RESUMO
A side effect of the raised consumption of Greek yogurt is the generation of massive amounts of yogurt acid whey (YAW). The dairy industry has tried several methods for handling these quantities, which constitute an environmental problem. Although the protein content of YAW is relatively low, given the huge amounts of produced YAW, the final protein amount in the produced YAW should not be underestimated. Taking into consideration the increased interest for bioactive peptides and the increased demand for dietary proteins, combined with protein and peptides content of YAW, efforts should be made toward reintroducing the latter in the food supply chain. In this context and in view of the prevalent dietary iron deficiency problem, the objective of the present study was the investigation of YAW fractions' effect on Fe bioavailability. With this purpose, an in vitro digest approach, following the INFOGEST protocol, was coupled with the Caco2 cell model. To evaluate whether YAW digest fractions exert positive, negative or neutral effect on Fe bioavailability, they were compared with the ones derived from milk, a well-studied food in this context. Milk and YAW showed the same effectiveness on both Fe bioavailability and the expression of relative genes (DCYTB, DMT1, FPN1, and HEPH). Focusing further on YAW fractions, by comparison with their blank digest control counterparts, it resulted that YAW 3- to 10-kDa digests fraction had a superior effect over the 0- to 3-kDa fraction on Fe-uptake, which was accompanied by a similar effect on the expression of Fe metabolism-related genes (DCYTB, FPN1, and HEPH). Finally, although the 3- to 10-kDa fraction of bovine YAW digests resulted in a nonsignificant increased Fe uptake, compared with the ovine and caprine YAW, the expression of DCYTB and FPN1 genes underlined this difference by showing a similar pattern with statistically significant higher expression of bovine compared with ovine and bovine compared with both ovine and caprine, respectively. The present study deals with the novel concept that YAW may contain factors affecting Fe bioavailability. The results show that it does not exert any negative effect and support the extensive investigation for specific peptides with positive effect as well as that YAW proteins should be further assessed on the prospect that they can be used in human nutrition.
Assuntos
Ferro , Soro do Leite , Animais , Ovinos , Bovinos , Humanos , Ferro/metabolismo , Soro do Leite/química , Disponibilidade Biológica , Iogurte , Células CACO-2 , Cabras/metabolismo , Proteínas do Soro do Leite/análise , Peptídeos/metabolismoRESUMO
Yogurt acid whey (YAW) is a by-product of Greek strained yogurt production. The disposal of YAW constitutes an environmental problem, and given the increasing demand of Greek yogurt worldwide, its handling is a challenge. However, whey-derived peptides, resulting from microbial fermentation as well as those resulting from further hydrolysis during the digestion process, have been linked to enhanced biological activities. In this study, the antioxidant capacity of 33 samples of YAW obtained from Greek dairy companies of bovine, ovine or caprine origin was investigated using both cell-free and cell-based assays. The YAW samples, their in vitro digestion products (YAW-Ds) and a fraction of the digests (less than 3 kDa; YAW-D-P3) were assessed using four biochemical assays, namely ORAC, ABTS, FRAP and P-FRAP. Our data revealed a higher antioxidant capacity for digested samples compared with undigested samples, with all four methods. ORAC values after in vitro digestion were higher for the ovine samples compared to their bovine (YAW-D and YAW-D-P3) and caprine (YAW-D-P3) counterparts. Furthermore, the YAW-D-P3 fraction derived from samples collected in the summer months exhibited higher ORAC values when compared to the respective fraction from the winter months' samples. The cellular antioxidant activity of ovine YAW-D-P3 was improved in H2O2-treated HT29 cells compared to the control H2O2-treated cells. However, YAW-D-P3 could not trigger either the pathways involving the transcription factors NF-κB or NFE2L2 or the gene expression of SOD1, CAT and HMOX1 in LPS-challenged THP-1-derived macrophages. These results suggest that YAW, and particularly YAW from ovine origin, could be used as a natural source for its antioxidant potential in human and animal nutrition.
RESUMO
In addition to being essential for gene expression, transcription is crucial for the maintenance of genome integrity. Here, we undertook a systematic approach, to monitor the assembly kinetics of the pre-initiating RNA Polymerase (Pol) II at promoters at steady state and different stages during recovery from UV irradiation-stress, when pre-initiation and initiation steps have been suggested to be transiently shut down. Taking advantage of the reversible dissociation of pre-initiating Pol II after high salt treatment, we found that de novo recruitment of the available Pol II molecules at active promoters not only persists upon UV at all times tested but occurs significantly faster in the early phase of recovery (2 h) than in unexposed human fibroblasts at the majority of active genes. Our method unveiled groups of genes with significantly different pre-initiation complex (PIC) assembly dynamics after UV that present distinct rates of UV-related mutational signatures in melanoma tumours, providing functional relevance to the importance of keeping transcription initiation active during UV recovery. Our findings uncover novel mechanistic insights further detailing the multilayered transcriptional response to genotoxic stress and link PIC assembly dynamics after exposure to genotoxins with cancer mutational landscapes.
