Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

High expression of human carboxypeptidase M in Pichia pastoris: purification and partial characterization.

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.

Identification of serine proteinases with tonin-like activity in the rat submandibular and prostate glands.

Two enzymes with tonin-like activity, designated rSMT3 and rSMT4, were purified from rat submandibular glands and another, rPT1, was obtained from the prostate. The three enzyme fractions hydrolysed angiotensin I, angiotensinogen (AG) and synthetic AG(1-14) to form angiotensin II. With angiotensin I as substrate, pH optima were 6.5 for rSMT3, 6.8 for rSMT4 and 7.5 for rPT1. With AG(1-14), the three enzymes had optimal activity at pH 7.5. The three enzymes had negligible activity upon a kallikrein substrate, Ac-Phe-Arg-Nan. The enzymes were inhibited by aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride but not by two angiotensin converting enzyme inhibitors, ethylenediaminetetracetic acid or enalaprilat. N-tosyl-L-phenylalanine chloromethyl ketone (1 mM) inhibited rPT1 and rSMT4 but not rSMT3. Molecular weights (SDS-PAGE) were 31,700 for rSMT3, 29,800 for rSMT4 and 28,100 for rPT1. Total activity in the prostate is 150-times lower than in the submandibular gland, where 92% of the tonin activity is related to rSMT4. Physical and chemical properties suggest that rSMT4 is tonin, whereas rSMT3 and rPT1 are tonin-like enzymes which can generate angiotensin II from different substrates.

Distribution of tonin- and kallikrein-like activities in rat brain.

Tonin- and kallikrein-like activities were investigated in different regions of the rat brain. The highest values of specific tonin activity, expressed as picomoles of angiotensin II liberated per minute per milligram of protein, were found in the neurohypophysis (359 +/- 190) and in the archicerebellum (200 +/- 68). The highest level of total tonin activity (picomoles of angiotensin II liberated per minute) was observed in the archicerebellum (902 +/- 308) which retained 97% of total tonin activity of whole cerebellum. Tonin activity was not detected in the cortex of cerebellum and in the choroid plexus. Low to intermediate values of specific (1.09 +/- 0.33 to 5.32 +/- 2.37) and total (1.38 +/- 0.55 to 93.00 +/- 49.30) tonin activity were observed in adenohypophysis, cerebellar nuclei, hypothalamus, thalamus, midbrain, pons, medulla and neurohypophysis. The lowest values of specific (0.11 +/- 0.05) and total (0.69 +/- 0.31) activities were observed in the hippocampus. Kallikrein-like activity was expressed as picomoles of p-nitroaniline liberated per minute per milligram of protein. No activity was detected in the neurohypophysis. For other regions, the values of the specific activity ranged between 72 +/- 18 and 282 +/- 14 except for the choroid plexus which was 5 +/- 2. The total kallikrein activity was also homogeneous ranging from 330 +/- 100 to 1870 +/- 112. For the choroid plexus and adenohypophysis the total kallikrein activity was 2.0 +/- 0.8 and 27 +/- 11, respectively.

Characterization for virulence of an axenic strain of Entamoeba histolytica: evidence for overexpression of two proteins.

The pattern of protein from membrane and crude homogenate of Entamoeba histolytica strain 462 axenically cultivated (462ac) and submitted to hamster liver passage (462hp) was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrains 462ac and 462hp were compared by zymodeme analysis, erythrophagocytosis, cytopathic effect upon mammalian cells and the capability to induce abscess in hamster liver. The results showed no differences for erythrophagocytosis, cytopathic effect or zymodene for substrains 462ac and 462hp. A type II pathogenic zymodene was observed. Substrain 462ac did not induce liver abscess, but 462hp induced abscesses in 70% of the inoculated animals. The pattern of proteins from plasma membrane and crude homogenate were different. One protein of approximately 45kDa and another of 23 kDa showed at no detectable levels in the membrane of 462ac. A third component of approximately 90 kDa showed more intensively expressed in the 462ac.

