Pharmacokinetic aspects of ter-butylaminoethyl dissulfide, an experimental drug against schistosomiasis in mice.

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resein. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUC i.v.):671 ± 14; AUC oral: 150 ± 40 µg.min. ml [raised to the power of -1]; elimination rate constant: 0.071 min [raised to the power of -1]; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undertermined model. Combining the results, the occurence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 µg/ml.Um estudo preliminar da farmacocinética do t-butilamino-etil-dissulfeto foi conduzido utilizando droga fria ou radioativa em duas diferentes doses (35 e 300 mg/Kg). Amostras de plasma ou sangue foram tratadas com ditiotreiol, ácido perclórico, e, após filtração, submetidas a uma subseqüente purificação em um "batch" de resina aniônica. Na etapa final, a droga foi retida em coluna de resina catiônica, eluída com NaCl 1 M e detectada pelo método de Ellman (1958). Alternativamente, a droga radioativa foi detectada por cintilação líquida. Os resultados correspondentes a droga total administrada na menor dose sugeriram um comportamento farmacocinético relacionado ao modelo de um compartimento aberto, com os seguintes parâmetros: área sob a curva intravenosa (ASCI.V.): 671 ± 14; ASCoral: 150 ± 40 microng.min.ml [elevado a -1]; constante de eliminação: 0,071 min [elevado a -1]; meia-vida biológica: 9,8 min; volume de distribuição: 0,74 ml/g. Para a dose mais alta, os resultados indicaram aparentemente a ocorrência de um modelo mais complexo e não adequadamente classificado. Analisados em conjunto os resultados sugerem a ocorrência de um comportamento farmacocinético dose-dependente. A droga é absorvida e eliminada rapidamente, sendo este último processo relacionado principalmente a metabolização. A droga parece mais tóxica quando administrada via I.V. porque por esta via ela não sobre metabolismo de primeira passagem e, é, por outro lado rapidamente distribuída para os tecidos. O nível sanguíneo máximo tolerado pelos animais parece ser de 16 µg/ml

Elimination of turbidity in serum iron colorimetric assay by enzymatic proteolysis.

We describe a modification in the commercial colorimetric method for the determination of serum iron by using Ferrozine® . The modification was proposed because during the conventional procedure, turbidity observed when the serum of animals submitted to surgery was used interfered with the assay. We added to the original method, a previous treatment of the serum with proteolytic enzymes. This modification was also tested using plasma samples, although this was not recommended when the original method was used. The results demonstrated that: a) the treatment with a mixture of trypsin and chymotrypsin was effective in order to eliminate turbidity; b) there was no difference between the standard curves obtained by the conventional and the modified method for control assays; c) the absorbencies of the samples of serum and plasma submitted to proteolysis, estimated by the addition of different concentrations of iron, were directly proportional to iron concentrations; d) the pre-treatment with enzymes allowed the utilization of plasma; e) the pre-treatment with guanidine. HCl was not effective.Descreveu-se modificação no método colorimétrico comercial para dosagem de ferro sérico que utiliza Ferrozine® como reagente de cor. A modificação foi proposta porque durante o procedimento observava-se turvação quando o soro de animais submetidos à cirurgia era utilizado, comprometendo os resultados. Foi acrescentado ao método um tratamento prévio do soro com enzimas proteolíticas. Avaliou-se também a modificação utilizando amostras de plasma, o que não é recomendado na metodologia original. O tratamento das amostras com cloreto de guanidina, descrito anteriormente para amostras de pacientes submetidos à hemodiálise, também foi avaliado. Os resultados demonstraram que: a) tratamento das amostras com tripsina e quimotripsina eliminou a turvação; b) não houve diferenças entre as curvas padrão obtidas pelo método original ou modificado para amostras de soro provenientes de animais controle; c) as absorbâncias das amostras de soro e plasma submetidas à proteólise foram proporcionais às concentrações de ferro, estimada pela adição de diferentes concentrações do íon; d) o tratamento enzimático permitiu a utilização de plasma; e) o tratamento prévio dos soros, de animais submetidos à cirurgia, com guanidina. HCl, não foi eficaz

Bowman-Birk inhibitors, proteasome peptidase activities and colorectal pre neoplasias induced by 1,2-dimethylhydrazine in Swiss mice.

