Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

Within- and between-laboratory reproducibility and predictive capacity of amino acid derivative reactivity assay (ADRA) using a 0.5 mg/mL test chemical solution: Results of the study for reproducibility confirmation implemented in five participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Detection system of the intracellular nitric oxide in yeast by HPLC with a fluorescence detector.

Nitric oxide (NO) is an important signaling molecule involved in various biological phenomena in many organisms. The physiological functions and metabolism of NO in yeast, a unicellular microorganism, are still unknown, mainly because it is difficult to analyze the intracellular NO levels accurately. Here, we developed a new method of more accurately measuring NO content in yeast cells with the detection limit of 6 nM, by treating the cells with an NO-specific fluorescence probe followed by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). This approach successfully detected and quantified the NO content inside yeast cells treated with an NO donor. Moreover, the HPLC/FLD analysis indicates that the fluorescence induced under some environmental stress conditions, such as ethanol, vanillin, and heat-shock, was not derived from NO. The HPLC/FLD method developed in this study provides a new strategy for measuring the intracellular NO concentration with higher accuracy.

Oxidation of a cysteine-derived nucleophilic reagent by dimethyl sulfoxide in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

Combined intravitreal methotrexate and immunochemotherapy followed by reduced-dose whole-brain radiotherapy for newly diagnosed B-cell primary intraocular lymphoma.

Primary intraocular lymphoma (IOL) has a propensity for central nervous system (CNS) relapse within 2 years of initial diagnosis, affecting clinical outcome. To reduce CNS relapse, we performed the combination treatment protocols of intravitreal methotrexate injections, methotrexate-based systemic induction chemotherapy and consolidation high-dose cytarabine and reduced-dose whole brain radiation therapy (rdWBRT, 23·4 Gy) for B-cell primary IOL with or without newly diagnosed CNS involvement. All patients underwent longitudinal brain magnetic resonance imaging (MRI) and cognitive assessment for evaluation of treatment-induced leucoencephalopathy. Seventeen patients initiated and 16 completed the protocol treatment. CNS relapse occurred in 2 patients and intraocular relapse in 3. Four-year progression-free survival (PFS) was 74·9% and 4-year overall survival (OS) was 86·3%, with a median follow-up period of 48·9 months. Of 11 patients without CNS involvement, 1 had CNS relapse and 3 intraocular relapse, and 4-year PFS and OS was 72·7% and 88·9%, respectively. Although white matter abnormalities shown by MRI were significantly increased at 4 years after rdWBRT, only one patient developed mild cognitive impairment. The combination of intravitreal chemotherapy, prophylactic systemic chemotherapy and rdWBRT for primary IOL showed a potential to reduce CNS relapse rate and improved 4-year PFS and OS without increase of cognitive dysfunction.

Effects and Limitations of Naldemedine for Opioid-Induced Urinary Retention: A Case Report.

A 78-year-old man with postoperative recurrence of esophageal cancer was admitted to the hospital due to chest pain and dyspnea. Oral short-acting opioids provided some relief, but chest pain persisted and worsened, leading to the initiation of a transdermal fentanyl patch. However, the patient developed opioid-induced urinary retention, which was treated with a naldemedine, a medication used for opioid-induced constipation and urinary retention. Opioid switching led to recurrent urinary retention, requiring placement of a urinary catheter. The patient ultimately required continuous deep sedation for refractory symptoms and died several days later.

The antinociceptive and anti-inflammatory action of the CHCl3-soluble phase and its main active component, damnacanthal, isolated from the root of Morinda citrifolia.

Morinda citrifolia (Rubiaceae, Noni) is a traditional medicine with various pharmacological activities. We investigated if the MeOH-, CHCl(3)- and BuOH-soluble phase and its main active component, damnacanthal, isolated from the Noni root, have antinociceptive and anti-inflammatory actions in mice. The CHCl(3)-soluble phase (3 g/kg, per os (p.o.)) significantly reduced pain-related behavior observed in the formalin test. These effects were not suppressed by pretreatment with naloxone (1 mg/kg, intraperitoneally (i.p.)), an opioid receptor antagonist. The CHCl(3)-soluble phase (3 g/kg, p.o.) significantly reduced histamine-induced paw edema. The MeOH- and BuOH-soluble phase had no effect in either test. Furthermore, damnacanthal (10-100 mg/kg, p.o.) exerted an antinociceptive effect on chemical nociceptive stimuli, and decreased histamine-induced paw edema. Damnacanthal was weakly bound to the histamine H(1) receptor. These data suggest that the CHCl(3)-soluble phase of the Noni root has antinociceptive and anti-inflammatory effects. Furthermore, these effects of damnacanthal isolated from the Noni root is mediated in part by the histamine H(1) receptor.

