Production of bioactive diterpenoids in the Euphorbiaceae depends on evolutionarily conserved gene clusters

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Intestinal microbiota associated with differential feed conversion efficiency in chickens

Analysis of model systems, for example in mice, has shown that the microbiota in the gastrointestinal tract can play an important role in the efficiency of energy extraction from diets. The study reported here aimed to determine whether there are correlations between gastrointestinal tract microbiota population structure and energy use in chickens. Efficiency in converting food into muscle mass has a significant impact on the intensive animal production industries, where feed represents the major portion of production costs. Despite extensive breeding and selection efforts, there are still large differences in the growth performance of animals fed identical diets and reared under the same conditions. Variability in growth performance presents management difficulties and causes economic loss. An understanding of possible microbiota drivers of these differences has potentially important benefits for industry. In this study, differences in cecal and jejunal microbiota between broiler chickens with extreme feed conversion capabilities were analysed in order to identify candidate bacteria that may influence growth performance. The jejunal microbiota was largely dominated by lactobacilli (over 99% of jejunal sequences) and showed no difference between the birds with high and low feed conversion ratios. The cecal microbial community displayed higher diversity, and 24 unclassified bacterial species were found to be significantly (<0.05) differentially abundant between high and low performing birds. Such differentially abundant bacteria represent target populations that could potentially be modified with prebiotics and probiotics in order to improve animal growth performance.

Up-regulated Desaturase and Elongase Gene Expression Promoted Accumulation of Polyunsaturated Fatty Acid (PUFA) but Not Long-Chain PUFA in Lates calcarifer, a Tropical Euryhaline Fish, Fed a Stearidonic Acid- and γ-Linoleic Acid-Enriched Diet

The limited activity of Δ6 fatty acid desaturase (FAD6) on α-linolenic (ALA, 18:3n-3) and linoleic (LA, 18:2n-6) acids in marine fish alters the long-chain (≥C₂₀) polyunsaturated fatty acid (LC-PUFA) concentration in fish muscle and liver when vegetable oils replace fish oil (FO) in aquafeeds. Echium oil (EO), rich in stearidonic acid (SDA, 18:4n-3) and γ-linoleic acid (GLA, 18:3n-6), may enhance the biosynthesis of n-3 and n-6 LC-PUFA by bypassing the rate-limiting FAD6 step. Nutritional and environmental modulation of the mechanisms in LC-PUFA biosynthesis was examined in barramundi, Lates calcarifer, a tropical euryhaline fish. Juveniles were maintained in either freshwater or seawater and fed different dietary LC-PUFA precursors present in EO or rapeseed oil (RO) and compared with FO. After 8 weeks, growth of fish fed EO was slower compared to the FO and RO treatments. Irrespective of salinity, expression of the FAD6 and elongase was up-regulated in fish fed EO and RO diets, but did not lead to significant accumulation of LC-PUFA in the neutral lipid of fish tissues as occurred in the FO treatment. However, significant concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), but not docosahexaenoic acid (DHA, 22:6n-3), appeared in liver and, to a lesser extent, in muscle of fish fed EO with marked increases in the phospholipid fraction. Fish in the EO treatment had higher EPA and ARA in their liver phospholipids than fish fed FO. Endogenous conversion of dietary precursors into neutral lipid LC-PUFA appears to be limited by factors other than the initial rate-limiting step. In contrast, phospholipid LC-PUFA had higher biosynthesis, or selective retention, in barramundi fed EO rather than RO.

Functional similarities between pigeon 'milk' and mammalian milk : induction of immune gene expression and modification of the microbiota

Pigeon ‘milk’ and mammalian milk have functional similarities in terms of nutritional benefit and delivery of immunoglobulins to the young. Mammalian milk has been clearly shown to aid in the development of the immune system and microbiota of the young, but similar effects have not yet been attributed to pigeon ‘milk’. Therefore, using a chicken model, we investigated the effect of pigeon ‘milk’ on immune gene expression in the Gut Associated Lymphoid Tissue (GALT) and on the composition of the caecal microbiota. Chickens fed pigeon ‘milk’ had a faster rate of growth and a better feed conversion ratio than control chickens. There was significantly enhanced expression of immune-related gene pathways and interferon-stimulated genes in the GALT of pigeon ‘milk’-fed chickens. These pathways include the innate immune response, regulation of cytokine production and regulation of B cell activation and proliferation. The caecal microbiota of pigeon ‘milk’-fed chickens was significantly more diverse than control chickens, and appears to be affected by prebiotics in pigeon ‘milk’, as well as being directly seeded by bacteria present in pigeon ‘milk’. Our results demonstrate that pigeon ‘milk’ has further modes of action which make it functionally similar to mammalian milk. We hypothesise that pigeon ‘lactation’ and mammalian lactation evolved independently but resulted in similarly functional products.

The role of the TP53 gene during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A nonsense mutation in the tyrosinase gene causes albinism in water buffalo

Background: Oculocutaneous albinism (OCA) is an autosomal recessive hereditary pigmentation disorder affecting humans and several other animal species. Oculocutaneous albinism was studied in a herd of Murrah buffalo to determine the clinical presentation and genetic basis of albinism in this species.Results: Clinical examinations and pedigree analysis were performed in an affected herd, and wild-type and OCA tyrosinase mRNA sequences were obtained. The main clinical findings were photophobia and a lack of pigmentation of the hair, skin, horns, hooves, mucosa, and iris. The results of segregation analysis suggest that this disease is acquired through recessive inheritance. In the OCA buffalo, a single-base substitution was detected at nucleotide 1,431 (G to A), which leads to the conversion of tryptophan into a stop codon at residue 477.Conclusion: This premature stop codon produces an inactive protein, which is responsible for the OCA buffalo phenotype. These findings will be useful for future studies of albinism in buffalo and as a possible model to study diseases caused by a premature stop codon.

