Commentary: Aldehyde dehydrogenase, redox balance and exercise physiology: What is missing?

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme involved in reactive aldehyde detoxification. Approximately 560 million people (about 8% of the world's population) carry a point mutation in the aldehyde dehydrogenase 2 gene (ALDH2), identified as ALDH2*2, which leads to decreased ALDH2 catalytic activity. ALDH2*2 variant is associated with an accumulation of toxic reactive aldehydes and consequent disruption of cellular metabolism, which contributes to the establishment and progression of several degenerative diseases. Consequences of aldehyde accumulation include impaired mitochondrial functional, hindered anabolic signaling in the skeletal muscle, impaired cardiovascular and pulmonary function, and reduced osteoblastogenesis. Considering that aldehydes are endogenously produced through redox processes, it is expected that conditions that have a high energy demand, such as exercise, might be affected by impaired aldehyde clearance in ALDH2*2 individuals. Despite the large body of evidence supporting the importance of ALDH2 to ethanol metabolism, redox homeostasis and overall health, specific research investigating the impact of ALDH2*2 on phenotypes relevant to exercise performance are notoriously scarce. In this commentary, we highlight the consolidated knowledge on the impact of ALDH2*2 on physiological processes that are relevant to exercise.

Role of Metabolism on Alcohol Preference, Addiction, and Treatment.

Studies presented in this chapter show that: (1) in the brain, ethanol is metabolized by catalase to acetaldehyde, which condenses with dopamine forming salsolinol; (2) acetaldehyde-derived salsolinol increases the release of dopamine mediating, via opioid receptors, the reinforcing effects of ethanol during the acquisition of ethanol consumption, while (3) brain acetaldehyde does not influence the maintenance of chronic ethanol intake, it is suggested that a learned cue-induced hyperglutamatergic system takes precedence over the dopaminergic system. However, (4) following a prolonged ethanol deprivation, the generation of acetaldehyde in the brain again plays a role, contributing to the increase in ethanol intake observed during ethanol re-access, called the alcohol deprivation effect (ADE), a model of relapse behavior; (5) naltrexone inhibits the high ethanol intake seen in the ADE condition, suggesting that acetaldehyde-derived salsolinol via opioid receptors also contributes to the relapse-like drinking behavior. The reader is referred to glutamate-mediated mechanisms that trigger the cue-associated alcohol-seeking and that also contribute to triggering relapse.

Expression of a Tumor Stem Cell Marker (Aldehyde Dehydrogenase 1-ALDH1) in Benign Epithelial Odontogenic Lesions.

The morphological diversity and different biological behaviors of human lesions has been attributed to the presence of cells with stem cell (SC) characteristics. Among SC markers, ALDH1 has been used in studies investigating different neoplasms and high expression of this marker was associated with clinicopathological features and prognosis in some groups. The aim of this study was to analyze the presence and distribution of SCs based on the expression of ALDH1 in epithelial odontogenic cysts and tumors. The sample consisted of 80 cases (20 dentigerous cysts (DCs), 20 odontogenic keratocysts (OKCs), 20 ameloblastomas (AMs), and 20 adenomatoid odontogenic tumors (AOTs). An immunoreactivity score was obtained from the percentage of positive cells and intensity of immunostaining. A level of 5% (p < 0.05) was adopted for the statistical tests. Immunoexpression of ALDH1 was observed in cytoplasm and nucleus-cytoplasm. The median scores indicated significantly higher expression in OKCs and DCs compared to AMs (p < 0.0001) and AOTs (p < 0.0001). In the tumor stroma and cystic capsule, immunoreactivity was detected in all odontogenic cysts studied and in 85% and 90% of AMs and AOTs, respectively. The expression of ALDH1 suggests the presence of SCs in the odontogenic lesions studied. Epithelial immunoexpression was higher in odontogenic cysts than in odontogenic tumors.

Structure Based Protein Engineering of Aldehyde Dehydrogenase from Azospirillum brasilense to Enhance Enzyme Activity against Unnatural 3-Hydroxypropionaldehyde.

