Homogentisic acid affects human osteoblastic functionality by oxidative stress and alteration of the Wnt/β-catenin signaling pathway

Alkaptonuria (AKU) is a rare disease correlated with deficiency of the enzyme homogentisate 1,2 dioxygenase, which causes homogentisic acid (HGA) accumulation. HGA is subjected to oxidation/polymerization reactions, leading to the production of a peculiar melanin-like pigmentation (ochronosis) after chronic inflammation, which is considered as a triggering event for the generation of oxidative stress. Clinical manifestations of AKU are urine darkening, sclera pigmentation, early severe osteoarthropathy, and cardiovascular and renal complication. Despite major clinical manifestations of AKU being observed in the bones and skeleton, the molecular and functional parameters are so far unknown in AKU. In the present study, we used human osteoblasts supplemented with HGA as a AKU cellular model. We observed marked oxidative stress, and for the first time, we were able to correlate HGA deposition with an impairment in the Wnt/β-catenin signaling pathway, opening a range of possible therapeutic strategies for a disease still lacking a known cure.     

Identification and Molecular Characterization of the Homogentisate Pathway Responsible for Pyomelanin Production,the Major Melanin Constituents in Aeromonas media WS

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.     

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila

The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.     

Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called “ochronosis” and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell–matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU.     

Increased toxin production by Vibrio cholerae O1 during survival with a green alga, Rhizoclonium fontanum, in an artificial aquatic environment

In the aquatic environment, the physiological state of Vibrio cholerae can be affected by various environmental conditions (e.g., sunlight, pH, temperature, competition with other bacteria for nutrients, etc.). The effect of these factors on the toxigenicity of V. cholerae was investigated. Toxin production by 5 toxigenic strains of V. cholerae incubated in laboratory microcosms containing Rhizoclonium fontanum was tested at different time intervals. The microcosms were exposed to sunlight, and the V. cholerae were in competition for nutrients with the resident bacterial flora of R. fontanum. The increase or decrease in toxin production by V. cholerae recovered at different time intervals was measured by ELISA and compared with the parent strains. Results of the study demonstrated an increase in toxin production by V. cholerae O1 during survival with R. fontanum. It is concluded that various environmental conditions in the aquatic environment affect toxin production by V. cholerae.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Synthesis, characterization, and DNA-binding properties of Ln(III) complexes containing gatifloxacin

Four novel water-soluble complexes of Ln(III) with gatifloxacin (HGA), [La(HGA)3Cl3] x 2H2O, [Nd(HGA)3Cl3] x 2H2O, [Eu(HGA)3Cl3] x 2H2O, [Tb(HGA)3Cl3] x 2H2O, have been synthesized and characterized by elemental analyses, molar conductivities, IR spectra, fluorescence spectra, and thermogravimetry/differential thermal analysis (TG-DTA). In addition, the DNA-binding properties of the ligand and its complexes have been investigated by absorption, fluorescence spectra, and viscosity measurements. The experimental results indicated that the complexes and ligand bind to DNA via groove binding mode.     

利用rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定替代结素皮试检出结核感染的可行性

评估rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定与结素皮试检出结核感染的敏感性及特异性。对疑似结核病患者共229例进行随机、双盲、平行、对照、前瞻性试验,后经细菌培养证实患结核病的病人共129人,没有结核病史的非结核病患者共100人。以某一特定的γ-IFN体外释放水平及结素皮试反应硬结直径?10 mm为阳性切割值,rCFP-10/ESAT-6融合蛋白刺激γ-IFN体外释放测定的敏感性为96%,显著高于结素皮试(89%)(χ2=4.92;0.025相似文献   

Increased immune gene expression and immune cell infiltration in high-grade astrocytoma distinguish long-term from short-term survivors

Survival in the majority of high-grade astrocytoma (HGA) patients is very poor, with only a rare population of long-term survivors. A better understanding of the biological factors associated with long-term survival in HGA would aid development of more effective therapy and survival prediction. Factors associated with long-term survival have not been extensively studied using unbiased genome-wide expression analyses. In the current study, gene expression microarray profiles of HGA from long-term survivors were interrogated for discovery of survival-associated biological factors. Ontology analyses revealed that increased expression of immune function-related genes was the predominant biological factor that positively correlated with longer survival. A notable T cell signature was present within this prognostic immune gene set. Using immune cell-specific gene classifiers, both T cell-associated and myeloid linage-associated genes were shown to be enriched in HGA from long-term versus short-term survivors. Association of immune function and cell-specific genes with survival was confirmed independently in a larger publicly available glioblastoma gene expression microarray data set. Histology was used to validate the results of microarray analyses in a larger cohort of long-term survivors of HGA. Multivariate analyses demonstrated that increased immune cell infiltration was a significant independent variable contributing to longer survival, as was Karnofsky/Lansky performance score. These data provide evidence of a prognostic anti-tumor adaptive immune response and rationale for future development of immunotherapy in HGA.     

Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism

Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.     

New insights and advances on pyomelanin production: from microbial synthesis to applications

Pyomelanin is a brown-black phenolic polymer and results from the oxidation of homogentisic acid (HGA) in the L-tyrosine pathway. As part of the research for natural and active ingredients issued from realistic bioprocesses, this work re-evaluates the HGA pigment and makes an updated inventory of its syntheses, microbial pathways, and properties, with tracks and recent advances for its large-scale production. The mechanism of the HGA polymerization is also well documented. In alkaptonuria, pyomelanin formation leads to connective tissue damage and arthritis, most probably due to the ROS issued from HGA oxidation. While UV radiation on human melanin may generate degradation products, pyomelanin is not photodegradable, is hyperthermostable, and has other properties better than L-Dopa melanin. This review aims to raise awareness about the potential of this pigment for various applications, not only for skin coloring and protection but also for other cells, materials, and as a promising (semi)conductor for bioelectronics and energy.     

Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis

Many passerine species lay eggs that are speckled with dark protoporphyrin pigmentation. Because protoporphyrin is mainly derived from the blood, we here formulate and test a new hypothesis that links an increase in anaemia along the laying sequence to within-clutch variation in egg pigmentation. More intense pigmentation is expected if pigments accumulate during enhanced red blood cell production in response to anaemia. Reduced pigmentation is expected if pigments are derived from the degradation of red blood cells that circulate in smaller numbers due to blood loss. To test this hypothesis, we manipulated anaemia in great tit (Parus major) females by infesting the nests with hen fleas (Ceratophyllus gallinae) prior to egg laying. Polychromatophil (i.e., immature red blood cells) percentage, as a measure of blood cell production, was positively correlated with parasite load confirming that female great tits experienced stronger anaemia when infested with haematophagous parasites during egg laying. We found a positive relationship between spot darkness and laying order that weakened under high parasite load. This result suggests that anaemia in females due to blood-sucking parasites led to diminished protoporphyrin from disintegrated red blood cells and hence a decreased deposition of protoporphyrin. However, the overall increase in pigment darkness along the laying sequence suggests that pigments also accumulate by enhanced red blood cell production caused by anaemia due to egg production itself.     

Seasonal Abundance and Distribution of Vibrio cholerae in Coastal Waters Quantified by a 16S-23S Intergenic Spacer Probe

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera and is indigenous to brackish waters. To advance our understanding of the ecology of this bacterium, we have developed a molecular probing method for detection of V. cholerae in coastal waters. Water samples from 7 locations in the Newport Bay watershed, California were sampled monthly for a whole year. V. cholerae concentrations were determined by membrane filtration-colony hybridization using an oligonucleotide probe targeting the 16S-23S intergenic spacer (ITS) region. In addition to V. cholerae concentrations, environmental parameters, including temperature, salinity, total bacterial direct counts, total viable counts, and chlorophyll a concentrations, were determined for each site. V. cholerae was detected year-round throughout the watershed. Regression analysis indicated that the concentration of V. cholerae inversely correlated with salinity (p <0.001). The sampling sites located nearest to the Pacific Ocean had lower concentrations, whereas sites located along the brackish San Diego Creek (salinity 0-12 per thousand) routinely had higher concentrations. V. cholerae concentrations also correlated with temperature (p <0.01) in the watershed, with concentrations ranging from less than 1 CFU mL-1 to 2,930 CFU mL-1 of water. The results of this study indicate that the dynamics of V. cholerae is mainly influenced, out of the parameters measured, by the temperature and salinity of the environment. This information is valuable for understanding the ecology of V. cholerae.     

