Development of an Analytical Method for Unsaturated Disaccharides from Heparan Sulfate Using Dansylhydrazine Derivatization and High-Performance Liquid Chromatography with Fluorescence Detection.

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play essential roles in living organisms. Understanding the functionality of HS and its involvement in disease progression necessitates the sensitive and quantitative detection of HS-derived unsaturated disaccharides. Conventionally, fluorescence derivatization precedes the HPLC analysis of these disaccharides. However, the presence of excess unreacted derivatization reagents can inhibit rapid and sensitive analysis in chromatographic determinations. In this study, we describe analytical methods that use dansylhydrazine as a derivatization agent for the detection and determination of HS-derived unsaturated disaccharides using HPLC. In addition, we have developed a straightforward method for removing excess unreacted reagent using a MonoSpin NH2 column. This method may be employed to remove excess pre-labeling reagents, thereby facilitating the analysis of HS-derived unsaturated disaccharides with satisfactory reproducibility.

The role of nutrition and oxidative stress as aging factors in Caenorhabditis elegans.

The molecular mechanism of aging, which has been a "black box" for many years, has been elucidated in recent years, and the nematode C. elegans, which is a model animal for aging research, has played a major role in its elucidation. From the analysis of C. elegans longevity-related mutant genes, many signal transduction systems, with the insulin/insulin-like growth factor signal transduction system at the core, have emerged. It has become clear that this signal transduction system is greatly affected by external nutrients and is involved in the downstream regulation of oxidative stress, which is considered to be one of the main causes of aging.

Mitochondrial Electron Transport Chain Complex II Dysfunction Causes Premature Aging of Hematopoietic Stem Cells.

Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.

Sex difference in clinical presentation of patients with infectious mononucleosis caused by Epstein-Barr virus.

INTRODUCTION: There are few studies on sex difference in patients with infectious mononucleosis caused by Epstein-Barr virus (EBV-IM). We performed a retrospective study to evaluate the sex difference in clinical presentation of patients with EBV-IM. METHODS: We performed a single-center retrospective study evaluating >14-year-old patients with serologically confirmed EBV-IM during 2006-2017. We compared the patients' age, symptoms, physical findings, and laboratory data between male and female patients. To adjust for confounding factors, we performed a logistic regression analysis based on the results of univariate comparisons. RESULT: Of the 122 eligible patients (56 male and 66 female, ratio: 1:1.2), the median ages were 26 years old (interquartile range [IR], 22-31.5 years old]) and 22 years old (IR, 20-25 years old) for males and females, respectively (p < 0.001). Headache was significantly more prevalent in males (25.0% vs. 10.6%, p = 0.036). Leukocyte count was also significantly higher in males (11,400/mm3 [IR, 7,600-14,100/mm3] vs. 9,400/mm3 [IR, 6,600-11,600/mm3], p = 0.021). The prevalence of periorbital edema (male: 3.6% vs. female: 18.1%, p = 0.012) and severity of transaminase elevation were significantly higher in females. The regression analysis evaluating clinical characteristics of male patients showed that age >30 years old, headache, and leukocyte >11,000/mm3 had high odds ratios. CONCLUSION: Our single-center retrospective study suggests that older age of onset, headache, and leukocytosis are more likely to be characteristics of male patients with EBV-IM. Our study also underscores the importance of periorbital edema as a clue for early diagnosis of EBV-IM, especially in female patients.

A Study of Morphological Changes in Renal Afferent Arterioles Induced by Angiotensin II Type 1 Receptor Blockers in Hypertensive Patients.