3-Hydroxypropionic acid (3HP) is a platform chemical and can be converted into other valuable C3-based chemicals. Because a large amount of glycerol is produced as a by-product in the biodiesel industry, glycerol is an attractive carbon source in the biological production of 3HP. Although eight 3HP-producing aldehyde dehydrogenases (ALDHs) have been reported so far, the low conversion rate from 3-hydroxypropionaldehyde (3HPA) to 3HP using these enzymes is still a bottleneck for the production of 3HP. In this study, we elucidated the substrate binding modes of the eight 3HP-producing ALDHs through bioinformatic and structural analysis of these enzymes and selected protein engineering targets for developing enzymes with enhanced enzymatic activity against 3HPA. Among ten AbKGSADH variants we tested, three variants with replacement at the Arg281 site of AbKGSADH showed enhanced enzymatic activities. In particular, the AbKGSADHR281Y variant exhibited improved catalytic efficiency by 2.5-fold compared with the wild type.

Estudo comparativo da expressão de ALDH1 em líquen plano oral, lesão liquenoide e leucoplasia em epitélio e no infiltrado inflamatório liquenoide / Comparative study of ALDH1 expression in oral lichen plane, lichenoid lesion and leucoplasia in epithelial and lichenoid inflammatory infiltrate

O líquen plano oral é uma doença potencialmente maligna e compartilha muitas características clínicas e histopatológicas com outras lesões semelhantes. O ALDH1 é um biomarcador específico na identificação de células-tronco, porém seu papel nas células estromais do infiltrado inflamatório imune não foi explorado. O objetivo deste estudo foi investigar a imunoexpressão de ALDH1 em células epiteliais e estromais de Líquen Plano Oral e outras lesões liquenóides. O estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de Minas Gerais (UFMG) sob o parecer no 5.410.374, CAAE: 52763021.0.0000.5149. Foi realizada uma busca no arquivo do laboratório de Patologia Oral e Bucomaxilofacial da UFMG em um período de 10 anos (2011 ­ 2021) de casos diagnosticados como Líquen Plano oral, Lesão Liquenoide, Leucoplasia e Processo Inflamatório Inespecífico (controle). Foram selecionados 633 casos e após a utilização dos critérios de inclusão e exclusão, a expressão imuno-histoquímica do ALDH1 foi investigada em 64 casos, 16 de cada diagnóstico. O ALDH1 foi avaliado tanto no epitélio quanto nas células estromais do infiltrado inflamatório liquenóide. Um percentual de 5% de células ALDH1+ foi considerado positivo no epitélio. A avaliação das células estromais foi feita de forma semiquantitativa. Os campos foram classificados em escores, segundo os critérios: 1 (0 a 10%); 2 (11 a 50%) e 3 (mais de 51%). O valor médio da soma dos campos foi o escore final. Diferenças estatísticas entre os grupos foram investigadas e a significância foi estabelecida considerando p < 0,05. A análise estatística foi realizada utilizando o software: SPSS ­ Statistical Package for the Social Science® (versão 26.0, 2019, Chicago, IL, USA). A expressão de ALDH1 no epitélio foi baixa em todos os grupos sem diferença entre eles, embora a maioria dos casos tenha sido encontrada na amostra de inflamação crônica inespecífica. O escore de células ALDH1+ na lâmina própria foi maior para LPO (2,0), seguido por Leucoplasia (1,3), LLO (1,2) e processo inflamatório inespecífico (1,1) (p<0,05). A imunoexpressão do ALDH1 no epitélio de lesões com um infiltrado liquenóide não mostrou uma ferramenta contributiva, porém o ALDH1 em células estromais no infiltrado inflamatório do líquen plano merece atenção, pois pode estar envolvido no complexo processo de regulação imune associado à patogênese desta doença.

