In vitro characterization of mitochondrial function and structure in rat and human cells with a deficiency of the NADH:ubiquinone oxidoreductase Ndufc2 subunit

Ndufc2, a subunit of the NADH:ubiquinone oxidoreductase, plays a key role in the assembly and activity of complex I within the mitochondrial OXPHOS chain. Its deficiency has been shown to be involved in diabetes, cancer and stroke. To improve our knowledge on the mechanisms underlying the increased disease risk due to Ndufc2 reduction, we performed the present in vitro study aimed at the fine characterization of the derangements in mitochondrial structure and function consequent to Ndufc2 deficiency. We found that both fibroblasts obtained from skin of heterozygous Ndufc2 knock-out rat model showed marked mitochondrial dysfunction and PBMC obtained from subjects homozygous for the TT genotype of the rs11237379/NDUFC2 variant, previously shown to associate with reduced gene expression, demonstrated increased generation of reactive oxygen species and mitochondrial damage. The latter was associated with increased oxidative stress and significant ultrastructural impairment of mitochondrial morphology with a loss of internal cristae. In both models the exposure to stress stimuli, such as high-NaCl concentration or LPS, exacerbated the mitochondrial damage and dysfunction. Resveratrol significantly counteracted the ROS generation. These findings provide additional insights on the role of an altered pattern of mitochondrial structure-function as a cause of human diseases. In particular, they contribute to underscore a potential genetic risk factor for cardiovascular diseases, including stroke

Afferent signalling from the acid-challenged rat stomach is inhibited and gastric acid elimination is enhanced by lafutidine

<p>Abstract</p> <p>Background</p> <p>Lafutidine is a histamine H₂receptor antagonist, the gastroprotective effect of which is related to its antisecretory activity and its ability to activate a sensory neuron-dependent mechanism of defence. The present study investigated whether intragastric administration of lafutidine (10 and 30 mg/kg) modifies vagal afferent signalling, mucosal injury, intragastric acidity and gastric emptying after gastric acid challenge.</p> <p>Methods</p> <p>Adult rats were treated with vehicle, lafutidine (10 – 30 mg/kg) or cimetidine (10 mg/kg), and 30 min later their stomachs were exposed to exogenous HCl (0.25 M). During the period of 2 h post-HCl, intragastric pH, gastric volume, gastric acidity and extent of macroscopic gastric mucosal injury were determined and the activation of neurons in the brainstem was visualized by c-Fos immunocytochemistry.</p> <p>Results</p> <p>Gastric acid challenge enhanced the expression of c-Fos in the nucleus tractus solitarii but caused only minimal damage to the gastric mucosa. Lafutidine reduced the HCl-evoked expression of c-Fos in the NTS and elevated the intragastric pH following intragastric administration of excess HCl. Further analysis showed that the gastroprotective effect of lafutidine against excess acid was delayed and went in parallel with facilitation of gastric emptying, measured indirectly via gastric volume changes, and a reduction of gastric acidity. The H₂receptor antagonist cimetidine had similar but weaker effects.</p> <p>Conclusion</p> <p>These observations indicate that lafutidine inhibits the vagal afferent signalling of a gastric acid insult, which may reflect an inhibitory action on acid-induced gastric pain. The ability of lafutidine to decrease intragastric acidity following exposure to excess HCl cannot be explained by its antisecretory activity but appears to reflect dilution and/or emptying of the acid load into the duodenum. This profile of actions emphasizes the notion that H₂receptor antagonists can protect the gastric mucosa from acid injury independently of their ability to suppress gastric acid secretion.</p

Lafutidine, a Protective H2 Receptor Antagonist, Enhances Mucosal Defense in Rat Esophagus

