Data processing optimization is one of the main concerns for developing of multiple-valued logic.... more Data processing optimization is one of the main concerns for developing of multiple-valued logic. An advantage could be achieved by realization of new functions existing in non-binary logic. These new logic functions could be implemented using quaternary look-up tables. In this work, a quaternary multiplexer circuit is designed to implement any n-variable quaternary logic function based on its truth table. Voltage-mode CMOS with multi-threshold transistors and multi-Vdd quaternary design was suggested. The multiplexer circuit consists of quaternary down literal circuits, binary inverters and binary pass transistor gates. All circuits were simulated with the Spice tool using TSMC 0.18 mum technology and have shown improvements in performance and power consumption and using less transistors than their equivalent binary circuits.
Background In the barley malting process, partial hydrolysis of β-glucans begins with seed germin... more Background In the barley malting process, partial hydrolysis of β-glucans begins with seed germination. However, the endogenous 1,3-1,4-β-glucanases are heat inactivated, and the remaining high molecular weight β-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous β-glucanases to reduce the β-glucans already present in the malt barley is highly desirable. Results The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of β-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the β-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-β-glucan (barley β-glucan), but showed only residual activity against 1,3-β-glucan (laminarin), or no activity at all against 1,4-β-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-β-glucan were in the ranges of 4–5, and 50–60°C, respectively. The Km and Vmax values for hydrolysis of β-glucan at pH 5.0 and 50°C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower. Conclusion The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze β-D-glucan that contains both the 1,3- and 1,4-bonds (barley β-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4–5, and temperatures in the range of 50–60°C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.
The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process ... more The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process that does not require supplementation with human or animal derived components in production, were assessed. Thus, the objective is to produce a safer vaccine at a lower cost. A total of 296 volunteers was divided into two groups: Group 1, which received the study vaccine, and Group 2, which received the Vero cells vaccine produced by Sanofi Pasteur. Five doses were given on days 0, 3, 7, 14 and 28. Blood samples for determination of rabies virus neutralizing antibodies were collected on days 0, 14, 38 and 90. The geometric mean titers (GMT) were much higher than 0.5 IU/ml in both groups on days 14, 38 and 90, indicating seroconversion according to the World Health Organization. In Group 1, however, the GMTs were higher than in Group 2, the difference being statistically significant in the two last samples. There was no statistical difference between the groups in the ratio of individuals with titers > or =0.5 IU/ml in each sample. Pain at the injection site was the most common adverse reaction and occurred most often in Group 1 (p < 0.001). All cases had a favorable evolution. There were no severe adverse reactions. It was concluded that the new vaccine is safe and immunogenic.
Automated protein sequencing is an important tool in protein characterization. Most instruments u... more 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.
The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bi... more The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.
Enterolobin is a plant cytolytic protein similar to the bacterial cytolysin aerolysin. Biochemica... more Enterolobin is a plant cytolytic protein similar to the bacterial cytolysin aerolysin. Biochemical and biophysical techniques were used to verify if enterolobin, like aerolysin, adopts a dimeric structure in solution. SDS^PAGE showed bands corresponding to enterolobin monomer, dimer and oligomers, whilst gel ¢ltration chromatography and electrospray mass spectrometry revealed preferred association of enterolobin as a dimer. Atomic force microscopy (AFM) of enterolobin showed images of a dimer assembly at a concentration as low as 10 W Wg/ml, similarly to aerolysin. The enterolobin in silico docked structure is coherent with AFM enterolobin dimer shapes. ß
The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cang... more The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations--episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements--and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8-12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.
Data processing optimization is one of the main concerns for developing of multiple-valued logic.... more Data processing optimization is one of the main concerns for developing of multiple-valued logic. An advantage could be achieved by realization of new functions existing in non-binary logic. These new logic functions could be implemented using quaternary look-up tables. In this work, a quaternary multiplexer circuit is designed to implement any n-variable quaternary logic function based on its truth table. Voltage-mode CMOS with multi-threshold transistors and multi-Vdd quaternary design was suggested. The multiplexer circuit consists of quaternary down literal circuits, binary inverters and binary pass transistor gates. All circuits were simulated with the Spice tool using TSMC 0.18 mum technology and have shown improvements in performance and power consumption and using less transistors than their equivalent binary circuits.
Background In the barley malting process, partial hydrolysis of β-glucans begins with seed germin... more Background In the barley malting process, partial hydrolysis of β-glucans begins with seed germination. However, the endogenous 1,3-1,4-β-glucanases are heat inactivated, and the remaining high molecular weight β-glucans may cause severe problems such as increased brewer mash viscosity and turbidity. Increased viscosity impairs pumping and filtration, resulting in lower efficiency, reduced yields of extracts, and lower filtration rates, as well as the appearance of gelatinous precipitates in the finished beer. Therefore, the use of exogenous β-glucanases to reduce the β-glucans already present in the malt barley is highly desirable. Results The zygomycete microfungus Rhizopus microsporus var. microsporus secreted substantial amounts of β-glucanase in liquid culture medium containing 0.5% chitin. An active protein was isolated by gel filtration and ion exchange chromatographies of the β-glucanase activity-containing culture supernatant. This isolated protein hydrolyzed 1,3-1,4-β-glucan (barley β-glucan), but showed only residual activity against 1,3-β-glucan (laminarin), or no activity at all against 1,4-β-glucan (cellulose), indicating that the R. microsporus var. microsporus enzyme is a member of the EC 3.2.1.73 category. The purified protein had a molecular mass of 33.7 kDa, as determined by mass spectrometry. The optimal pH and temperature for hydrolysis of 1,3-1,4-β-glucan were in the ranges of 4–5, and 50–60°C, respectively. The Km and Vmax values for hydrolysis of β-glucan at pH 5.0 and 50°C were 22.39 mg.mL-1 and 16.46 mg.min-1, respectively. The purified enzyme was highly sensitive to Cu+2, but showed less or no sensitivity to other divalent ions, and was able to reduce both the viscosity and the filtration time of a sample of brewer mash. In comparison to the values determined for the mash treated with two commercial glucanases, the relative viscosity value for the mash treated with the 1,3-1,4-β-glucanase produced by R. microsporus var. microsporus. was determined to be consistently lower. Conclusion The zygomycete microfungus R. microsporus var. microsporus produced a 1,3-1,4-β-D-glucan 4-glucanhydrolase (EC 3.2.1.73) which is able to hydrolyze β-D-glucan that contains both the 1,3- and 1,4-bonds (barley β-glucans). Its molecular mass was 33.7 kDa. Maximum activity was detected at pH values in the range of 4–5, and temperatures in the range of 50–60°C. The enzyme was able to reduce both the viscosity of the brewer mash and the filtration time, indicating its potential value for the brewing industry.
The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process ... more The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process that does not require supplementation with human or animal derived components in production, were assessed. Thus, the objective is to produce a safer vaccine at a lower cost. A total of 296 volunteers was divided into two groups: Group 1, which received the study vaccine, and Group 2, which received the Vero cells vaccine produced by Sanofi Pasteur. Five doses were given on days 0, 3, 7, 14 and 28. Blood samples for determination of rabies virus neutralizing antibodies were collected on days 0, 14, 38 and 90. The geometric mean titers (GMT) were much higher than 0.5 IU/ml in both groups on days 14, 38 and 90, indicating seroconversion according to the World Health Organization. In Group 1, however, the GMTs were higher than in Group 2, the difference being statistically significant in the two last samples. There was no statistical difference between the groups in the ratio of individuals with titers > or =0.5 IU/ml in each sample. Pain at the injection site was the most common adverse reaction and occurred most often in Group 1 (p < 0.001). All cases had a favorable evolution. There were no severe adverse reactions. It was concluded that the new vaccine is safe and immunogenic.
Automated protein sequencing is an important tool in protein characterization. Most instruments u... more 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.
The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bi... more The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.
Enterolobin is a plant cytolytic protein similar to the bacterial cytolysin aerolysin. Biochemica... more Enterolobin is a plant cytolytic protein similar to the bacterial cytolysin aerolysin. Biochemical and biophysical techniques were used to verify if enterolobin, like aerolysin, adopts a dimeric structure in solution. SDS^PAGE showed bands corresponding to enterolobin monomer, dimer and oligomers, whilst gel ¢ltration chromatography and electrospray mass spectrometry revealed preferred association of enterolobin as a dimer. Atomic force microscopy (AFM) of enterolobin showed images of a dimer assembly at a concentration as low as 10 W Wg/ml, similarly to aerolysin. The enterolobin in silico docked structure is coherent with AFM enterolobin dimer shapes. ß
The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cang... more The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations--episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements--and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8-12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.
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