Healing activity of proteolytic fraction (P1G10) from Vasconcellea cundinamarcensis in a cutaneous wound excision model.

The proteolytic enzymes from Vasconcellea cundinamarcensis have demonstrated efficacy to accelerate healing of skin lesions. We report here the efficacy of the proteolytic fraction - P1G10 during repair of excisional wounds in rodent model and analyze possible mediators involved. Using 0.05% P1G10 we observed on day 3rd increased wound contraction accompanied by an increase in activated neutrophils and VEGF relative to the control. On day 7th neutrophils returned to normal levels, and at 0.01% P1G10, an increase in NAG activity used to monitor monocyte/macrophage, was observed. On the other hand, on day 7th, we observed a decrease in TGF-ß at 0.05% P1G10, accompanied by an increased transformation of the latent TGF-ß to its active form. Also, on day 7th a reduction in MMP-9 activity and the number of apoptotic cells was observed along with an increase in fibroblast levels. Morphometrically, it appears that treatment with P1G10 accelerates the decline of initial inflammatory phase and reduces some unwanted effects likely caused by remaining TGF-ß or MMPs, thus enhancing the quality of scar. Overall, these data suggest that the active proteolytic fraction P1G10 enhances the efficacy of repair in excisional cutaneous wounds.

Malestar psicológico en familiares de pacientes con daño neurológico durante la fase subaguda de NeuroRehabilitación / Psychological distress in relatives of patients with neurological damage during the subacute phase of NeuroRehabilitation

Durante la última década la Neurorehabilitación ha comenzado a prestar mayor atención a las familias de personas que adquieren un daño neurológico. Este giro parece responder a un creciente número de estudios describiendo importantes niveles de malestar psicólogico en dichas familias y advirtiendo de su impacto en el proceso de rehabilitación. En Chile, lamentablemente, no contamos con estudios que exploren el estado emocional de familiares en ninguna de las etapas de rehabilitación, esto, a pesar de existir guías clínicas que sugieren el abordaje familiar como meta central. Dicha falta de información limita tanto la capacidad diagnóstica como interventiva de los equipos de rehabilitación. El objetivo de este artículo es describir, y comparar, el perfil de malestar psicológico en familiares de personas con lesión cerebral y medular en etapa subaguda de rehabilitación. Método. 89 familiares de personas con daño neurológico (Lesión Cerebral Adquirida = 50; Trauma Raquimedular = 39) respondieron el General Health Questionnaire-28 como medida de malestar psicológico. Este instrumento que se compone de cuatro subescalas: síntomas somáticos, ansiedad e insomnio, disfunción social y depresión grave. Resultados. En ambas poblaciones el puntaje total del GHQ-28 se observó por encima de los estándares poblacionales, sugiriendo niveles clínicos de malestar psicológico en 90 por ciento de la muestra. No se observaron diferencias entre ambas poblaciones en términos de puntaje total o puntaje de subescalas, sugiriendo similares perfiles de malestar psicólogico. Dicho perfil se caracterizó por altos niveles de ansiedad e insomnio, seguido en menor grado de síntomas somáticos...

During the last decade NeuroRehabilitation has begun to pay more attention to the families of people with neurological damage. This shift seems to respond to a growing number of studies describing significant levels of psychological distress in families, and warning professionals of its potential impact in the process of rehabilitation. In Chile, unfortunately, we have no studies that explore the emotional state of families in any stage of the rehabilitation process, this, despite the existence of clinical guidelines suggesting to address family needs as a central goal. This lack of information limits rehabilitation teams’ ability as well as their capacity to develop interventions. The aim of this article is to describe, and compare, the profile of psychological distress in relatives of people with brain damage and spinal cord injuries, during the sub-acute phase of rehabilitation. Method. 89 relatives of people with neurological damage (Acquired Brain Injury = 50; Spinal Cord Injury = 39) completed the General Health Questionnaire-28, a self-report measure of psychological distress. This instrument has four subscales, each of them screening for different types of symptoms: somatic, anxiety/insomnia, social dysfunction and severe depression. Results. Both neurological groups presented GHQ-28 total scored above population standards, thus suggesting clinical levels of psychological distress in 90 percent of the sample. No differences were observed between the two groups in terms of GHQ-28 total score or subscale score, thus, suggesting similar profiles of psychological distress. High levels of anxiety and insomnia, followed to a lesser degree by somatic symptoms, characterized this profile...

