Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy.

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

A case series of Fabry diseases with CKD in Japan.

BACKGROUND: It is well known that kidney injury is vital organ damage in Fabry disease (FD). Renin-angiotensin system (RAS) inhibitors are known to reduce proteinuria in patients with chronic kidney disease (CKD) by dilating the glomerular export arteries and reducing intraglomerular pressure. This improvement in intraglomerular pressure, although lowering the glomerular filtration rate, is thought to prevent renal damage and be renoprotective in the long term. RAS inhibitors may be effective in FD patients with proteinuria to prevent the progression of kidney disease, however, the degree to which they are used in clinical practice is unknown. METHODS: The J-CKD-DB-Ex is a comprehensive multicenter database that automatically extracts medical data on CKD patients. J-CKD-DB-Ex contains data on 187,398 patients in five medical centers. FD patients were identified by ICD-10. Clinical data and prescriptions of FD patients between January 1 of 2014, and December 31 of 2020 were used for the analysis. RESULTS: We identified 39 patients with FD from the J-CKD-DB-Ex including those with suspected FD. We confirmed 22 patients as FD. Half of the patients received RAS inhibitors. RAS inhibitors tended to be used in CKD patients with more severe renal impairment. CONCLUSIONS: This case series revealed the actual clinical practice of FD patients with CKD. In particular, we found cases in which patients had proteinuria, but were not treated with RAS inhibitors. The database was shown to be useful in assessing the clinical patterns of patients with rare diseases.

HOPS, CORVET and newly-identified Hybrid tethering complexes contribute differentially towards multiple modes of endocytosis.

Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic fusion and vacuole protein sorting (HOPS) and class C core endosomal vacuole tethering (CORVET) interact with Rab-GTPases and SNARE proteins to regulate vesicle transport, fusion, and maturation in autophagy and endocytosis pathways. In this study, we identified two novel "Hybrid" tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced with the subunit from the other. Substrates taken up by receptor-mediated endocytosis or pinocytosis were transported by distinctive pathways, and the newly identified hybrid complexes contributed to pinocytosis in the presence of HOPS, whereas receptor-mediated endocytosis was exclusively dependent on HOPS. Our study provides new insights into the molecular mechanisms of the endocytic pathway and the function of the vacuolar protein sorting-associated (VPS) protein family.

A case of Marfanoid-progeroid-lipodystrophy syndrome: experimental proof of skipping exons and escaping nonsense-mediated decay.

We report a Japanese patient with tall stature, dolichocephaly, prominent forehead, narrow nasal ridge, mild retrognathia, subcutaneous fat reduction, bilateral entropion of both eyelids, high arched palate, long fingers, and mild hyperextensible finger joints as a case of Marfanoid-progeroid-lipodystrophy syndrome. Genetic investigation revealed a heterozygous variant NC_000015.10(NM_000138.5):c.8226+5G>A in the FBN1 gene. Skipping of exon 65 and escaping nonsense-mediated decay followed by frameshift were experimentally confirmed in the proband's mRNA.

Prenatal Diagnosis of Mucopolysaccharidosis-Plus Syndrome (MPSPS).

Mucopolysaccharidosis-plus syndrome (MPSPS) is an autosomal-recessive disorder caused by c.1492C>T (p.R498W) in the VPS33A gene. MPSPS is a severe disorder that causes a short lifespan in patients. Currently, there is no specific treatment for patients. The Yakut population is more prone to this disease than others. Diagnosing MPSPS relies on clinical manifestations, and genetic testing (GT) is used to confirm the diagnosis. In this research, we examined two pregnancy cases, one of which involved a prenatal diagnosis for MPSPS. Notably, neither pregnant woman had a known family history of the disorder. During their pregnancies, both women underwent prenatal ultrasonography, which revealed increased prenasal thickness during the second trimester. In the first case, ultrasonography indicated increased prenasal thickness in the second trimester, but a definitive diagnosis was not made at that time. The patient was eventually diagnosed with MPSPS at 11 months of age. On the contrary, in the second case, GT uncovered that the parents were carriers of MPSPS. Consequently, a placental biopsy was performed, leading to an early diagnosis of MPSPS. This study emphasizes the importance of ultrasonography findings in prenatal MPSPS diagnosis. Combining ultrasonography with GT can be a valuable approach to confirming MPSPS at an early stage, allowing for the appropriate planning of delivery methods and medical care. Ultimately, this comprehensive approach can significantly enhance the quality of life of both affected patients and their parents.