Action of the saliva of Triatoma infestans (Heteroptera: Reduviidae) on sodium channels.

Saliva of Triatoma infestans (Klug) produced a progressive reduction in the amplitude of the compound action potential recorded from rat sciatic nerve. The saliva also inhibited the Na+ current on GH3 cells. The data demonstrate that the saliva of T. infestans has an inhibitory effect on Na+ channels. We conclude that this effect may decrease the generation and conduction of nerve action potential, thereby decreasing the sensitivity of the region in which the insect probes, in a manner similar to that of local anesthetics. This study demonstrates such activity in the saliva of hematophagous insects. The adaptive value of this activity is clear, because the quantity of blood obtained by triatomines is limited by the irritation caused during the feeding process.

Tonin and kallikrein-kinin system.

The kininogenase activity of tonin has been demonstrated by Ikeda and Arakawa, 1984. Tonin of the rat submandibular gland contracts the rat uterus independent of addition of the substrate. On repetition, the same dose of enzyme elicited desensitization. When a double dose was used the contraction again occurred. After desensitization to tonin the contraction to kallikrein was reduced about 80% of the control. The desensitization to kallikrein lightly reduced the contraction to tonin. When the muscle was desensitized to trypsin tonin did not evoke contraction. These experiments suggest the presence of two different substrates in the uterus, one more specific to kallikrein and the other for tonin. The experiments with the parallel uterus preparation strongly suggest release of kinin in the process of contraction of the uterus by tonin.

Effect of growth hormone and induced IGF-I release on germ cell population and apoptosis in the bovine testis.

Bovine growth hormone has been used in dairy cattle to increase milk production,but it also increases the twin parturition rate. This effect is mediated by insulin-like growth factor-I (IGF-I), which prevents follicular atresia by hindering apoptosis of granulosa cells. The action of GH and IGF-I on testicular function remains unclear. The goal of this study, therefore, was to verify the effects of short-term administration of GH and induced IGF-I release on the number of testicular germ cells, testicular morphology, and apoptosis in the bovine testis. Twenty Zebu bulls were split into 2 groups. The bulls in Group 1 (n = 10) were treated with 2 subcutaneous injections of bovine GH (500 mg/bull) 7 d apart. Group 2 bulls (n = 10) received placebos under the same protocol. All of the bulls were slaughtered 14 d after the start of treatment. Fragments of the testis were collected, fixed in Bouin's solution, embedded in paraffin, and the sections stained with hematoxilin and eosin. The paraffin-embedded sections were also used for in situ detection of apoptotic cells. Blood samples were collected at slaughter to measure serum levels of IGF-I, FSH and LH. Neither the number of Stage I seminiferous epithelium germ cells and the morphometric parameters (tubular diameter, seminiferous epithelium height, and volumetric proportions of structural components) nor the blood levels of FSH and LH showed a significant difference between the 2 groups. However, the treated animals showed an increase in serum IGF-I (P<0.01). Apoptotic germ cells were detected in the testis of both groups, showing the same pattern and a stage-specific apoptosis pattern. Most of the labeled cells were spermatocytes. The localization of apoptotic germ cells did not differ between groups. These results suggest that short-term administration of GH does not affect bovine spermatogenesis in adult bulls.

