Background In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference... more
Background In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction. Results In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated. Conclusion Good reference genes that reflect total RNA ...
Research Interests: RNA, Biology, Medicine, Gene expression, Biological Sciences, and 15 moreFemale, Animals, Early development, Blastocyst, Genes, Embryonic Development, Oocytes, Embryos, Ribosomal Protein, Biochemistry and cell biology, Gene expression profiling, Reference standards, real time polymerase chain reaction, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to... more
Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls. Some of us have previously shown that tyrosinase is involved in truffle development and differentiation. Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis[methylthio]methane, the main flavour compounds of black and whitish truffles. The MWr is 39 000. L-3,4-dihydroxyphenylalanine and L-tyrosine stain corresponding bands as expected for a true tyrosinase. Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L-tyrosine and L-3,4dihydroxyphenylalanine oxidation. < 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
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Research Interests: Biology, Medicine, Brain, Kidney, Liver, and 10 moreFemale, Animals, Glutathione, Muscles, Isoenzymes, Bufo Bufo, Glutathione Transferase, Ovary, Bufo, and Tissue distribution
1. Harding-Passey mouse-melanoma tyrosinase (EC 1.14.18.1) is inhibited during L-3,4-dihydroxyphenylalanine oxidation by reaction products. L-3,4-dihydroxyphenyl 3-[14C]alanine oxidation products bind to the enzyme, as demonstrated by gel... more
1. Harding-Passey mouse-melanoma tyrosinase (EC 1.14.18.1) is inhibited during L-3,4-dihydroxyphenylalanine oxidation by reaction products. L-3,4-dihydroxyphenyl 3-[14C]alanine oxidation products bind to the enzyme, as demonstrated by gel electrophoresis and radioactivity measurements. 2. The enzyme interacts with indoles and oxidizes dopamine and norepinephrine. 3. L-epinephrine activates tyrosinase at non hormonal concentrations and bovine serum albumin protects the enzyme from auto-inhibition. 4. The inhibition of the Harding-Passey mouse-melanoma tyrosinase, during substrate oxidation, is very similar to that of mushroom enzyme.
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It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate... more
It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.
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Research Interests: Physiology, Biology, Medicine, Pergamon, Animals, and 14 morePeptides, Protease Inhibitors, Substrate Specificity, Multienzyme complexes, Cysteine endopeptidases, Molecular Mass, Bufo Bufo, Molecular weight, Amino Acid Sequence, Hydrolysis, Biochemistry and cell biology, Chymotrypsin, Molecular Sequence Data, and ovum
White and black truffles of the genus Tuber are Ascomycotina as well as Neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. Tyrosinase expression dependent on reproductive differentiation has been also... more
White and black truffles of the genus Tuber are Ascomycotina as well as Neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. Tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. We present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. L-tyrosine 3-monooxygenase and L-DOPA oxidase activities colocalize as histochemically detected and are similarly located in white and black truffles, from the hypothecium through the sporogenic hyphae to asci and spores. Sulfur components of truffle flavours reversibly inhibit tyrosinase. The respiratory phenotype of truffle mitochondria is discussed in relation to reproductive differentiation and melanogenesis.
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Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since... more
Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.
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Research Interests: Biology, Mitochondria, Animals, Oxidative phosphorylation, Bufo Bufo, and 12 moreIndexation, Cell Proliferation, Bovine Serum Albumin, Embryos, Oxygen Consumption, Succinate Dehydrogenase, Biochemistry and cell biology, Nucleic Acid, Adenosine Triphosphate, Adenosine Diphosphate, Mitochondrion, and Reduced glutathione
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... Truffle tyrosinase: Properties and activity Michele Miranda&amp;#x27;, Antonella Bonfiglia, Osvaldo Zarivia, Anna Maria Ragnellia, Giovanni Pacionib and ... BC Malmstrom, LE Andreasson and B. Reinhammar, Copper-containing oxidases... more
... Truffle tyrosinase: Properties and activity Michele Miranda&amp;#x27;, Antonella Bonfiglia, Osvaldo Zarivia, Anna Maria Ragnellia, Giovanni Pacionib and ... BC Malmstrom, LE Andreasson and B. Reinhammar, Copper-containing oxidases and superoxide dismutase, in: PD Boyer (Ed.), The ...
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The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and... more
The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.
Research Interests: Phytochemistry, Melanin, Biology, Immunohistochemistry, Medicine, and 15 moreQuantitative PCR, Biological Sciences, Enzyme, CHEMICAL SCIENCES, Fungal genome size, Mycorrhizae, Amino Acid Sequence, Cell Wall, Tyrosinase, Sexual Differentiation, Ascomycota, Binding Site, Molecular Sequence Data, Mycelium, and Medical and Health Sciences
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Research Interests: Genomics, Molecular Evolution, Symbiosis, Multidisciplinary, Nature, and 15 moreCrop Production, Ectomycorrhizal fungi, Molecular Phylogenetics and Evolution, Enzyme, Sulfur, Fungal genome size, CARBOHYDRATES, Genome sequence, Plant Cell Wall, Genetic Predisposition, Cell Wall, Ascomycota, Gene Family, Molecular Sequence Data, and Haploidy
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The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution... more
The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.
Research Interests: Embryo, Humans, Kidney, Liver, Female, and 9 moreAnimals, Glutathione, Clinical Sciences, Rats, Isoenzymes, Bufo Bufo, Glutathione Transferase, Ovary, and Embryos
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We studied the levels of antioxidant and detoxifying enzymes in the livers and lungs of young and old rats kept under hypoxic or hyperoxic conditions as models of oxidative stress. In particular, we investigated the levels of enzymes... more
We studied the levels of antioxidant and detoxifying enzymes in the livers and lungs of young and old rats kept under hypoxic or hyperoxic conditions as models of oxidative stress. In particular, we investigated the levels of enzymes directly involved in active oxygen species scavenging (superoxide dismutase, catalase and glutathione peroxidase-selenium dependent) and enzymes challenged with detoxification processes (glutathione transferase, glyoxalase I and glutathione reductase) in order to obtain a wide comparative view of the defence strategies used with respect to the age of the animals. The results show that the responses of some protective enzymes in young rats are opposite to those of old ones. Some of the changes found appear mainly due to age, while others appear to be due only to the oxygen tensions and are independent of the aging process. The glutathione contents of the liver and lung from young and old rats under hypoxic and hyperoxic conditions were measured.
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Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be... more
Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.
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The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in... more
The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism. A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development. In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney. No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.
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Research Interests: Free Radicals, Transcription Factors, Apoptosis, Signal Transduction, Free Radical, and 15 moreCaspases, Cell line, Humans, Reactive Oxygen Species, Glioblastoma, Glutathione, Catalase, Glutathione Peroxidase, Cancer Chemotherapy, Signal Transduction Pathway Models, Superoxide Dismutase, Neuroblastoma, Antioxidant enzyme, Biochemistry and cell biology, and Free Radical Biology
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Changes in melanosome protein composition duringBufo bufo development have been investigated. The number of bands in SDS-PAGE varies from stage 7 (sixth cleavage) to stage 19 (heart beat), with a minimum at stages 17 (tail bud) and 19... more
Changes in melanosome protein composition duringBufo bufo development have been investigated. The number of bands in SDS-PAGE varies from stage 7 (sixth cleavage) to stage 19 (heart beat), with a minimum at stages 17 (tail bud) and 19 (heart beat). At least ...