BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects.

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 microM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 microM; guanylin - 0.2 microM) it promoted increases in urine flow (DeltaUF of 0.25 +/- 0.09 mL.g(-1)/min, P < 0.05) and Na+ excretion (% Delta ENa+ of 18.20 +/- 2.17, P < 0.05). BTCI (1.0 microM) also increased %ENa+ (from 22.8 +/- 1.30 to 34.4 +/- 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 microM) induced increases in glomerular filtration rate (GFR; from 0.96 +/- 0.02 to 1.28 0.02 mL.g(-1)/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

BTCI enhances guanylin-induced natriuresis and promotes renal glomerular and tubular effects

Guanylin and uroguanylin are small cysteine-rich peptides involved in the regulation of fluid and electrolyte homeostasis through binding and activation of guanylyl cyclases signaling molecules expressed in intestine and kidney. Guanylin is less potent than uroguanylin as a natriuretic agent and is degraded in vitro by chymotrypsin due to unique structural features in the bioactive moiety of the peptide. Thus, the aim of this study was to verify whether or not guanylin is degraded by chymotrypsin-like proteases present in the kidney brush-border membranes. The isolated perfused rat kidney assay was used in this regard. Guanylin (0.2 µM) induced no changes in kidney function. However, when pretreated by the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI - 1.0 µM; guanylin - 0.2 µM) it promoted increases in urine flow (deltaUF of 0.25 ± 0.09 mL.g-1/min, P < 0.05) and Na+ excretion ( percent delta ENa+ of 18.20 ± 2.17, P < 0.05). BTCI (1.0 µM) also increased percentENa+ (from 22.8 ± 1.30 to 34.4 ± 3.48, P < 0.05, 90 minutes). Furthermore, BTCI (3.0 µM) induced increases in glomerular filtration rate (GFR; from 0.96 ± 0.02 to 1.28 0.02 mL.g-1/min, P < 0.05, 60 minutes). The present paper strongly suggests that chymotrypsin-like proteases play a role in renal metabolism of guanylin and describes for the first time renal effects induced by a member of the Bowman-Birk family of protease inhibitors.

Guanilina e uroguanilina são peptídeos pequenos, ricos em cisteína, envolvidos na regulação da homeostase de fluidos e eletrólitos através da ligação e ativação da guanilato ciclase expressa no intestino e nos rins. A guanilina é menos potente do que a uroguanilina como agente natriurético e é degradada in vitro pela quimiotripsina devido a características estruturais únicas no domínio bioativo do peptídeo. Portanto o objetivo deste trabalho foi verificar se a guanilina é degradada por proteases tipo quimiotripsina, presentes na membrana da borda em escova dos rins. Para esta investigação, foi usado o modelo do rim isolado de rato perfundido. A Guanilina (0,2 µM) não induziu mudanças na função renal. Entretanto, quando pré-tratada com inibidor de tripsina e de quimiotripsina de black-eyed pea (BTCI - 1,0 µM; guanilina - 0,2 µM) promoveu um aumento no fluxo urinário (deltaUF de 0,25 ± 0,09 mL.g-1/min, P < 0,05) e na excreção de Na+ ( por centoDENa+ de 18,20 ± 2,17, P < 0,05). BTCI (1,0 µM) também aumenta por centoENa+ (de 22,8 ± 1,30 a 34,4 ± 3,48, P < 0,0590 minutos). Além disto, BTCI (3,0 µM) induziu um aumento da taxa de filtração glomerular (GFR; de 0,96 ± 0,02 para 1,28 ± 0,02 mL.g-1/min, P < 0,05, 60 minutos). O presente trabalho sugere fortemente que proteases semelhantes à quimiotripsina desempenham um papel no metabolismo renal de guanilinas e descreve, pela primeira vez, os efeitos renais induzidos por um membro da família de inibidores de proteases do tipo Bowman-Birk.

[Pharmacological effects of arginine supplementation in rats with Walker 256 solid tumor]. / Efeitos farmacológicos da suplementação dietética com arginina em ratos com tumor sólido de Walker 256.

The effects of diet arginine supplementation for those with cancer are controversial. We evaluate the effects of dietetic supplementation with arginine over body weight, growth of tumor, metastatic dissemination, surviving time, amino acid metabolism, haematological changes of the rats with Walker 256 solid tumor. Intragastrical solutions with arginine at 4% and 6%, a standard diet (control) were administered to the animals. The supplementation with arginine was associated with a lower weight gain during the study period (p < 0.05). Surviving time of the rats with solid tumor did not vary significantly between the groups. The rate of metastase was lower in animals with Walker 256 solid tumor supplemented with arginine. The amino acid metabolism was estimulate in the animals after arginine supplementation at 4% and 6%, demonstrated by significant increases in blood levels of arginine, ornitine, citruline, proline and histidine when compared to the control group. Anaemia was less severe in the rats with Walker 256 solid tumor that received arginine supplementation. The results suggest that arginine 6% supplementation may have pharmacologic effect in rats with Walker 256 solid beyond the nutritional one.

