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2.
Int J Mol Sci ; 20(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769810

RESUMO

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP (p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.


Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Derivados de Hidroxietil Amido/farmacologia , Antígeno de Macrófago 1/genética , Neutrófilos/efeitos dos fármacos , Quimiotaxia de Leucócito/genética , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Derivados de Hidroxietil Amido/química , Interleucina-8/química , Interleucina-8/metabolismo , Antígeno de Macrófago 1/química , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/química
3.
Chem Biol Interact ; 294: 167-177, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30170107

RESUMO

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (µM), although pre-treatment with 100 µM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.


Assuntos
6-Aminonicotinamida/farmacologia , Leishmania/metabolismo , Metabolômica , Via de Pentose Fosfato/efeitos dos fármacos , Aminoácidos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Interações Hidrofóbicas e Hidrofílicas , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fosfogluconato Desidrogenase/metabolismo
4.
Mediators Inflamm ; 2016: 3648719, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27872513

RESUMO

Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2 level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n = 57) or treated with LF 4 hours before amniocentesis (n = 54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p < 0.01, p < 0.005, and p < 0.001, resp.). Conversely, active MMP-2 (p < 0.0001) and MMP-2/TIMP-2 molar ratio (p < 0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial number NCT02695563.


Assuntos
Líquido Amniótico/metabolismo , Dinoprostona/metabolismo , Lactoferrina/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Vagina/metabolismo , Aborto Espontâneo/prevenção & controle , Adulto , Amniocentese , Anti-Inflamatórios/uso terapêutico , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Gravidez , Risco
5.
Dis Markers ; 2016: 3672353, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27555667

RESUMO

Matrix Metalloproteases (MMPs) and cytokines have been involved in the pathogenesis of multiple sclerosis (MS). However, no studies have still explored the possible associations between the two families of molecules. The present study aimed to evaluate the contribution of active MMP-9, active MMP-2, interleukin- (IL-) 17, IL-18, IL-23, and monocyte chemotactic proteins-3 to the pathogenesis of MS and the possible interconnections between MMPs and cytokines. The proteins were determined in the serum and cerebrospinal fluid (CSF) of 89 MS patients and 92 other neurological disorders (OND) controls. Serum active MMP-9 was increased in MS patients and OND controls compared to healthy subjects (p < 0.001 and p < 0.01, resp.), whereas active MMP-2 and ILs did not change. CSF MMP-9, but not MMP-2 or ILs, was selectively elevated in MS compared to OND (p < 0.01). Regarding the MMPs and cytokines intercorrelations, we found a significant association between CSF active MMP-2 and IL-18 (r = 0.3, p < 0.05), while MMP-9 did not show any associations with the cytokines examined. Collectively, our results suggest that active MMP-9, but not ILs, might be a surrogate marker for MS. In addition, interleukins and MMPs might synergistically cooperate in MS, indicating them as potential partners in the disease process.


Assuntos
Interleucinas/sangue , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Esclerose Múltipla/sangue , Adulto , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Quimiocina CCL3/sangue , Quimiocina CCL3/líquido cefalorraquidiano , Feminino , Humanos , Interleucinas/líquido cefalorraquidiano , Masculino , Metaloproteinase 2 da Matriz/líquido cefalorraquidiano , Metaloproteinase 9 da Matriz/líquido cefalorraquidiano , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano
6.
Mult Scler ; 21(9): 1121-30, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25662349

RESUMO

BACKGROUND: The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown. OBJECTIVE: To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis. METHODS: We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing-remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls. RESULTS: Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively). CONCLUSION: Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity.


