Chemical modification of Arthrobacter sarcosine oxidase by N-methylisothiazolinone reduces reactivity toward oxygen.

N-Methylisothiazolinone (MIT) is a thiol group modifier and antimicrobial agent. Arthrobacter sarcosine oxidase (SoxA), a diagnostic enzyme for assaying creatinine, loses its activity upon the addition of MIT, and its inactivation mechanism remains unclear. In this study, SoxA was chemically modified using MIT (mo-SoxA), and its structural and chemical properties were characterized. Spectral analysis data, oxygen consumption rates, and reactions were compared between intact SoxA and mo-SoxA. These demonstrate that the oxidative half-reaction toward oxygen is inhibited by MIT modification. The oxidase activity of mo-SoxA was approximately 2.1% of that of intact SoxA, and its dehydrogenase activity was approximately 4.2 times higher. The C-to-S mutants revealed that cooperative modification of 2 specific cysteine residues caused a drastic change in the enzyme reaction mode. Based on the modeled tertiary structures, the putative entrance for oxygen uptake is predicted to be blocked by the chemical modification of the 2 cysteine residues.

Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I.

Site-specific suppressors of superoxide production (named S1QELs) in the quinone-reaction site in mitochondrial respiratory complex I during reverse electron transfer have been previously reported; however, their mechanism of action remains elusive. Using bovine heart submitochondrial particles, we herein investigated the effects of S1QELs on complex I functions. We found that the inhibitory effects of S1QELs on complex I are distinctly different from those of other known quinone-site inhibitors. For example, the inhibitory potencies of S1QELs significantly varied depending on the direction of electron transfer (forward or reverse). S1QELs marginally suppressed the specific chemical modification of Asp160 in the 49-kDa subunit, located deep in the quinone-binding pocket, by the tosyl chemistry reagent AL1. S1QELs also failed to suppress the binding of a photoreactive quinazoline-type inhibitor ([125I]AzQ) to the 49-kDa subunit. Moreover, a photoaffinity labeling experiment with photoreactive S1QEL derivatives indicated that they bind to a segment in the ND1 subunit that is not considered to make up the binding pocket for quinone or inhibitors. These results indicate that unlike known quinone-site inhibitors, S1QELs do not occupy the quinone- or inhibitor-binding pocket; rather, they may indirectly modulate the quinone-redox reactions by inducing structural changes of the pocket through binding to ND1. We conclude that this indirect effect may be a prerequisite for S1QELs' direction-dependent modulation of electron transfer. This, in turn, may be responsible for the suppression of superoxide production during reverse electron transfer without significantly interfering with forward electron transfer.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches.

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.

Multi-center study on the prevalence of hypothyroidism in patients with hypercholesterolemia.

Hypercholesterolemia is one of the most representative disorders of the common diseases. To evaluate the prevalence of hypothyroidism in the population of adult hypercholesterolemia, we prospectively examined the thyroid function in patients with untreated or treated hypercholesterolemia as a multi-center survey. Subjects were the patients who were treated with some antilipemic agents or the untreated patients whose total cholesterol (TC) was over 220 mg/dL and/or LDL-cholesterol (LDL-C) over 140 mg/dL. Among 737 cases recruited, 725 cases (300 males and 425 females) participated in the survey including the thyroid function test. The patient's backgrounds include hypertension (51%), diabetes mellitus (49%), fatty liver (17%), smoking (15%), and habitual drinking (10%). The 72% of the patients were treated with some antilipemic agents and the mean values of TC, LDL-C, triglyceride (TG), HDL-cholesterol (HDL-C), and LDL-C/HDL-C ratio (L/H) were 204.5 mg/dL, 119.6 mg/dL, 144.4 mg/dL, 60.7 mg/dL and 2.25, respectively. The primary hypothyroidism was seen in 27 cases (3.7%) (11 males, 16 females) with subclinical hypothyroidism in 17 cases (2.4%) and overt hypothyroidism in 10 cases (1.4%). The central hypothyroidism was seen in 4 cases (0.6%). The prevalence of hypothyroidism was 4.3% in patients with hypercholesterolemia. Taking account of the large number of patients with dyslipidemia and importance of avoiding unnecessary administration and associated adverse effects, evaluation of the thyroid function could be warranted in patients with dyslipidemia although cost-benefit issues waits further investigation.

Evaluation of Nutrition in the Healing of Pressure Ulcers: Are the EPUAP Nutritional Guidelines Sufficient To Heal Wounds?

