Kinetic properties of the rat intestinal microsomal 1-naphthol:UDP-glucuronosyl transferase. Inhibition by UDP and UDP-N-acetylglucosamine

The kinetic properties of the rat intestinal microsomal 1-naphthol:UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic constants: Vmax = 102 +/- 6 nmol/min per mg microsomal protein, Km (UDPGlcUA) = 1.26 +/- 0.10 mM, Km (1-naphthol) = 96 +/- 10 microM and Ki (1-naphthol) = 25 +/- 7 microM. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso-Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP-N-acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl2 and Triton X-100.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Smad gene expression in pulmonary fibroblasts: indications for defective ECM repair in COPD

Background

Endothelial dysfunction is a major complication of pulmonary endarterectomy (PTE) that can lead to pulmonary edema and persistent pulmonary hypertension. We hypothesized that endothelial dysfunction is related to increased endothelial-cell (EC) death.

Methods

In piglets, the left pulmonary artery (PA) was ligated to induce lung ischemia then reimplanted into the main PA to reperfuse the lung. Animals sacrificed 5 weeks after ligation (n = 5), 2 days after reperfusion (n = 5), or 5 weeks after reperfusion (n = 5) were compared to a sham-operated group (n = 5). PA vasoreactivity was studied and eNOS assayed. EC apoptosis was assessed by TUNEL in the proximal and distal PA and by caspase-3 activity assay in the proximal PA. Gene expression of pro-apoptotic factors (thrombospondin-1 (Thsp-1) and plasminogen activator inhibitor 1 (PAI-1)) and anti-apoptotic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was investigated by QRT-PCR.

Results

Endothelium-dependent relaxation was altered 5 weeks after ligation (p = 0.04). The alterations were exacerbated 2 days after reperfusion (p = 0.002) but recovered within 5 weeks after reperfusion. EC apoptosis was increased 5 weeks after PA ligation (p = 0.02), increased further within 2 days after reperfusion (p < 0.0001), and returned to normal within 5 weeks after reperfusion. Whereas VEGF and bFGF expressions remained unchanged, TSP and PAI-1 expressions peaked 5 weeks after ligation (p = 0.001) and returned to normal within 2 days after reperfusion.

Conclusion

Chronic lung ischemia induces over-expression of pro-apoptotic factors. Lung reperfusion is followed by a dramatic transient increase in EC death that may explain the development of endothelial dysfunction after PE. Anti-apoptotic agents may hold considerable potential for preventing postoperative complications.     

Cell surface antigen expression by peripheral blood monocytes in allergic asthma: results of 2.5 years therapy with inhaled beclomethasone dipropionate

At present, inhaled glucocorticoids are widely accepted as the therapy of choice in chronic asthma. Treatment with inhaled glucocorticoids significantly suppresses local airway inflammation in asthmatics, but may also have systemic effects, e.g. a reduction of the number of circulating hypodense eosinophils or a down-modulation of HLA-DR antigen (Ag) expression by T lymphocytes in peripheral blood. However, the effect of long-term therapy with inhaled glucocorticoids on peripheral blood monocytes (PBM), which are the precursors of the most numerous cell type in the lung, the alveolar macrophage, have not yet been evaluated. We therefore investigated the expression of various cell surface Ag on PBM from non-smoking patients with allergic asthma who were treated for 2.5 years with a beta(2)-receptor agonist plus either an inhaled glucocorticoid (beclomethasone dipropionate, BDP) (n = 4) or an anticholinergic or placebo (n = 8). We compared the results with healthy volunteers (n = 7). Long-term treatment of allergic asthmatics with inhaled BDP, but not anticholinergic or placebo therapy, was associated with a significantly lower CDllb Ag expression (p < 0.04) and higher expression of CD13, CD14 and CD18 Ag (p < 0.05, p < 0.02 and p < 0.04, respectively) when compared with the healthy control subjects (n = 7). Most interestingly, PBM of asthmatics treated with inhaled BDP expressed an almost two-fold higher level of CD14 Ag on their cell surface than PBM of patients treated with anticholinergic or placebo (p < 0.03). No significant differences in the expression of CD16, CD23, CD25, CD32 and CD64 Ag or HLA-DR were observed between PBM from the different patient groups or healthy controls. Taken together, this study shows that long-term local therapy with inhaled BDP coincides with an altered expression of at least one cell surface Ag on PBM from allergic asthmatics.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.     

Rapid formation of N-hydroxymethylpentamethylmelamine by mitochondria from rat small intestinal epithelium

Isolated rat intestinal mitochondria showed a considerable capacity to convert hexamethylmelamine to its monodemethylated metabolite pentamethylmelamine. Mitochondrial turnover rate is about the same as compared with microsomal preparations. Only in mitochondrial incubations N-hydroxymethylpentamethylmelamine could be identified as a metabolic intermediate. The known chemical reactivity of carbinolamines means that this activation pathway in mitochondria could be involved in the pharmacological or toxic effects of hexamethylmelamine.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.