BACKGROUND: Renin-angiotensin-aldosterone system blockers are known to reduce hypertrophy of vascular smooth muscle cells (SMCs) in hypertensive cases. However, we have reported marked proliferative changes of renal afferent arteriolar SMCs in rats induced by a long-term administration of angiotensin II type 1 receptor blockers (ARBs) and an angiotensin-converting enzyme inhibitor (ACEI). In this study, we examined the morphological changes of afferent arteriolar walls in human kidneys with or without ARBs/ACEIs. METHODS: Forty-four wedge resections were taken from patients aged 45-74 years from 92 nephrectomized kidneys due to malignancy at Toho University Omori Medical Center between 2013 and 2016. They were divided into the following three groups: 18 hypertensive patients treated with antihypertensive agents including ARBs or ACEIs (the HTARB group), 6 hypertensive patients treated with calcium channel blockers without ARBs/ACEIs (the HTCCB group), and 20 normotensive patients (the normotensive group) as a control. Cases expecting vascular changes such as diabetes were excluded. In each case renal arterioles were measured as the ratio of inner/outer arteriolar diameter, and pathologists estimated morphological abnormal changes, scoring each specimen independently. RESULTS: The ratio in the HTARB group was 0.39 ± 0.05 (mean ± SD), and was significantly the lowest among the three groups (0.46 ± 0.02 in the HTCCB, 0.53 ± 0.02 in the normotensive group; p = 0.0107 vs. HTCCB, p = 0.00001 vs. normotensive). The ratio in the three groups significantly correlated with the estimated glomerular filtration rate (r = 0.4915, p < 0.0007). The afferent arteriolar SMCs in the HTARB group frequently showed marked proliferative and irregular changes. The score of SMC abnormalities estimated regarding the proliferation, irregularity of the arrangement, and size in hilar afferent arteriolar SMCs was highest in the HTARB group and showed statistical significance (p = 0.0088, p = 0.00001, and p = 0.025 versus other two groups). CONCLUSIONS: We consider that these morphological changes in arterioles are induced by ARBs/ACEIs. These changes could induce an important suppression of glomerular hyperfiltration and could lead to glomerular ischemia. However, the clinical consequences of these morphological changes in correlation with ARBs/ACEIs were not sufficiently clear and require further analysis. We should consider renal arteriolar morphological changes when using ARBs/ACEIs.

Clinical differentiation of acute appendicitis and right colonic diverticulitis: A case-control study.

BACKGROUND: Acute right colonic diverticulitis (ARCD) is an important differential diagnosis of acute appendicitis (AA) in Asian countries because of the unusually high prevalence of right colonic diverticula. Due to qualitative improvement and the high penetration rate of computed tomography (CT) scanning in Japan, differentiation of ARCD and AA mainly depends on this modality. But cost, limited availability, and concern for radiation exposure make CT scanning problematic. Differential findings of ARCD from AA are based on several small studies that used univariate comparisons from Korea and Taiwan. Previous studies on clinical and laboratory differences between AA and ARCD are limited. AIM: To determine clinical differences between AA and ARCD for differentiation of these two diagnoses by creating a logistic regression model. METHODS: We performed an exploratory single-center retrospective case-control study evaluating 369 Japanese patients (age ≥ 16 years), 236 (64.0%) with AA and 133 (36.0%) with ARCD, who were hospitalized between 2012 and 2016. Diagnoses were confirmed by CT images. We compared age, sex, onset-to-visit interval, epigastric/periumbilical pain, right lower quadrant (RLQ) pain, nausea/vomiting, diarrhea, anorexia, medical history, body temperature, blood pressure, heart rate, RLQ tenderness, peritoneal signs, leukocyte count, and levels of serum creatinine, serum C-reactive protein (CRP), and serum alanine aminotrans-ferase. We subsequently performed logistic regression analysis for differentiating AA from ARCD based on the results of the univariate analyses. RESULTS: In the AA and ARCD groups, median ages were 35.5 and 41.0 years, respectively (p=0.011); median onset-to-visit intervals were 1 [interquartile range (IQR): 0-1] and 2 (IQR: 1-3) days, respectively (P < 0.001); median leukocyte counts were 12600 and 11500/mm3, respectively (P = 0.002); and median CRP levels were 1.1 (IQR: 0.2-4.1) and 4.9 (IQR: 2.9-8.5) mg/dL, respectively (P < 0.001). In the logistic regression model, odds ratios (ORs) were significantly high in nausea/vomiting (OR: 3.89, 95%CI: 2.04-7.42) and anorexia (OR: 2.13, 95%CI: 1.06-4.28). ORs were significantly lower with a longer onset-to-visit interval (OR: 0.84, 95%CI: 0.72-0.97), RLQ pain (OR: 0.28, 95%CI: 0.11-0.71), history of diverticulitis (OR: 0.034, 95%CI: 0.005-0.20), and CRP level > 3.0 mg/dL (OR: 0.25, 95%CI: 0.14-0.43). The regression model showed good calibration, discrimination, and optimism. CONCLUSION: Clinical findings can differentiate AA and ARCD before imaging studies; nausea/vomiting and anorexia suggest AA, and longer onset-to-visit interval, RLQ pain, previous diverticulitis, and CRP level > 3.0 mg/dL suggest ARCD.

