Effect of high pressure on the structure and antibacterial activity of bovine lactoferrin treated in different media.

The effect of high-pressure treatment (400, 500 and 650 MPa) on the structure and activity of bovine lactoferrin in different iron-saturation forms has been studied by several techniques. The structural changes produced in lactoferrin by high-pressure were analysed by differential scanning calorimetry and fluorescence spectroscopy, and the immunoreactivity by ELISA. The effect of high-pressure was also studied on some biological properties of lactoferrin, such as iron binding capacity, retention of the bound iron, and antibacterial activity against Escherichia coli O157:H7. Results obtained indicate that treatment at 400 MPa does not substantially modify the conformation of lactoferrin, meanwhile treatments at 500 and 650 MPa greatly affect some of its properties. With respect to the antibacterial activity, the apo and native forms of lactoferrin maintain that activity against Esch. coli only after 400 MPa treatment.

Kinetic and thermodynamic parameters for heat denaturation of human recombinant lactoferrin from rice.

Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. D(t) values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf.

Effects of hydrostatic high pressure on the structure and antibacterial activity of recombinant human lactoferrin from transgenic rice.

High pressure was applied to recombinant human lactoferrin obtained from rice (rhLF) and its effect was evaluated on the structure and activity of the protein. Treatments of 400, 500, and 650 MPa for 15 min (20 °C), were applied to rhLF at 2 mg/mL in three iron-saturation forms. The structural characteristics of the treated proteins were analyzed by differential scanning calorimetry (DSC) and by fluorometric analysis, and immunoreactivity by ELISA. Iron retention and binding properties and antibacterial activity against Escherichia coli O157:H7 were also studied. The results obtained indicate that the treatments at 400 and 500 MPa did not greatly modifiy the conformation of lactoferrin, meanwhile treatment at 650 MPa affected in different degrees the three forms of rhLF. With respect to antibacterial activity, only apo rhLF showed antibacterial activity against E. coli, activity that was maintained after treatment at 400 MPa, while holo and AsIs rhLF did not inhibit the growth of E. coli.

Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii.

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

Effect of technological treatments on bovine lactoferrin: An overview.

Lactoferrin (LF) is a multifunctional protein that exerts important activities in the neonate through its presence in milk, and also in other external mucosas, acting as a defense protein of innate immunity. The addition of bovine LF to infant formula and also to other functional products and cosmetics has increased during the last decades. Consequently, it is essential to know the effect that the technological processes, necessary to elaborate those products, have on LF activity. In this study, we have revised the effect of classical treatments on lactoferrin structure and activity, such as heat treatment or drying, and also of emerging technologies, like high pressure or pulsed electric field. The results of the studies included in this review indicate that LF stability is dependent on its level of iron-saturation and on the characteristics of the treatment media. Furthermore, the studies revised here reveal that the non-thermal treatments are interesting alternatives to the traditional ones, as they protect better the structure and activity of lactoferrin. It is also clear the need for research on LF encapsulation by different ways, to protect its properties before it reaches the intestine. All this knowledge would allow designing processes less harmful for LF, thus maintaining all its functionality.

High expression of XIAP and Bcl-2 may inhibit programmed cell death in glioblastomas.

Glioblastoma (GBM) is the most malignant glioma and represents 29% of all brain tumors. Tumorigenesis is intimately connected with characteristics acquired in the physiologic pathway of cellular death. OBJECTIVE: In the present study, the expression of anti-apoptotic (XIAP and Bcl-2) and apoptotic (cytochrome C, caspase 9, APAF-1), caspase 3 and the Smac/DIABLO genes related to the apoptosis pathway were evaluated in 30 samples of glioblastoma. METHODS: The gene expression was evaluated in 30 glioblastomas (WHO grade IV) and compared to 10 white matter control samples with real-time PCR. RESULTS AND CONCLUSION: There were higher expressions of XIAP (p = 0.0032) and Bcl-2 (p = 0.0351) in the glioblastoma samples compared to the control samples of normal brain. These results raise the question of whether Bcl-2 and XIAP genes can be responsible for the inhibition of programmed cell death in glioblastomas. Moreover, they provide additional information capable of allowing the development of new target therapy strategies.

High expression of anti-apoptotic genes in grade I and II meningiomas.

Objective: To evaluate the expression of c-FLIP, XIAP, Bcl-2, caspase 3, 8 and 9, cytochrome c, APAF 1 and Smac/DIABLO genes related to apoptosis pathways. Methods: The gene expression was evaluated in 30 meningiomas (WHO grades I and II) and in 10 normal samples (from arachnoid tissue) through PCR-RT. Results: The results showed higher expression of anti-apoptotic genes in meningiomas when compared to the control group, which had a low expression of pro-apoptotic genes. Conclusion: There is a possible block in the activation of caspases through the intrinsic apoptosis pathway in meningiomas. c-FLIP modulates caspase 8 and, by inhibiting its activation due to the lack of connection with the receiver, there is a block to the FAS activation of apoptosis by its extrinsic pathway.

