylulose kinase was mapped in the 80.2 min region of the chromosome by conjugation and transduction. Cloning and comparison of the restriction map with the Kohara map (Kohara, Y., Akiyame, K., and Isono, K. (1987) Cell 50, 495-501) located the gene between positions 3963 and 3965 kilobases. The molecular and functional features of L-xylulose kinase together with the location of the corresponding gene indicate that this enzyme did not derive from mutation of any other known kinase. The new kinase opens a route for the utilization of L-lyxose through the action of rhamnose permease, rhamnose isomerase, and the phosphorylation of the L-xylulose formed to L-xylulose 5-phosphate, which is then introduced into the pentose phosphate pathway for subsequent metabolism.

Molecular characterization of Escherichia coli malate synthase G. Differentiation with the malate synthase A isoenzyme.

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.

Nucleotide sequence of the rhaR-sodA interval specifying rhaT in Escherichia coli.

The precise location of the rhaT gene, encoding rhamnose permease, has been established between sodA and rhaC at 3605-3607 kb of Kohara's physical map, which corresponds to 88.4 min on the Escherichia coli chromosomal map. The dependence of the activity of the rhaT product on the function of rhaC, the rhamnose operon regulatory gene, was established by measuring rhamnose transport in wild-type and rhaC-deficient strains. The sequence of the sodA-rhaC interval displayed a single ORF corresponding to rhaT, which is transcribed counterclockwise on the E. coli chromosome. The ORF was shown to be preceded by a ribosome binding consensus sequence and a catabolite repression protein consensus sequence. The derived amino acid sequence displayed very low homology with any other permease and was clearly dissimilar to the homologous group formed by the xylose, arabinose, galactose and several glucose transporters. Analysis of the rhaT primary sequence identified potential membrane-spanning regions, possibly defining a protein structure model different from the one corresponding to the above-mentioned homologous group.

Inactivation of propanediol oxidoreductase of Escherichia coli by metal-catalyzed oxidation.

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.

Aldehyde dehydrogenase induction by glutamate in Escherichia coli. Role of 2-oxoglutarate.

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of L-lactaldehyde to L-lactate is induced not only by L-fucose, L-rhamnose or D-arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4-Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2-oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2-oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2-oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2-oxoglutarate formation. Evidence is presented showing that L-fucose- and 2-oxoglutarate-inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose-repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerol-mediated repression. These results indicate that ald is under carbon regulation mediated by at least two different mechanisms.

L-lyxose metabolism employs the L-rhamnose pathway in mutant cells of Escherichia coli adapted to grow on L-lyxose.

Escherichia coli cannot grow on L-lyxose, a pentose analog of the 6-deoxyhexose L-rhamnose, which supports the growth of this and other enteric bacteria. L-Rhamnose is metabolized in E. coli by a system that consists of a rhamnose permease, rhamnose isomerase, rhamnulose kinase, and rhamnulose-1-phosphate aldolase, which yields the degradation products dihydroxyacetone phosphate and L-lactaldehyde. This aldehyde is oxidized to L-lactate by lactaldehyde dehydrogenase. All enzymes of the rhamnose system were found to be inducible not only by L-rhamnose but also by L-lyxose. L-Lyxose competed with L-rhamnose for the rhamnose transport system, and purified rhamnose isomerase catalyzed the conversion of L-lyxose into L-xylulose. However, rhamnulose kinase did not phosphorylate L-xylulose sufficiently to support the growth of wild-type E. coli on L-lyxose. Mutants able to grow on L-lyxose were analyzed and found to have a mutated rhamnulose kinase which phosphorylated L-xylulose as efficiently as the wild-type enzyme phosphorylated L-rhamnulose. Thus, the mutated kinase, mapped in the rha locus, enabled the growth of the mutant cells on L-lyxose. The glycolaldehyde generated in the cleavage of L-xylulose 1-phosphate by the rhamnulose-1-phosphate aldolase was oxidized by lactaldehyde dehydrogenase to glycolate, a compound normally utilized by E. coli.

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12.

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.

Oxygen regulation of L-1,2-propanediol oxidoreductase activity in Escherichia coli.

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.

Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12.

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.

Aerobic excretion of 1,2-propanediol by Salmonella typhimurium.

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.