Unique protonation states of aspartate and topaquinone in the active site of copper amine oxidase

The oxidative deamination of biogenic amines, crucial in the metabolism of a wealth of living organisms, is catalyzed by copper amine oxidases (CAOs). In this work, on the ground of accurate molecular modeling, we provide a clear insight into the unique protonation states of the key catalytic aspartate residue Asp298 and the prosthetic group of topaquinone (TPQ) in the CAO of Arthrobacter globiformis (AGAO). This provides both extensions and complementary information to the crystal structure determined by our recent neutron diffraction (ND) experiment. The hybrid quantum mechanics/molecular mechanics (QM/MM) simulations suggest that the ND structure closely resembles a state in which Asp298 is protonated and the TPQ takes an enolate form. The TPQ keto form can coexist in the fully protonated state. The energetic and structural analyses indicate that the active site structure of the AGAO crystal is not a single state but rather a mixture of the different protonation and conformational states identified in this work.

Copper amine oxidases catalyze the oxidative deamination of biogenic amines. We investigated the unique protonation states in the active site using first-principle calculations.     

Bacillus Calmette–Guérin-unresponsive non-muscle-invasive bladder cancer: Its definition and future therapeutic strategies

Bacillus Calmette–Guérin induction with or without maintenance is the gold standard therapy for intermediate-/high-risk non-muscle-invasive bladder cancer; however, one-third of patients treated with adequate bacillus Calmette–Guérin therapy do not achieve sufficient responses, and this is referred to as “bacillus Calmette–Guérin failure.” The term, bacillus Calmette–Guérin failure, is ambiguous and includes a very heterogeneous population of patients. By strictly focusing on patients who are unlikely to benefit from additional bacillus Calmette–Guérin therapy and who need to be treated with radical cystectomy, the new concept of “bacillus Calmette–Guérin unresponsive” was recently proposed, and might accelerate the development of novel therapeutic options for bacillus Calmette–Guérin-unresponsive disease. A promising therapeutic strategy for bacillus Calmette–Guérin-unresponsive disease is the blockade of the programmed cell death-1/programmed cell death-ligand 1 pathway, which is considered to be activated by bacillus Calmette–Guérin therapy. Several large clinical trials have been carried out to assess the potential of programmed cell death-1/programmed cell death-ligand 1 blockade in bacillus Calmette–Guérin-naïve high-risk non-muscle-invasive bladder cancer and bacillus Calmette–Guérin-unresponsive disease. Furthermore, clinical trials that are targeting bacillus Calmette–Guérin-unresponsive disease with other strategies, such as vaccines, gene therapy, and targeted and cytotoxic therapies, are ongoing. The findings of these trials are awaited in order to establish appropriate bladder-sparing approaches for patients with bacillus Calmette–Guérin-unresponsive disease.     

Evaluation of 5-Fluorouracil Metabolic Enzymes as Predictors of Response to Adjuvant Chemotherapy Outcomes in Patients with Stage II/III Colorectal Cancer: A Decision-Curve Analysis

Background

The effectiveness of 5-fluorouracil (5-FU)-based adjuvant chemotherapy is reported in patients with colorectal cancer (CRC), but the usefulness of 5-FU metabolic enzymes as predictive biomarkers of the efficacy of this chemotherapy remains unclear.

Objective

This study aims to verify whether 5-FU metabolic enzymes are predictive biomarkers in the clinical setting of adjuvant chemotherapy for stage II/III CRC.

Methods

In total, 179 patients with stage II/III CRC who were treated at our institute between 2000 and 2010 were enrolled. Messenger RNA (mRNA) expression of major 5-FU metabolic enzymes, namely thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase (TP), orotate phosphoribosyl transferase, and β-actin (control) was evaluated using the Danenberg Tumor Profile method. mRNA expression and other clinicopathological data were investigated with regard to CRC relapse.

Results

A total of 78 patients underwent surgery alone, while 101 underwent adjuvant chemotherapy (5-FU plus leucovorin [LV] or tegafur plus uracil /LV) following surgery. Relapse-free survival was longer and risk of recurrence was lower in association with high TP mRNA expression than in association with low TP mRNA expression in the adjuvant chemotherapy group (hazard ratio 0.66; 95 % confidence interval 0.47–0.92; p = 0.016), but not in the surgery alone group. mRNA expression of no other enzymes was associated with relapse in both groups. In decision-curve analyses, the predictive efficiency of TP mRNA expression plus clinicopathological factors was slightly better than that of clinicopathological factors only.