Metabolic Biomarkers in Nematode C. elegans During Aging.

Changes in energy metabolism occur not only in diseases such as cancer but also in the normal development and aging processes of various organisms. These metabolic changes result to lead to imbalances in energy metabolism related to cellular and tissue homeostasis. In the model organism C. elegans, which is used to study aging, an imbalance in age-related energy metabolism exists between mitochondrial oxidative phosphorylation and aerobic glycolysis. Cellular lactate and pyruvate are key intermediates in intracellular energy metabolic pathways and can indicate age-related imbalances in energy metabolism. Thus, the cellular lactate/pyruvate ratio can be monitored as a biomarker during aging. Moreover, recent studies have proposed a candidate novel biomarker for aging and age-related declines in the nematode C. elegans.

Clinical differentiation of infectious mononucleosis that is caused by Epstein-Barr virus or cytomegalovirus: A single-center case-control study in Japan.

INTRODUCTION: Infectious mononucleosis (IM) is a common viral infection that typically causes fever, pharyngitis, and lymphadenopathy in young patients. The Epstein-Barr virus (EBV) is the most common cause of IM, followed by cytomegalovirus (CMV). Given that serological testing is associated with limitations regarding its accuracy, availability, and time to receive results, clinical differentiation based on symptoms, signs, and basic tests would be useful. We evaluated whether clinical findings could be used to differentiate EBV-IM from CMV-IM. METHODS: In this single-center retrospective case-control study, we evaluated >14-year-old patients with serologically confirmed EBV-IM or CMV-IM during 2006-2017. We compared the patients' symptoms, physical findings, blood counts, and serum biomarkers to create three regression models: model 1 (symptoms and signs), model 2 (model 1 plus sonographic hepatosplenomegaly and blood counts), and model 3 (model 2 plus hepatobiliary biomarkers). RESULTS: Among the 122 patients (72.6%) with EBV-IM and 46 patients (27.4%) with CMV-IM, the median age was 25 years and 82 patients (48.8%) were male. The median age was 10 years older in the CMV-IM group (p < 0.001) and the median interval from onset to visit was 5 days longer in the CMV-IM group (p < 0.001). Logistic regression revealed that EBV-IM was predicted by younger age, short onset-to-visit interval, lymphadenopathy, tonsillar white coat, hepatosplenomegaly, atypical lymphocytosis, and elevations of lactate dehydrogenase and gamma-glutamyl transferase. All regression models had areas under the curve of >0.9. CONCLUSION: History and physical findings, especially when used with atypical lymphocytosis and sonographic hepatosplenomegaly, can help physicians differentiate EBV-IM from CMV-IM.

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain.