Assuntos
RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Dano ao DNA , Mutagênese , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Raios Ultravioleta , Fibroblastos/metabolismo , Reparo do DNARESUMO
G-Quadruplex structures are non-B DNA structures that occur in regions carrying short runs of guanines. They are implicated in several biological processes including transcription, translation, replication and telomere maintenance as well as in several pathological conditions like cancer and thus have gained the attention of the scientific community. The rise of the -omics era significantly affected the G-quadruplex research and the genome-wide characterization of G-Quadruplexes has been rendered a necessary first step towards applying genomics approaches for their study. While in human and several model organisms there is a considerable number of works studying genome-wide the DNA motifs with potential to form G-quadruplexes (G4-motifs), there is a total absence of any similar studies regarding livestock animals. The objectives of the present study were to provide a detailed characterization of the bovine genic G4-motifs' distribution and properties and to suggest a possible mechanism for the delivery of G4-motifs in the genes. Our data indicate that the distribution of G4-motifs within bovine genes and the annotation of said genes to Gene Ontology terms are similar to what is already shown for other organisms. By investigating their structural characteristics and polymorphism, it is obvious that the overall stability of the putative quadruplex structures is in line with the current notion in the G4 field. Similarly to human, the bovine G4-motifs are overrepresented in specific LINE repeat elements, the L1_BTs in the case of cattle. We highlight the potential role of these elements as vehicles for delivery of G4-motifs in the introns of the bovine genes. Lastly, it seems that a basis exists for connecting traits of agricultural importance to the genetic variation of G4-motifs, thus, the value of cattle as an interesting new model organism for G4-related genetic studies might be worth investigating.
Assuntos
Quadruplex G , Animais , Bovinos/genética , DNA/química , Genoma , GenômicaRESUMO
BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the plasminogen activation system. Although several lines of evidence support a significant role of PAI-1 in the brain, the regulation of its expression in neurons is poorly understood. In the present study we tested the hypothesis that NGF induces the upregulation of PAI-1 via the calcineurin/nuclear factor of activated T cells (NFAT) pathway and analysed whether the overexpression of the Down syndrome-related proteins DYRK1A and RCAN1 modulated the effect of NGF on PAI-1 expression. RESULTS: NGF upregulated PAI-1 mRNA levels in primary mouse hippocampal neurons cultured for 3 days in vitro and in the rat pheochromocytoma cell line PC12. Reporter gene assays revealed that NGF activated the calcineurin/NFAT pathway in PC12 cells. Induction of PAI-1 by NGF was sensitive to the calcineurin inhibitor FK506 and the specific inhibition of NFAT activation by the cell permeable VIVIT peptide. Activation of calcineurin/NFAT signalling through other stimuli resulted in a much weaker induction of PAI-1 expression, suggesting that other NGF-induced pathways are involved in PAI-1 upregulation. Overexpression of either DYRK1A or RCAN1 negatively regulated NFAT-dependent transcriptional activity and reduced the upregulation of PAI-1 levels by NGF. CONCLUSION: The present results show that the calcineurin/NFAT pathway mediates the upregulation of PAI-1 by NGF. The negative effect of DYRK1A and RCAN1 overexpression on NGF signal transduction in neural cells may contribute to the altered neurodevelopment and brain function in Down syndrome.
Assuntos
Síndrome de Down/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Calcineurina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fatores de Transcrição NFATC/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Ratos , Regulação para Cima/efeitos dos fármacos , Quinases DyrkRESUMO
BACKGROUND: Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells). We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. RESULTS: The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 µM). UTP, 4-benzoylbenzoyl ATP and α,ß-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS). This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin) and BDNF (brain derived neurotrophic factor). CONCLUSION: The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein kinase cascade.
Assuntos
Trifosfato de Adenosina/fisiologia , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição NFATC/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Receptores Purinérgicos P2X/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/fisiologia , Neurônios/efeitos dos fármacos , Nifedipino/farmacologia , Células PC12 , Ratos , Receptores Purinérgicos P2X/genéticaRESUMO
While in human and rodents lipogenesis occurs predominantly in the liver, adipose tissue is the major site in ruminants. Here we report the nucleotide sequence of the 5'-flanking region of the ovine malic enzyme gene (ME1). The ME1 promoter is located within a GC-rich region fulfilling the criteria of CpG islands and lacks a TATA-box. Deletion analysis identified a region (-231/-170) that suppressed promoter activity in luciferase assays in HepG2 hepatoma cells but not in 3T3-L1 adipocytes. This region contains a putative triiodothyronine response element (T3RE) that differs from the human ME1 T3RE by two nucleotides. When the human ME1 T3RE was introduced into the ovine ME1 promoter context, transcriptional activity was increased in the hepatic cell lines HepG2 and H4IIE but not in differentiated 3T3-L1 cells. Our results suggest that the sequence of the T3RE in the ME1 promoter determines differences in the tissue/species activity of malic enzyme in ruminants and human.