A phytopathogenic cysteine peptidase from latex of wild rubber vine Cryptostegia grandiflora.

A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 µg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins.

Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.

Molecular characterization by LSSP-PCR and DNA sequencing of a pathogenic isolate of Leptospira interrogans from Brazil.

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31-21%), Hardjo Norma (46-40%), Hardjo hardjobovis (18-10%), Mini (8-4%) and Wolffi (7-4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three-dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single-specific primer polymerase chain reaction (LSSP-PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.

Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom.

A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bß-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh(-1)nmol(-1) ± 0.1031 and K m = 14.59 mg/mL ± 4.610).

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis.

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

[Anesthetic management during removal of a giant intrathoracic mass]. / Manejo anestésico en la exéresis de un tumor intratorácico gigante.

We report the case of a young woman with a giant intrathoracic angiomyolipoma accounting for 10% of her weight and occupying 75% of the right hemithorax and 30% of the left. Before anesthetic induction, an arterial line and a central venous catheter were applied for monitoring; neck and thoracic punctures were avoided. The trachea was intubated with a double lumen tube after provision of sedation and analgesia with remifentanil-midazolam and topical anesthesia of the larynx. A rigid bronchoscope and extracorporeal circulation were available at all times and muscle relaxants were avoided. Ventilation was maintained with pressure support until the mass effect was resolved. The patient was transferred to the intensive care unit, extubated after 24 hours, and discharged 5 days after surgery. We describe the recommendations for perioperative management in cases involving this type of tumor and the complications that can develop. Recent symptoms, diagnostic images, and the results of lung function tests provide information for guiding the anesthetic approach. The obstructive ventilatory compromise caused by a giant mass depends more on location than size. Extracorporeal circulation or rigid bronchoscopy might be needed at any time during surgery.

Manejo anestésico en la exéresis de un tumor intratorácico gigante / Anesthetic management during removal of a giant intrathoracic mass

Se describe el caso de una mujer joven con un angiomiolipomagigante intratorácico correspondiente al 10%de su peso, que ocupaba 75% del hemitórax derecho y30% del izquierdo. Previo a la inducción anestésica semonitorizó a la paciente de forma invasiva, evitando losaccesos centrales en cuello o tórax. La intubación se realizócon tubo de doble luz izquierdo, previa sedaciónanalgesiacon remifentanilo-midazolam y anestesia tópicasobre las estructuras laríngeas. Se mantuvodisponibilidad permanente de broncoscopio rígido y circulaciónextracorpórea y se evitó el uso de relajantesmusculares manteniendo la ventilación con soporte depresión hasta que el efecto de masa fue resuelto. Lapaciente fue trasladada a UCI, extubada a las 24 horas ydada de alta a los 5 días del postoperatorio. Se describenlas recomendaciones perioperatorias en este tipo detumores y los posibles tipos de complicaciones.Al enfrentarnos a este tipo de pacientes, la sintomatologíareciente y la evaluación mediante imágenes y pruebasfuncionales nos guiarán en el enfoque anestésico. Elcompromiso obstructivo generado por estas masasgigantes depende principalmente de su localización másque de su tamaño. Cualquier momento del intraoperatoriopuede requerir la necesidad de circulación extracorpóreao broncoscopia rígida

We report the case of a young woman with a giantintrathoracic angiomyolipoma accounting for 10% of herweight and occupying 75% of the right hemithorax and30% of the left. Before anesthetic induction, an arterialline and a central venous catheter were applied for monitoring;neck and thoracic punctures were avoided. Thetrachea was intubated with a double lumen tube afterprovision of sedation and analgesia with remifentanilmidazolamand topical anesthesia of the larynx. A rigidbronchoscope and extracorporeal circulation were availableat all times and muscle relaxants were avoided. Ventilationwas maintained with pressure support until themass effect was resolved. The patient was transferred tothe intensive care unit, extubated after 24 hours, and discharged5 days after surgery. We describe the recommendationsfor perioperative management in cases involvingthis type of tumor and the complications that can develop.Recent symptoms, diagnostic images, and the resultsof lung function tests provide information for guiding theanesthetic approach. The obstructive ventilatory compromisecaused by a giant mass depends more on locationthan size. Extracorporeal circulation or rigid bronchoscopymight be needed at any time during surgery

Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis.