Negative pressure induces dedifferentiation of hepatocytes via RhoA/ROCK pathway.

Biomechanical forces are known to regulate the biological behaviors of cells. Although negative pressure has been used for wound healing, it is still unknown about its role in regulating cell plasticity. We investigated whether negative pressure could induce the dedifferentiation of hepatocytes. Using a commercial device, we found that the exposure of primary human hepatocytes to -50 mmHg quickly induced the formation of stress fibers and obviously changed cell morphology in 72 h. Moreover, the exposure of hepatocytes to -50 mmHg significantly upregulated RhoA, ROCK1, and ROCK2 in 1-6 h, and dramatically enhanced the expression of marker molecules on "stemness", such as OCT4, SOX2, KLF4, MYC, NANOG, and CD133 in 6-72 h. However, all these changes in hepatocytes induced by -50 mmHg stimulation were almost abrogated by ROCK inhibitor Y27623. Our data suggest that an appropriate force of negative pressure stimulation can effectively induce the dedifferentiation of hepatocytes via RhoA/ROCK pathway activation.

A Case of Mucopolysaccharidosis II Caused by a Novel Variant with Skin Linear Hyperpigmented Streaks along Blaschko's Lines.

We report a case of an eight-year-old boy with mucopolysaccharidosis (MPS) II with atypical skin lesions of hyperpigmented streaks along Blaschko's lines. This case presented with mild symptoms of MPS such as hepatosplenomegaly, joint stiffness, and quite mild bone deformity, which was the reason for the delay in diagnosis until the age of seven years. However, he showed an intellectual disability that did not meet the diagnostic criteria for an attenuated form of MPS II. Iduronate 2-sulfatase activity was reduced. Clinical exome sequencing of DNA from peripheral blood revealed a novel pathogenic missense variant (NM_000202.8(IDS_v001):c.703C>A, p.(Pro235Thr)) in the IDS gene, which was confirmed in the mother with a heterozygous state. His brownish skin lesions differed from the Mongolian blue spots or "pebbling" of the skin that are observed in MPS II.

SPRTN and TDP1/TDP2 Independently Suppress 5-Aza-2'-deoxycytidine-Induced Genomic Instability in Human TK6 Cell Line.

DNA-protein cross-links (DPCs) are generated by internal factors such as cellular aldehydes that are generated during normal metabolism and external factors such as environmental mutagens. A nucleoside analog, 5-aza-2'-deoxycytidine (5-azadC), is randomly incorporated into the genome during DNA replication and binds DNA methyltransferase 1 (DNMT1) covalently to form DNMT1-DPCs without inducing DNA strand breaks. Despite the recent progress in understanding the mechanisms of DPCs repair, how DNMT1-DPCs are repaired is unclear. The metalloprotease SPRTN has been considered as the primary enzyme to degrade protein components of DPCs to initiate the repair of DPCs. In this study, we showed that SPRTN-deficient (SPRTN-/-) human TK6 cells displayed high sensitivity to 5-azadC, and the removal of 5-azadC-induced DNMT1-DPCs was significantly slower in SPRTN-/- cells than that in wild-type cells. We also showed that the ubiquitination-dependent proteasomal degradation, which was independent of the SPRTN-mediated processing, was also involved in the repair of DNMT1-DPCs. Unexpectedly, we found that cells that are double deficient in tyrosyl DNA phosphodiesterase 1 and 2 (TDP1-/-TDP2-/-) were also sensitive to 5-azadC, although the removal of 5-azadC-induced DNMT1-DPCs was not compromised significantly. Furthermore, the 5-azadC treatment induced a marked accumulation of chromosomal breaks in SPRTN-/- as well as TDP1-/-TDP2-/- cells compared to wild-type cells, strongly suggesting that the 5-azadC-induced cell death was attributed to chromosomal DNMT1-DPCs. We conclude that SPRTN protects cells from 5-azadC-induced DNMT1-DPCs, and SPRTN may play a direct proteolytic role against DNMT1-DPCs and TDP1/TDP2 also contributes to suppress genome instability caused by 5-azadC in TK6 cells.

A novel nonsense variant in ARID1B causing simultaneous RNA decay and exon skipping is associated with Coffin-Siris syndrome.