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle-aged Tet-mev-1 mice with the SDHCV69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle-aged Tet-mev-1 mice overproduced MitoSOX Red-detectable mitochondrial ROS compared to age-matched wild-type C57BL/6J mice, only young adult Tet-mev-1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn- and Cu/Zn-SODs) activities to eliminate the MitoSOX Red-detectable mitochondrial ROS. In contrast, middle-aged Tet-mev-1 mice accumulated both MitoSOX Red-detectable mitochondrial ROS and CM-H2 DCFDA-detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle-aged Tet-mev-1 mice relative to age-matched wild-type C57BL/6J mice. Furthermore, only middle-aged Tet-mev-1 mice showed JNK/SAPK activation and Ca2+ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100ß in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age-dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.

Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models.

Oxidative stress is associated with some forms of both male and female infertility. However, there is insufficient knowledge of the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. There are a number of animal models for understanding age-dependent decrease of reproductive ability and diabetic embryopathy, especially abnormal spermatogenesis, oogenesis and embryogenesis with mitochondrial dysfunctions. Several important processes occur in mitochondria, including ATP synthesis, calcium ion storage, induction of apoptosis and production of reactive oxygen species (ROS). These events have different effects on the several aspects of reproductive function. Tet-mev-1 conditional transgenic mice, developed after studies with the mev-1 mutant of the nematode C. elegans, offer the ability to carefully regulate expression of doxycycline-induced mutated SDHC(V69E) levels and hence modulate endogenous oxidative stress. The mev-1 models have served to illuminate the effects of complex II deficiency-dependent mitochondrial ROS production, although interestingly they maintain normal mitochondrial and intracellular ATP levels. In this review, the reproductive dysfunctions are presented focusing on fertility potentials in each gamete, early embryogenesis, maternal conditions with placental function and neonatal development.

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice.

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.

Alteration of Breath Hydrogen and Methane in Ethanol-Fed Rats.

Chronic alcohol consumption can cause dysbiosis, but it is difficult to determine the effect of alcohol on the structure and activity of gastrointestinal tract microbiota. We therefore designed a noninvasive hydrogen breath test (HBT) to investigate the alteration in the chemical profile of gut microbiota in ethanol-fed rats. Thirteen F344/DuCri rats were fed on a commercial mash food with 16% ethanol solution drinking fluid from 4 weeks of age. HBTs were carried out on six 8-week-old and seven 24-week-old ethanol-fed rats. As controls, HBTs were carried out on sixteen 8-week-old, six 24-week-old, and five 48-week-old male rats. Six 24-week- old male rats were examined twice at 1-week intervals. HBTs were performed after fasting for 24 hr. Rats were orally administrated 4 mL/kg of 65% lactulose solution and housed in an animal chamber. The expired air was collected in a breath-sampling bag at 10-min intervals for 180 min. The hydrogen (H2) and methane (CH4) levels in the breath sample were measured using a breath analyzer and were expressed.as parts- per million (ppm). Elevated H2 and CH4 levels were more frequent in male rats. Maximal values of H2 and CH4 were highest in 8-week- old rats, followed by 48-week-old and 24-week-old rats. No ethanol-fed rat exhaled more than 2 ppm of H2 or CH4 until 180 min after the oral administration of lactulose, unlike the controls. This alteration was more obvious than that of aging or gender differentiation. We conclude that there is a close association between chronic ethanol consumption and H2 and CH4 production. An asymptomatic heavy drinker might have dysbiosis that involves gut microbiota with lower fermentation performance.

Oxidative stress tolerance of early stage diabetic endothelial progenitor cell.