Luminal acid or CO2 induces a hyperemic response in the esophagus, via activation of acid sensors on capsaicin-sensitive afferent nerves (CSAN). Since disruption of the hyperemic response to luminal CO2 acidifies the interstitium of the esophageal mucosa, the hyperemic response may maintain interstitial pH (pHint). We hypothesized that acid-related hyperemia maintains pHint, preventing acid-induced injury in the esophageal mucosa. We examined the effects of capsaicin (Cap) or lafutidine (Laf), a mucosal protective H2 antagonist, on the regulation of pHint and blood flow in rat esophagus using ratiometric microimaging and laser-Doppler measurements of the lower esophageal mucosa of living rats. The esophagus was topically superfused with pH 7.0 buffer, or a pH 1.0 or pH 1.0 + pepsin (1 mg/ml) solution with or without Laf. Cap (30 or 100 µM) or Laf (0.1 or 1 mM) dose-dependently increased blood flow, accompanied by increased pHint. The pH 1.0 solution increased blood flow without pHint change, whereas Laf (1 mM) increased blood flow and pHint during acid exposure. The effects of Laf were abolished by ablation of CSAN. Perfusion of the acidified pepsin solution gradually decreased pHint, inhibited by Laf perfusion. Activation of CSAN by Laf with or without acid, accompanied by hyperemia, increased pHint, preventing acidified pepsin-induced interstitial acidification. Stimulation of the capsaicin pathway with compounds such as Laf enhances mucosal protection from acid-related injury in the upper gastrointestinal tract

Age-Related Neuronal Degeneration: Complementary Roles of Nucleotide Excision Repair and Transcription-Coupled Repair in Preventing Neuropathology

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR–deficient Csa−/− and Csb−/− CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER–deficient Xpa−/− and Xpc−/− XP mice, but also occurred in XpdXPCS mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR–deficient mice are compatible with focal dysmyelination in CS patients. Both TCR–deficient and NER–deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa−/−, Csb−/−) or highly sporadic (Xpa−/−, Xpc−/−) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR–deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa−/− and Csb−/− TCR–deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR–deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival

Avaliação da toxicidade das favas de Stryphnodendron fissuratum (Mimosoideae) em vacas gestantes

Com o objetivo de avaliar a toxicidade de diferentes concentrações das favas de Stryphnodendron fissuratum em vacas prenhes, as favas desta árvore foram moídas, misturadas à ração comercial e fornecidas a oito vacas nas doses totais de 6,5g/kg, 7,5g/kg, 9g/kg e 10g/kg. Os animais que receberam doses de 6,5g/kg pariram bezerros normais e aqueles que receberam 7,5g/kg pariram bezerros fracos que não sobreviveram. Doses de 9g/kg resultaram no nascimento de um bezerro imaturo e de outro bezerro com distiquíase, opacidade congênita das córneas e microftalmia. Ambas as vacas que ingeriram 10g/kg morreram, porém uma vaca abortou antes de morrer. Nas vacas que morreram, as lesões macroscópicas e histológicas do sistema digestivo e fígado foram semelhantes às descritas anteriormente na intoxicação por S. fissuratum. Nos bezerros e no feto abortado não foram observadas lesões macroscópicas ou histológicas significantes. A análise fitoquímica dos extratos metanólicos das favas de S. fissuratum revelou a presença de taninos hidrossolúveis, proantocianidinas, leucoantocianidinas e da saponina triterpénica β-amirina. Saponinas triterpénicas têm sido associadas com a toxicidade das favas de Stryphnodendron spp. e Enterolobium spp., que causam sinais clínicos semelhantes aos observados na intoxicação por S. fissuratum. Esta pesquisa confirmou a toxicidade das favas de S. fissuratum para bovinos, no entanto não foram confirmados os efeitos abortivos das mesmas, pois o aborto e as mortes neonatais observadas podem ser decorrentes dos efeitos tóxicos da planta nas mães. Novas pesquisas são necessárias para pesquisar se as favas da planta causam malformações semelhantes às observas em um dos bezerros nascidos vivos

Studies in Dhāraṇī Literature II: Pragmatics of Dhāraṇīs

This article is one of a series that reassesses the dhāraṇī texts of Mahāyāna Buddhism. The article seeks to examine dhāraṇī texts by using the linguistic tools of pragmatics, especially historical pragmatics, to assist the understanding of their statements. Rather than the meaning of the term dhāraṇī as a subject term, the domain of truth-conditional semantics, this paper examines statements in texts labelled dhāraṇī. Pragmatics examines meaning in context, and the categories of speech acts developed by Searle has been especially helpful in mapping out differences within such texts and the formalization of statements across texts. The grammaticalization of specific speech elements, especially interjections, in the context of mantra-dhāraṇīs is also discussed