Oral lichen planus (OLP) is a potentially malignant lesion and shares clinical and histopathological features with similar lesions. ALDH1 is a specific biomarker in the identification of stem cells, but its role in the stromal cells of the immune inflammatory infiltrate has not been elucidated yet. The aim of this study was to investigate the ALDH1 immunoexpression in epithelial and stromal cells from OLP and other lichenoid lesions. The study was approved by the Research Ethics Committee of the Universidade Federal de Minas Gerais (UFMG) under protocol no 5.410.374, CAAE: 52763021.0.0000.5149. An search of the biopsy records of the Oral and Maxillofacial Pathology Laboratory at UFMG in a period of 10 years (2011 - 2021) was performed including the diagnosis of Oral Lichen Planus (OLP), Oral Lichenoid Lesion (OLL), Oral Leukoplakia (OLK) and Unspecific Inflammatory Process (UIP) ­ as control. Initially were obtained 633 cases. After inclusion and exclusion criteria a total of 64 cases were investigated for the ALDH1 immunohistochemical expression, 16 cases of each diagnose. ALDH1 was evaluated both in the epithelium and in the stromal cells. A percentage of ≤5% of ALDH1+ cells was considered positive in the epithelium. The evaluation of stromal cells was performed semi-quantitatively. The fields were classified into scores, according to the following criteria: 1 (0 to 10%); 2 (11 to 50%) and 3 (more than 51%). The mean value of the sum of the fields represented the final score. Statistical differences between groups were investigated and significance was established considering p < 0.05. Statistical analysis was performed using the software: SPSS ­ Statistical Package for the Social Science® (version 26.0, 2019, Chicago, IL, USA). The expression of ALDH1 in the epithelium was low in all groups with no difference between them, although most cases were found in the UIP samples. ALDH1+ cells in the lamina propria was higher for OLP (2.0) than others followed by OLK (1.3), OLL (1.2) and UIP (1.1) (p<0.05). The ALDH1 immunoexpression in the epithelium of lichenoid diseases did not prove to be contributory to show a malignant potential, but ALDH1 in stromal cells from OLP infiltrate might be involved in the complex immune regulation process associated with the pathogenesis of this disease.

Participação da aldeído desidrogenase-2 na neuroinflamação induzida pela constrição do nervo isquiático

Background: Aldehyde dehydrogenase-2 (ALDH2) is a mitochondrial enzyme responsible for detoxifying aldehydes generated from the lipid peroxidation. We previously showed that Alda-1, a small molecule that activates ALDH2, has a potent antinociceptive effect in a chronic neuropathic pain model induced by the chronic constriction injury of the sciatic nerve (CCI) in mice, by decreasing reactive aldehydes, such as 4-hydroxynonenal (4-HNE) at the injured site. One of the most important mechanisms involved in the neuropathic pain onset and chronification is the activation of immune and glial cells. Considering that reactive aldehydes are pro- inflammatory and pro-nociceptive, we hypothesized that 4-HNE is involved in the central sensitization in CCI-induced neuropathy model. Methods: The nociceptive threshold was assessed by the up and down method using von Frey filaments, before (0), 7-, and 14-days post-injury (DPI). The ALDH2 function was modulated by Alda-1 (16 mg/Kg/day, s.c. osmotic pump) or by using a transgenic mice model with an inactivating mutation found commonly in Asiatic populations that impairs ~95% in ALDH2 activity (ALDH2*2). The 4-HNE protein adducts and the activation of glial cells in the dorsal horn of the spinal cord was assessed by using immunofluorescence techniques, followed by microglia morphology, and co- localization analysis. Moreover, axonal degeneration was assessed by evaluating the cell infiltration and fragmentation of myelin in histological preparation of sciatic nerve. Results: CCI decreases the nociceptive threshold in 7 and 14DPI, in wildtype (WT) and ALDH2*2 animals, and Alda-1 prevents CCI-induced hypernociception in both periods and genotypes. Also, CCI increases the immunostaining for 4-HNE adducts, GFAP (astrocyte), Iba-1 (microglia) along with a change in microglia morphology to pro-inflammatory profile in wild type mice. Alda-1 prevents 4-HNE adducts formation and glial cells activation, as measured in microglia morphology. Interestingly, the impairment in ALDH2 activity contributes to changes in glial activation dynamic. Alda- 1 reduces 4-HNE levels in ALDH2*2 mice, without interfering with IBA-1 and GFAP expressions. However, the microglia morphology analysis revealed that Alda-1 might induce a shift from inflammatory to anti-inflammatory profile. The co-localization study showed that 4-HNE accumulates in afferent axons, Schwann cells, as well as spinal cord neurons and oligodendrocytes. Finally, we also showed that CCI induced sciatic nerve degeneration in both genotypes, which was prevented by the treatment with Alda-1. Conclusion: The data suggest that 4-HNE plays an important role in neuropathic hypernociception by controlling aldehyde levels and central neuroinflammation, as well as regulates axonal degeneration and peripheral inflammation in this model. Interestingly, the genetic impairment in ALDH2 activity changes the dynamics of glial activation upon nerve injury. Finally, Alda-1 is a promising molecule for neuropathic pain treatment.

