Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE

PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE Objectives: Materno-fetal transplacental transport is crucial for the fetal well-being. The altered expression of placental transport proteins under specific pathophysiological conditions may affect the intrauterine environment. Pre-eclampsia is often associated with high maternal uric acid serum levels. The regulation of the placental uric transport system and its transporter glucose transporter (GLUT)-9 are not fully understood yet. The aim of this study was to investigate the placental urate transport and to characterize its transporter GLUT9. Methods: In this study we used a transepithelial transport (Transwell®) model to assess uric acid transport activity. Electrophysiological techniques and radioactive ligand up-take assays were used to measure transport activity of GLUT9 expressed in Xenopus oocytes. Results: In the Transwell/model uric acid is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min at the linear stage. We could successfully over-express GLUT9 using the Xenopus laevis oocytes expression system. Chloride modulates the urate transport system: interestingly replacing chloride with iodine resulted in a complete loss of urate transport activity.We determined the IC50 of iodine at 30uM concentration. In radioactive up-take experiments iodinehad noeffect on uric acid transport. Conclusions: In vitro the “materno-fetal” transport of uric acid is slow. This indicates that in vivo the child is protected from short-term fluctuations of maternal uric acid serum concentrations. The different results regarding iodine-mediated regulation of GLUT9 transport activity between electrophysiological and radioactive ligand uptake experiments may suggest that iodine does not directly inhibit uric acid transport, but changes the mode of up-take from an electrogenic to an electroneutral transport. GLUT9 is not an uric acid uniporter, there are more ions involved in the transport. This may allow regulating uric acid transport by the change from an active to a passive transport.

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9)

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9). INTRODUCTION: Pre-eclampsia, a pregnancy-specific disease, contributes substantially to perinatal morbidity and mortality of both the mother and her child. Pre-eclampsia is often associated with high maternal urate serum levels, which in turn has been shown to play a role in the pathogenesis of this disease. The aim of this study was to investigate the glucose transporter GLUT9-mediated placental uric acid transport system. METHODS: In this study western blot, immunofluorescence techniques as well as a transepithelial transport (Transwell) model were used to assess GLUT9 protein expression and, respectively, uric acid transport activity. Electrophysiological techniques and transmission electron microscopy (TEM) were used to characterize the properties and the structure of GLUT9. RESULTS: Uric acid is transported across a BeWo choriocarcinoma cell monolayer with 530 pmol/min. We could successfully overexpress and for the first time purify the GLUT9b isoform using the Xenopus laevis oocytes expression system. Chloride seems to modulate the urate transport system. TEM revealed that GLUT9b isoform is present as monomer and dimmer in the Xenopus laevis overexpression model. A class average of all the particles allowed us to develop a first model of human GLUT9b structure, which was derived from the published crystal structure of the bacterial homologue of GLUT1-4. CONCLUSIONS: In vitro the “materno-fetal” transport of uric acid is slow indicating that in vivo the fetus might be protected from short-term fluctuations of maternal urate serum levels. The low-resolution structure obtained from TEM validates the proposed homology model regarding the structure of human GLUT9b. In ongoing studies this model is used to perform virtual screening to identify novel modulators of the urate transport system enabling the development of novel therapies in pregnancy complications.

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-DL-methyl-tryptophane (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Infiltration of Natural Caries Lesions in Relation to Their Activity Status and Acid Pretreatment in vitro

This study aimed at testing how active and inactive enamel caries lesions differ by their degree of resin infiltration, and whether the choice of acid pretreatment plays a crucial role. Four examiners assessed 104 human molars and premolars with noncavitated enamel lesions and classified them as 'active' or 'inactive' using the Nyvad criteria. Forty-five teeth were included in this study after independent unanimous lesion activity assessment. Lesions were cut perpendicularly into 2 halves. Each half lesion was pretreated with either 15% hydrochloric acid or 35% phosphoric acid. The lesions were infiltrated after staining with rhodamine isothiocyanate. Thin sections of 100 µm were prepared and the specimens were bleached with 30% hydrogen peroxide. The specimens were then counterstained with sodium fluorescein, subjected to confocal laser scanning microscopy and analyzed quantitatively. Outcome parameters were maximum and average infiltration depths as well as relative penetration depths and areas. In active lesions no significant difference of percentage maximum penetration depth and percentage average penetration depth between lesions pretreated with hydrochloric or phosphoric acid could be observed. In inactive lesions, however, phosphoric acid pretreatment resulted in significantly lower penetration compared to hydrochloric acid pretreatment. Surface conditioning with hydrochloric acid led to similar infiltration results in active and inactive lesions. Moreover, inactive lesions showed greater variability in all assessed infiltration parameters than did active lesions. In conclusion, caries lesion activity and acid pretreatment both influenced the infiltration. The use of phosphoric acid to increase permeability of the surface layer of active lesions should be further explored.

Mycophenolate acid inhibits endothelial NAD(P)H oxidase activity and superoxide formation by a Rac1-dependent mechanism

Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins

The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.

Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.