The pyrazinamidase gene coding for the enzyme that activates the bactericidal drug pyrazinamide contains a polymorphic site that is preserved in Mycobacterium bovis. We synthesized two sets of primers, one encompassing a 180 bp fragment, and the second spanning a 726 bp fragment including the full pncA gene. Following PCR of Mycobacterium tuberculosis and M. bovis samples, it is possible to discriminate by this polymorphism between these species by digestion with Eco065 I. Digestion of the 180 bp fragment results in two fragments of 101 and 79 bp, specific for M. tuberculosis. Alternatively, digestion of the 726 bp fragment yields three fragments of 452, 165 and 109 bp for M. tuberculosis, but only two fragments of 561 and 165 bp for M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73 percent of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96 percent of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant.

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73% of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96% of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

A mitogenic protein fraction in latex from Carica candamarcensis.

Latex from Caricaceae contains a number of proteins believed to be part of a defense mechanism that protects these plants from wounding. Prior evidence suggests that some components in Carica papaya improve healing of ulcerous wounds in mammals. This study shows the chromatographic isolation of a protein fraction from C. candamarcensis that stimulates cell proliferation of mammalian cells by measuring MTT reduction and thymidine incorporation. The effect appears to be cell specific as L929, MDA-MB231 and BHK-21 cells are stimulated while no effect is seen on CHO cells. The maximal stimulatory effect reaches 2.2-fold 72 h after addition of the active fraction to L929, 1.8-fold in MDA-MB231 cells and 1.6-fold in BHK cells. Proteolytic inactivation of the active fraction suggests that a protein is responsible for the proliferative activity and its size is estimated between 10 and 25 kDa. A potential candidate for this function is a 23 kDa protein found in the fraction that reacts with human EGF antibody.

Mycobacterium bovis: polymerase chain reaction identification in bovine lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism.

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70 percent of the samples. PCR confirmed the identification of 23 samples (100 percent) that grew in culture, 9 samples (60 percent) that failed to grow in culture, plus 6 (37.5 percent) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes

Purification of a cysteine proteinase from Carica candamarcensis L. and cloning of a genomic putative fragment coding for this enzyme.

We describe the purification of a cysteine proteinase from latex of Carica candamarcensis, hereby designated CC23. The enzyme has been purified by ion-exchange chromatography and behaves electrophoretically as a monomer of M(r) 23,000 and optimal pH of 8.0. It displays a basic isoelectric point, has one cysteine residue in the active site by titration with E-64, confirmed by DNA sequencing, and responds to proteinase inhibitors as a classic cysteine proteinase. The K(m) and k(cat)/K(m) for CC23 using BAPNA were respectively 14.7 +/- 1.8 x 10(-4) M and 1.3 x 10(3) M(-1) s(-1). Therefore, the catalytic efficiency of CC23 is sixfold higher than that of CC-I, another proteinase from the same plant. DNA primers were designed to amplify by PCR a genomic sequence related to this enzyme. An 895-bp DNA fragment was cloned and sequenced. It shows strong homology with chymopapain isoform IV from C. papaya. The translated sequence is similar to that of chymopapain isoform II (73%) and CC-III (77%) from C. candamarcensis.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis.

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100 degrees C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Chemical characterization and biological activity of Macfadyena unguis-cati (Bignoniaceae).

Macfadyena unguis-cati (L.) has been widely used in folk medicine as an anti-inflammatory, antimalarial and antivenereal. The purpose of this study was to chemically characterize the main plant components, and to evaluate the biological properties of some of the fractions derived from leaves (MACb) and liana (MACa) of this plant. Chemical characterization allowed the identification of the compounds corymboside, vicenin-2, quercitrin, chlorogenic acid, isochlorogenic acid, lupeol, beta-sitosterol, beta-sitosterylglucoside, allantoin and lapachol. The biological screening of fractions and/or purified substances derived from fractions revealed antitumoral and antitrypanosomal activities in fractions MACa/lapachol and MACb/MACb21, respectively. The anti-lipoxygenase and anti-cyclooxygenase effect seen in fractions MACa and MACb showed a partial correlation with the anti-inflammatory property attributed to this plant.