Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model / [Avaliação do efeito imunomodulador da talidomida em macrófagos associados a tumores no modelo murino de câncer de mama metastático 4T1]

The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.(AU)

No presente trabalho, foi avaliado o efeito imunomodulador de diferentes doses de talidomida em macrófagos associados ao tumor (TAMs), em um modelo murino de câncer de mama. Camundongos foram inoculados com células 4T1, na região do flanco esquerdo, e tratados com talidomida, uma vez ao dia, nas doses de 50, 100 e 150mg/k, por massa corporal, do quinto dia ao 28º dia de inoculação tumoral. Os tumores foram medidos, o índice de proliferação celular e a contagem de TAMs foram avaliados nos tumores primários e nos pulmões com metástases. Além disso, a taxa de metástases pulmonares também foi avaliada. A talidomida na dose de 150mg/kg diminuiu significativamente o crescimento tumoral, o índice de proliferação celular e a infiltração de TAMs nos tumores primários. Por outro lado, maior número de TAMs e menor índice de proliferação celular foram observados nos pulmões metastáticos, em camundongos tratados com 150mg/kg de talidomida. Ademais, a talidomida na dose de 150mg/kg diminuiu significativamente os nódulos metastáticos nos pulmões. Os resultados demonstraram que o tratamento com talidomida diminuiu o crescimento tumoral e as metástases pulmonares em camundongos, associado com diferentes efeitos na infiltração de TAMs nesses locais.(AU)

Protease-activated receptor 4 plays a role in lipopolysaccharide-induced inflammatory mechanisms in murine macrophages.

The role of protease-activated receptor (PAR)4 in thrombin-induced platelet aggregation has been studied, and PAR4 blockade is thought to be useful as a new and promising approach in antiplatelet therapy in humans. In recent years, studies have been conducted to clarify the role of PAR4 in the host defense against invading microorganisms and pathogen-induced inflammation; however, to date, the role of PAR4 in mediating the LPS-induced inflammatory repertoire in macrophages remains to be elucidated. Here, we investigated the effects of the synthetic PAR4 agonist peptide (PAR4-AP) AYPGKF-NH2 on the phagocytosis of zymosan-FITC particles; NO, ROS, and iNOS expression; and cytokine production in C57/BL6 macrophages cocultured with PAR4-AP/LPS. The PAR4-AP impaired LPS-induced and basal phagocytosis, which was restored by pharmacological PAR4 blockade. Coincubation with the PAR4-AP/LPS enhanced NO and ROS production and iNOS expression; decreased IL-10, but not TNF-α, in the culture supernatant; and increased translocation of the p65 subunit of the proinflammatory gene transcription factor NF-κ-B. Our results provide evidence for a complex mechanism and new approach by which PAR4 mediates the macrophage response triggered by LPS through counter-regulating the phagocytic activity of macrophages and innate response mechanisms implicated in the killing of invading pathogens.

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.

Distinct types of tumors exhibit differential grade of inflammation and angiogenesis in mice.

Inflammation, angiogenesis and cytokine production are common features of almost, if not all tumors. However, the extent of these processes induced by different types of tumors has not been evaluated. We investigated the growth pattern of the experimental metastatic tumors, B16F10 melanoma, CT26.WT colon and 4T1 mammary cells inoculated in the flank of syngeneic mice and determined the degree of inflammation, angiogenesis, and production level of pro-inflammatory and pro-angiogenic cytokines within the tumors. In addition, we have analyzed vascular changes in the interface between the tumors and the adjacent cutaneous tissue and levels of relevant pro-inflammatory and pro-angiogenic cytokines systemically. The weight of tumors 15 days post-inoculation of 10(6) cells was markedly different. Melanomas were 2 and 10-fold heavier than colon and mammary tumors, respectively. Locally, CT26.WT tumor cells induced more vessels in cutaneous tissue adjacent to the tumors but systemically, the plasma levels of VEGF were higher (approximately 2-fold) in 4T1 tumor-bearing mice compared with the other two tumors. Mammary tumors presented the most prominent inflammatory content as assessed by a range of markers (inflammatory enzymes and cytokines). The vascular index, as determined by the intra-tumor amount of hemoglobin and number of vessels in hot spot areas, was also higher (approximately 2-fold) in melanomas compared with the other two tumors. These findings showing that distinct tumor types determine differential grade of inflammation, angiogenesis and host interaction in mice may provide new insights to tailor differential therapeutic approach based on the status of tumor biomarkers.