A aldeído desidrogenase-2 (ALDH2) é uma enzima mitocondrial responsável pela detoxicação de aldeídos gerados a partir da peroxidação lipídica. Mostramos anteriormente que a Alda-1, uma pequena molécula ativadora a ALDH2, inibe a hipernocicepção em um modelo de dor neuropática induzida pela constrição crônica do nervo isquiático (CCI) em camundongos, por levar a diminuição de aldeídos reativos, como 4-hidroxinonenal (4-HNE) no local lesionado. Sabe-se que um dos principais mecanismos envolvidos na instalação e na cronificação da dor neuropática é a sensibilização central e periférica, que ocorre por ativação de células imunes e gliais. Considerando que os aldeídos reativos são pró-inflamatórios e pró- nociceptivos, levantamos a hipótese que o 4-HNE participa dos processos de sensibilização central e periférica no modelo de neuropatia induzida por CCI. Métodos: O limiar nociceptivo foi determinado pelo método up and down por meio de filamentos de von Frey, que foi aplicado antes (0), 7 e 14 dias após a cirurgia. A função da ALDH2 foi modulada pelo tratamento com a Alda-1 (16 mg/Kg/dia, s.c. por bomba osmótica), ou por meio de camundongos transgênicos contendo uma mutação encontrada em populações asiáticas que reduz (~ 95%) a atividade de ALDH2 (ALDH2*2). A análise dos níveis de adutos de 4-HNE, ativação de células imunes e gliais e liberação de citocinas foram avaliados por meio de western blot, imunofluorescência e ensaio de multiplex, respectivamente. Ainda, a degeneração axonal foi determinada pela infiltração celular e fragmentação de mielina no preparo histológico do nervo isquiático. Resultados: a CCI diminui o limiar nociceptivo em 7 e 14 dias após a cirurgia, em animais selvagens (WT) e ALDH2*2. A administração de Alda-1 previne a hipernocicepção induzida pela CCI em ambos os períodos e genótipos. Adicionalmente, demonstramos que a CCI aumenta a imunomarcação para adutos de 4-HNE, GFAP (astrócitos), Iba-1 (microglia), juntamente com uma mudança na morfologia da micróglia para um perfil pró-inflamatório, no corno dorsal da medula espinal de animais selvagens. A Alda-1 previne o aumento nos níveis de 4-HNE e na ativação de células gliais. Curiosamente, o prejuízo na atividade da ALDH2 contribui para mudanças na dinâmica de ativação glial, já que a Alda-1 reduz os níveis de 4-HNE em camundongos ALDH2*2, sem interferir nas expressões de IBA-1 e GFAP. No entanto, a análise da morfologia da micróglia revelou que Alda-1 pode induzir uma mudança para um perfil anti-inflamatório. O estudo de co- localização mostrou que o 4-HNE se acumula em axônios aferentes, células de Schwann e macrófagos na periferia, bem como neurônios e oligodendrócitos da medula espinal. Por fim, também mostramos que a CCI induziu degeneração do nervo isquiático em ambos os genótipos, que foi prevenida pelo tratamento com Alda-1. Conclusão: Os dados sugerem que o 4-HNE desempenha um papel importante na hipernocicepção neuropática controlando os níveis de aldeídos e a neuroinflamação central, bem como regula a degeneração axonal e a inflamação periférica neste modelo. Curiosamente, o comprometimento genético na atividade da ALDH2 altera a dinâmica da ativação glial frente à lesão do nervo. Finalmente, a Alda-1 é uma molécula promissora para o tratamento da dor neuropática.