Biallelic variants in LIG3 cause a novel mitochondrial neurogastrointestinal encephalomyopathy

none67si: Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.This work was supported by Telethon Grant GGP15171 to E.B. and R.D.G. and by a donation from Kobe city to the Department of General Pediatrics, Kobe University Graduate School of Medicine (K550003302). S.C. was supported by a Dutch Cancer Foundation grant (KWF11011). V.C. and A.M. were supported by the Italian Ministry of Health (“Ricerca Corrente” funding). R.D.G. is the recipient of grants from University of Ferrara (FAR and FIR funds).openBonora, Elena; Chakrabarty, Sanjiban; Kellaris, Georgios; Tsutsumi, Makiko; Bianco, Francesca; Bergamini, Christian; Ullah, Farid; Isidori, Federica; Liparulo, Irene; Diquigiovanni, Chiara; Masin, Luca; Rizzardi, Nicola; Cratere, Mariapia Giuditta; Boschetti, Elisa; Papa, Valentina; Maresca, Alessandra; Cenacchi, Giovanna; Casadio, Rita; Martelli, Pierluigi; Matera, Ivana; Ceccherini, Isabella; Fato, Romana; Raiola, Giuseppe; Arrigo, Serena; Signa, Sara; Sementa, Angela Rita; Severino, Mariasavina; Striano, Pasquale; Fiorillo, Chiara; Goto, Tsuyoshi; Uchino, Shumpei; Oyazato, Yoshinobu; Nakamura, Hisayoshi; Mishra, Sushil K; Yeh, Yu-Sheng; Kato, Takema; Nozu, Kandai; Tanboon, Jantima; Morioka, Ichiro; Nishino, Ichizo; Toda, Tatsushi; Goto, Yu-Ichi; Ohtake, Akira; Kosaki, Kenjiro; Yamaguchi, Yoshiki; Nonaka, Ikuya; Iijima, Kazumoto; Mimaki, Masakazu; Kurahashi, Hiroki; Raams, Anja; MacInnes, Alyson; Alders, Mariel; Engelen, Marc; Linthorst, Gabor; de Koning, Tom; den Dunnen, Wilfred; Dijkstra, Gerard; van Spaendonck, Karin; van Gent, Dik C; Aronica, Eleonora M; Picco, Paolo; Carelli, Valerio; Seri, Marco; Katsanis, Nicholas; Duijkers, Floor A M; Taniguchi-Ikeda, Mariko; De Giorgio, RobertoBonora, Elena; Chakrabarty, Sanjiban; Kellaris, Georgios; Tsutsumi, Makiko; Bianco, Francesca; Bergamini, Christian; Ullah, Farid; Isidori, Federica; Liparulo, Irene; Diquigiovanni, Chiara; Masin, Luca; Rizzardi, Nicola; Cratere, Mariapia Giuditta; Boschetti, Elisa; Papa, Valentina; Maresca, Alessandra; Cenacchi, Giovanna; Casadio, Rita; Martelli, Pierluigi; Matera, Ivana; Ceccherini, Isabella; Fato, Romana; Raiola, Giuseppe; Arrigo, Serena; Signa, Sara; Sementa, Angela Rita; Severino, Mariasavina; Striano, Pasquale; Fiorillo, Chiara; Goto, Tsuyoshi; Uchino, Shumpei; Oyazato, Yoshinobu; Nakamura, Hisayoshi; Mishra, Sushil K; Yeh, Yu-Sheng; Kato, Takema; Nozu, Kandai; Tanboon, Jantima; Morioka, Ichiro; Nishino, Ichizo; Toda, Tatsushi; Goto, Yu-Ichi; Ohtake, Akira; Kosaki, Kenjiro; Yamaguchi, Yoshiki; Nonaka, Ikuya; Iijima, Kazumoto; Mimaki, Masakazu; Kurahashi, Hiroki; Raams, Anja; MacInnes, Alyson; Alders, Mariel; Engelen, Marc; Linthorst, Gabor; de Koning, Tom; den Dunnen, Wilfred; Dijkstra, Gerard; van Spaendonck, Karin; van Gent, Dik C; Aronica, Eleonora M; Picco, Paolo; Carelli, Valerio; Seri, Marco; Katsanis, Nicholas; Duijkers, Floor A M; Taniguchi-Ikeda, Mariko; De Giorgio, Robert