 Malnutrition is a significant factor in the development of pressure ulcers and many nutritional guidelines for preventing pressure ulcers have been published. However, few clinical investigations have examined the energy required to heal pressure ulcers. The aim of the present study was to investigate the relationship between nutritional intake and improvement of pressure ulcers. Total calories, which were supplied by mouth through a feeding tube and via venous alimentation were examined for 40 hospitalized bedridden inpatients who had pressure ulcers. Of these patients, 21 whose wounds improved or healed and 19 whose wounds became worse or did not improve were eligible for this retrospective study. Pressure ulcers in patients who received more than 30 kcal/kg per day improved or healed, while those of patients who received less than 20 kcal/kg per day worsened or failed to improve. Furthermore, intake of 30 kcal/kg per day enabled serum albumin levels to improve. Energy intake of 30 kcal/kg per day is comparable to the predicted total energy expenditure and is thought to be essential for improving pressure ulcers in bedridden patients .

Toll-like receptor expression in lupus peripheral blood mononuclear cells.

OBJECTIVE: To investigate expression of members of the Toll-like receptor (TLR) family in peripheral blood mononuclear cells (PBMC) in patients with systemic lupus erythematosus (SLE). METHODS: We analyzed PBMC from 14 patients with SLE and 15 healthy subjects. The surface expressions of TLR2 and TLR4 and intracellular expression of TLR9 on PBMC were analyzed by flow cytometry. RESULTS: Although TLR4 expressions on CD14+ monocytes were not significantly different between healthy subjects and patients with SLE, TLR2 expressions on monocytes were reduced in patients with SLE compared to healthy subjects. Intracellular TLR9 expression levels of CD19+ B lymphocytes were significantly elevated in patients with SLE. However, the TLR9 expression levels of plasmacytoid dendritic cells were not significantly different between these patients and healthy subjects. CONCLUSION: Our results show that human peripheral blood B cells express TLR9 and that its expression is increased in patients with SLE. This upregulated expression of TLR9 in B cells may be related to the abnormal B cell hyperactivity in patients with SLE.

Relationship between serum resistin concentrations and inflammatory markers in patients with type 2 diabetes mellitus.

To examine whether serum resistin concentrations are associated with metabolic or inflammatory markers in patients with type 2 diabetes mellitus, we examined serum concentrations levels and metabolic or inflammatory markers in 56 patients with type 2 diabetes mellitus and 41 healthy subjects. Serum levels of resistin, serum amyloid A, and soluble vascular cell adhesion molecule-1 were measured by enzyme-linked immunosorbent assay. Serum resistin levels were significantly elevated in diabetic patients compared with those in healthy subjects. Serum resistin concentrations did not correlate with body mass index; however, there was a significant positive correlation between resistin and soluble vascular cell adhesion molecule-1 in diabetic patients. Based on the present results, we conclude that resistin appears to be associated with vascular inflammatory markers in patients with type 2 diabetes mellitus.

Idiopathic portal hypertension associated with systemic sclerosis and Sjögren's syndrome.

We report a patient with idiopathic portal hypertension (IPH) associated with systemic sclerosis (SSc) and Sjögren's syndrome. A 72-year-old Japanese woman was admitted to our hospital because of Raynaud's phenomenon, sclerodactyly, and dyspnea. The patient had splenomegaly, esophageal varices in the absence of extrahepatic portal obstruction, and cirrhosis of the liver. Immunological studies revealed positive anti-nuclear antibodies and high titers of anti-Scl-70, anti-SS-A, anti-centromere, and anti-mitochondrial M2 antibodies. Histological examinations of the liver biopsy specimen revealed stenosis and loss of small portal veins without findings of primary biliary cirrhosis. The patient was diagnosed as having IPH associated with SSc and Sjögren's syndrome. These observations suggest an immunological role in the pathogenesis of IPH.

Catastrophic transverse myelitis in a patient with systemic lupus erythematosus.

A 24-year-old Japanese woman was admitted to our hospital suffering from high fever and progressive paralysis in both legs. Magnetic resonance imaging of the spinal cord showed high-intensity signals from C5 to Th4 and from Th7 to L1 on T2-weighted images. The patient was diagnosed as having acute transverse myelitis trade mark, which was a complication of systemic lupus erythematosus based on the serological findings. Despite aggressive immunosuppressive treatments including corticosteroid pulse therapy, plasmapheresis, and intravenous cyclophosphamide, the paralysis of her lower extremities did not improve. In the catastrophic type of lupus-associated TM, which develops extensively and longitudinally along the spinal cord, the prognosis still seems to be poor despite intensive treatments.