Conclusions

TP mRNA expression in tumors predicted the effects of adjuvant chemotherapy for stage II/III CRC, although the beneficial effects were marginal.     

Abnormal glutathione metabolism and increased cytotoxicity caused by H2O2 in human umbilical vein endothelial cells cultured in high glucose medium

Summary To determine whether increased oxidative stress in diabetes mellitus is due to an impaired freeradical scavenger function in endothelial cells, GSH-dependent H₂O₂ degradation in human umbilical vein endothelial cells was studied. The GSH-dependent, NaN₃-uninhibitable H₂O₂-degradation in endothelial cells was reduced by 48% (p <0.001) when the cells were exposed to 33 mmol/l d-glucose vs 5.5 mmol/l d-glucose. This impairment was dependent not only on the d-glucose concentration in the medium but also on d-glucose specific metabolism, since neither 27.5 mmol/l l-glucose nor 27.5 mmol/l d-raffinose had any effect on the peroxide degradation activity. Activation of the glutathione redox cycle by H₂O₂ in cells exposed to high glucose concentrations was attenuated as compared with 5.5 mmol/l d-glucose because of: 1) a 42% decrease (p <0.001) in intracellular NADPH content, and 2) a 34% reduction (p <0.01) in glutathione release into the media. This results in an accumulation of GSSG in the cells following exposure to H₂O₂. Both H₂O₂-evoked ⁵¹Cr-release and H₂O₂-induced endothelial cell damage were significantly (p <0.01) greater in the 33 mmol/l d-glucose group than in the 5.5 mmol/l d-glucose group. These results indicate that the abnormal glutathione redox cycle observed in endothelial cells is induced by high glucose concentrations in the medium, resulting in an impairment of reduced GSH-dependent H₂O₂-degradation. These abnormalities may associate with the increased cellular damage following an exogenous exposure to H₂O₂.Abbreviations GSH Reduced glutathione - GSSG oxidized glutathione - BSO L-buthionine-[S,R]-sulfoximine     

Vasopressin responsiveness of subclinical Cushing's syndrome due to ACTH-independent macronodular adrenocortical hyperplasia

OBJECTIVE: Vasopressin (AVP) is reported to be an important factor for regulating cortisol secretion in patients with Cushing's syndrome due to ACTH-independent macronodular adrenocortical hyperplasia (AIMAH). Recently, there have been several case reports of subclinical Cushing's syndrome due to AIMAH, in which the pathophysiological role of AVP has been unknown. The aim was to conduct an extensive investigation of AVP in the autonomous secretion of cortisol in subclinical Cushing's syndrome due to AIMAH. PATIENTS AND MEASUREMENTS: Five cases of AIMAH with subclinical Cushing's syndrome underwent prospective study including physical examination, imaging (MRI, CT and 131I-adosterol scintigraphy) and endocrinological evaluation that comprised basal plasma cortisol levels and urinary excretions of steroid metabolites, a dexamethasone suppression test and an AVP stimulation test. In case 1, left adrenalectomy was performed and the pathological diagnosis of AIMAH was established. An in vitro experiment using the cultured AIMAH adrenal cells was conducted to investigate cortisol secretion and expression of the V1-AVP receptor, mRNA by RT-PCR. RESULTS: All five cases were discovered incidentally to have bilateral adrenal nodules. Imaging by MRI and CT revealed large multinodular lesions in both adrenal glands, which showed positive uptake on 131I-adosterol scintigraphy. Although the basal values of plasma cortisol and urinary excretions of steroid metabolites were within normal limits, autonomous secretion of cortisol was assumed to occur because of lack of suppression during dexamethasone suppression. The five patients had no overt signs of Cushing's syndrome, and they were therefore diagnosed with subclinical Cushing's syndrome due to AIMAH. In all five patients, AVP stimulated cortisol secretion in vivo, whereas desmopressin acetate failed to affect cortisol secretion. In case 1, AVP stimulated cortisol secretion from cultured AIMAH adrenal cells, but this secretion had no relationship with cAMP production. In addition, over-expression of V1-AVP receptor mRNA in AIMAH tissue was determined by RT-PCR. CONCLUSION: Patients with subclinical Cushing's syndrome due to AIMAH commonly exhibit cortisol responsiveness to AVP, and this is probably mediated through activation of overexpressed V1-AVP receptors.