Coffin-Siris syndrome (CSS) is a congenital disorder that is characterized by an absent/hypoplastic fifth distal phalanx, psychomotor developmental delay, and coarse facial features. One of the causative genes, ARID1B (AT-rich interactive domain-containing protein 1B), encodes components of the BAF chromatin remodeling complexes. Here, we report a case of a 3-year 8-month-old male with a novel nonsense variant (NM_001374820.1:c.4282C > T, p.(Gln1428*)) in the ARID1B gene, which was identified with whole-exome sequencing. He showed clinical symptoms of cleft soft palate, distinctive facial features (flat nasal bridge, thick eyebrows, and long eyelashes), right cryptorchidism, and hypertrichosis that partially overlapped with CSS. One of the most characteristic features of CSS is absent/hypoplastic fifth distal phalanx. He showed no obvious clinical finding in the lengths of his fingers or in the formation of his fingernails. However, radiographic analyses of the metacarpophalangeal bones revealed shortening of all the distal phalanges and fifth middle phalanges, suggesting brachydactyly. We performed mRNA analyses and revealed that both nonsense-mediated decay and nonsense-associated altered splicing were simultaneously caused by the c.4282C > T nonsense variant. The proband's clinical manifestations fit the previously reported criteria of disease for CSS or intellectual disability with ARID1B variant. Altogether, we suggest that c.4282C > T is a pathogenic variant that causes this clinical phenotype.

Hematopoietic Disorders, Renal Impairment and Growth in Mucopolysaccharidosis-Plus Syndrome.

Mucopolysaccharidoses (MPS) are rare lysosomal storage disorders (LSD) characterized by the excessive accumulation of glycosaminoglycans (GAG). Conventional MPS, caused by inborn deficiencies of lysosomal enzymes involved in GAG degradation, display various multisystemic symptoms-including progressive neurological complications, ophthalmological disorders, hearing loss, gastrointestinal and hepatobiliary issues, cardiorespiratory problems, bone and joint abnormalities, dwarfism, and coarse facial features. Mucopolysaccharidosis-Plus Syndrome (MPSPS), an autosomal recessive disease caused by a mutation in the endo-lysosomal tethering protein VPS33A, shows additional renal and hematopoietic abnormalities ("Plus symptoms") uncommon in conventional MPS. Here, we analyze data from biochemical, histological, and physical examinations-particularly of blood counts and kidney function-to further characterize the clinical phenotype of MPSPS. A series of blood tests indicate hematopoietic symptoms including progressive anemia and thrombocytopenia, which correlate with histological observations of hypoplastic bone marrow. High urinary excretion of protein (caused by impairments in renal filtration), hypoalbuminemia, and elevated levels of creatinine, cholesterol, and uric acid indicate renal dysfunction. Histological analyses of MPSPS kidneys similarly suggest the extensive destruction of glomerular structures by foamy podocytes. Height and weight did not significantly deviate from the average, but in some cases, growth began to decline at around six months or one year of age.

High-Dose Irradiation Inhibits Motility and Induces Autophagy in Caenorhabditis elegans.

Radiation damages many cellular components and disrupts cellular functions, and was previously reported to impair locomotion in the model organism Caenorhabditis elegans. However, the response to even higher doses is not clear. First, to investigate the effects of high-dose radiation on the locomotion of C. elegans, we investigated the dose range that reduces whole-body locomotion or leads to death. Irradiation was performed in the range of 0-6 kGy. In the crawling analysis, motility decreased after irradiation in a dose-dependent manner. Exposure to 6 kGy of radiation affected crawling on agar immediately and caused the complete loss of motility. Both γ-rays and carbon-ion beams significantly reduced crawling motility at 3 kGy. Next, swimming in buffer was measured as a motility index to assess the response over time after irradiation and motility similarly decreased. However, swimming partially recovered 6 h after irradiation with 3 kGy of γ-rays. To examine the possibility of a recovery mechanism, in situ GFP reporter assay of the autophagy-related gene lgg-1 was performed. The fluorescence intensity was stronger in the anterior half of the body 7 h after irradiation with 3 kGy of γ-rays. GFP::LGG-1 induction was observed in the pharynx, neurons along the body, and the intestine. Furthermore, worms were exposed to region-specific radiation with carbon-ion microbeams and the trajectory of crawling was measured by image processing. Motility was lower after anterior-half body irradiation than after posterior-half body irradiation. This further supported that the anterior half of the body is important in the locomotory response to radiation.

