Oleic Acid Activates Mitochondrial Energy Metabolism and Reduces Oxidative Stress Resistance in the Pancreatic ß-Cell Line INS-1.

Elevated concentration of saturated fatty acids in plasma adversely affects pancreatic ß-cells, but the effects of unsaturated fatty acids are controversial. In this study, we examined the effects of oleic acid (OA), a monounsaturated fatty acid, on mitochondrial function, which is important for insulin secretion, using INS-1 cells, a pancreatic ß-cell line derived from rats. Observations of mitochondrial membrane potential and intracellular ATP concentration showed that the electron transport chain was enhanced and ATP production increased in cells treated with OA, indicating that the response that occurs from sensing an increase in glucose concentration to the production of ATP was accelerated. Measurements of intracellular reactive oxygen species (ROS) indicated that the rate of increase in ROS after glucose stimulation was significantly higher in OA-treated cells. The mRNA expression levels of superoxide dismutase 1 and 2, which are responsive to ROS and other substances, were significantly increased in OA 1-d treated cells, but decreased in OA 7-d treated cells. It can be inferred that continued exposure to high concentrations of OA reduced ROS processing capacity and increased intracellular ROS levels. The mRNA expression of apoptosis-inducing enzyme Caspase-3 was significantly increased in OA-treated cells, although its activity was not high. However, the apoptosis induction rate after H2O2 stimulation was significantly higher in OA-treated cells. The high OA environment was shown to promote mitochondrial energy metabolism, leading to an increase in glucose sensitivity and a decrease in oxidative stress resistance.

Effectiveness of integrated bovine leukemia virus eradication strategies utilizing cattle carrying resistant and susceptible major histocompatibility complex class II DRB3 alleles.

Bovine leukemia virus (BLV) has spread worldwide and causes serious problems in the cattle industry owing to the lack of effective treatments and vaccines. Bovine leukemia virus is transmitted via horizontal and vertical infection, and cattle with high BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk, are considered major infectious sources within herds. The PVL strongly correlates with highly polymorphic bovine lymphocyte antigen (BoLA)-DRB3 alleles. The BoLA-DRB3*015:01 and *012:01 alleles are known susceptibility-associated markers related to high PVL, and cattle with susceptible alleles may be at a high risk of BLV transmission via direct contact with healthy cows. In contrast, the BoLA-DRB3*009:02 and *014:01:01 alleles comprise resistant markers associated with the development of low PVL, and cattle with resistant alleles may be low-risk spreaders for BLV transmission and disrupt the BLV transmission chain. However, whether polymorphisms in BoLA-DRB3 are useful for BLV eradication in farms remains unknown. Here, we conducted a validation trial of the integrated BLV eradication strategy to prevent new infection by resistant cattle and actively eliminate susceptible cattle in addition to conventional BLV eradication strategies to maximally reduce the BLV prevalence and PVL using a total of 342 cattle at 4 stall-barn farms in Japan from 2017 to 2019. First, we placed the resistant milking cattle between the BLV-positive and BLV-negative milking cattle in a stall barn for 3 yr. Interestingly, the resistant cattle proved to be an effective biological barrier to successfully block the new BLV infections in the stall-barn system among all 4 farms. Concomitantly, we actively eliminated cattle with high PVL, especially susceptible cattle. Indeed, 39 of the 60 susceptible cattle (65%), 76 of the 140 neutral cattle (54%), and 20 of the 41 resistant cattle (48.8%) were culled on 4 farms for 3 years. Consequently, BLV prevalence and mean PVL decreased in all 4 farms. In particular, one farm achieved BLV-free status in May 2020. By decreasing the number of BLV-positive animals, the revenue-enhancing effect was estimated to be ¥5,839,262 ($39,292.39) for the 4 farms over 3 yr. Our results suggest that an integrated BLV eradication program utilization of resistant cattle as a biological barrier and the preferential elimination of susceptible cattle are useful for BLV infection control.

Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice.

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer's solution (control) or CO (60-125 µM) in Ringer's solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20 min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p = 0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.

Fluorescence Analysis of the Mitochondrial Effect of a Plasmalemmal Na+/Ca2+ Exchanger Inhibitor, SEA0400, in Permeabilized H9c2 Cardiomyocytes.

We investigated the effect on mitochondrial Ca2+ of SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX) which reduces mitochondrial Ca2+ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca2+ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca2+ concentration caused by an increase in extramitochondrial Ca2+ concentration was inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca2+ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.

An autopsy case of death by combined use of benzodiazepines and diphenidine.

We present an autopsy case involving benzodiazepines and diphenidine. Quantitative toxicological analysis showed concentrations of 7-aminoflunitrazepam (a flunitrazepam metabolite), 7-aminonimetazepam (a nimetazepam metabolite), chlorpheniramine and diphenidine in femoral blood of 0.086 µg/ml, 0.027 µg/ml, 0.066 µg/ml, and 0.073 µg/ml, respectively. Death was attributed to combined toxicity due to the influence of multiple drug interactions.

Ethanol and Acetaldehyde After Intraperitoneal Administration to Aldh2-Knockout Mice-Reflection in Blood and Brain Levels.

This paper reports, for the first time, on the analysis of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the blood and brains of Aldh2-knockout (Aldh2-KO) and C57B6/6J (WT) mice. Animals were administrated EtOH (1.0, 2.0 or 4.0 g/kg) or 4-methylpyrazole (4-MP, 82 mg/kg) plus AcH (50, 100 or 200 mg/kg) intraperitoneally. During the blood tests, samples from the orbital sinus of the eye were collected. During the brain tests, dialysates were collected every 5 min (equal to a 15 µl sample) from the striatum using in vivo brain microdialysis. Samples were collected at 5, 10, 15, 20, 25, 30 and 60 min intervals post-EtOH and -AcH injection, and then analyzed by head-space GC. In the EtOH groups, high AcH levels were found in the blood and brains of Aldh2-KO mice, while only small traces of AcH were seen in the blood and brains of WT mice. No significant differences in EtOH levels were observed between the WT and the Aldh2-KO mice for either the EtOH dose. EtOH concentrations in the brain were comparable to the EtOH concentrations in the blood, but the AcH concentrations in the brain were four to five times lower compared to the AcH concentrations in the blood. In the AcH groups, high AcH levels were found in both WT and Aldh2-KO mice. Levels reached a sharp peak at 5 min and then quickly declined for 60 min. Brain AcH concentrations were almost equal to the concentrations found in the blood, where the AcH concentrations were approximately two times higher in the Aldh2-KO mice than in the WT mice, both in the blood and the brain. Our results suggest that systemic EtOH and AcH administration can cause a greater increase in AcH accumulation in the blood and brains of Aldh2-KO mice, where EtOH concentrations in the Aldh2-KO mice were comparable to the EtOH concentrations in the WT mice. Furthermore, detection of EtOH and AcH in the blood and brain was found to be dose-dependent in both genotypes.

Search and removal of radioactive seeds: another application of postmortem computed tomography prior to autopsy.

We herein introduce 3 cases illustrating a new application for pre-autopsy postmortem computed tomography (PMCT). In all 3 cases, there was insufficient background information about the victims provided to the forensic pathologists' department. PMCT showed metallic particles in the prostate gland, an indication of metallic seeds containing radioactive isotopes. In 2 of 3 cases, migrated seeds were also detected by CT imaging in the lungs and the heart. Also in 2 of 3 cases, authorities reinvestigated the victim's history before autopsy was completed, which resulted in following appropriate procedure for dealing with the seeds. Although all 3 cadavers were cremated after autopsy, the International Commission on Radiological Protection (ICRP) discourages cremation for deaths soon after radioactive seeds implantation to prevent air pollution by radioactive isotopes in the ash. Our opinion from the present cases is that pre-autopsy PMCT can be recommended for use by forensic pathologists and guidelines for investigating deaths after permanent brachytherapy should include how to deal with cadavers when medical history is limited.