Assuntos
Metaloproteinase 9 da Matriz/sangue , Esclerose Múltipla Recidivante-Remitente/enzimologia , Esclerose Múltipla Recidivante-Remitente/patologia , Adulto , Encéfalo/patologia , Feminino , Humanos , Isoenzimas/sangue , Imageamento por Ressonância Magnética , Masculino , Esclerose Múltipla Recidivante-Remitente/sangue , Inibidor Tecidual de Metaloproteinase-1/metabolismo
7.
Inflamm Res ; 63(5): 325-8, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24458309

RESUMO

OBJECTIVES AND DESIGN: We investigated the effect of balanced (BS) and unbalanced (UBS) solutions in the absence or presence of hydroxyethyl starch (HES) on neutrophil functionality, evaluating the release of matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), and MMP-8. MATERIALS AND METHODS: Neutrophils were isolated by gradient centrifugation and dextran sedimentation and incubated in BS or UBS without or with HES, in the absence or presence of Interleukin-8 (IL-8) or Lipopolysaccharide (LPS). MMP-9, MPO, and MMP-8 were assayed by commercially available ELISA kits. RESULTS: There was not any influence of volume replacement solutions on the release of the enzymes from resting neutrophils. After IL-8 stimulation, the release of MMP-9 was higher in BS than in UBS or RPMI-1640, whereas HES enhanced its release regardless of the composition. After LPS stimulation, the release of MMP-9 was higher in both UBS and BS than RPMI-1640, but HES brought its release back to physiological conditions. No difference was found in the release of MPO and MMP-8 after stimulation with IL-8 or LPS. CONCLUSION: Volume replacement solutions might have an impact on the release of MMP-9 depending on the inflammatory milieu, suggesting that the use of balanced or unbalanced solutions is not a neutral choice.


Assuntos
Inflamação/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Neutrófilos/enzimologia , Humanos , Derivados de Hidroxietil Amido/farmacologia , Interleucina-8/farmacologia , Lipopolissacarídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Soluções
8.
J Inflamm (Lond) ; 10(1): 29, 2013 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-24059479

RESUMO

BACKGROUND: Administration of normal saline might increase circulating levels of pro-inflammatory cytokines and may cause variation of plasmatic electrolytic and hyperchloremic acidosis, which in turn can impair renal function. Hence the use of balanced solutions could influence the inflammatory cascade triggered by the surgical procedures, the plasmatic electrolyte concentration, the acid-base equilibrium, and the renal function. METHODS: This is a double blind randomized trial. Forty patients undergoing major abdominal surgery (bowel cancer) were allocated in two groups, the balanced solution (BS) group in which the fluids administered were balanced solutions (colloids and crystalloids); and the unbalanced solution (UBS) group in which the fluids administered were unbalanced solutions (colloids and crystalloids). Measurements were performed after anaesthesia induction (T0), at the end of surgery (T1), within 2 h after surgery (T2) and 24 h after the beginning of surgery (T3). The following data were collected: 1) active matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1), IL-6, IL-8, IL-10; 2) blood gases variables; 3) electrolytes, albumin, total serum protein and the strong ion difference; 4) neutrophil gelatinase-associated lipocalin (NGAL) from urinary sample. RESULTS: The BS group exhibited higher circulating level of IL-10 and TIMP-1 and lower level of active MMP-9. The UBS group experienced hypercloremia, hypocalcemia, hypomagnesemia, worse acid-base equilibrium and higher level of NGAL. CONCLUSIONS: The use of balanced solutions was responsible of less alteration of plasmatic electrolytes, acid-base equilibrium, kidney function and it might be associated with an early anti-inflammatory mechanisms triggering. TRIAL REGISTRATION: ClinicalTrials.gov (Ref: NCT01320891).

9.
Mol Cell Biochem ; 381(1-2): 243-55, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23737137

RESUMO

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.


Assuntos
Membrana Celular/metabolismo , Antígenos HLA-G/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteólise , Sequência de Aminoácidos , Anticorpos Bloqueadores/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Ácido Edético/farmacologia , Antígenos HLA-G/química , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Metaloproteinase 2 da Matriz/química , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Dados de Sequência Molecular , Proteólise/efeitos dos fármacos
10.
PLoS One ; 7(11): e48781, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23152803

RESUMO

Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.


