Ischemia and reperfusion of the soleus muscle of rats with pentoxifylline

BACKGROUND: Reperfusion of the skeletal muscle worsens existing lesions during ischemia, since the production of reactive oxygen species, associated with intense participation of neutrophils, increases the inflammatory reaction that induces tissue changes. OBJECTIVE: To evaluate the morphological and immunohistochemical changes of the skeletal (soleus) muscle of rats submitted to ischemia and reperfusion with pentoxifylline. METHODS: Sixty rats were submitted to ischemia of the pelvic limb for 6 hours induced by clamping the left common iliac artery. After ischemia, group A animals (n = 30) were observed for 4 hours and group B animals (n = 30) for 24 hours. Six animals constituted the sham group. Pentoxifylline was applied only in the reperfusion period A2 (n = 10) and B2 (n = 10), and in ischemia and reperfusion periods in A3 (n = 10) and B3 (n = 10). The soleus muscle was evaluated by histological (fiber disruption, leukocyte infiltrate, necrosis) and immunohistochemical (apoptosis through caspase-3 expression) analysis. The non-parametric tests Kruskal-Wallis and Mann-Whitney (p < 0.05) were applied. RESULTS: The changes were more intense in group B1, with fiber disruption mean scores of 2.16±0.14; neutrophilic infiltrate of 2.05±0.10; and caspase-3 expression in the perivascular area of 4.30±0.79; and less intense in group A3, with means of 0.76±0.16; 0.92±0.10; 0.67±0,15, respectively (p < 0.05). Caspase-3 was more expressive in group B1 in the perivascular area, with mean of 4.30±0.79 when compared with group B1 in the perinuclear area, with mean of 0.91±0.32 (p < 0.05) CONCLUSIONS: The lesions were more intense when observation time was longer after reperfusion, and pentoxifylline attenuated these lesions, above all when used in the beginning of ischemia and reperfusion phases.CONTEXTO: A reperfusão de músculo esquelético piora as lesões já presentes no período de isquemia, pois a produção de espécies reativas de oxigênio, associadas à intensa participação de neutrófilos, amplia a reação inflamatória que induz alterações teciduais. OBJETIVO: Avaliar as alterações morfológicas e imuno-histoquímicas de músculo esquelético (sóleo) de ratos submetidos a isquemia e reperfusão com pentoxifilina. MÉTODOS: Sessenta ratos foram submetidos a isquemia do membro pélvico, por 6 horas, pelo clampeamento da artéria ilíaca comum esquerda. Após isquemia, os animais do grupo A (n = 30) foram observados por 4 horas, e os do grupo B (n = 30), por 24 horas. Seis animais constituíram o grupo simulado. Administrou-se pentoxifilina apenas no período de reperfusão em A2 (n = 10) e B2 (n = 10) e nos períodos de isquemia e reperfusão em A3 (n = 10) e B3 (n = 10). O músculo sóleo foi avaliado por análise histológica (dissociação de fibras, infiltrado leucocitário, necrose) e imuno-histoquímica (apoptose pela expressão da caspase-3). Foram aplicados os testes não-paramétricos de Kruskal-Wallis e Mann-Whitney (p < 0,05). RESULTADOS: As alterações foram mais intensas no grupo B1, com médias de escore da dissociação de fibras musculares de 2,16 ± 0,14, infiltrado neutrofílico de 2,05 ± 0,10 e expressão da caspase-3 na área perivascular de 4,30 ± 0,79; e menos intensas no grupo A3, com respectivas médias de 0,76 ± 0,16, 0,92 ± 0,10 e 0,67 ± 0,15 (p < 0,05). A caspase-3 mostrou-se mais expressiva no grupo B1 na área perivascular, com média de 4,30 ± 0,79, em comparação com o grupo B1 na área perinuclear, com média de 0,91 ± 0,32 (p < 0,05). CONCLUSÕES: As lesões são mais intensas quando o tempo de observação é maior após a reperfusão, e a pentoxifilina atenua essas lesões, sobretudo quando usada no início das fases de isquemia e de reperfusão.SBACVUniversidade Federal de Mato Grosso do Sul Hospital UniversitárioUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUFMSUNIFESP-EPM Departamento de PatologiaUFMS Departamento de Clínica CirúrgicaUFMS Hospital Universitário Comissão de Residência MédicaUNIFESP, EPM, Depto. de PatologiaSciEL