[Involvement of glycemic control in the inhibiting effect of Morinda citrifolia on cerebral ischemia-induced neuronal damage].

Fruit juice of Morinda citrifolia (Rubiaceae, Noni juice) is a well-known healthy drink and has various pharmacological properties including antioxidant and anti-inflammatory effects. We have hitherto found the protective effect of Noni juice on neuronal damage caused by ischemic stress in mice. In addition, we have also recently reported that suppression of post-ischemic glucose intolerance might be important for good prognosis. Here, we focused on the effect of Noni juice on the development of the post-ischemic glucose intolerance as a cerebral protective mechanism. Noni juice was obtained from the mature fruit grown in Okinawa (ONJ). Male ddY mice were given 10% ONJ in drinking water for 7 d. Then, mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). We found that 10% ONJ treatment suppressed the development of neuronal damage after MCAO. Interestingly, glucose intolerance observed on 1 d after MCAO completely disappeared by 10% ONJ. Furthermore, 10% ONJ treatment significantly increased serum insulin levels much further than the control group on 1 d, while serum adiponectin levels were not affected at all. These results suggest that ONJ could facilitate insulin secretion and may attenuate the development of glucose intolerance under ischemic stress. These functions may contribute to the neuronal protective effect of ONJ against ischemic stress.

[Protective effect of Morinda citrifolia on the ischemic neuronal damage].

The fruits juice of Morinda citrifolia (Noni juice) is a well known healthy drink and has various pharmacological effects such as antioxidant. Here, we focused on the protective effect of Okinawa Noni juice (ONJ) against ischemic stress using cerebral ischemia-reperfusion model mice. We found that free intake of 10% ONJ for 7 days showed the protective effect on neuronal damage after ischemic stress, while 3% ONJ did not show such effects. It has recently reported that glucose intolerance might be induced by ischemic stress and controlling of the blood glucose levels in the early phase of ischemic stress seems to be important for good prognosis. Interestingly, we have found the glucose intolerance was developed on 1st day after ischemic stress, while it disappeared on 3rd day. On the other hand, neuronal damage gradually developed from 1st to 5th day after ischemic stress. These results indicate that development of glucose intolerance might be one of the exacerbating factors of neuronal damage after ischemic stress. Noteworthy, the development of glucose intolerance on 1st day after ischemic stress significantly suppressed by ONJ in a dose-dependent manner. These results suggest that protective effect of ONJ might be mediated by suppression of ischemic stress-induced glucose intolerance.

[Isorhythmic dissociation during sevoflurane anesthesia].

Arrhythmia during general anesthesia occurrs occasionally as a result of the effect of inhalation anesthetic agent on cardiac conduction. We experienced a case of atrioventricular junctional rhythm (AVJR) during maintenance of general anesthesia with sevoflurane. A 61-year-old woman with normal preoperative electrocardiogram was scheduled for right total knee arthroplasty and autologous iliac crest bone graft under general and epidural anesthesia. After inserting the lumbar epidural catheter, endotracheal intubation was performed following anesthesia induction with fentanyl, thiopental, and vecuronium, and anesthesia was maintained using inhalation of nitrous oxide and sevoflurane, and epidural anesthesia with ropivacaine. Forty minutes after starting operation, the dissociated and upright P-wave approached the QRS complex, remaining within QRS complex for several beats, and then reappeared from it electrocardiographically. ST segment was transiently depressed from baseline. The R-R interval and waveform of the QRS complex did not change. The sequence was repeated. The blood pressure had been held slightly depressed but waveform of pulse oxymetry (SpO2) did not change during this phenomenon. We diagnosed it as AVJR, which is classified into isorhythmic dissociation. After discontinuation of sevoflurane at the end of the operation, isorhythmic dissociation returned to sinus rhythm. Sevoflurane could induce atrioventricular conduction disturbance leading to isorhythmic dissociation of AVJR. We should be aware that hypotension can result from isorhythmic dissociation during sevoflurane anesthesia.