Yeast Cyc8p and Tup1p proteins function as coactivators for transcription of Stp1/2p-dependent amino acid transporter genes.

The yeast Cyc8p-Tup1p complex is known to serve primarily as a transcriptional corepressor in a variety of biological processes. However, less is known about its function as a coactivator. Herein, we found tryptophan transporter genes, TAT1 and TAT2, that, when overexpressed, suppressed the slow growth of Δcyc8. We observed that the addition of tryptophan to Δcyc8 cultures partially restored cell growth, and the deletion of CYC8 and TUP1 reduced transcriptional levels of TAT1 and TAT2. Tup1p bound to the promoter region of TAT1 and TAT2 genes that were dependent on STP1 and STP2 (encoding DNA-binding activator proteins) for expression. Similarly, transcription of the other Stp1/2p-dependent amino acid transporter (AAT) genes also required CYC8 and TUP1 gene functions. These data indicate that Cyc8p-Tup1p plays a role as a transcriptional coactivator for AAT genes via Stp1/2p activators and that lowering intracellular tryptophan by CYC8 deletion causes slow growth.

Novel Compression Tool to Prevent Hematomas and Skin Erosions After Device Implantation.

BACKGROUND: The incidence of hematoma formation following implantation of a cardiovascular implantable electronic device (CIED) is estimated to be 5% even if a pressure dressing is applied. It is unclear whether a pressure dressing can really compress the pocket in different positions. Furthermore, the adhesive tape for fixing pressure dressings can tear the skin. We developed a new compression tool for preventing hematomas and skin erosions. METHODS AND RESULTS: We divided 46 consecutive patients receiving anticoagulation therapy who underwent CIED implantation into 2 groups (Group I: conventional pressure dressing, Group II: new compression tool). The pressure on the pocket was measured in both the supine and standing positions. The incidence of hematomas was compared between the 2 groups. The pressure differed between the supine and standing positions in Group I, but not in Group II (Group I: 14.8±7.1 mmHg vs. 11.3±9.9 mmHg, P=0.013; Group II: 13.5±2.8 mmHg vs. 13.5±3.5 mmHg, P=0.99). The incidence of hematomas and skin erosions was documented in 2 (8.7%) and 3 (13%) Group I patients, respectively. No complications were documented in Group II. CONCLUSIONS: The new compression tool can provide adequate continuous pressure on the pocket, regardless of body position. This device may reduce the incidence of hematomas and skin erosions after CIED implantation.

Correlation between suicide and meteorological parameters.

OBJECTIVE: The aim of this study was to investigate the link between suicide and meteorological parameters in the 23 wards of Tokyo, Japan. MATERIALS AND METHODS: Monthly data (from January 2008 to December 2012) of suicide stratified by the type of suicide, i.e. hanging, drowning and jumping, were obtained from the Tokyo Medical Examiner's Office official web site. Monthly meteorological parameters (atmospheric pressure, air temperature, humidity and daylight hours) in the 23 wards of Tokyo were also used for the required period. The effects of meteorological parameters on suicide were explored. RESULTS: The number of suicides was 110.4±14.7 (80-149) for men and 55.6±9.1 (41-87) for women in the 23 wards of Tokyo, Japan. The mean air temperature was 16.6°C±7.7 °C (4.8-29.6 °C). The number of suicides by drowning for men was significantly and positively correlated with air temperature, and weakly and positively correlated with humidity. In addition, the number of suicides by drowning for men was significantly and negatively correlated with atmospheric pressure. CONCLUSIONS: The number of suicides by drowning was associated with meteorological parameters, especially in men, in the 23 wards of Tokyo, Japan.