ALDH1 expression and potential clinical implications in chronic inflammatory periapical lesions

Abstract: Aldehyde dehydrogenase 1 (ALDH-1) is a marker of stem cells in a variety of diseases, but its role in individuals with chronic inflammatory periapical lesions remains unknown. The aim of this study was to investigate the presence of cells with a stem cell profile based on the immunoexpression of ALDH-1 in periapical granulomas (PGs) and radicular cysts (RCs). A total of 51 cases of periapical lesions (25 PGs and 26 RCs) were subjected to immunohistochemical study. The anti-ALDH-1 antibody was applied using the immunoperoxidase technique. An immunoexpression score (intensity vs. percentage of cells) was used, with the cases being classified as low expression (score: 0 to 4) and high expression (score: 6 to 9). The Chi-square test was used with a 5% level of significance. Immunoexpression of ALDH-1 was detected in all cases of PGs and RCs. In PG cases, the expression was diffuse in connective tissue cells, with most cases exhibiting high expression (n = 18; 69.2%), while in RC cases the expression revealed focal distribution in cells of the capsule and epithelial cells of the cystic lining, with most cases classified as low expression (n = 18; 72%). Significant differences in the expression scores of ALDH-1 were observed in PGs (p = 0.003). The variable expression of ALDH-1 suggests the presence of cells with stem cell profiles in PGs and RCs. These findings suggest that periapical tissues infiltrated by chronic inflammation can recruit important cells for the repair or evolution of periapical lesions.

Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BACKGROUND The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. RESULTS We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41C. CONCLUSIONS Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore rovides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets

Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2.

Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium.

Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.

Outcomes of Japanese patients with non-alcoholic fatty liver disease according to genetic background and lifestyle-related diseases.

INTRODUCTION AND OBJECTIVES: Genetic background may be involved in the mechanisms of liver injury and the development of non-alcoholic fatty liver disease (NAFLD). However, its contributions to the long-term outcome of NAFLD have been unclear. METHODS: We enrolled 314 Japanese patients with biopsy-confirmed NAFLD from 2000 to 2018 (161 men [51.3%]; median age, 53 [14-84] years; 114 with advanced fibrosis [37.5%]) in the patients without hepatocellular carcinoma at diagnosis. Genomic DNA was extracted from peripheral blood and single nucleotide polymorphisms (SNPs) were analyzed. Associations of mortality with patatin-like phospholipase 3 (PNPLA3) and aldehyde dehydrogenase 2 (ALDH2) were analyzed. Finally, a subgroup analysis according to lifestyle-related disease was performed. RESULTS: During the median 7 years of follow-up, 20 patients (6.4%) died (13 liver-related [4.1%] and 7 non-liver-related deaths [2.2%]). Patients with ALDH2 (non-GG genotype) who had reduced alcohol metabolism tended to have a poor prognosis (p = 0.06). Patients carrying both risk SNPs of PNPLA3 (GG) and ALDH2 (non-GG) had a significantly poor prognosis (p = 0.01). In the subgroup analysis, patients with PNPLA3 (GG) who were non-diabetics (p = 0.06) or non-dyslipidemic (p = 0.03), with ALDH2 (non-GG) who were non-dyslipidemic (p = 0.01) or hypertensive (p = 0.03), also had a poor prognosis. The Cox analysis revealed that ALDH2 (non-GG) was associated with a poor prognosis (Hazard ratio: 4.568, 95% Confidence Interval: 1.294-16.131, p = 0.02) similar to the liver function tests. CONCLUSIONS: Genetic background may affect NAFLD prognosis and ALDH2 SNP could predict the outcome.

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour.

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

Development and application of two novel functional molecular markers of BADH2 in rice

BACKGROUND: Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. RESULTS: In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2- E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F2 population. CONCLUSIONS: Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.

Role of potassium levels in pkBADH heterogeneity of NAD+ binding site.

Betaine aldehyde dehydrogenase (BADH) catalyzes the oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Studies in porcine kidney BADH (pkBADH) suggested that the enzyme exhibits heterogeneity of active sites and undergoes potassium-induced conformational changes. This study aimed to analyze if potassium concentration plays a role in the heterogeneity of pkBADH active sites through changes in NAD+ affinity constants, in its secondary structure content and stability. The enzyme was titrated with NAD+ 1 mM at fixed-variable KCl concentration, and the interaction measured by Isothermal Titration Calorimetry (ITC) and Circular Dichroism (CD). ITC data showed that K+ increased the first active site affinity in a manner dependent on its concentration; KD values to the first site were 14.4, 13.1, and 10.4 µM, at 25, 50, and 75 mM KCl. ΔG values showed that the coenzyme binding is a spontaneous reaction without changes between active sites or depending on KCl concentration. ΔH and TΔSb values showed that NAD+ binding to the active site is an endothermic process and is carried out at the expense of changes in entropy. α-Helix content increased as KCl increased, enzyme (Tm)app values were 2.6 °C and 3.3 °C higher at 20 mM and 200 mM K+. PkBADH molecular model showed three different interaction K+ sites. Results suggested K+ can interact with pkBADH and cause changes in the secondary structure, it provokes changes in the enzyme affinity by the coenzyme, and in the thermostability.

