Alexandre D. M. Cavagis

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at μM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.

Endogenous lectin as a possible regulator of the hydrolysis of physiological substrates by soybean seed acid phosphatase

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 AP1 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.

Defining the molecular basis of tumor metabolism: a continuing challenge since Warburg's discovery.

Cancer cells are the product of genetic disorders that alter crucial intracellular signaling pathways associated with the regulation of cell survival, proliferation, differentiation and death mechanisms. The role of oncogene activation and tumor suppressor inhibition in the onset of cancer is well established. Traditional antitumor therapies target specific molecules, the action/expression of which is altered in cancer cells. However, since the physiology of normal cells involves the same signaling pathways that are disturbed in cancer cells, targeted therapies have to deal with side effects and multidrug resistance, the main causes of therapy failure. Since the pioneering work of Otto Warburg, over 80 years ago, the subversion of normal metabolism displayed by cancer cells has been highlighted by many studies. Recently, the study of tumor metabolism has received much attention because metabolic transformation is a crucial cancer hallmark and a direct consequence of disturbances in the activities of oncogenes and tumor suppressors. In this review we discuss tumor metabolism from the molecular perspective of oncogenes, tumor suppressors and protein signaling pathways relevant to metabolic transformation and tumorigenesis. We also identify the principal unanswered questions surrounding this issue and the attempts to relate these to their potential for future cancer treatment. As will be made clear, tumor metabolism is still only partly understood and the metabolic aspects of transformation constitute a major challenge for science. Nevertheless, cancer metabolism can be exploited to devise novel avenues for the rational treatment of this disease.Cancer cells are the product of genetic disorders that alter crucial intracellular signaling pathways associated with the regulation of cell survival, proliferation, differentiation and death mechanisms. The role of oncogene activation and tumor suppressor inhibition in the onset of cancer is well established. Traditional antitumor therapies target specific molecules, the action/expression of which is altered in cancer cells. However, since the physiology of normal cells involves the same signaling pathways that are disturbed in cancer cells, targeted therapies have to deal with side effects and multidrug resistance, the main causes of therapy failure. Since the pioneering work of Otto Warburg, over 80 years ago, the subversion of normal metabolism displayed by cancer cells has been highlighted by many studies. Recently, the study of tumor metabolism has received much attention because metabolic transformation is a crucial cancer hallmark and a direct consequence of disturbances in the activities of oncogenes and tumor suppressors. In this review we discuss tumor metabolism from the molecular perspective of oncogenes, tumor suppressors and protein signaling pathways relevant to metabolic transformation and tumorigenesis. We also identify the principal unanswered questions surrounding this issue and the attempts to relate these to their potential for future cancer treatment. As will be made clear, tumor metabolism is still only partly understood and the metabolic aspects of transformation constitute a major challenge for science. Nevertheless, cancer metabolism can be exploited to devise novel avenues for the rational treatment of this disease.

Cyanogenic Residues: Environmental Impacts, Complexation with Humic Substances, and Possible Application as Biofertilizer

The disposal of cyanogenic residues from the processing of cassava, during the flour production in certain regions of the Brazilian northeastern, has been a cause of concern in recent years, since this practice may lead to environmental imbalances. The results obtained in this work show a possible impact caused by the release of this kind of waste into water bodies, as well as its potential use as biofertilizer, mainly due to its high nutrient content. Humic substances (HS) from water and soil showed high interaction with cyanide ions (CN−), being the main responsible for the bioavailability of these ions into the environment. Furthermore, studies in microcosms propose viable and low-cost alternatives to decrease the levels of CN− ions in the liquid waste (called “manipueira”), as well as its potential use as biofertilizer.

Essential sulfhydryl groups in the active site of castor bean (Ricinus communis) seed acid phosphatase

Abstract In order to determine which amino acids are involved in substrate binding, castor bean seeds acid phosphatase was treated with amino acid-modifying reagents, such as phenylglyoxal (PGO), iodoacetic acid (IAA), N-bromosuccinimide (NBS), N-acetylimidazole (NAI), diethylpyrocarbonate (DEPC), N,N′-dicyclohexylcarbodiimide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), specific for arginine, cysteine, tryptophan, tyrosine, histidine, and aspartic and glutamic acids, respectively. Enzyme activity was determined using p -nitrophenylphosphate ( p NPP) as substrate. Enzyme inhibition was observed with IAA, NBS, EDC and DCCD. In the presence of the reaction products, p -nitrophenol ( p NP) and inorganic phosphate (Pi), which are competitive inhibitors, the enzyme was protected from inactivation by IAA, indicating involvement of the enzyme active site. The inhibition by IAA was time- and concentration-dependent, with an apparent bimolecular rate constant of 48×10 −4 M −1 s −1 , and two molecules of IAA bound per active site. Our results suggest that sulfhydryl groups are essential for enzyme catalysis, and are located in or near the substrate-binding domain. Other amino acids such as tryptophan, aspartic and glutamic acids, were also important for the enzyme activity, but were probably located outside of the active site.

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

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 (30 min and 2 h 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 30 min 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 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. 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 Y397 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.

Influence of the extractant on the complexing capacity of humic substances from peat for macro and micronutrients using continuous flow: agricultural application and environmental impacts

The aim of the present work was to optimize a procedure for extraction of humic substances (HS) from peat, using continuous flow with different extractants (NaOH, KOH and Na4P2O7). The influence of extractant on the complexing capacity of humic substances for macro and micronutrients was also investigated. Our results showed that the extraction by flow is efficient, reducing the time required in the extraction step and, furthermore, indicating that the extracted HS have a high affinity for the essential nutrients Ca and Mg. Moreover, an enhanced complexation was achieved by using Na4P2O7, demonstrating not only the efficiency of this compound as extractant, but also its promising application in the process, since its high complexing capacity allows to make available the complexed nutrients as well as diminishing the amount of these nutrients that could be lixiviated, thus contributing to prevent environmental contaminations.

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 45 °C 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 55 °C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 °C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 °C, with the reaction products inorganic phosphate—P (10 mM) and p-nitrophenol—p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 °C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule (Mol Cell Biochem 266: 11–15, 2004)

Osteoblast Adhesion Dynamics: A Possible Role for ROS and LMW-PTP: LMW-PTP AND ROS SIGNALING IN OSTEOBLAST ADHESION

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 (30 min and 2 h 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 30 min 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 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. 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 Y397 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.

Osteoblast Adhesion Dynamics

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 (30 min and 2 h 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 30 min 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 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. 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 Y397 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.