Avaliação da atividade citotóxica das sementes de Annona cornifolia A. St.-Hil. (Annonaceae) / Evaluation of the cytotoxic activity of seeds of Annona cornifolia A. St.-Hil. (Annonaceae)

Na família Annonaceae, especialmente o gênero Annona é muito apreciado por fornecer frutos comestíveis. Espécies desse gênero são utilizadas na medicina popular contra diabetes, malária e infecções. Muitas dessas atividades biológicas têm sido relacionadas às acetogeninas de anonáceas. O objetivo deste estudo foi avaliar a atividade citotóxica dos grupos e de uma acetogenina pura (cornifolina) obtidos a partir do extrato etanólico das sementes de Annona cornifolia A. St.-Hil. (Annonaceae). Esta atividade foi avaliada pelo ensaio colorimétrico MTT. Cornifolina (1), a única substância pura testada, apresentou citotoxicidade positiva sobre todas as linhagens tumorais avaliadas. Os grupos testados, todos caracterizados por espectroscopia no infravermelho (IV), apresentaram 68,7% dos valores de CI50 menores que 20,0 µg mL-1, sendo também considerados citotóxicos. As amostras testadas foram mais ativas que o taxol sobre melanoma humano (MeWo) e, ainda, o grupo G10-5 apresentou melhor atividade sobre fibroblasto tumorigênico de camundongo (L929). Além disso, os grupos mostraram menor citotoxidade do que o taxol sobre a linhagem normal (CHO).

The family Annonaceae, especially the genus Annona, is greatly appreciated for providing edible fruits. Species of this genus are used in folk medicine against diabetes, malaria and infections. Many of these biological activities have been related to annonaceous acetogenins. The aim of this study was to evaluate the cytotoxic activity of groups and a pure acetogenin (cornifolin) obtained from the ethanol extract of the seeds of Annona cornifolia A. St.-Hil. (Annonaceae). This activity was evaluated by using MTT colorimetric assay. Cornifolin (1), the only tested substance that was pure, showed positive cytotoxicity on all evaluated tumor cell lines. The tested groups, all characterized by infrared spectroscopy (IR), showed 68.7% of the IC50 values lower than 20.0 µg mL-1, also considered cytotoxic. The tested samples were more active than taxol on human melanoma (MeWo) and the group G10-5 showed better activity on mouse tumorigenic fibroblast (L929). In addition, the tested groups showed less cytotoxicity than taxol on the normal line (CHO).

Preparation and cytotoxicity of cisplatin-containing liposomes.

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 microM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0 percent, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70 percent cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 æM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas...

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.

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.

Reduced production of hyperalgesic substances by mononuclear cells from aged rats incubated with carrageenan: role of interleukin 2 and prostaglandins.

OBJECTIVE AND DESIGN: To compare the production of hyperalgesic substances by cells from aged (A; 24-month) and juvenile (J; 2-month) rats. MATERIAL AND METHODS: 4 x 10(5) purified mononuclear cells from J and A were 2 h-stimulated (test) or not (control) by 250 microg lambda-carrageenan/well. Supernatants (0.1 ml) were intraplantarly (ipl) injected in rat paws and development of mechanical hyperalgesia, in grams, evaluated. Rat interleukin 2 (IL 2) and prostaglandin E2 (PGE2) were also assessed for hyperalgesia development. RESULTS: Test supernatants from A compared with J induced significantly less hyperalgesia (-56 +/- 8.1 and -88.4 +/- 4.6 g, respectively, p < 0.05, ANOVA t test). Local injection of a specific, but not a control, antiserum against IL 2 significantly blocked both pure IL 2- and stimulated supernatants-derived hyperalgesia. In contrast to PGE-like materials, IL 2 content in supernatants was compatible with hyperalgesia development. CONCLUSIONS: Hyperalgesia induced by test supernatants was significantly less intense when derived from aged animals. IL 2 may have accounted for such hyperalgesia.

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.