INTRODUCTION: One of the causes for poor vasculogenesis of diabetes mellitus (DM) is known to rise from the dysfunction of bone marrow-derived endothelial progenitor cells (BM EPCs). However, the origin of its cause is less understood. We aimed to investigate the effect of oxidative stress in early stage of diabetic BM-EPC and whether its vasculogenic dysfunction is caused by oxidative stress. METHODS: Bone marrow c-Kit+Sca-1+Lin- (BM-KSL) cells were sorted from control and streptozotocin-induced diabetic C57BL6J mice by flow cytometry. BM-KSLs were then assessed for vasculogenic potential (colony forming assay; EPC-CFA), accumulation of intracellular ROS (CM-H2DCFDA), carbonylated protein (ELISA), anti-oxidative enzymes expression (RT-qPCR) and catalase activity (Amplex Red). RESULTS: Compared to control, DM BM-KSL had significantly lower EPC-CFUs in both definitive EPC-CFU and total EPC-CFU (p < 0.05). Interestingly, the oxidative stress level of DM BM-KSL was comparable and was not significantly different to control followed by increased in anti-oxidative enzymes expression and catalase activity. CONCLUSIONS: Primitive BM-EPCs showed vasculogenic dysfunction in early diabetes. However the oxidative stress is not denoted as the major initiating factor of its cause. Our results suggest that primitive BM-KSL cell has the ability to compensate oxidative stress levels in early diabetes by increasing the expression of anti-oxidative enzymes.

Genetically induced oxidative stress in mice causes thrombocytosis, splenomegaly and placental angiodysplasia that leads to recurrent abortion.

Historical data in the 1950s suggests that 7%, 11%, 33%, and 87% of couples were infertile by ages 30, 35, 40 and 45, respectively. Up to 22.3% of infertile couples have unexplained infertility. Oxidative stress is associated with male and female infertility. However, there is insufficient evidence relating to the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. Recently, we have established Tet-mev-1 conditional transgenic mice, which can express the doxycycline-induced mutant SDHC(V69E) transgene and experience mitochondrial respiratory chain dysfunction leading to intracellular oxidative stress. In this report, we demonstrate that this kind of abnormal mitochondrial respiratory chain-induced chronic oxidative stress affects fertility, pregnancy and delivery rates as well as causes recurrent abortions, occasionally resulting in maternal death. Despite this, spermatogenesis and early embryogenesis are completely normal, indicating the mutation's effects to be rather subtle. Female Tet-mev-1 mice exhibit thrombocytosis and splenomegaly in both non-pregnant and pregnant mice as well as placental angiodysplasia with reduced Flt-1 protein leading to hypoxic conditions, which could contribute to placental inflammation and fetal abnormal angiogenesis. Collectively these data strongly suggest that chronic oxidative stress caused by mitochondrial mutations provokes spontaneous abortions and recurrent miscarriage resulting in age-related female infertility.

Predicting longevity in C. elegans: fertility, mobility and gene expression.

Expression level of an hsp-16.2::gfp transgene is a predictor of longevity in Caenorhabditis elegans. Here we examine fertility, movement and longevity, comparing high-expressing ("bright") and low-expressing ("dim") animals. There was no differential fertility between bright and dim individuals, suggesting that dim worms were not excessively frail. Worms with high hsp-16.2::gfp expression had improved mobility, consistent with improved health span. We predicted that the increased longevity of the bright worms would be associated with increased expression of protective genes such as those shown to be upregulated in Age mutants. However, few genes were differentially transcribed, although internal controls (hsp-16.2 and family members) were differentially expressed. Quite surprising was the observation that expression level of the transgenic reporter was inherited by the progeny: in seven experiments bright worms consistently produced progeny that were brighter. We tested and ruled out possible artifacts such as differential copy-number of the transgene as an explanation of this differential brightness. These results suggest that a robust physiological state does not depend heavily upon transcriptional differences for its establishment, consistent with proteostatic mechanisms underlying the differential longevity.

Model animals for the study of oxidative stress from complex II.