Assuntos
Células Dendríticas/virologia , Anticorpos Anti-HIV/metabolismo , HIV-1/metabolismo , Integrinas/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação , Células Dendríticas/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Integrinas/imunologia , Macaca fascicularis , Masculino , Simulação de Acoplamento Molecular , Testes de Neutralização , Oligopeptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Internalização do Vírus , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia
11.
J Biochem ; 151(5): 493-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22343748

RESUMO

In vitro activation of matrix metalloproteinase-9 (MMP-9) (Gelatinase B) with MMP-3 shows the presence of two different forms: an 82 kDa, N-terminal truncated form, and a 65 kDa, N- and C-terminal truncated form. So far the presence of the 65 kDa form has not been reported in vivo. Affinity chromatography was performed to separate MMP-9 from MMP-2 and immunoprecipitation to isolate ∼65 kDa MMP-9 from 82 kDa MMP-9 in sera of healthy donors. The presence of ∼65 kDa active MMP-9 was demonstrated both with gelatin zymography and western blot analysis. The ∼65 kDa MMP-9 lacks the haemopexin domain required for the high-affinity binding of the tissue inhibitor TIMP-1, and can be evaluated by activity assay in the presence of TIMP-1. This opens the possibility to investigate the role of this form of MMP-9 that escapes physiological regulation.


Assuntos
Líquidos Corporais/enzimologia , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Adulto , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/sangue , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/sangue , Inibidores de Metaloproteinases de Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Valores de Referência , Inibidor Tecidual de Metaloproteinase-1/metabolismo
12.
J Biol Chem ; 285(28): 21366-71, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20452987

RESUMO

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Fosfogluconato Desidrogenase/metabolismo , Sítio Alostérico , Bioquímica/métodos , Candida/enzimologia , Carbono/química , Catálise , Dimerização , Gluconatos/química , Cinética , Modelos Químicos , Especificidade por Substrato
13.
Biochemistry ; 47(7): 1862-70, 2008 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-18205398

RESUMO

A recent study suggested sheep liver 6-phosphogluconate dehydrogenase (6PGDH) sees the oxidized and reduced cofactor differently [Cervellati, C., Dallocchio, F., Bergamini, C. M., and Cook, P. F. (2005) Biochemistry 44, 2432-2440]. Data were consistent with a rotation into the active site of the nicotinamide ring of NADP upon its reduction, resulting in a displacement of the 1-carboxylate of 3-keto-6PG better positioning it for decarboxylation, and further suggested a role of the cofactor in generating the precatalytic conformation of the enzyme. To further probe the role of the cofactor, multiple isotope effects were measured for the enzyme with mutations of the two residues that directly interact with the nicotinamide ring of NADP+, methionine 13 and glutamate 131. Kinetic and isotope effect data obtained in this study will thus be interpreted in terms of a mechanism that includes the rotation of the nicotinamide ring. The M13V, M13Q, M13C, and E131A mutant enzymes were characterized with respect to their kinetic parameters, deuterium, 13C, multiple deuterium/13C isotope effects, and the kinetics of utilization of 2-deoxy-6PG. Data suggest the position of the nicotinamide ring is important in identifying the open and closed conformations of the enzyme, with the latter optimal for catalysis. The 6PGDH reaction provides an excellent example of the use of substrate binding energy to drive catalysis.


Assuntos
NADP/química , Niacinamida/química , Fosfogluconato Desidrogenase/química , Sequência de Bases , Catálise , Primers do DNA , Modelos Moleculares , Fosfogluconato Desidrogenase/metabolismo , Conformação Proteica
14.
FEBS J ; 274(24): 6426-35, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18021252

RESUMO

6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state.


