RESUMO
Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway
Assuntos
Animais , Apoptose , Caspases , Músculo Esquelético , Deficiência de Vitamina E , Galinhas , Fragmentação do DNA , Ativação Enzimática , Músculo EsqueléticoRESUMO
Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 æg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 æM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 æM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 æM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM)
Assuntos
Animais , Humanos , Bovinos , Fator XII , Fibrinolíticos , Glicosaminoglicanos , Calicreína Plasmática , Plasminogênio , Inibidores de Cisteína Proteinase , Calicreína Plasmática , Inibidor da Proteína CRESUMO
Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.
Assuntos
Apoptose , Caspases/metabolismo , Músculo Esquelético/citologia , Selênio/deficiência , Deficiência de Vitamina E/enzimologia , Animais , Apoptose/genética , Inibidores de Caspase , Galinhas , Fragmentação do DNA , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Músculo Esquelético/enzimologiaRESUMO
Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).
Assuntos
Fator XII/efeitos dos fármacos , Fibrinolíticos/farmacologia , Glicosaminoglicanos/farmacologia , Calicreína Plasmática/efeitos dos fármacos , Plasminogênio/efeitos dos fármacos , Animais , Bovinos , Proteínas Inativadoras do Complemento 1/efeitos dos fármacos , Proteína Inibidora do Complemento C1 , Inibidores de Cisteína Proteinase/farmacologia , Fator XII/fisiologia , Humanos , Calicreína Plasmática/antagonistas & inibidores , Calicreína Plasmática/fisiologiaRESUMO
Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Calicreínas/antagonistas & inibidores , Calicreínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Calicreínas/sangue , Calicreínas/química , Cinética , Cininas/metabolismoRESUMO
Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His.