Análise da imunoexpressão da proteína aldeído desidrogenase 1 em lesões odontogênicas epiteliais benignas / Analysis of the immunoexpression of aldehyde dehydrogenase 1 protein in benign epithelial odontogenic lesions

Os cistos e tumores odontogênicos constituem lesões heterogêneas que se originam de algum remanescente da odontogênese. Dentre essas, o cisto dentígero (CD), o ceratocisto odontogênico (CO), o ameloblastoma (AM) e o tumor odontogênico adenomatóide (TOA) são lesões odontogênicas de origem epitelial apresentando diversos perfis biológicos e sendo alvos de constantes investigações. As células-tronco (CT) são a principal fonte para a geração e manutenção da diversidade celular e distúrbios na regulação podem levar a produção de CT alteradas, as células-tronco tumorais (CTT). Entre os marcadores das CTT, a ALDH1 vem sendo usada em estudos em diversas neoplasias e a sua alta expressão esteve associada a informações de diagnóstico e prognóstico. O objetivo deste estudo foi analisar a presença, quantidade e distribuição de CTT no parênquima tumoral, cápsula cística e estroma, através do padrão de expressão imuno-histoquímico da proteína ALDH-1 em cistos e tumores odontogênicos de origem epitelial. A amostra foi constituida por 80 casos (20 CDs, 20 COs, 20 AMs e 20 TOAs). O sistema de pontuação de imunorreatividade, utilizado foi o de percentual de células positivas e de intensidade da imunomarcação, com escores de 0 a 3. O escore final foi determinado através da fórmula ''Escore de imunoexpressão = intensidade × percentual de células'', sendo definido como baixa expressão, os escores de 0 a 4 e, alta expressão, os escores de 6 a 9 para a cápsula cística e parênquima tumoral. Também, foi analisada a expressão da proteína no estroma das lesões, sendo estabelecido 0 = negativo e 1 = positivo. Em todas as variáveis, os testes não-paramétricos de Kruskal-Wallis (KW) e Mann-Whitney (U) foram realizados com nível de significância de 5% (p < 0,05). A imunoexpressão da ALDH-1 exibiu marcação no núcleo e núcleo-citoplasma. As medianas dos escores obtidos, do padrão de expressão e da intensidade de imunoexpressão de ALDH-1 no componente epitelial das lesões, demonstrou expressão significativamente superior de ALDH-1 em COs em comparação aos AMs (p < 0,0001) e aos TOAs (p < 0,0001). Foi observada maior expressão de ALDH-1 em CDs em comparação aos AMs (p < 0,0001) e TOAs (p < 0,0001). No estroma e na cápsula cística, foi evidenciada imunoreatividade em todos os casos de cistos odontogênicos estudados e em 85% e 90% dos AMs e TOAs, respectivamente. A expressão da ALDH-1 nas lesões odontogênicas estudadas sugere a presença de células com perfil de CT nas mesmas, destacando-se os cistos odontogênicos que apresentaram expressões epiteliais muito superiores aos tumores (AU).

