Riboflavin:A multifunctional vitamin.

Riboflavin, a component of the B-2 vitaminic complex, plays important roles in biochemistry, especially in redox reactions, due to the ability to participate in both one- and two-electron transfers as well as acting as a photosensitizer. Accordingly, low intakes of this vitamin have been associated with different diseases, including cancer and cardiovascular diseases. Riboflavin is thought to contribute to oxidative stress through its capacity to produce superoxide but, interestingly, it can also promote the reduction of hydroperoxides. This peculiar and multifunctional behavior allows riboflavin to take part in various biochemical pathways as a nucleophile and an electrophile, turning it into a versatile and important biological compound

Cytotoxicity of Dehydrocrotonin (a Nor-Clerodane from Croton cajucara) on Human Lymphocyte's

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Trans-Dehydrocrotonin, a 19-nor-clerodane, is the major norditerpene obtained from Croton cajucara, a Brazilian medicinal plant which presents important biological effects, such as antineoplastic and antiulcerogenic activities. In this work, we analyzed the effect of this sesquiterpene lactone on normal human lymphocytes. The cell viability was verified after treatment for 24 and 72 h with trans-dehydrocrotonin, in the presence and absence of phytohemagglutin (specific mitogen for this cell), through three end-points to assess cytotoxicity in vitro: MTT reduction (mitochondrial function), protein quantification (cell number) and phosphatase activity (cell metabolism). When the cells were treated with dehydrocrotonin in the presence of mitogen, no toxic effect was observed. Nevertheless, in the absence of mitogen, the IC50 was 450,mu M for MTT reduction and phosphatase activity. Moreover, in this condition, trans-dehydrocrotonin caused stimulation of protein content from 100 mu M. Our results suggest that phytohemagglutin protects human lymphocytes against the trans-dehydrocrotonin toxic effect.276914917Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

K. G. WETTERLUND: Några tull- och handelspolitiska spörsmål. Udg. af Skånes Handelskammare, -U S. Malmø 1918.

The effects of two lectins concanavalin A (conA) and soybean agglutinin, on soybean seed acid phosphatase activity were investigated using p-nitrophenylphosphate (pNPP), pyrophosphate (PPi) and phosphoenolpyruvate (PEP) as substrates. Of the four acid phosphatase isoforms (AP1, AP2, AP3A and AP3B) purified from soybean seeds, only API was activated 40 and 60% by conA and soybean agglutinin, respectively. Both lectins affected some of the kinetic parameters of AP1. The activation by lectins was not affected by 1 mM Ca2+ or Mn2+ but glucose and methylmannopyranoside (100 mM) prevented activation by conA. Under the same conditions, galactose had no effect. These results suggest that plant acid phosphatases may be regulated by lectins, the effects vary according to the substrate used. (C) 2001 Elsevier Science Ltd. All rights reserved.58222122

Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaG(u), was calculated at 25 degreesC as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole. (C) 2004 Elsevier Ltd. All rights reserved.65783183

Kinectic characterization and flavonoid effect on human lymphocyte protein tyrosyne phosphatase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The aim of this work was to determine the kinetic properties and evaluate the effect of some flavonoids on human lymphocyte protein tyrosine phosphatase. Tyrosine-, serine- and threonine-phosphate were hydrolyzed by this phosphatase 60%, 20% and 10%, respectively. In the kinetic studies the enzymatic activity was determined by using p-nitrophenylphosphate (pNPP) as substrate. The enzyme presented optimum pH around 5.0 and was inhibited by 100 mu M p-chloromercuribenzoate (pCMB) (80%), 10 mM fluoride (35%), 10 mM vanadate (100%), and 5mM Cu+2 (85%). The enzyme was also strongly inhibited by a tyrosine phosphatase inhibitor cocktail, but was unaffected by okadaic acid. These results confirm that the major phosphatase activity in human lymphocytes is a protein tyrosine phosphatase. Among the bioflavonoids tested only fisetin showed an inhibitory effect in order of 80% on the enzymatic activity.273436439Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45degreesC using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55degreesC. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60degreesC. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60degreesC, with the reaction products inorganic phosphate - P (10 mM) and p-nitrophenol - p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (T-m) value of 75degreesC and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.26641671111

TNF alpha contributes for attenuating both Y(397)FAK and Y(416)Src phosphorylations in osteoblasts

ObjectiveOur poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor (TNF)-induced focal adhesion kinase (FAK) and Src phosphorylation. Material and MethodsMC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNF treatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNF content at conditioned medium. Tumor necrosis factor--neutralizing antibodies also were used. ResultsIt was possible to show that TNF provokes attenuation at Y-phosphorylation of both FAK (at Y-397) and Src (at Y-416) proteins (P<0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide (LPS)-stimulated macrophages, it being significantly different than control (FAK and Src, P<0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti-TNF neutralizing antibody (2gml(-1)) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated (P<0.05). Moreover, we also showed that TNF impairs osteoblast adhesion, suggesting an interesting role on osteoblast performance. ConclusionsAltogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNF on osteoblast performance

Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling

Objective. Tetrahydroxyquinone is a molecule best known as a primitive anticataract drug but is also a highly redox active molecule that can take part in a redox cycle with semiquinone radicals, leading to the formation of reactive oxygen species (ROS). Its potential as an anticancer drug has not been investigated. Methods. The effects of tetrahydroxyquinone on HL60 leukemia cells are investigated using fluorescein-activated cell sorting-dependent detection of phosphatidylserine exposure combined with 7-amino-actinomycin D exclusion, via Western blotting using phosphospecific antibodies, and by transfection of constitutively active protein kinase B. Results. We observe that in HL60 leukemia cells tetrahydroxyquinone causes ROS production followed by apoptosis through the mitochondrial pathway, whereas cellular physiology of normal human blood leukocytes was not affected by tetrahydroxyquinone. The antileukemic effect of tetrahydroxyquinone is accompanied by reduced activity of various antiapoptotic survival molecules including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells and thus artificially increasing protein kinase B activity inhibits tetrahydroxyquinone-dependent cytotoxicity. Conclusion. Tetrahydroxyquinone provokes cytotoxic effects on leukemia cells by reduced protein kinase B-dependent survival signaling followed by apoptosis through the mitochondrial pathway. Thus, tetrahydroxyquinone may be representative of a novel class of chemotherapeutic drugs, inducing apoptosis in cancer cells through diminished survival signaling possibly as a consequence of ROS generation. (C) 2006 International Society for Experimental Hematology. Published by Elsevier Inc

Osteoblast Adhesion Dynamics: A Possible Role for ROS and LMW-PTP

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very-known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW-PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW-PTP activity. Our results showed that during osteoblast adhesion/spreading (30min and 2h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti-oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y-397). Moreover, after 2h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW-PTP expression at 30min or 2h. In order to validate our hypothesis that LMW-PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW-PTP in this context. Our results showed that FAK phosphorylation at Y-397 was increased and decreased in osteoblasts with silenced or overexpressed LMW-PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW-PTP/FAK supra-molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063-1069, 2014. (c) 2013 Wiley Periodicals, Inc.115610631069Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP