DECBI - Departamento de Ciências Biológicas

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American trypanosomiasis (chagas' disease) in conventional and germfree rats and mice.
(1987) Silva, Marcelo Eustáquio; Evangelista, Elísio Alberto; Nicoli, Jacques Robert; Bambirra, Eduardo Alves; Vieira, Enio Carlos
Pharmacokinetic aspects of ter-butylaminoethyl dissulfide, an experimental drug against schistosomiasis in mice.
(1989) Andrade, Milton Hércules Guerra de; Freire, Antonio Carlos Teixeira; Nelson, David Lee
Pharmacokinetic profile of tert-butylaminoethanetiol.
(1990) Andrade, Milton Hércules Guerra de; Freire, Antonio Carlos Teixeira; Nelson, David Lee
Tyrosine 151 is part of the substrate activation binding site of bovine trypsin.
(1993) Oliveira, Maria Goreti de Almeida; Rogana, Edyr; Rosa, José César; Reinhold, Bruce B.; Andrade, Milton Hércules Guerra de; Greene, Lewis Joel; Guia, Marcos Luíz dos Mares
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.
Infection with Leishmania major induces interleukin-12 production in vivo.
(1994) Vieira, Leda Quercia; Hondowicz, Brian D.; Afonso, Luís Carlos Crocco; Wysocka, Maria; Trinchieri, Giorgio; Scott, Phillip
Experimental infections of mice with the protozoan parasite Leishmania major provide an excellent model for defining the conditions required for generation of CD4 ÷ Thl and Th2 cells in vivo. Since interleukin-12 (IL-12) has been implicated in the development of Thl cells, we investigated whether L. major stimulates IL-12 production in vitro or in vivo. Surprisingly, macrophages cultured in vitro failed to produce IL-12 following L. major infection. In contrast, lymph node cells from C3H mice infected for 2 days with L. major produced elevated levels of IL-12. In order to determine if the inability to stimulate IL-12 production was limited to in vitro infections, we infected macrophages in vivo by inoculating L. major into the peritoneal cavity. Peritoneal cells isolated 24 h later exhibited a significant increase in the number of cells producing IL-12. In addition, supernatants harvested from these cells following culture contained elevated levels of IL-12. These data indicate that L. major infection induces increased IL-12 production in mice.
Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.
(1995) Nabors, Gary S.; Afonso, Luís Carlos Crocco; Farrell, Jay P.; Scott, Phillip
Successful treatment in allergic, autoimmune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development of CD4+ Thi and Th2 cells, the requirements for switching an established response are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infected mice and that healing is associated with a switch from a Th2 to a Thl response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a Th2 response and enhance a Thi response. These observations are important for treatment of nonhealing forms of human leishmaniasis and also demonstrate that in a chronic infectious disease an inappropriate Th2 response can be switched to an effective Thi response.
NMDA receptors in NTS are involved in the bradycardic but not in the pressor response of chemoreflex.
(1995) Haibara, Andréa Siqueira; Colombari, Eduardo; Chianca Júnior, Deoclécio Alves; Bonagamba, Leni Gomes Heck; Machado, Benedito Honório
Activation of carotid chemoreceptors with intravenous potassium cyanide (KCN) produces increases in arterial pressure, bradycardia, and tachypnea. In the present study, we activated carotid chemoreceptors with KCN and the neurotransmission of the chemoreceptor reflex into the commissural nucleus tractus solitarii (NTS) was blocked with phosphonovaleric acid @P-5), an N-methyl-D-aspartate (NMDA)-selective antagonist. The aim of this study was to evaluate the involvement of NMDA receptors in the cardiovascular and respiratory responses produced by chemoreceptor activation in unanesthetized rats. The pressor response to KCN was not changed after microinjection of three different doses of AP-5 into the NTS, whereas the bradycardic response was reduced in a dosedependent manner. The increase in respiratory frequency in response to carotid chemoreceptor activation was also not affected by AP-5 microinjected into the NTS. The data indicate that the activation of the cardiovagal component of the chemoreflex in the commissural NTS is mediated by NMDA receptors, whereas pressor and ventilatory responses are not.
Microinjection of NMDA antagonist into the NTS of conscious rats blocks the Bezold-Jarisch reflex.
(1996) Chianca Júnior, Deoclécio Alves; Machado, Benedito Honório
The purpose of the present study was to evaluate whether or not cardiovagal excitatory and sympatho-inhibitory pathways of the Bezold-Jarischreflex at the NTS level were mediated by NMDA receptors. The Bezold-Jarischreflex was activated by intravenous (i.v.) injection of serotonin in consciousrats before and after microinjection of phosphonovaleric acid (AP-5), a selective NMDAantagonist, into the NTS. The Bezold-Jarischreflex was also activated before and after methyl-atropine (i.v.) in order to evaluate if the changes in mean arterial pressure were dependent on the bradycardic response. The data showed that AP-5 into the NTS produced a dose-dependent reduction in both bradycardic and hypotensive responses to activation of the Bezold-Jarischreflex. Methyl-atropine also blocked the bradycardic and hypotensive responses to Bezold-Jarischreflex activation. The data show that in consciousrats the cardiovagal component of the Bezold-Jarischreflex plays a major role in the cardiovascular changes produced by the activation of this reflex and suggest that the neurotransmission of the cardiovagal component of the Bezold-Jarischreflex is mediated by NMDA receptors.
Vaccine for prophylaxis and immunotherapy, Brazil.
(1996) Genaro, Odair; Toledo, Vicente de Paulo Coelho Peixoto de; Costa, Carlos Alberto da; Hermeto, Marco Victor; Afonso, Luís Carlos Crocco; Mayrink, Wilson
Autonomic processing of the cardiovascular reflexes in the nucleus tractus solitarii.
(1997) Machado, Benedito Honório; Chianca Júnior, Deoclécio Alves; Haibara, Andréa Siqueira; Mauad, Helde; Colombari, Eduardo
The nucleus tractus solitarii (NTS) receives afferent projections from the arterial baroreceptors, carotid chemoreceptors and cardiopulmonary receptors and as a function of this information produces autonomic adjustments in order to maintain arterial blood pressure within a narrow range of variation. The activation of each of these cardiovascular afferents produces a specific autonomic response by the excitation of neuronal projections from the NTS to the ventrolateral areas of the medulla (nucleus ambiguus, caudal and rostral ventrolateral medulla). The neurotransmitters at the NTS level as well as the excitatory amino acid (EAA) receptors involved in the processing of the autonomic responses in the NTS, although extensively studied, remain to be completely elucidated. In the present review we discuss the role of the EAA L-glutamate and its different receptor subtypes in the processing of the cardiovascular reflexes in the NTS. The data presented in this review related to the neurotransmission in the NTS are based on experimental evidence obtained in our laboratory in unanesthetized rats. The two major conclusions of the present review are that a) the excitation of the cardiovagal component by cardiovascular reflex activation (chemo- and Bezold-Jarisch reflexes) or by L-glutamate microinjection into the NTS is mediated by N-methyl-D-aspartate (NMDA) receptors, and b) the sympatho-excitatory component of the chemoreflex and the pressor response to L-glutamate microinjected into the NTS are not affected by an NMDA receptor antagonist, suggesting that the sympatho-excitatory component of these responses is mediated by non-NMDA receptors.
Neurotransmission of the Bezold-Jarisch reflex in the nucleus tractus solitarii of sino-aortic deafferentated rats.
(1997) Chianca Júnior, Deoclécio Alves; Bonagamba, Leni Gomes Heck; Machado, Benedito Honório
The Bezold-Jarisch _B-J. reflex was activated by serotonin _5-HT, i.v.. before and 10 min after bilateral microinjection of increasing doses of kynurenic acid, a non-selective antagonist of excitatory amino acid _EAA. receptors, into the commissural nucleus tractus solitarii _NTS. of sino-aortic deafferentated _SAD. and sham-operated _SO. unanesthetized rats. Increasing doses of kynurenic acid produced a dose-dependent blockade of the bradycardic and hypotensive responses to B-J reflex activation in both SO _from 0.1 to 10.0 nmolr100 nl. and SAD _from 0.1 to 2.0 nmolr100 nl.. Comparison of the effect of kynurenic acid on the hypotension and bradycardic dose–response curves showed a significant difference between SO and SAD rats, indicating that smaller doses of kynurenic acid are required in SAD rats than in SO rats to block the neurotransmission of the B-J reflex in the NTS. The data also showed that bilateral microinjection of kynurenic acid into the NTS at doses of 0.5 _131"7 vs. 115"8 mmHg. and 2.0 nmolr100 nl _140"11 vs. 116"9 mmHg. produced an acute and significant increase in the basal mean arterial pressure of SAD rats similar to that observed with the same doses in SO rats, which was back to control values 5–10 min later. The increase in basal mean arterial pressure immediately after kynurenic acid microinjection into the NTS of SAD rats suggests that in the absence of the arterial baroreceptors, the B-J reflex plays an important role in the autonomic regulation of the circulation. The data also show different dose–response curves for hypotension and bradycardia in response to B-J reflex activation in SAD than in SO rats in the presence of increasing doses of kynurenic acid into the NTS, indicating that the neurotransmission of the B-J reflex in the NTS of SAD rats is more sensitive to the blockade of the EAA receptors than in SO rats.
Morphological and molecular characterization of the Poxvirus BeAn 58058.
(1998) Fonseca, Flávio Guimarães da; Lanna, Maria Célia da Silva; Campos, Marco Antônio da Silva; Kitajima, Elliot Watanabe; Péres, José Noronha; Golgher, Romain Rolland; Ferreira, Paulo César Peregrino; Kroon, Erna Geessien
BeAn 58058 virus (BAV) was isolated from an Oryzomis rodent in Brazil. BAV was shown to be antigenically related to another poxvirus also isolated in Brazil, the Cotia virus, but it remained ungrouped. Electron microscopy revealed that BAV has a typical poxvirus morphology. The Hind III DNA profile of BAV genome was similar with that of VV WR and Lister, but some differences in the profile were detected. We have also detected the presence of genes homologous to vaccinia virus (VV WR) genes in the genome of BAV. Genes related to vaccinia thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene were found. The patterns of TK and VGF mRNA transcripts described for vaccinia virus infected cells were observed in BAV infected cells. Nucleotide sequence of BAV VGF homologous gene was similar to VVWRVGF sequences. This similarity was further seen when cross-hybridization of total genomes of BAV and VV was done. Polypeptide synthesis of BAV and vaccinia in infected cells also showed similar profiles. The genetic data was used to construct a phylogenetic tree where BAV and VV were placed at the same cluster. Based on our findings we propose that BAV is a vaccinia virus variant.
Kinin receptors mediating the effect of bradykinin on gastric acid secretion.
(1998) Alzamora, Andréia Carvalho; Ferreira, Patrícia Maria; Oliveira, César Nonato de; Alzamora, Fernando; Vieira, Maria Aparecida Ribeiro
Kinins, and bradykinin in particular, can affect electrolyte transport in different segments of the intestine, thus being able to stimulate chloride secretion. Since the stomach is the main chloride secretory unit in the gastrointestinal tract, we have investigated the effect of 2 8 2 6 bradykinin on acid secretion in the isolated frog (Rana catesbeiana) gastric mucosa. Bradykinin (2 3 10 to 2 3 10 M) and 9 2 9 2 7 des-Arg -bradykinin (2 3 10 to 23 10 M) were able to stimulate acid secretion in a dose-dependent manner. The bradykinin 2 79 2 85,872 7 (2 3 10 M) and des-Arg -bradykinin (2 3 10 M)-induced acid secretion was unaffected by Thi ,D -Phe -bradykinin (2 3 10 to 2 5 98 2 7 2 3 10 M), a B -kinin receptor antagonist. Interestingly, the B -kinin receptor antagonist, des-Arg -(Leu )-bradykinin (2 3 10 to 21 2 59 2 3 10 M) blocked both bradykinin- and des-Arg -bradykinin-stimulated acid secretion. Although the kininase I inhibitor, D -L -2 6 2 59 mercapto-methyl-3-guanidino-ethyl-propanoic acid (23 10 and 23 10 M) had no effect on des-Arg -bradykinin-induced acid 9 secretion, it inhibited the response to bradykinin. We conclude that bradykinin requires, at least in part, hydrolysis to des-Arg –bradykinin to increase gastric acid secretion and that its effect is mediated by B -kinin receptors.
Coinfection with Toxoplasma gondii inhibits antigen-specific Th2 immune responses, tissue inflamation, and parasitism in BALB/c mice infected with Leishmania major.
(1999) Santiago, Helton da Costa; Oliveira, Milton Adriano Pelli de; Bambirra, Eduardo Alves; Faria, Ana Maria Caetano de; Afonso, Luís Carlos Crocco; Oliveira, Leda Quercia; Gazzinelli, Ricardo Tostes
Lesion size, cellular infiltration, and tissue parasitism in the footpads of BALB/c mice infected with Leishmania major were all dramatically inhibited during acute but not chronic infection with Toxoplasma gondii. Similarly, acute but not chronic toxoplasmosis at the time of infection with L. major had a strong inhibitory effect on development of acquired immune responses mediated by Th2 lymphocytes. In contrast, no m
Differential expression of cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat spinal preganglionic nuclei.
(2000) Dun, S. L.; Chianca Júnior, Deoclécio Alves; Dun, Nae J.; Yang, J.; Chang, Jaw-Kang
The distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was investigated in the rat spinal cords with the use of an antiserum against the CART peptide fragment 55±102. CART-LI ®bers were concentrated in the super®cial layers of the dorsal horn of all segments. In addition to CART-LI ®bers, intensely labeled somata were detected in the intermediolateral cell column (IML) and other sympathetic preganglionic nuclei of the thoracolumbar segments. In the lumbosacral segments, CART-LI ®bers but not somata were seen in the sacral parasympathetic nucleus. Double-labeling the spinal sections with choline acetyltransferase (ChAT)-antisera and CARTantisera revealed that the large majority of ChAT-positive somata in the sympathetic preganglionic nuclei were CARTpositive, whereas ChAT-positive somata in the parasympathetic preganglionic nuclei were CART-negative. Our results show that CART-LI is selectively expressed in a population of sympathetic preganglionic neurons (SPNs), but not in parasympathetic preganglionic neurons (PPNs) of the rat.
Immunochemotherapy in american cutaneous leishmaniasis : immunological aspects before and after treatment.
(2001) Toledo, Vicente de Paulo Coelho Peixoto de; Mayrink, Wilson; Gollob, Kenneth John; Oliveira, Milton Adriano Pelli de; Costa, Carlos Alberto da; Genaro, Odair; Pinto, J. A.; Afonso, Luís Carlos Crocco
In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.
Role of ubiquitin-proteasome-dependent proteolytic process in degradation of muscle proteic from diabetic rabbits.
(2001) Galban, Victor Diaz; Evangelista, Elísio Alberto; Migliorini, Renato Hélios; Kettelhut, Isis do Carmo
The activity of ATP, ubiquitin (Ub)-dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ub-dependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ub-dependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ub-proteasomedependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.
Effects of low-protein diet on the baroreflex and Bezold-Jarisch reflex in conscious rats.
(2001) Costa, Fernanda Tropia; Cardoso, Leonardo Máximo; Pedrosa, Maria Lúcia; Silva, Marcelo Eustáquio; Haibara, Andréa Siqueira; Moraes, Márcio Flávio Dutra; Chianca Júnior, Deoclécio Alves
The present study evaluated the effects of a lowprotein diet (LP, 6% protein) on cardiovascular reflexes of Male Fisher rats. Three experimental groups, and their respective controls (15% protein), were used: (1) Baroreceptor reflex (BAR); (2) Bezold-Jarisch reflex (BJR); and (3) Prazosin treated. Dietary restriction began after weaning (three weeks) and lasted for a period of five weeks, after which animais were subjected to the experimental protocols. The BAR group was evaluated through injections of phenylephrine (0.5-S.O)Jg!Kg, i.v.) and sodium nitroprusside (0.7- 7.0J1g/Kg, i.v.) while the BJR was evaluated through injections of serotonin (2.5-10)Jg!Kg, i.v.). Our results showed an increased baroreflex gain bradycardia for the LP group (-0.96 ± 0.34 vs. -2.12 ± 1.06 bpm/ mmHg) and a larger bradycardia for the BJR the LP group (160 ± 18% greater than controls). Basal cardiovascular parameters were not different between LP and control rats, however LP animais treated with prazosin resulted in a larger fall of blood pressure (-19±3 vs. -28±5mmHg). In conclusion, LP rats present an increased responsiveness of BAR and BJR, which could contribute to their normal levels of cardiovascular parameters, in spite of the possible increase in the sympathetic vasomotor tonus observed with the prazosin protocol.
Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).
(2001) Pimenta, Adriano Monteiro de Castro; Eauclaire, Marie France Martin; Rochat, Hervé; Figueiredo, Suely Gomes de; Kalapothakis, Evanguedes; Afonso, Luís Carlos Crocco; Lima, Maria Elena de
We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house ¯ies (LD50 80.0 ng/house ¯y). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house ¯y). The amino-acid sequences of these toxins were established and found to be similar (60±95%) to previously described b-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure±function relationships that may be responsible for the differences in bioactivity displayed by these toxins.
Screening of expression libraries using ELISA : identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom.
(2001) Kalapothakis, Evanguedes; Jardim, S.; Magalhães, Ana Cristina Ribeiro; Mendes, Thais Melo; Marco, Luiz de; Afonso, Luís Carlos Crocco; Olórtegui, Carlos Delfin Chávez
The present report describes the use of ELISA with cDNA expression libraries in the identification of immunogenic proteins. The methodology described was applied using libraries constructed with mRNA isolated from Tityus serrulatus and Tityus bahiensis venom glands. In addition we describe for the first time the sequence of a neurotoxin from Tityus bahiensis venom gland named TbTx5 whose amino acid sequencing showed 93% similarity with the Tityus bahiensis TbTx IV-5 neurotoxin. The methodology described can be used for the generation of an immunogenic bank in order to contribute to genome and proteome projects.

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