Freqüência da mutação 844ins68 do gene da cistationina beta-sintetase em pacientes com trombose venosa profunda

A hiper-homocisteinemia, resultante da deficiência na conversão da homocisteína em cistationina, constitui em fator de risco isolado para doenças vasculares. A mutação 844ins68 do gene da cistationina beta-sintetase é um fator adicional de risco para a trombose venosa profunda. O objetivo deste estudo foi avaliar a freqüência da mutação 844ins68 do gene da cistationina beta-sintetase em pacientes com trombose venosa profunda. Foram avaliados em estudo caso-controle 95 pacientes com trombose venosa profunda, a presença da mutação 844ins68 no éxon 8 do gene da cistationina beta-sintetase. Como critério de inclusão foi adotada a presença de trombose venosa profunda confirmada pelo dúplex ou flebografia. O grupo controle constituiu-se de 95 doadores de sangue, sem história familiar prévia de trombose venosa, com sexo, grupo étnico e idades pareados aos do grupo de estudo. Foram coletados 5 mL de sangue venoso com o uso de anticoagulante EDTA de cada participante. O DNA foi extraído dos leucócitos pelo método DTAB e CTAB. A detecção da mutação do gene foi realizada por amplificação de um segmento gênico por PCR, com iniciadores que flanqueiam a região de inserção e com revelação em gel de agarose a 2%, corado com brometo de etídio, sob luz UV. O fragmento correspondente ao alelo normal contém 184 pares de base e o correspondente ao alelo mutante, 252 pares de base. O teste exato de Fisher foi utilizado na análise dos resultados. A condição heterozigota para a mutação foi encontrada em 14,73% dos pacientes e em 3,1% dos indivíduos do grupo controle (p = 0,009). A freqüência do alelo mutante mostrou diferença significativa (p = 0,01), sendo 0,074 para os pacientes versus 0,016 para o grupo controle. Não foram encontrados casos de homozigose.

Effect of glycerol on GHR and IGF-1 gene expression in breast muscle and on growth of Japanese meat quails

We evaluated messenger RNA (mRNA) expression of the growth-hormone (GHR) and insulin-like growth factor (IGF-1) genes in 28-day-old Japanese meat quails fed diets containing 0, 8, or 12% dietary glycerol in substitution of corn. Total RNA was extracted from the breast muscle and the DNA was amplified with specific primers using real-time PCR. Feed conversion ratio and feed intake were evaluated. The birds fed 8 and 12% glycerol presented higher IGF-1 mRNA expression [0.059 and 0.049 arbitrary units (AU), respectively] relative to those not fed with glycerol (0.029 AU), while 12% glycerol reduced GHR mRNA expression (0.022 AU). Dietary inclusion of 8% glycerol promoted similar performance results (feed conversion) as the diet with no glycerol. We conclude that inclusion of glycerol in the diet affects GHR and IGF-1 gene expression in Japanese meat quails. However, considering the performance results and the expression of the GHR and IGF-1 genes, 8% glycerol may be safely included in the diet of meat quails. © FUNPEC-RP.

Single CpG island methylation is not sufficient to maintain the silenced expression of CASPASE-8 apoptosis-related gene among women with epithelial ovarian cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise de Alterações Cromossômicas, Mutações no Éxon 1 e do Padrão de Metilação da Região Promotora do Gene FOXO3 em Síndrome Mielodisplásica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

Isolamento, variabilità ed espressione del gene PIMT in Girasole

Il vigore è un aspetto rilevante della qualità delle sementi, strettamente connesso al loro status fisiologico, al genotipo e alle condizioni di stoccaggio. Stress ossidativi e danni alle macromolecole sono alla base del deterioramento del seme, che è equipaggiato con sistemi protettivi e di riparazione. Uno di questi coinvolge l’L-isoaspartil metiltransferasi (PIMT) che ripara i misfolding proteici catalizzando la riconversione in aspartato dell’isoaspartile anomalo accumulato. Scopo di questo studio era valutare il possibile ruolo del meccanismo di riparazione di PIMT nel vigore del seme in girasole. Per questo il relativo gene è stato isolato e caratterizzato, la variabilità allelica determinata su un campione di linee inbred e l’espressione genica misurata in risposta all’invecchiamento accelerato (aging) e al priming. La sequenza codificante ottenuta è costituita da 4 esoni e contiene i 5 domini caratteristici delle metiltransferasi. Il gene mostra elevata similarità con gli ortologhi vegetali e scarsa diversità nucleotidica nei genotipi coltivati rappresentativi della variabilità della specie. Nella sequenza aminoacidica, comunque, sono state rinvenute tre sostituzioni che potrebbero influenzare la funzionalità enzimatica. Dal punto di vista fisiologico i genotipi considerati hanno esibito notevole variabilità di risposte ai trattamenti, sia in termini di vigore che di espressione genica. Aging e priming hanno prodotto generalmente gli effetti attesi, rispettivamente negativi e positivi, sulla germinabilità e sulla sua velocità. In generale l’espressione di PIMT è risultata massima nel seme secco, come riportato altrove, e ridotta dall’aging. Anche il priming ha diminuito l’espressione rispetto al seme quiescente, mentre il suo effetto dopo l’aging è risultato genotipo-dipendente. Tuttavia, nelle condizioni descritte, non si sono evidenziate correlazioni significative tra vigore ed espressione di PIMT, tali da suggerire un chiaro ruolo di questo meccanismo nella qualità fisiologica del seme.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.