A color test for the convenient identification of an ingested surface activating agent.

Color tests are easy, simple and inexpensive methods for the qualitative identification of chemicals. A color test was applied to the stomach contents of a forensic autopsy case. The result of the test, using bromophenol blue reagent, indicated the ingestion of a commercial cleaning product containing a cationic surface activating agent. Our findings suggest that forensic investigators should consider the additives used in commercial chemical products, such as surface activating agents, when determining the cause of death.

A fatal case of poisoning with ethanol and psychotropic drugs with putrefactive changes.

We present a fatal case involving poisoning with paroxetine, flunitrazepam, and ethanol, with putrefactive changes. Quantitative toxicological analysis showed that the concentrations of paroxetine and 7-aminoflunitrazepam, a metabolite of flunitrazepam, in the femoral blood were 0.28 µg/ml and 0.17 µg/ml, respectively. We also detected an ethanol level of 2.90 mg/ml and an n-propanol level of 0.10 mg/ml. We concluded that the cause of death was due to the interaction of paroxetine, flunitrazepam, and ethanol. The effects of putrefactive changes should be considered during forensic toxicological evaluation.

Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency.

Somatic mosaicism has been described in several primary immunodeficiency diseases and causes modified phenotypes in affected patients. X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the NF-κB essential modulator (NEMO) gene and manifests clinically in various ways. We have previously reported a case of XL-EDA-ID with somatic mosaicism caused by a duplication mutation of the NEMO gene, but the frequency of somatic mosaicism of NEMO and its clinical impact on XL-EDA-ID is not fully understood. In this study, somatic mosaicism of NEMO was evaluated in XL-EDA-ID patients in Japan. Cells expressing wild-type NEMO, most of which were derived from the T-cell lineage, were detected in 9 of 10 XL-EDA-ID patients. These data indicate that the frequency of somatic mosaicism of NEMO is high in XL-ED-ID patients and that the presence of somatic mosaicism of NEMO could have an impact on the diagnosis and treatment of XL-ED-ID patients.

Effect of terfenadine and pentamidine on the HERG channel and its intracellular trafficking: combined analysis with automated voltage clamp and confocal microscopy.

The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

Lower air temperature is associated with ambulance transports and death in Takamatsu area, Japan.

OBJECTIVE: The aim of this study was to investigate the linkage among ambulance transports, the number of death and air temperature in Takamatsu area, Japan. METHODS: Monthly data of ambulance transports (total and acute disease) and the number of death from 2004 to 2012 were obtained from Fire Department Service in Takamatsu and Takamatsu city official website, Japan. Climate parameters for required period were also obtained from Japan Meteorological Agency. Population data in Takamatsu area were also used to adjust ambulance transports and the number of death. The linkage among ambulance transports, the number of death and climate parameters was evaluated by ecological analysis. RESULTS: Total ambulance transports (/a hundred thousand people/day) and ambulance transports due to acute disease (/a hundred thousand people/day) were 12.3 ± 0.9 and 6.8 ± 0.7, respectively. The number of death (/a hundred thousand people/day) was 2.5 ± 0.4. By quadratic curve, ambulance transports due to acute disease and the number of death were significantly correlated with the parameters of air temperature. However, the number of death was the highest in January and the lowest in August. CONCLUSION: Although higher air temperature was only associated with higher ambulance transports, lower air temperature was associated with both higher ambulance transports and the number death in Takamatsu area, Japan.

Barley consumption under a high-fat diet suppresses lipogenic genes through altered intestinal bile acid composition.

We evaluated whether barley flour consumption in a high-fat environment affects lipid metabolism through signals mediated by bile acids. Four-week-old mice were fed a high-fat diet supplemented with cellulose (HC) or ß-glucan-rich barley flour (HB) for 12 weeks. Bile acid composition in the intestinal tract and feces was measured by GC/MS. Gene expression levels involved in bile acid metabolism in the liver and intestinal tract were determined by RT-PCR. Similar parameters were measured in mice treated with antibiotics (antibiotics-cellulose [AC] and antibiotics-barley [AB]) to reduce the activity of intestinal bacteria. The Results showed that the HB group had lower liver blood cholesterol and triglyceride levels than the HC group. The HB group showed a significant decrease in primary bile acids in the gastrointestinal tract compared to the HC group. On the other hand, the concentration of secondary bile acids relatively increased in the cecum and feces. In the liver, Fxr activation suppressed gene expression levels in synthesizing bile acids and lipids. Furthermore, in the gastrointestinal tract, Tgr5 was activated by increased secondary bile acids. Correspondingly, AMP levels were increased in the HB group compared to the HC group, AMPK was phosphorylated in the liver, and gene expression involved in lipid synthesis was downregulated. A comparison of the AC and AB groups treated with antibiotics did not confirm these effects of barley intake. In summary, our results suggest that the prevention of lipid accumulation by barley consumption involves signaling through changes in bile acid composition in the intestinal tract.

Crystal structure of the N-terminal domain of the yeast general corepressor Tup1p and its functional implications.

The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) → Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.

Crystal structure of Rcl1, an essential component of the eukaryal pre-rRNA processosome implicated in 18s rRNA biogenesis.

Rcl1 is an essential nucleolar protein required for U3 snoRNA-guided pre-rRNA processing at sites flanking the 18S rRNA sequence. A potential catalytic role for Rcl1 during pre-rRNA cleavage has been suggested based on its primary structure similarity to RNA 3'-terminal phosphate cyclase (Rtc) enzymes, which perform nucleotidyl transfer and phosphoryl transfer reactions at RNA ends. Here, we report the 2.6 Å crystal structure of a biologically active yeast Rcl1, which illuminates its modular 4-domain architecture and overall homology with RNA cyclases while revealing numerous local differences that account for why Rtcs possess metal-dependent adenylyltransferase activity and Rcls do not. A conserved oxyanion-binding site in Rcl1 was highlighted for possible catalytic or RNA-binding functions. However, the benign effects of mutations in and around the anion site on Rcl1 activity in vivo militate against such a role.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein.

Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4-deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Low-dose nicotine facilitates spatial memory in ApoE-knockout mice in the radial arm maze.

Here, we investigated the effects of nicotine on spatial memory in ApoE-knockout (ApoE-KO) and wild-type (WT) mice in a radial arm maze. Training occurred on three consecutive days and the test was performed on day 4, with one trial per day. Then on day 4, animals were administered nicotine (0.1, 0.25, 0.5, and 1.0 mg/kg) or the antagonist of nicotinic receptors (nAChRs) mecamylamine (MEC 2 mg/kg) alone or together with 0.1 mg/kg nicotine. The number of errors in the first eight choices was recorded. The results were that 0.1 mg/kg nicotine decreased errors in ApoE-KO mice, while 0.1 and 0.25 mg/kg nicotine reduced errors in WT mice, indicating that lower doses of nicotine elicit a memory improvement. In contrast, 1.0 mg/kg nicotine increased errors in WT mice, but not in ApoE-KO mice. MEC alone had no noticeable effect on errors in either strain of mice. However, co-administration of 0.1 mg/kg nicotine and MEC increased errors and reduced the effects of nicotine in WT mice, but not in ApoE-KO mice. Our study found a biphasic effect of nicotine in WT mice: it improves spatial memory at lower doses and impairs it at a higher dose. In ApoE-KO mice, nicotine improves memory at a low dose and has no effect at a higher dose, suggesting that the ApoE deficiency may influence the efficacy of nicotine. Moreover, a reversal of nicotinic effects with MEC was seen in WT mice, indicating the likelihood of the involvement of nAChRs in the spatial-memory response to nicotine.