PRDX1 is essential for the viability and maintenance of reactive oxygen species in chicken DT40.

BACKGROUND: Peroxiredoxin 1 (PRDX1) is a member of a ubiquitous family of thiol peroxidases that catalyze the reduction of peroxides, including hydrogen peroxide. It functions as an antioxidant enzyme, similar to catalase and glutathione peroxidase. PRDX1 was recently shown act as a sensor of reactive oxygen species (ROS) and play a role in ROS-dependent intracellular signaling pathways. To investigate its physiological functions, PRDX1 was conditionally disrupted in chicken DT40 cells in the present study. RESULTS: The depletion of PRDX1 resulted in cell death with increased levels of intracellular ROS. PRDX1-depleted cells did not show the accumulation of chromosomal breaks or sister chromatid exchange (SCE). These results suggest that cell death in PRDX1-depleted cells was not due to DNA damage. 2-Mercaptoethanol protected against cell death in PRDX1-depleted cells and also suppressed elevations in ROS. CONCLUSIONS: PRDX1 is essential in chicken DT40 cells and plays an important role in maintaining intracellular ROS homeostasis (or in the fine-tuning of cellular ROS levels). Cells deficient in PRDX1 may be used as an endogenously deregulated ROS model to elucidate the physiological roles of ROS in maintaining proper cell growth.

Nicaraven Attenuates Postoperative Systemic Inflammatory Responses-Induced Tumor Metastasis.

BACKGROUND: Inflammation has been demonstrated to promote cancer metastasis. Due to the well-known systemic inflammatory responses (SIR) after major surgery, it is critical to investigate and attenuate SIR-induced tumor metastasis of cancer patients suffering surgical procedures. METHODS: C57BL/6 mice were intravenously injected with Lewis lung cancer cells at 6, 24, and 72 h after the induction of intestinal ischemia/reperfusion (I/R) injury. We found that the number of tumor nodules significantly increased in lungs of mice injected with cancer cells at 6 h but not at 24 and 72 h after I/R injury. The administration of nicaraven 30 min before and 24 h after I/R injury effectively attenuated the enhanced tumor metastasis to lungs. Protein array showed the increase of various cytokines in plasma of mice at 6 h after I/R injury, but many of them were attenuated by the administration of nicaraven. Immunostaining indicated the increase of Ly6g-, CD206-, and CD11c-positive inflammatory cells in the lungs, but it was also attenuated by nicaraven administration. CONCLUSIONS: Postoperative SIR-induced tumor metastasis have been clearly evidenced in our experimental model, and the administration of nicaraven may ameliorate the SIR-induced tumor metastasis by suppressing inflammatory responses.

CiApex1 has AP endonuclease activity and abrogated AP site repair disrupts early embryonic development in Ciona intestinalis.

Apurinic/apyrimidinic (AP) sites are the most common form of cytotoxic DNA damage. Since AP sites inhibit DNA replication and transcription, repairing them is critical for cell growth. However, the significance of repairing AP sites during early embryonic development has not yet been clearly determined. Here, we focused on APEX1 from the ascidian Ciona intestinalis (CiApex1), a homolog of human AP endonuclease 1 (APEX1), and examined its role in early embryonic development. Recombinant CiApex1 protein complemented the drug sensitivities of an AP endonuclease-deficient Escherichia coli mutant, and exhibited Mg2+-dependent AP endonuclease activity, like human APEX1, in vitro. Next, the effects of abnormal AP site repair on embryonic development were investigated. Treatment with methyl methanesulfonate, which alkylates DNA bases and generates AP sites, induced abnormal embryonic development. This abnormal phenotype was also caused by treatment with methoxyamine, which inhibits AP endonuclease activity. Furthermore, we constructed dominant-negative CiApex1, which inhibits CiApex1 action, and found that its expression impaired embryonic growth. These results suggested that AP site repair is essential for embryonic development and CiApex1 plays an important role in AP site repair during early embryonic development in C. intestinalis.

ATM Induces Cell Death with Autophagy in Response to H2O2 Specifically in Caenorhabditis elegans Nondividing Cells.