Odontogenic cysts and tumors are heterogeneous lesions that arise from remnants of odontogenesis. Among these, the dentigerous cyst (DC), the odontogenic keratocyst (OK), the ameloblastoma (AM), and the adenomatoid odontogenic tumor (AOT) are odontogenic lesions of epithelial origin that have different biological profiles and have been extensively investigated. Stem cells (SC) are the main source for the generation and maintenance of cell diversity and disturbances in their regulation can lead to the production of altered SC, the socalled tumor stem cells (TSC). Among the markers of TSC, the aldehyde dehydrogenase-1 protein (ALDH1) has been evaluated in several neoplasms and its high expression has been associated with diagnostic and prognostic information. The aim of this study was to analyze the presence, quantity and distribution of TSC in the tumor parenchyma, cystic capsule and stroma, through the immunohistochemical expression of ALDH1 in odontogenic cysts and tumors of epithelial origin. The sample consisted of 80 cases (20 DCs, 20 OKs, 20 AMs and 20 AOTs) and the immunoreactivity was evaluated through the percentage of positive cells and intensity of immunostaining, with scores from 0 to 3. The final score was determined by the formula ''immunoexpression score = intensity × percentage of cells '', being defined as low expression the scores from 0 to 4 and high expression the scores from 6 to 9, in cystic capsule and tumor parenchyma. The expression of ALDH1 was also analyzed in the stroma of the lesions, with scores 0 = negative and 1 = positive. The data were submitted to nonparametric tests, considering a level of significance of 5% (p<0.05). Immunoexpression of ALDH-1 exhibited nuclear and nucleo-cytoplasmic marking. When the lesions were compared regarding the medians of the obtained scores, the expression pattern and the intensity of immunoexpression of ALDH-1 in the epithelial component of the lesions, a significantly higher expression of ALDH-1 was observed in KOs compared to AMs (p < 0.0001) and AOTs (p <0.0001), and higher expression of ALDH-1 was observed in DCs compared to AMs (p <0.0001) and AOTs (p <0.0001). When evaluating stroma and cystic capsule, immunoreactivity was observed in all cases of odontogenic cysts studied and in 85% and 90% of AMs and AOTs, respectively. The expression of ALDH-1 in the odontogenic lesions studied suggests the presence of SCs in them, highlighting the odontogenic cysts for presenting higher epithelial expressions compared to odontogenic tumors (AU).

Decreased malondialdehyde levels in fish (Astyanax altiparanae) exposed to diesel: Evidence of metabolism by aldehyde dehydrogenase in the liver and excretion in water.

Increased malondialdehyde (MDA) levels are commonly considered an indicator of lipid peroxidation derived from oxidative stress insults promoted by exposure of fish to pollutants. However, a decrease in MDA levels after xenobiotic exposure has been also reported, an effect that is mostly attributed to enhanced antioxidant defenses. In this study, we assessed whether pollutant-mediated MDA decrease would be associated with antioxidant enhancement or with its metabolism by aldehyde dehydrogenase (ALDH) in the liver and gills of lambari (Astyanax altiparanae) exposed to diesel oil (0.001, 0.01, and 0.1 mL/L). MDA levels were decreased in the liver of lambari exposed to diesel. The activities of the antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx), were unchanged in the liver, while that of glucose-6-phosphate dehydrogenase (G6PDH) was decreased. In contrast, levels of total glutathione (tGSH) and the activity of glutathione S-transferase (GST) were increased in the liver, which partly support antioxidant protection against lipid peroxidation. More importantly, ALDH activity increased in a concentration-dependent manner, being negatively correlated with MDA levels, indicating MDA metabolism by ALDH. In the gills, diesel exposure increased MDA and lipid hydroperoxide levels, and promoted increases in antioxidant defenses, indicating oxidative stress. Curiously, ALDH activity was undetectable in the gills, supporting the possibility of direct MDA excretion in the water by the gills. Analyses of MDA in the water revealed increased levels of MDA in the aquaria in which the fish were exposed to diesel, compared to control aquaria. A second experiment was carried out in which the fish were intraperitoneally injected with MDA (10 mg/kg) and analyzed after 1, 6, and 12 h. MDA injection caused a time-dependent decrease in hepatic MDA levels, did not alter ALDH, CAT, GPx, and GST activities, and decreased G6PDH activity and tGSH levels. In the gills, MDA injection caused a slight increase in MDA levels after 1 h, but did not alter GPx, G6PDH, and GST activities. MDA injection also enhanced CAT activity and tGSH levels in the gills. MDA concentration in water increased progressively after 1, 6, and 12 h, supporting the hypothesis of direct MDA excretion as an alternative route for MDA elimination in fish. Our results suggest that the decreased MDA levels after exposure of lambari to diesel oil pollutant probably reflects an association between enhanced antioxidant protection, MDA metabolism, and MDA excretion in water.

Effect of salinity on the synthesis and concentration of glycine betaine in osmoregulatory tissues from juvenile shrimps Litopenaeus vannamei.