Structural and functional insights into S-thiolation of human serum albumins.

Human serum albumin (HSA) is the most abundant serum protein, contributing to the maintenance of redox balance in the extracellular fluids. One single free cysteine residue at position 34 is believed to be a target of oxidation. However, the molecular details and functions of oxidized HSAs remain obscure. Here we analyzed serum samples from normal subjects and hyperlipidemia patients and observed an enhanced S-thiolation of HSA in the hyperlipidemia patients as compared to the control individuals. Both cysteine and homocysteine were identified as the low molecular weight thiols bound to the HSAs. Intriguingly, S-thiolations were observed not only at Cys34, but also at multiple cysteine residues in the disulfide bonds of HSA. When the serum albumins from genetically modified mice that exhibit high levels of total homocysteine in serum were analyzed, we observed an enhanced S-homocysteinylation at multiple cysteine residues. In addition, the cysteine residues in the disulfide bonds were also thiolated in recombinant HSA that had been treated with the disulfide molecules. These findings and the result that S-homocysteinylation mediated increased surface hydrophobicity and ligand binding activity of HSA offer new insights into structural and functional alternation of serum albumins via S-thiolation.

Beta-sitosterol-3-O-beta-D-glucopyranoside: a eukaryotic DNA polymerase lambda inhibitor.

Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.

Chemical constituents of Morinda citrifolia fruits inhibit copper-induced low-density lipoprotein oxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in the genesis of arteriosclerosis. The present study focused on the effects of the fruits of Morinda citrifolia on preventing arteriosclerosis. The MeOH extract and CHCl(3)-, EtOAc-, n-BuOH-, and H(2)O-soluble phases derived from the fruits of M. citrifolia were evaluated for their inhibitory activity on copper-induced LDL oxidation by the thiobarbituric acid-reactive substances (TBARS) method. The MeOH extract and EtOAc-soluble phase showed 88 and 96% inhibition, respectively. Six lignans were isolated by repeated column chromatography from the EtOAc-soluble phase. These compounds were determined by spectroscopic analysis to be 3,3'-bisdemethylpinoresinol (1), americanol A (2), americanin A (3), americanoic acid A (4), morindolin (5), and isoprincepin (6), of which 4 and 5 are novel compounds. These compounds inhibited copper-induced LDL oxidation in a dose-dependent manner. 1, 2, 5, and 6 exhibited remarkably strong activities, which were the same or better than that of the known antioxidant 2,6-di-tert-butyl-p-cresol. The IC(50) values for 1, 2, 5, and 6 were 1.057, 2.447, 2.020, and 1.362 microM, respectively. The activity of these compounds is mainly due to their number of phenolic hydroxyl groups.

Long-term effect of repeated lidocaine injections into the external oblique for upper camptocormia in Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is occasionally complicated by camptocormia. In a previous study, we classified camptocormia into upper and lower types based on the inflection point, and reported that lidocaine injection into the external oblique muscle, but not into the internal oblique or rectus abdomen, improved upper camptocormia in PD. The effect of a single lidocaine injection disappeared over a period of few days. In this study, we used repeated lidocaine injections into the external oblique for 4-5 days and evaluated the effects of such treatment for up to 90 days. METHODS: The study subjects were 12 patients with PD and upper camptocormia who were treated with repeated lidocaine injections into the bilateral external oblique followed by rehabilitation. The effect of treatment was evaluated by measuring the angle of truncal flexion before and after the injection. Patients who showed improvement with repeated injections were evaluated during a 90-day period. RESULTS: Eight out of 12 patients showed significant improvement in posture after a single lidocaine injection. However, the effect subsided several days after treatment. Repeated injections produced long-term improvement in 9 out of 12 patients, which was maintained during the 90-day observation period in eight of these patients. CONCLUSIONS: Our results showed that repeated lidocaine injections into the external oblique improved upper camptocormia, and that the effect was maintained in the majority of patients during the 90-day observation period, indicating that repeated lidocaine injections into the external oblique have therapeutic effect on upper camptocormia in patients with Parkinson's disease.

