Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ñ 83 µg/kg

A new brain metalloendopeptidase which degrades the Alzheimer beta-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human brain using successive steps of chromatography on DEAE-Trisacryl, hydroxylapatite and Sephacryl S-200. The purified enzyme cleaved the Gly(33)-Leu(34) bond of the 25-35 neurotoxic sequence of the Alzheimer Beta-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects. This enzyme activity was only inhibited by divalent cation chelators such as EDTA, AGTA and o-phenanthroline (1 mM) and was insensitive to phosphoramidon and captopril (1 muM concentration), specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. The high affinity of this human brain endopeptidase for Beta-amyloid 1-40 peptide (Km = 5 muM) suggests that it may play a physiological role in the degradation of this substance produced by normal cellular metabolism. It may also be hypothesized that the abnormal accumulation of the amyloid Beta-protein in Alzheimer´s disease may be initiated by a defect or an inactivation of this enzyme.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing oaminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid rsidues, Abz-Darg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8muM, Kcat = 5.3 min-1, Kcat/Km = 2 min-1 muM-1 and Km = 5.0 muM, Kcat = 7.0 min-1, Kcat/Km = 1.4 min-1 muM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE;EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.

Metabolism of diglycine and triglycine by non-filtering kidneys

We have studied the metabolism of diglycine and triglycine in the isolated non-filtering rat kidney. Kidneys from adult male Wistar Kyoto rats weighing 250-350 g were perfused with Krebs-Henseleit solution containing either 1 mM diglycine or triglycine. The analysis of the peptide residues and their components was performed using an amino acid microanalyzer utilizing ion exchange chromatography. Diglycine was degraded to a final concentration of 0.09 mM after 120 min (91 per cent); this degradation occurred predominantly during the first hour, with a 56 per cent reduction of the initial concentration. The metabolism of triglycine occurred similarly, with a final concentration of 0.18 mM (82 per cent); during the first hour there was a 67 per cent reduction of the initial concentration of the tripeptide. Both peptides produced glycine in increasing concentrations, but there was a slightly lower recovery of glycine, suggesting its utilization by the kidney as fuel. The hydrolysis of triglycine also produced diglycine, which was also hydrolyzed to glycine. The results of the present study show the existence of functional endothelial or contraluminal membrane peptidases which may be important during parenteral nutrition.

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11)

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGdFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 µM,Kcat = 127 / min and Kcat / Km = 42 / min µM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demostrated by incubating Abz-GGdFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragments Abz-GGdFLRRnp accounted for more than 95 percent of the products wich were totally inhibited by 1 µM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.

Isolation and properties of a multicatalytic proteinase complex from Xenopus laevis skin secretion

A multicatalytic proteinase complex present in the skin secretion of Xenopus laevis was purified and its enzymatic activity towards natural and synthetic peptides was investigated. We identified three activities: i) a C-terminal deamidation enzyme activity which exhibited selectivity for the Asp-Phe-NH2 and Phe-Leu-NH2 motifs of cerulein, minigastrin Leu-enkephalinamide, (des-Tyr1)Leu-enkephalinamide and diaminobenzylthiocyanate-DVDERDVRGFASFL NH2 (DABTCDR8kermit); ii) an endopeptidase activity that cleaves peptide bonds on the carboxyl side of hydrophobic amino acid residues such as Tyr-Gly of LHRH, Ile-Ala of PGLa and Leu-Ala of buccalin; iii) an enzyme activity that cleaves peptide bonds at the dibasic sites of peptides of the dynorphin family. The molecular weight determined by Sephacryl S-400 molecular sieve filtration indicated an Mr about 600 kDa. The activities characterized here exhibit and optimal pH of about 7.4. The activities of the multicatalytic complex were differentially inhibited by the classical inhibitors of proteases

Human neuroblastoma cells express a novel metallo-endopeptidase activity able to inactivate atrial natriuretic factor: inhibition during retinoic acid-induced differentiation

A new metallo-endopeptidase which hydrolyzes atrium natriuretic factor (ANF) has been isolated from human neuroblastoma NB-OK-1 cells. In the present study we show that this metallo-endopeptidase is also present in several other human neuroblastoma cell lines, which include CHP 100, SH-SY5Y, SK-N-BE(2), BE(2)-C and BE(2)M-17. Additionally, we show that this endopeptidase activity is reduced to about 20 per cent of the control during retinoic acid (RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells, but not in the RA-resistant BE(2)-M17 cells. This suggests that the inhibition is related to neuronal differentiation and not to a direct effect of 5 microM RA on the enzyme activity. This new enzyme is clearly distinct from neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-converting enzyme (ACE,EC 3.4.15.1), since specific inhibitors for these endopeptidases (10 microM phosphoramidon and 1 mM captopril, respectively) had no effect on their activity. However, this enzyme was inhibited 100 per cent by 10 mM o-phenanthroline showing an inhibitory spectrum similar to that of another novel metallo-endopeptidase recently isolated in our laboratory from Xenopus laevis skin secretion. Although the physiological function of this new enzyme in human neuroblastoma cells is not known at the present time, we suggest that it may participate in inactivation of neuropeptides such as atrium natriuretic factor (ANF), substance P, somatostatin-14 and bradykinin in vivo

Metabolism of dipeptides by isolated and perfused nonfiltering kidneys

Rehal metabolism of Glycyl-glycine (Gly-gly), Glycyl-proline (Gly-pro) and Prolyl-glycine (Pro-gly) was studied in the non-filtering, isolated perfused rat kidney. Gly-gly is metabolized by more than 90% after 120 min of perfusion. Gly-pro is more resistant to degradation and about 75% of the original peptide can be found intact in the perfusate at the end of perfusion. For Pro-gly only 25% reamins intact at the end of the experiment. Glycine was also monitored as another marker for dipepptide degratation and ins production increased throughout the perfusion time. In some experiments we also determined the production of proline. We conclude from these experiments that the basolateral membrane, or perhaps the kidney blood vessels, posses an efficient apparatus for the hydrolysis of Gly-gly and Pro-gly. This mechanism is less efficient in the case of Gly-pro. This confirms an earlier hypothesis that dipeptide metabolism does not occur solely in the brush-border membranes

Causas de excisao de globos oculares pos-ferimento perfurante. / Causes of ocular excision following penetrating injuries

Os autores apresentam as causas de excisao de 39 olhos de 768 pacientes com ferimento perfurante ocular. Lembram que nenhuma excisao ocorreu por receio de oftalmia simpatica. A excisao em l8 casos foi determinada pela ocorrencia de FPO com prolapso irremediavel do material intra-ocular, em 17 casos por endoftalmite nao controlavel clinicamente, em 3 por "phthisis bulbi" e em 1 por hemoftalmo e CEIO. Ressaltam que a conduta inicial deve ser conservadora, destacando que mesmo olhos severamente lesados podem ser recuperados atraves de cirurgias combinadas