Assuntos
Inibidores Enzimáticos/química , Fosfogluconato Desidrogenase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Calorimetria/métodos , Inibidores Enzimáticos/farmacologia , Gluconatos/química , Gluconatos/metabolismo , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Cinética , Modelos Químicos , Estrutura Molecular , NADP/química , NADP/metabolismo , Fosfogluconato Desidrogenase/antagonistas & inibidores , Fosfogluconato Desidrogenase/química , Ligação Proteica , Proteínas de Protozoários/química , Especificidade por Substrato , Açúcares Ácidos/química , Açúcares Ácidos/farmacologia , Fosfatos Açúcares/química , Fosfatos Açúcares/farmacologia , Termodinâmica
15.
Clin Biochem ; 40(16-17): 1272-6, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17904541

RESUMO

OBJECTIVES: To identify the best procedure for preanalytical blood collection in the determination of matrix metalloproteinase (MMP)-2 and -9 by testing the effects of anticoagulants on their activity. DESIGN AND METHODS: Active forms of both gelatinases were measured by specific activity assay systems in serum, plasma EDTA, plasma-heparin and plasma-citrate obtained from 20 healthy volunteers, as well as in a pooled serum sample before and after anticoagulant treatment. RESULTS: : Active MMP-2 and MMP-9 mean concentrations were similar in serum and in plasma-citrate, higher in plasma EDTA than in serum, in plasma-heparin and in plasma-citrate, and lower in plasma-heparin than in serum and plasma-citrate. A similar trend was observed in untreated and treated pooled serum samples. CONCLUSIONS: Our results indicate that MMP-2 and MMP-9 in their active forms are not released by platelets during blood clotting, whereas the use of calcium chelating anticoagulants can profoundly alter the activity of endogenous gelatinases. This suggests that the determination of active forms of MMP-2 and MMP-9 in serum samples represents a suitable procedure.


Assuntos
Anticoagulantes/farmacologia , Gelatinases/metabolismo , Adulto , Coleta de Amostras Sanguíneas/métodos , Citratos/farmacologia , Ácido Cítrico/farmacologia , Ácido Edético/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Gelatinases/sangue , Heparina/farmacologia , Humanos , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/sangue , Metaloproteinase 9 da Matriz/metabolismo , Plasma/metabolismo , Soro/metabolismo
16.
Biochim Biophys Acta ; 1770(10): 1437-40, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17693024

RESUMO

We investigated the mechanisms underlying severe bleeding occurring upon consumption of Ferula communis. The prenylated coumarin ferulenol extracted from this plant did not directly affect blood coagulation but showed hepatocyte cytotoxicity and, at non-cytotoxic concentrations (<100 nM), impaired factor X biosynthesis (40% reduction). Studies with ferulenol derivatives indicated the prenyl residue as major determinant of ferulenol activity.


Assuntos
Anticoagulantes/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/toxicidade , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cumarínicos/efeitos adversos , Cricetinae , Fator X/biossíntese , Humanos , Relação Estrutura-Atividade
17.
Anesthesiology ; 106(1): 85-91, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17197849

RESUMO

BACKGROUND: Excessive production of matrix metalloproteinase 9 (MMP-9) is linked to tissue damage and anastomotic leakage after large bowel surgery. Hence, the aim of this study was to verify whether different strategies of fluids administration can reduce MMP-9 expression. METHODS: In the in vitro experiment, the authors tested the hypothesis of a direct inhibition of MMP-9 by the fluids used perioperatively, i.e., lactated Ringer's solution, 3.4% poligeline, and hydroxyethyl starch 130/0.4. In the in vivo experiment, 36 patients undergoing surgery for colon cancer were randomly assigned to three groups to receive lactated Ringer's solution, poligeline, or hydroxyethyl starch. MMP-9 and tissue inhibitor of metalloproteinases were measured from venous blood samples; the MMP-9/tissue inhibitor of metalloproteinases ratio was calculated as an index of equilibrium between the action of MMP-9 and its inhibition. RESULTS: In the in vitro experiment, the presence of hydroxyethyl starch 130/0.4 in the MMP-9 assay system showed a strong inhibition of the enzymatic activity compared with lactated Ringer's solution. In the in vivo experiment, MMP-9 and tissue inhibitor of metalloproteinases plasma levels did not differ among the three groups at baseline, whereas those levels increased significantly at the end of surgery. At that time, the MMP-9 plasma levels and the MMP-9/tissue inhibitor of metalloproteinases ratio were significantly higher in the lactated Ringer's solution and poligeline groups than in the hydroxyethyl starch group. These results were confirmed 72 h after surgery. CONCLUSIONS: This study demonstrates that hydroxyethyl starch 130/04 decreases the circulating levels of MMP-9 in patients undergoing abdominal surgery.