Amino acid sequence of a myotoxic Lys49-phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmani.

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A2 (PLA2) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA2 showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA2s (His-48, Tyr-52 and Asp-99) are conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA2 s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA2. The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca(2+)-independent membrane damaging activity.

Improving the recovery of lysine in automated protein sequencing.

Automated protein sequencing is an important tool in protein characterization. Most instruments use tetrahydrofuran (THF) as the HPLC eluent for separation of the derivatized amino acids residues. THF is highly perishable when exposed to air, generating peroxides which can degrade amino acids, mainly lysine, leading to uncertainty in chromatogram interpretations. Modifications of the existing HPLC equipment were introduced to create a permanent inert atmosphere inside the bottle of THF solution. This was carried out by changing the argon plumbing and some electrical connections and by reprogramming the software of the protein sequencer. The positive results of this procedure were demonstrated by comparing the decreasing lysine peak area during 28 days before and after the modifications. In the modified instrument, lysine recovery was much better as a function of the age of the THF eluent. Since these modifications improved the instrument performance, they have been adopted for routine use in our laboratory.

Determination of the amino acid sequence of the plant cytolysin enterolobin.

The cytolytic seed protein enterolobin from seeds of Enterolobium contortisiliquum was purified by using FPLC on a Mono Q column giving a single peak in capillary electrophoresis. The complete amino acid sequence of the plant cytolysin was determined by an automated method, yielding a molecular mass of 54,806 Da. Databank searches and sequence alignment demonstrated a high degree of sequence identity and similarity between enterolobin and bacterial aerolysins from Aeromonas hydrophila and A. sobria. Several key residues involved in oligomerization of A. hydrophila aerolysin are conserved in enterolobin. Circular dichroism measurements and structural predictions revealed that enterolobin is very rich in beta sheet, like aerolysin. Light-scattering studies revealed that enterolobin oligomerizes as a hexamer at pH levels below 7.0. NaCl concentrations above 50 mM caused dimerization of enterolobin. Dithiothreitol did not cause oligomerization.

Amino acid sequence of the Phaseolus vulgaris var. "fogo na serra" inhibitor and interactive surface modeling for the enzyme-inhibitor complex.

A trypsin and chymotrypsin inhibitor from seeds of Phaseolus vulgaris var. "Fogo na Serra" (PFSI) was purified and its complete amino acid sequence was determined using Edman degradation methods. The inhibitor was found to belong to the Bowman-Birk family of enzymatic inhibitors; it has 82 amino acid residues and a 8.985-kDa molecular mass. The PFSI/alpha-chymotrypsin binary complex has been modeled using the Turkey ovomucoid inhibitor third domain (OMTKY3) bound to alpha-chymotrypsin [Fujinaga et al. (1987), J. Mol. Biol., 195, 397-418. template. The model allowed identification of the binding surface.

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae).

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 of Loxosceles gaucho, L. laeta, or L. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components. B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa in L. gaucho and L. intermedia, but 32 kDa in L. laeta venom. These toxins were isolated from venoms of L. gaucho, L. laeta, and L. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of the L. reclusa (North American spider) toxin. A multiple sequence alignment of the Loxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho and L. reclusa) to 61.1% (L. intermedia and L. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping after in situ partial hydrolysis of the blotted samples. The results obtained suggest that L. intermedia protein is more similar to L. laeta toxin than L. gaucho toxin and revealed a smaller homology between L. intermedia and L. gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms of Loxosceles species have a molecular mass of 32-35 kDa and are probably homologous proteins.

Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the Brazilian tree Dipteryx alata (Leguminosae).