Hancornia speciosa Gomes induces hypotensive effect through inhibition of ACE and increase on NO.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Hancornia speciosa Gomes are popularly used in Brazil to treat diabetes and hypertension. Cardiovascular diseases are the main cause of death worldwide and their incidences are increasing in Brazilian population. The present study aimed to investigate the hypotensive effect and the mechanism of action of Hancornia speciosa Gomes. METHODS: A fraction of the ethanolic extract of leaves from Hancornia speciosa (SFH) was obtained and standardized by its content on rutin, bornesitol and quinic acid. Systolic blood pressure (SBP) of normotensive mice was measured by tail plethysmography. SFH was given orally and SBP was monitored for 5h. Angiotensin-converting enzyme (ACE) inhibitor activity of SFH (1mg/kg) or captopril (10mg/kg) was measured by colorimetric methods. Serum nitrite levels were measured by spectrophotometry. RESULTS: SFH induced a dose-dependent hypotensive effect in normotensive mice. The serum activity of ACE and the level of angiotensin II were significantly reduced by SFH and by captopril. Administration of SFH induced a significant increase on plasmatic level of nitrites and the systemic inhibition of nitric oxide synthase by L-NAME (20mg/kg) reduced the hypotensive effect of SFH. CONCLUSIONS: The present work demonstrated that Hancornia speciosa has a potent hypotensive effect in normotensive mice. The inhibition of ACE leading to reduction on angiotensin II and increase on NO levels might account for the hypotensive effect. These results support the use of Hancornia speciosa by traditional medicine as antihypertensive.

Leucurogin, a new recombinant disintegrin cloned from Bothrops leucurus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth.

Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC50 = 0.65 µM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 µg/day. No effects were observed upon adenosine 5'-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.

Effect of type 2 diabetes on plasma kallikrein activity after physical exercise and its relationship to post-exercise hypotension.

AIM: The present study was undertaken to determine the effects of type 2 diabetes (T2D) on plasma kallikrein activity (PKA) and postexercise hypotension (PEH). METHODS: Ten T2D patients (age: 53.6±1.3 years; body mass index: 30.6±1.0kg/m(2); resting blood glucose: 157.8±40.2mgdL(-1)) and 10 non-diabetic (ND) volunteers (age: 47.5±1.0 years; body mass index: 28.3±0.9kg/m(2); resting blood glucose: 91.2±10.5mgdL(-1)) underwent two experimental sessions, consisting of 20min of rest plus 20min of exercise (EXE) at an intensity corresponding to 90% of their lactate threshold (90LT) and a non-exercise control (CON) session. Blood pressure (BP; Microlife BP 3AC1-1 monitor) and PKA were measured during rest and every 15min for 135min of the postexercise recovery period (RP). RESULTS: During the RP, the ND individuals presented with PEH at 30, 45 and 120min (P<0.05) while, in the T2D patients, PEH was not observed at any time. PKA increased at 15min postexercise in the ND (P<0.05), but not in the T2D patients. CONCLUSION: T2D individuals have a lower PKA response to exercise, which probably suppresses its hypotensive effect, thus reinforcing the possible role of PKA on PEH.

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor.

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.

Kint3-4 promotes apoptosis and inhibition of angiogenesis in solid Ehrlich Tumor / Kint3-4 promove apoptose e inibição da angiogênese no tumor sólido de Ehrlich

A proteína Kint3-4 (PKint3-4), codificadora da angiostatina, é reconhecida por sua potencialidade antiangiogênica. O presente estudo teve como objetivo avaliar o efeito da proteína Kint3-4 no crescimento do tumor sólido de Ehrlich. Para isso, foram analisados a curva de desenvolvimento tumoral, o índice apoptótico e a dosagem de hemoglobina, a fim de se avaliar a angiogênese, em 20 camundongos Swiss fêmeas, inoculadas com o tumor sólido de Ehrlich em seus coxins plantares. Os resultados demonstraram a participação de PKint3-4 na indução à apoptose de células neoplásicas, na diminuição da concentração de hemoglobina e, principalmente, na diminuição do desenvolvimento tumoral. Sugere-se que a ação antitumoral, determinada pela sequência proteica utilizada, possa estar associada ao papel antiangiogênico da angiostatina, que indiretamente aumentaria o índice apoptótico das células neoplásicas, e/ou a uma ação direta da proteína Kint3-4 sobre essas células, estimulando-as a sofrerem apoptose.

A new method for bovine pepsinogen purification. Preparation of a specific antibody.

Bovine pepsinogen was purified from abomasum by ammonium sulphate precipitation and ionic exchange chromatography on DEAE-cellulose and Mono Q columns. Purified pepsinogen was shown to be homogeneous by analytical electrophoresis, having an estimated molecular mass of 46,000 Daltons. The isoelectric point, determined by analytical chromatofocusing was 4.6. Using this pepsinogen preparation to immunize rabbits, a specific antiserum of high titer was obtained.