Stable-isotope dilution gas chromatography-mass spectrometric measurement of 3-hydroxyglutaric acid, glutaric acid and related metabolites in body fluids of patients with glutaric aciduria type 1 found in newborn screening

We developed a simple and sensitive stable-isotope dilution method for the quantification of 3-hydroxyglutaric acid (3HGA) and glutaric acid (GA) in body fluids. In our method, tert-butyldimethylsilyl (tBDMS) derivatives of 3HGA and GA were measured with a conventional electron-impact ionization (EI) mode in gas chromatography-mass spectrometry (GC-MS). The control values for 3HGA in nmol/ml were 0.15+/-0.08 (serum; n=10) and 0.07+/-0.03 (CSF; n=10). In addition, glutarylcarnitine and free carnitine were quantified by electrospray tandem mass spectrometry. Using these methods, we monitored 3HGA, GA, and glutarylcarnitine in the body fluids of three patients with glutaric aciduria type 1 found during newborn screening. None of the patients had experienced neurological strokes, which are possibly caused by the accumulation of 3HGA, at 15-24 months of age under a disease-specific treatment, including carnitine supplementation. Our data showed that 3HGA levels were relatively high in some serum samples with lower glutarylcarnitine and carnitine levels, suggesting that carnitine supplementation may play a role in preventing the accumulation of 3HGA in patients with this disease.     

Comparative in vivo sensitizing efficacy of porphyrin and chlorin dimers joined with ester,ether, carbon–carbon or amide bonds

The anti-cancer activity of dimers joined with ether, ester or carbon–carbon bonds by photodynamic therapy (PDT) was compared by using DBA/2 mice transplanted with SMT/F tumors. Dimers with ether and carbon–carbon linkages were found to be more effective than those linked with ester bonds. Variation of the substituents at peripheral positions made a significant difference in in vivo efficacy. Among the ether and carbon–carbon linked dimers, the divinyl analogs were found to be most effective. The preliminary in vivo results also suggest that the position(s) of the hydrophilic substituents in the molecules make a remarkable difference in photosensitizing activity. An unsymmetrical dimer with an amide linkage, obtained from 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) was found to be less effective than HPPH.     

Characterization of a Deep-Sea Sediment Metagenomic Clone that Produces Water-Soluble Melanin in Escherichia coli

To access to the microbial genetic resources of deep-sea sediment by a culture-independent approach, the sediment DNA was extracted and cloned into fosmid vector (pCC1FOS) generating a library of 39,600 clones with inserts of 24–45 kb. The clone fss6 producing red-brown pigment was isolated and characterized. The pigment was identified as melanin according to its physico-chemical characteristics. Subcloning and sequences analyses of fss6 demonstrated that one open reading frame (ORF2) was responsible for the pigment production. The deduced protein from ORF2 revealed significant amino acid similarity to the 4-hydroxyphenylpyruvate dioxygenase (HPPD) from deep-sea bacteria Idiomarina loihiensis. Further study demonstrated that the production of melanin was correlated with homogentistic acid (HGA). The p-hydroxyphenylpyruvate produced by the Escherichia coli host was converted to HGA through the oxidation reaction of introduced HPPD. The results demonstrate that expression of DNA extracted directly from the environment might generate applicable microbial gene products. The construction and analysis of the metagenomic library from deep-sea sediment contributed to our understanding for the reservoir of unexploited deep-sea microorganisms.     

The effects of phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin on cellular glucose transport

Glucose is the principal fuel for brain metabolism and its movement across the blood-brain barrier depends on Glut1. Impaired glucose transport to the brain may have deleterious consequences. For example, Glut1 deficiency syndrome (Glut1DS) is the result of heterozygous loss of function Glut1 mutation leading to energy failure of the brain and subsequently, epileptic encephalopathy. To preserve the integrity of the energy supply to the brain in patients with compromised glucose transport function, consumption of compounds with glucose transport inhibiting properties should be avoided. Phenytoin is a widely used anticonvulsant that affects carbohydrate metabolism. In this study, the hypothesis that phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) affect cellular glucose transport was tested. With a focus on Glut1, the effects of phenytoin and HPPH on cellular glucose transport were studied. Glucose uptake assay measuring the zero-trans influx of radioactive-labeled glucose analogues showed that phenytoin and HPPH did not exert immediate effects on erythrocyte Glut1 activity or glucose transport in Hs68 control fibroblasts, Glut1DS primary fibroblasts isolated from two patients, or in rat primary astrocytes. Prolonged exposure to the two compounds could stimulate glucose transport by up to 30-60% over the control level (p <0.05) in Hs68 and Glut1DS fibroblasts as well as in rat astrocytes. The stimulation of glucose transport by HPPH was dose-dependent and accompanied by an up-regulation of GLUT1 mRNA expression (p <0.05). In conclusion, phenytoin and HPPH do not compromise cellular glucose transport. Prolonged exposure to these compounds can modify carbohydrate homeostasis by up-regulating glucose transport in both normal and Glut1DS conditions in vitro.     

Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding

Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.