In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60 ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35 ppt salinity. In muscle, the BADH activity decreased at 35 ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60 ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60 ppt salinity, BADH activity increased 1.5-fold respect to 35 ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35 ppt. Glycine betaine concentration also increased at 20 ppt salinity, while at 10 ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20 ppt and 10 ppt (1.1-fold relative to 35 ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.

Brain ethanol-metabolizing enzymes are differentially expressed in lead-exposed animals after voluntary ethanol consumption: Pharmacological approaches.

Developmentally-lead (Pb)-exposed rats showed an enhanced vulnerability to the stimulating and motivational effects of ethanol (EtOH). This is accompanied by differential activity of the brain EtOH-metabolizing enzymes catalase (CAT) and mitochondrial aldehyde dehydrogenase (ALDH2). Based on the theory that brain acetaldehyde accumulation is associated with the reinforcing properties of EtOH, this study sought to determine brain CAT and ALDH2 expression in limbic areas of control and Pb-exposed animals after voluntary EtOH intake. Thirty-five-day-old rats perinatally exposed to 220 ppm Pb were offered with water or increasing EtOH solutions (2-10% v/v) during 28 days until postnatal day (PND) 63. Once intake was stable, the animals were administered: 1) saline (SAL; test days 21-24 or 21-28, as corresponds), or 2) a CAT inhibitor: 3-amine 1, 2, 4-triazole (AT; 250 mg/kg intraperitoneally [i.p.], 5 h before the last eight EtOH intake sessions -test days 21-24 and 25-28), or 3) a CAT booster: 3-nitropropionic acid (3NPA; 20 mg/kg subcutaneously [s.c.], 45 min before the last four EtOH intake sessions -test days 25-28). Two additional groups were centrally-administered cyanamide (CY, an ALDH2 inhibitor, 0.3 mg i.c.v. immediately before the last four EtOH sessions, test days 25-28) or its corresponding vehicle (VEH). Lead exposure increased EtOH intake, an effect potentiated in both groups by 3NPA or CY pretreatments and reduced by AT, albeit selectivity in the Pb group. Catalase abundance in limbic areas parallels these observations in the Pb group, showing higher CAT expression in all areas after EtOH consumption respect to the controls, an effect prevented by AT administration. In contrast, ALDH2 expression was reduced in the Pb animals after EtOH intake, with CY potentiating this effect in all brain areas under study. Based on these results and on previous evidences, we suggest that Pb exposure promotes acetaldehyde accumulation in limbic regions, providing some insights into the mechanism of action that underlies the vulnerability to the excessive EtOH consumption reported in these animals.

Pilot Study of an Integrative New Tool for Studying Clinical Outcome Discrimination in Acute Leukemia.

Acute leukemia is a heterogeneous set of diseases affecting children and adults. Current prognostic factors are not accurate predictors of the clinical outcome of adult patients and the stratification of risk groups remains insufficient. For that reason, this study proposes a multifactorial analysis which integrates clinical parameters, ex vivo tumor characterization and behavioral in vivo analysis in zebrafish. This model represents a new approach to understand leukemic primary cells behavior and features associated with aggressiveness and metastatic potential. Xenotransplantation of primary samples from patients newly diagnosed with acute leukemia in zebrafish embryos at 48 hpf was used to asses survival rate, dissemination pattern, and metastatic potential. Seven samples from young adults classified in adverse, favorable or intermediate risk group were characterized. Tumor heterogeneity defined by Leukemic stem cell (LSC) proportion, was performed by metabolic and cell membrane biomarkers characterization. Thus, our work combines all these parameters with a robust quantification strategy that provides important information about leukemia biology, their relationship with specific niches and the existent inter and intra-tumor heterogeneity in acute leukemia. In regard to prognostic factors, leukemic stem cell proportion and Patient-derived xenografts (PDX) migration into zebrafish were the variables with highest weights for the prediction analysis. Higher ALDH activity, less differentiated cells and a broader and random migration pattern are related with worse clinical outcome after induction chemotherapy. This model also recapitulates multiple aspects of human acute leukemia and therefore is a promising tool to be employed not only for preclinical studies but also supposes a new tool with a higher resolution compared to traditional methods for an accurate stratification of patients into worse or favorable clinical outcome.