Tonin-like activity present in the human submandibular gland.

An enzyme which is able to liberate angiotensin II from angiotensin I, angiotensinogen(1-14) fragment and angiotensinogen was purified from human submandibular gland. Its molecular weight is 110,000; is inhibited by PMSF but not by EDTA or enalaprilat. The pH optima for angiotensin II liberation were 4.0 for angiotensin I, 7.0 for angiotensinogen(1-14) fragment and 8.0 for angiotensinogen. The total amount of angiotensin II generating activity in the human submandibular gland is 5,000-times smaller than that in the rat gland.

On the mechanism of rat uterus desensitization to kallikrein.

An inactive form of kallikrein prepared by iodination with cold iodine, did not show any enzymatic or oxytocic action. However, a competitive pattern between this inactive and active kallikrein was observed in rat uterus preparation: When the inactive form was applied several times in the muscle, a single dose of active kallikrein was unable to cause contraction, but a double dose elicited a response. The rhythmic movement caused by a singular dose of active kallikrein, had its time curtailed by adding the inactive kallikrein to the bath. The inactive kallikrein did not interfere with bradykinin activity.

Kininogenase activity of rSMT3, a rat submandibular gland tonin.

rSMT3, a tonin like angiotensin II generating enzyme present in rSMG presents a potent oxytocic effect on the isolated rat uterus, which was blocked by a B2 bradykinin receptor antagonist. Under optimal conditions of pH, rSMT3 liberates kinin at rate 19-fold greater than angiotensin II.

The action of iodinated kallikrein on the rat uterus.

Iodinated kallikrein appeared inactive on synthetic substrates, was unable to liberate kinins from rat plasma, did not cause contraction of the rat uterus and did not potentiate bradykinin activity. After several doses of iodinated kallikrein were applied to the muscle, the contraction caused by kallikrein was blocked, but the response of the preparation to bradykinin was unaltered. If a double dose of kallikrein was applied, the contraction appeared again, and the response to bradykinin was also potentiated. These results support a kinin-mediated oxytocic action of kallikrein, and suggest a new interpretation for the mechanism of kallikrein desensitization in this preparation.

Molecular cloning and functional characterization of a mouse bradykinin B1 receptor gene.

The gene encoding a putative mouse bradykinin B1 receptor was cloned from a genomic library by low stringency screening. Analysis of two isolated clones revealed a region which contains an open reading frame uninterrupted by introns and encodes a 334 amino acid protein, which exhibits seven potential transmembrane domains and is 68% identical to the human and rabbit bradykinin B1 receptors. Lipopolysaccharide-treatment induces B1 receptor transcripts in the heart, liver, and lung. Stable expression of the coding region in COS-7 cells resulted in high levels of binding sites for the specific B1 ligand des-ARG10 kallidin (Kd = 1.3 nM; Bmax = 51 fmol/mg protein). The rank order of affinity of the receptor for the agonists and antagonists was: des-Arg9BKdes-Arg9Leu8BKdes- Arg10kallidin >> Hoe-140=bradykinin. Functional coupling of the cloned receptor was demonstrated by the dose-dependent effects of des-Arg(9)BK on the extracellular acidification rate in stably transfected COS-7 cells. This effect was not produced by bradykinin and could be blocked by the B1 antagonist des-Arg9Leu8BK.

An improved method to distinguish Entamoeba histolytica and Entamoeba dispar.

A 482 base pair gene fragment from samples of amoebae E. histolytica and E. dispar was amplified by PCR. The amplification products of fragments from the 2 species of amoebae presented differences in mobility in non-denaturing polyacrylamide gel, probably due to sequence-dependent conformational alterations in the DNA fragments. The method described here permits E. histolytica and E. dispar to be distinguished with greater sensitivity and rapidity.