Dipteryx alata trypsin inhibitor (DATI) has been purified and completely sequenced. It showed homology to members of the Bowman-Birk family of inhibitors. The last step of DATI purification by RP-HPLC (narrow-borc C18 column) suggested the existence of some isoforms of the inhibitor due to the presence of a cluster of very close peaks in the chromatogram. By using electrospray ionization mass spectrometry (ESIMS) and laser desorption mass spectrometry (LDIMS), the identification of DATI isoforms was made possible. From the ESIMS data, the following molecular masses were found: 6803.22 +/- 0.92 for isoform a; 6890.94 +/- 0.73 for b; 6977.58 +/- 0.39 for c; 7065.07 +/- 0.67 for d; 7151.42 +/- 0.86 for e; and 7291.70 +/- 0.43 for f. Similar masses were found when using LDIMS. Isoform b was the most abundant and its molecular mass matched the molecular mass of 6893 calculated from the sequence of DATI. The mass differences between a and b, b and c, c and d, and d and e were equal to 87, which corresponds to Ser. Isoform a might not have the N-terminal Ser present in isoform b, while the other additional Ser residues might comprise a row localized in the C- or N-terminal. The appearance of all these isoforms could result from posttranslational N- and C-terminal processing.

Amino acid sequence and disulphide-bridge pattern of three gamma-thionins from Sorghum bicolor.

The complete primary structure of a new alpha-amylase inhibitor from Sorghum bicolor belonging to the gamma-thionin family has been determined and the amino acid sequences of two components of the family already elucidated have been corrected by combining the classical Edman degradation with advanced mass spectrometric procedures. The same integrated approach allowed us to define the pattern of the disulphide bridges in the three isoinhibitors. The arrangement of the cysteine pairing was determined as Cys3-Cys47, Cys14-Cys34, Cys20-Cys41 and Cys24-Cys43. The amino acid sequences of the alpha-amylase inhibitors share a high degree of similarity with the related plant gamma-thionins. All these proteins consist of 47 residues, contain eight cysteine residues forming four disulphide bridges, and show the presence of two clusters of basic amino acids located at both ends of the polypeptide chain. The pattern of S-S bridges determined for the isoinhibitors is identical to that inferred by NMR analysis in two related gamma-thionins, thus suggesting a highly conserved organization of the disulphide pairing. These results indicate that the structural similarities among the different gamma-thionins extend far beyond the primary structure and possibly concern the secondary structure and the general folding of the entire gamma-thionin family.

Homology between the seed cytolysin enterolobin and bacterial aerolysins.

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian tree Enterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins from Aeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences of A. hydrophila and A. sobria aerolysins showed several similar regions with many residue identities. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.

Pro-inflammatory activity of enterolobin: a haemolytic protein purified from seeds of the Brazilian tree Enterolobium contortisiliquum.

The pro-inflammatory activity of enterolobin, a haemolytic protein from Enterolobium contortisiliquum seeds, was investigated. In doses ranging from 1 to 20 micrograms/site, enterolobin induced a dose-dependent paw oedema and pleurisy in rats. The effect was apparent after 15 min, peaked at 6 hr and decreased 24 hr after enterolobin was administered. One hour after the intrathoracic injection of enterolobin, the total leukocyte content of the pleural cavity increased significantly, mainly due to mononuclear and neutrophil accumulation. At 24 hr, although the number of mononuclear and neutrophil cells tended to decrease, a great rise in eosinophil counts was noted. Intraperitoneal treatment with the dual lipoxygenase and cyclooxygenase blockers, BW 755c (25 mg/kg) and NDGA (50 mg/kg) or the corticosteroid dexamethasone (0.1 mg/kg) inhibited enterolobin-induced paw oedema by 35, 38 and 47% respectively, whereas indomethacin (2 mg/kg) was inactive. The H1 antagonist, meclizine (25 mg/kg), was also effective against enterolobin oedema while the PAF-antagonists WEB 2086 and PCA 4248 (20 mg/kg) did not modify the reaction. It was concluded that enterolobin is a potent inducer of pleural exudation, cellular infiltration and paw oedema. Furthermore, enterolobin-induced oedema is partially dependent on lipoxygenase metabolites and histamine, while PAF and prostaglandins did not seem to be important in this reaction.

Enterolobin induces rat paw oedema independently of PAF-acether.

The potential participation of PAF-acether (PAF) on the paw oedema triggered by enterolobin was investigated. Intraplantar injections of enterolobin (5-20 micrograms/paw) yielded a dose response curve for oedema which appeared after 30 min, peaked in the interval between 2-4 h and faded after 24 h. The pre-treatment with BN 52021, but not with other PAF antagonists such as PCA 4248 or WEB 2086, significantly blocked enterolobin-induced oedema. To clarify better the discrepant results obtained with the PAF antagonists, desensitization to PAF was performed. The oedema triggered by enterolobin was not modified in PAF desensitized animals. It was concluded that the paw inflammation induced by enterolobin does not require PAF mechanism.