INTRODUCTION: Ataxia-telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response and is directly activated by reactive oxygen species (ROSs) in addition to DNA double-stranded breaks. However, the physiological function of the response to ROSs is not understood. PURPOSE: In the present study, we investigated how ATM responds to ROSs in Caenorhabditis elegans (C. elegans). MATERIALS AND METHODS: First, we measured sensitivities of larvae to DNA-damaging agents and ROSs. Next, we analyzed the drug sensitivities of fully matured adult worms, which consist of nondividing somatic cells. Dead cell staining with acridine orange was performed to visualize the dead cells. In addition, we performed GFP reporter assays of lgg-1, an autophagy-related gene, to determine the types of cell death. RESULTS: atm-1(tm5027) larvae showed a wide range of sensitivities to both DNA-damaging agents and ROSs. In contrast, fully matured adult worms, which consist of nondividing somatic cells, showed sensitivity to DNA-damaging agent, NaHSO3, but they showed resistance to H2O2. Dead cell staining and GFP reporter assays of lgg-1 suggest that C. elegans ATM-1 induces the cell death with autophagy in intestinal cells in response to H2O2. CONCLUSION: We revealed that ATM induces cell death in response to H2O2.

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O2) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O2) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed fancc and fancl cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in fancc and fancl cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.

Cytotoxicity of Tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide)-Induced DNA Damage in Chicken DT40 Cells.

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II-DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1-/-, tdp2-/-, parp1-/-, and aptx1-/- cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase-DNA complexes and unligatable DNA ends under hypoxic conditions.

A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells.

DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death.

Caenorhabditis elegans EXO-3 contributes to longevity and reproduction: differential roles in somatic cells and germ cells.

Apurinic/apyrimidinic (AP) sites are the major DNA damage generated continuously even under normal conditions, and inhibit DNA replication/transcription. AP endonucleases are ubiquitous enzymes required for the repair of AP sites and 3' blocking ends, but their physiological roles in multicellular organisms are not fully understood. In this study, we investigated how an AP endonuclease functions in a multicellular organism (Caenorhabditis elegans (C. elegans)). EXO-3 is one of the AP endonucleases in C. elegans. Using an exo-3 mutant worm, we found that deletion of the exo-3 gene caused shortened lifespan in an ung-1-dependent manner. UNG-1 is a uracil DNA glycosylase in C. elegans, and the present finding suggested that UNG-1 is the major producer of AP sites that affects lifespan, and EXO-3 contributes to longevity by completing the repair of uracil. Next we found that the exo-3 gene was abundantly expressed in the gonads, and AP sites in the gonad were efficiently repaired, suggesting that EXO-3 functioned particularly in the gonad. Deletion of the exo-3 gene resulted in a significant decrease in self-brood size. This was rescued by deficiency of NTH-1, which is a bifunctional DNA glycosylase in C. elegans that recognizes oxidative base damage. This result suggested that the major substrate of EXO-3 in the gonad was 3' blocking end generated by NTH-1, and that EXO-3 played an important role in reproduction. A contribution of EXO-3 to reproduction was also suggested by our finding here that the decrease of self-brood size of the exo-3 mutant became more marked when worms were treated with methyl methanesulfonate (MMS) and sodium bisulfite (NaHSO3). This study demonstrated differential roles of EXO-3 in somatic cells and germ cells.

Extension of lifespan and protection against oxidative stress by an antioxidant herb mixture complex (KPG-7) in Caenorhabditis elegans.

Excessive generation of reactive oxygen species within cells results in oxidative stress. Furthermore, accumulation of reactive oxygen species has been shown to reduce cell longevity. Many dietary supplements are believed to have anti-aging effects. The herb mixture KPG-7 contains several components with antioxidant activity. We aim to clarify the mechanisms responsible for the antioxidant activity of KPG-7 and to establish whether KPG-7 has an anti-aging effect. We examined whether dietary supplementation with KPG-7 could provide protection against oxidative stress, extend lifespan, and delay aging in Caenorhabditis elegans (C. elegans). We found that KPG-7 extended lifespan and delayed aging in adult C. elegans. The expression of oxidation resistance 1 protein was induced by juglone and this effect was significantly suppressed in KPG-7-treated. In addition, the amount of oxidized protein was significantly lower in KPG-7-treated worms than untreated worms. Furthermore, locomotive activity was increased in C. elegans at 3 days of age following the treatment with KPG-7. On the other hand, the level of cellular ATP was lower at 3 days of age in worms treated with KPG-7 than in untreated worms. KPG-7 increases lifespan and delays aging in C. elegans, well corresponding to its activity to protect against oxidative stress.