Weak Spatial and Temporal Population Genetic Structure in the Rosy Apple Aphid, Dysaphis plantaginea, in French Apple Orchards

We used eight microsatellite loci and a set of 20 aphid samples to investigate the spatial and temporal genetic structure of rosy apple aphid populations from 13 apple orchards situated in four different regions in France. Genetic variability was very similar between orchard populations and between winged populations collected before sexual reproduction in the fall and populations collected from colonies in the spring. A very small proportion of individuals (∼2%) had identical multilocus genotypes. Genetic differentiation between orchards was low (FST<0.026), with significant differentiation observed only between orchards from different regions, but no isolation by distance was detected. These results are consistent with high levels of genetic mixing in holocyclic Dysaphis plantaginae populations (host alternation through migration and sexual reproduction). These findings concerning the adaptation of the rosy apple aphid have potential consequences for pest management

Detection of platypus-type l/d-peptide isomerase activity in aqueous extracts of papaya fruit

Peptide isomerase catalyses the post-translational isomerisation of the l- to the d-form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In the process of delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used substrate segments from the N-terminus of the natriuretic peptide (OvCNP) and defensin-like peptides-2 and -4 (DLP-2 and DLP-4) from the venom. Synthetic hexapeptides containing the first three amino acid residues (IMF) of DLP-4 and DLP-2 were linked to the tripeptide srs (lower case letters denote the corresponding D-amino acid), to increase peptide stability and water solubility. The DLP analogues IMFsrs and ImFsrs were found to be effective substrates for the isomerase. Mutants of these hexapeptides were synthesized with the second amino acid replaced, respectively, by the L- and D-forms of Abu, Ala, His, Ile, Leu, Lys, Nle, Phe, Trp, Tyr, and Val. The relative rates of peptide isomerization were measured by using partially purified isomerase extracted from venom glands and separating the reactants with high performance liquid chromatography (HPLC). The isomerase was active with the mutants that contained L- or D-forms of Phe or Nle residues at the second position; but the amino acids that contained shorter, bta-branched- or long side-chains with terminal polar groups, viz., Ala, Abu, Ile, Leu, Val, Lys and Tyr respectively, were not substrates. For each of these non-substrates, newly formed peptides that eluted from HPLC earlier than the substrate were isolated and were deduced to have been formed through the loss of N-terminal Ile in each case. None of the hexapeptides based on LLH, the first three amino acid residues of OvCNPalpha were substrates, but when LLH was attached to full-length OvCNP it became a substrate for the isomerase; thus modulation of the substrate specificity is brought about by other sections of the longer peptide. Based on the action of the isomerase with the various substrates a model of the 'van der Waals outline' of the active site is proposed

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In the process of delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used substrate segments from the N-terminus of the natriuretic peptide (OvCNP) and defensin-like peptides-2 and -4 (DLP-2 and DLP-4) from the venom. Synthetic hexapeptides containing the first three amino acid residues (IMF) of DLP-4 and DLP-2 were linked to the tripeptide srs (lower case letters denote the corresponding D-amino acid), to increase peptide stability and water solubility. The DLP analogues IMFsrs and ImFsrs were found to be effective substrates for the isomerase. Mutants of these hexapeptides were synthesized with the second amino acid replaced, respectively, by the L- and D-forms of Abu, Ala, His, Ile, Leu, Lys, Nle, Phe, Trp, Tyr, and Val. The relative rates of peptide isomerization were measured by using partially purified isomerase extracted from venom glands and separating the reactants with high performance liquid chromatography (HPLC). The isomerase was active with the mutants that contained L- or D-forms of Phe or Nle residues at the second position; but the amino acids that contained shorter, bta-branched- or long side-chains with terminal polar groups, viz., Ala, Abu, Ile, Leu, Val, Lys and Tyr respectively, were not substrates. For each of these non-substrates, newly formed peptides that eluted from HPLC earlier than the substrate were isolated and were deduced to have been formed through the loss of N-terminal Ile in each case. None of the hexapeptides based on LLH, the first three amino acid residues of OvCNPalpha were substrates, but when LLH was attached to full-length OvCNP it became a substrate for the isomerase; thus modulation of the substrate specificity is brought about by other sections of the longer peptide. Based on the action of the isomerase with the various substrates a model of the ‘van der Waals outline’ of the active site is proposed