High expression of XIAP and Bcl-2 may inhibit programmed cell death in glioblastomas / Alta expressão de XIAP e Bcl-2 pode inibir morte celular programada em glioblastomas

ABSTRACT Glioblastoma (GBM) is the most malignant glioma and represents 29% of all brain tumors. Tumorigenesis is intimately connected with characteristics acquired in the physiologic pathway of cellular death. Objective: In the present study, the expression of anti-apoptotic (XIAP and Bcl-2) and apoptotic (cytochrome C, caspase 9, APAF-1), caspase 3 and the Smac/DIABLO genes related to the apoptosis pathway were evaluated in 30 samples of glioblastoma. Methods: The gene expression was evaluated in 30 glioblastomas (WHO grade IV) and compared to 10 white matter control samples with real-time PCR. Results and Conclusion: There were higher expressions of XIAP (p = 0.0032) and Bcl-2 (p = 0.0351) in the glioblastoma samples compared to the control samples of normal brain. These results raise the question of whether Bcl-2 and XIAP genes can be responsible for the inhibition of programmed cell death in glioblastomas. Moreover, they provide additional information capable of allowing the development of new target therapy strategies.

RESUMO O glioblastoma (GBM) é o glioma mais maligno e representa 29% de todos os tumores cerebrais. A tumorigênese está intimamente ligada à características adquiridas na via fisiológica de morte celular. Objetivo: Avaliar a expressão de genes anti-apoptóticos (XIAP e Bcl-2) e apoptóticos (citocromo C, a caspase 9, APAF-1), caspase 3 e SMAC/DIABLO, relacionados à apoptose, em 30 amostras de tecido de pacientes com glioblastoma. Métodos: A expressão gênica foi avaliada em trinta glioblastomas e comparada a dez amostras controles de substância branca por PCR em tempo real. Resultados e Conclusão: Houve maior nível de expressão de XIAP (p = 0,0032) e Bcl-2 (p = 0,0351) em comparação com as amostras controle, de cérebro normal. Estes resultados levantam a questão de que os genes Bcl-2 e XIAP podem ser responsáveis pela inibição da morte celular programada em glioblastomas, além disso, proporcionam informação adicional capaz de permitir o desenvolvimento de novas estratégias de terapia alvo.

High expression of anti-apoptotic genes in grade I and II meningiomas / Alta expressão de genes anti-apoptóticos em meningiomas graus I e II

ABSTRACT One of the different genetic mechanisms involved in the carcinogenesis of meningiomas is influenced by interactions between proteins that induce and inhibit apoptosis. Objective To evaluate the expression of c-FLIP, XIAP, Bcl-2, caspase 3, 8 and 9, cytochrome c, APAF 1 and Smac/DIABLO genes related to apoptosis pathways. Methods The gene expression was evaluated in 30 meningiomas (WHO grades I and II) and in 10 normal samples (from arachnoid tissue) through PCR-RT. Results The results showed higher expression of anti-apoptotic genes in meningiomas when compared to the control group, which had a low expression of pro-apoptotic genes. Conclusion There is a possible block in the activation of caspases through the intrinsic apoptosis pathway in meningiomas. c-FLIP modulates caspase 8 and, by inhibiting its activation due to the lack of connection with the receiver, there is a block to the FAS activation of apoptosis by its extrinsic pathway.

RESUMO Um dos diferentes mecanismos genéticos envolvidos na carcinogênese de meningiomas é influenciado por interações entre proteínas que induzem e inibem a apoptose. Objetivos Avaliar a expressão de c-FLIP, XIAP, Bcl-2, caspase 3, 8 e 9, citocromo C, APAF 1 e Smac/DIABLO, genes relacionados com as vias da apoptose. Métodos A expressão gênica foi avaliada em trinta amostras de meningiomas (OMS grau I e II) e em dez amostras normais (de aracnóide) por PCR em tempo real. Resultados Os resultados mostraram maior expressão de genes antiapoptóticos em meningiomas quando comparados com controle, em contraste com a menor expressão de genes próapoptóticos. Conclusão Há um possível bloqueio na ativação de caspases através da via intrínseca da apoptose em meningiomas. O c-FLIP modula a caspase 8 e, desse modo, inibindo a sua ativação pela ausência de ligação com o receptor, há um bloqueio na ativação de FAS pela via extrínseca da apoptose.