Bowman-Birk inhibitors (BBIs) are protein molecules containing two inhibitory domains for enzymes similar to trypsin and chymotrypsin. Interest in these inhibitors arose from their properties against the cancer chemically induced by 1,2-dimethylhydrazine (DMH). In this study the effect of two BBI preparations (from Glycine max and Macrotyloma axillare) were evaluated for the prevention of colorectal neoplasia induced by intraperitoneal injections of DMH, given at a dose of 30 mg/kg, during 12 weeks. Mice treated with DMH presented histopathological alterations consistent with tumor development, augmented CD44 expression and increased proteasome peptidase activities. Lysosomal fractions, obtained from the intestines, were chromatographed in a Sepharose-BBI column and increased activity for trypsin and chymotrypsin-like proteases recovered from DMH-treated animals. In parallel, mice treated for eight weeks with BBIs showed a decrease in the chymotrypsin and trypsin-like proteasome activities compared to animals fed on normal diet. For the groups receiving simultaneous treatment with DMH and BBIs, dysplasic lesions were not observed and proteasome peptidase activities were similar to the control group after the 24th week. These results suggest that the mechanism by which BBIs could prevent the appearance of pre neoplastic lesions is associated with inhibition of both the lysosomal and proteasome- dependent proteolytic pathways

A novel and efficient and low-cost methodology for purification of Macrotyloma axillare (Leguminosae) seed lectin.

The N-acetyl-galactosamine specific lectin fromMacrotyloma axillare seeds (LMA)was purified by precipitation and ion exchange chromatography. The LMA 0.2 mol L−1 fraction showed hemagglutinating activity on erythrocytes A1. The results for molecular mass determinations were about 28 kDa. The LMA pHdependent assays showed best hemagglutinating activity at pH 6.0–8.0; being decreased at acidic/alkaline conditions and by EDTA treatment. LMA is a tetramer at pH 8.2 and a dimer at pH 4.0. Human erythrocytes from ABO system confirmed the A1 specificity for LMA. This new methodology is useful and easy, with low costs, for lectin purification in large amounts

Tyrosine 151 is part of the substrate activation binding site of bovine trypsin.

Identification of the substrate activation site of p-trypsin by a 1:l reacwtiotnh p-diazoniumbenzamidine chloride was confirmebdy spectral anaylsisP. roteolysis of Cm-p-benzamidino-azo-@-trypsipnr ovided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at '&r-151, was recovereidn 35%y ield, and its structure was demonstrated by amino acid analysis, Edman deg radation, and mass spectrometry. Yielodfs labeled Tyr- 151, Tyr-39, and Tyr-172,i dentified by peptide analysis, were in the proportion of 100:7:3. Tyr-l51-(p-benzamidinol- azo-j3-trypsinis permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kc,,. vary from twot o three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitoorf ptrypsin, was ah yperbolic competitive inhibitor of azo-p-trypsin. Thus, Tyr-151, part of subsite 5'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium bindinwgi th berenil supportedt he kinetic data obtainedw ith substrates. This permitst he integration of protein modification, kinetics, equilibrium binding, and crystallographic datao demonstrate a fine interaction between subsites S1-S3 and5 '2 in trypsin and azo-p-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Qr-151

A new extraction and purification methodology of Bowman-Birk inhibitors from seeds and germinated seeds of Macrotyloma axillare.

The Macrotyloma axillare Bowman-Birk inhibitors from seeds and germinated seeds (120 h) were purified by an alternative methodology and revealed the presence of different contents of inhibitors for each treatment. Mass analyses of inhibitors, from seeds and germinated seeds, showed masses of about 7 and 8 kDa. The 120 h-germinated seed fractions presented higher activity isoforms than seeds without treatment

Antigenic peptides capable of inducing specific antibodies for detection of the major alterations found in type 2B von willebrand disease.

VonWillebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides amongwhich threewere representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura

Synthesis of amides and sulfonamides of d-galactopyranosylamine and lactosylamine and evaluation of their interactions with the lectins from erythrina cristagalli and ricinus communis.

We report herein the synthesis of some -D-galactopyranosylamine and -lactosylamine amides and sulfonamides. The interactions of these compounds with lectins from the seeds of Erythrina cristagalli (LEC) and Ricinus communis (RCA120) were evaluated in a hemagglutination inhibitory activity assay. D-Galactose and lactose were used as reference compounds. The -lactosylamine amides and sulfonamides were nearly as active as lactose in inhibiting LEC mediated hemagglutination and were less active against RCA120 agglutinin. The -D-galactopyranosylamine amides and sulfonamides were, with one exception, considerably less active than Dgalactose in the assay with both lectins