Enterolobin induces rat paw oedema independently of PAF-acether

The potential participation of PAF-acether (PAF) on the paw oedema triggered by enterolobin was investigated. Intraplantar injections of enterolobin )5-20 µg/paw) yielded a dose response curve for edema which appeared after 30 min, peaked in the interval between 2-4 h and faded after 24h. The pre-treatment with BN 52021, but not with other PAF antagonists such as PCA 4248 or WEB 2086, significantly blocked enterolobin-induced oedema. To clarify better the discrepant results obtained with the PAF antagonists, desensitization to PAF was performed. The oedema triggered by enterolobin was not modified in paf desensitized animals. It was concluded that the paw inflammation induced by enterolobin does not require PAF mechanism.

Enterolobin, a hemolytic protein from Enterolobium contortisiliquum seeds (Leguminosae--Mimosoideae). Purification and characterization.

Hemolytic and phospholipase D activities were found in the saline extract of Enterolobium contortisiliquum seeds. The hemolytic activity is due to a protein which was named enterolobin. This protein was highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of saturation, batch separation by adsorption on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 or G-150. In the batch separation the fraction showing hemolytic activity was not adsorbed by the resin while the fraction with phospholipase activity was. In this manner it was shown that those two activities were due to different proteins. Mouse erythrocytes were less susceptible to hemolysis by enterolobin than human and rabbit erythrocytes. The hemolytic activity was rapidly lost at or above 55 degrees C and in extreme acid (1.6) and basic (10.8) pHs. The following characteristics of purified enterolobin were determined: molecular weights of 55.000 D (by SDS-PAGE), 59.800 D (by gel filtration) and 51.300 D (by HPLC); pI = 7.0; Gln as the N-terminal amino acid residue; high levels of Asp(Asx), Glu(Glx), Ser and Thr residues and low levels of Cys and Met residues. Similarities were noticed between enterolobin and crotin, a hemolytic protein of Croton tiglium seeds.

The complete amino acid sequence of the Vigna unguiculata (L.) Walp. seed trypsin and chymotrypsin inhibitor.

The amino acid sequence of the "double-headed" trypsin and chymotrypsin inhibitor (BTCI), purified from the cowpea Vigna unguiculata (L.) Walp. cv "Seridó" was determined in our laboratory. Tryptic and chymotryptic peptides were sequenced by the Edman-Gray and Edman-Chang N-terminal manual method as well as the carboxypeptidase C-terminal method. The complete amino acid sequence of the BTCI is: Ser-Gly-His-His-Glx-Asx-Ser-Thr-Asx-Glx-Ala-Ser-Glx-Ser-Ser-Lys-Pro-Cys- Cys-Arg- Glx-Cys-Ala-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Glx-Cys-Arg-Cys-Ser-Asx-Val-Arg- Leu-Asn- Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys-Ala-Cys-Thr-Phe-Ser-Ile-Pro-Ala-Glx- Cys-Phe- Cys-Gly-Asx-Ile-Asx-Asx-Phe-Cys-Tyr-Lys-Pro-Cys-Lys-Ser-Ser-His-Ser-Asx- Asx-Asx-Asx-Trp-Asn. BTCI presents a high degree of homology with the Bowman-Birk proteinase inhibitor family.

The presence of histamine in Cnidosculus oligandrus (Euphorbiaceae).

1) The presence of histamine in prickles and lead and stem tissues of Cnidosculus oligandrus (Mull. Arg) Pax & Hoffm (Euphorbiaceae) is reported. 2) Prickles on stems and leaves have larger amounts of histamine, as determined by the isolated guinea-pig ileum and by fluorimetry of the condensation product of the histamine and ortho-phthalaldehyde (OPT). 3) Additionally, thin-layer chromatography and the increase of vascular permeability bioassay were used to confirm the presence of histamine.

culus oligandrus (Euphorbiaceae).culus oligandrus (Euphorbiaceae).

Os espectros de absorcao de segunda derivada e de derivada-de-diferenca do inibidor triptico e quimotriptico de sementes de Vigna unguiculata (L) Walp. (var. Serido) foram obtidos para determinar o estado dos residuos de fenilalanina na proteina.O espectro de segunda derivada do inibidor nativo, comparado aquele do inibidor reduzido e carboximetilado e ao de uma mistura-modelo de N-acetil etil esteres de fenilalanina, tirosina e triptofanio (em relacao molar de 3:1:1, para imitar a proteina ana), sugere que os residuos de fenilalanina sao extensamente expostos ao solvente. Alem disso, a primeira derivada dos espectros de diferenca do inibidor nativo e do reduzido indica que os tres residuos de fenilalanina por molecula de inibidor nativo estao expostos ao solvente em grau aproximadamente igual ao da proteina reduzida e carboximetilada