Chemical constituents of Baeckea frutescens leaves inhibit copper-induced low-density lipoprotein oxidation.

Oxidative modification of LDL plays an important role in the genesis of arteriosclerosis. This study focused on the effects of the leaves of Baeckea frutescens in the prevention of arteriosclerosis. The leaves of B. frutescens have afforded, besides known flavonoid and chromone glycosides, a novel biflavonoid glycoside, characterized as 3-O-alpha-L-rhamnopyranosylmyricetinyl-(I-2'',II-2'')-3-O-alpha-L-rhamnopyranosylmyricetin on the basis of chemical and spectral evidence. This compound exhibited marked inhibition of copper-induced LDL oxidation.

Morinda citrifolia fruit juice prevents ischemic neuronal damage through suppression of the development of post-ischemic glucose intolerance.

Fruit juice of Morinda citrifolia (Noni juice) is a well-known health drink and has various pharmacological properties including antioxidant and anti-inflammatory effects. We have hitherto found the protective effect of Noni juice on brain damage caused by ischemic stress in mice. In addition, we also recently reported that regulation of post-ischemic glucose intolerance might be important for good prognosis. Here, we focused on the effect of Noni juice on the development of the post-ischemic glucose intolerance as a cerebral protective mechanism. Noni juice was obtained from the mature fruit grown in Okinawa (about 1.5 L/4 kg of fruit; 100% ONJ). Male ddY mice were given 10% ONJ in drinking water for 7 days. Then, mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). Ingestion of 10% ONJ suppressed the development of neuronal damage after MCAO. Interestingly, glucose intolerance observed on the 1st day after MCAO completely disappeared after 10% ONJ administration. Furthermore, ONJ treatment significantly increased serum insulin levels much further than the control group on the 1st day, while serum adiponectin levels were not affected at all. These results suggest that ONJ could facilitate insulin secretion after ischemic stress and may attenuate the development of glucose intolerance. These mechanisms may contribute to the neuronal protective effect of ONJ against ischemic stress.

New anthraquinone glycosides from the roots of Morinda citrifolia.

Six new anthraquinone glycosides: digiferruginol-1-methylether-11-O-beta-gentiobioside (1); digiferruginol-11-O-beta-primeveroside (2); damnacanthol-11-O-beta-primeveroside (3); 1-methoxy-2-primeverosyloxymethyl-anthraquinone-3-olate (4); 1-hydroxy-2-primeverosyloxymethyl-anthraquinone-3-olate (5); and 1-hydroxy-5,6-dimethoxy-2-methyl-7-primeverosyloxyanthraquinone (6) were isolated from Morinda citrifolia (Rubiaceae) roots together with four known anthraquinone glycosides. The structures of the new compounds were established using spectral methods. For five of the new compounds, the sugar is attached via the hydroxymethyl group of the anthraquinone C-2 carbon. This type of bond is rarely found for anthraquinone glycosides isolated from natural sources.

Preventive effect of Morinda citrifolia fruit juice on neuronal damage induced by focal ischemia.

It is known that the fruit juice of Morinda citrifolia (M. citrifolia, Noni, Rubiaceae) has various pharmacological effects such as antioxidant or anti-inflammatory activities, which may help the inhibition of ischemic neuronal damage. Here, we examined the effect of the fruit juice of M. citrifolia (Noni juice) on the brain damage caused by ischemic stress in mice. Noni juice was obtained from the mature fruit grown in Okinawa (about 1.5 l/4 kg of fruit; 100% Okinawa Noni juice (ONJ). Male ddY mice were supplied with 3% or 10% juice in the drinking water for 7 d, and compared to the control group. On the 7th day, mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). Interestingly, the intake of juice reduced the infarct volume as analyzed by 2,3,5-triphenyltetrazolium chloride (TTC) staining on the 3rd day of MCAO when compared to the control group. Furthermore, we found that the neurological deficit scores (NDS) were decreased after the reperfusion in the juice-supplied mice. On the other hand, the intake of juice did not affect the expression levels of antioxidant such as Cu/Zn superoxide dismutase. The present study suggests that Noni juice may have a preventive effect against cerebral ischemic stress, while further studies are needed to explain the detailed mechanism.