Mitochondria play a role of energy production and produce intracellular reactive oxygen species (ROS), especially superoxide anion (O2(-)) as a byproduct of energy metabolism at the same time. O2(-) is converted from oxygen and is overproduced by excessive electron leakage from the mitochondrial respiratory chain. It is well known that mitochondrial complexes I and III in the electron transport system are the major endogenous ROS sources. We have previously demonstrated that mutations in complex II can result in excessive ROS (specifically in SDHC: G71E in Caenorhabditis elegans, I71E in Drosophila and V69E in mouse). Moreover, this results in premature death in C. elegans and Drosophila as well as tumorigenesis in mouse embryonic fibroblast cells. In humans, it has been reported that mutations in SDHB, SDHC or SDHD, which are the subunits of mitochondrial complex II, often result in inherited head and neck paragangliomas (PGLs). Recently, we established Tet-mev-1 conditional transgenic mice using our uniquely developed Tet-On/Off system, which can induce the mutated SDHC gene to be equally and competitively expressed compared to the endogenous wild-type SDHC gene. These mice experienced mitochondrial respiratory chain dysfunction that resulted in oxidative stress. The mitochondrial oxidative stress caused excessive apoptosis in several tissues leading to low-birth-weight infants and growth retardation during neonatal developmental phase in Tet-mev-1 mice. Tet-mev-1 mice also displayed precocious age-dependent corneal physiological changes, delayed corneal epithelialization, decreased corneal endothelial cells, thickened Descemet's membrane and thinning of parenchyma with corneal pathological dysfunctions such as keratitis, Fuchs' corneal dystrophy (FCD) and probably keratoconus after the normal development and growth phase. Here, we review the relationships between mitochondrial oxidative stress and phenomena in mev-1 animal models with mitochondrial complex II SDHC mutations. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

Oxidative stress induced inflammation initiates functional decline of tear production.

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b(560) large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease.

A new mouse model of dry eye disease: oxidative stress affects functional decline in the lacrimal gland.

PURPOSE: Oxidative damage and inflammation are proposed to be involved in the age-related functional decline of lacrimal glands. The molecular mechanism(s) of how oxidative stress affects the secretory function of lacrimal glands was investigated because this is currently unclear. METHODS: We used a novel mev-1 conditional transgenic mouse model (Tet-mev-1) with a modified tetracycline system. The mev-1 gene encodes the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria. RESULTS: Expression of the mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in aqueous secretory function. Tear volume in Tet-mev-1 mice was lower than in wild-type mice, and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. CONCLUSIONS: This new model provides evidence that mitochondria-induced oxidative damage in the lacrimal gland induces lacrimal dysfunction, resulting in dry eye disease. Our findings strongly suggest that oxidative stress can be a causative factor in the development of dry eye disease.

Mitochondrial superoxide anion overproduction in Tet-mev-1 transgenic mice accelerates age-dependent corneal cell dysfunctions.

PURPOSE: The Tet-mev-1 mouse expressing a mitochondrial complex-II mutated SDHC(V69E) gene controlled by a tetracycline (Tet)-On/Off system can overproduce O(2)(·-) and is a versatile whole-animal model for studying mitochondrial oxidative stress. Here we report a series of age-dependent variations in corneal epithelium, endothelium, and parenchymal cells of the Tet-mev-1 mice relative to wild-type C57BL/6j mice. METHODS: Measurements of (1) mitochondrial electron transport enzyme activities; (2) O(2)(·-) production; (3) carbonylated protein, and 8-hydroxydeoxyguanosine (8-OHdG) levels as markers of oxidative stress; (4) pathologic analyses under optical and electron microscopy; (5) hematoxylin-eosin or toluidine-blue staining; and (6) immunohistochemistry with an anti-ß-catenin antibody were performed in the eye, especially the cornea. RESULTS: Complex II-III activity was decreased by electron leakage between complex II and CoQ. This resulted in increased age-dependent intracellular oxidative stress in the eye of Tet-mev-1 mice. Corneal epithelialization was delayed in Tet-mev-1 mice after 20% ethanol treatment, as the number of cells and mitotic cells decreased in the corneal epithelium of Tet-mev-1 mice compared with that of wild type. The age-dependent decrease in cell number accelerated in the corneal endothelium cells. Moreover, it was suggested that the corneal thickness was decreased by thinning of parenchymal cells with age in Tet-mev-1 mice. CONCLUSIONS: These results suggest that mitochondrial oxidative stress with electron transport chain dysfunction can influence pathogenesis and progression of age-related corneal diseases, as well as generalized corneal aging acceleration.