Assuntos
Derivados de Hidroxietil Amido/uso terapêutico , Metaloproteinase 9 da Matriz/sangue , Substitutos do Plasma/uso terapêutico , Adulto , Idoso , Feminino , Humanos , Soluções Isotônicas/uso terapêutico , Masculino , Inibidores de Metaloproteinases de Matriz , Pessoa de Meia-Idade , Oxigênio/sangue , Poligelina/uso terapêutico , Lactato de Ringer , Inibidor Tecidual de Metaloproteinase-1/sangue
18.
Biochemistry ; 44(7): 2432-40, 2005 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-15709755

RESUMO

The crystal structure of sheep liver 6-phosphogluconate dehydrogenase (6PGDH) shows marked differences in the position of the nicotinamide mononucleotide (NMN) moiety of NADP(+) and NADPH (Adams, J. M., Grant, H. E., Gover, S., Naylor, C. E., and Phillips, C. (1994) Structure 2, 651-668). A methionine side chain (Met13) interacts with the si face of NADP(+) in the complex with the oxidized coenzyme, is likely to affect the binding mode of the nicotinamide ring of NADP(+), and may play a role in catalysis in the 6PGDH reaction. To check this possibility we performed site-directed mutagenesis, changing M13 to a number of residues including V, I, C, F, and Q. Mutant enzymes were characterized with respect to their kinetic parameters and primary deuterium isotope effects. All mutations resulted in a decrease in affinity of the enzyme for NADP(+), but not NADPH. In addition, the M13 to C (M13C), M13F, and M13Q mutant enzymes exhibited a decrease of at least an order of magnitude in V/E(t). The deuterium isotope effects on V and V/K(6PG) were decreased to about 1.2 for the M13F and M13C mutant enzymes, while they were increased to about 2.4 for the M13Q enzyme (a value of 1.8-1.9 is obtained for the wild-type enzyme). In at least three instances changes in the overall rate of the oxidative decarboxylation reaction relative to other steps along the reaction pathway were observed. Isotope effects indicate that the hydride transfer steps can become either more or less rate-determining dependent on the substitution. Data are consistent with a significant role of M13 in the orientation of the cofactor nicotinamide ring in the mechanism of 6PGDH, likely with respect to geometry and distance of the ring from C3 of 6PG.


Assuntos
Fígado/enzimologia , Metionina/química , Fosfogluconato Desidrogenase/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Dicroísmo Circular , Cisteína/genética , Medição da Troca de Deutério , Glutamina/genética , Cinética , Metionina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADP/química , Fenilalanina/genética , Fosfogluconato Desidrogenase/antagonistas & inibidores , Fosfogluconato Desidrogenase/genética , Ovinos
19.
Curr Med Chem ; 11(19): 2639-50, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15544466

RESUMO

New drugs are urgently required for Human African Trypanosomiasis (sleeping sickness), a disease which has re-emerged as a major health threat in Sub-Saharan Africa. The third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, has been shown to be a good target for drugs. The enzyme is essential to the trypanosomes that causes sleeping sickness and structural differences when compared to its mammalian counterpart allow for selective inhibition. Three series of inhibitors have been designed, these include phosphorylated carbohydrate substrate and transition state analogues, non-carbohydrate substrate analogues and also triphenylmethane-based compounds. All have shown selective inhibition of the trypanosomal 6-phosphogluconate dehydrogenase and representatives of each have trypanocidal activity.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fosfogluconato Desidrogenase/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Fosfogluconato Desidrogenase/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico
20.
Bioorg Med Chem ; 11(7): 1207-14, 2003 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-12628648

RESUMO

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.


Assuntos
Carboidratos/síntese química , Carboidratos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Fosfogluconato Desidrogenase/antagonistas & inibidores , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Animais , Sequência de Carboidratos , Escherichia coli/metabolismo , Indicadores e Reagentes , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Ovinos , Estereoisomerismo , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos
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