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1.
An. acad. bras. ciênc ; 89(3,supl): 2155-2165, 2017. tab, graf
Article in English | LILACS | ID: biblio-886808

ABSTRACT

ABSTRACT Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.


Subject(s)
Plant Extracts/pharmacology , Plant Extracts/chemistry , Plant Leaves/chemistry , Enzyme Inhibitors/pharmacology , Antioxidants/pharmacology , Obesity/drug therapy , Phenols/analysis , Water/analysis , Trypsin/pharmacology , Chromatography, High Pressure Liquid , Psidium/chemistry , alpha-Amylases/pharmacology , alpha-Glucosidases/pharmacology , Lipase/pharmacology
2.
Int. braz. j. urol ; 42(4): 817-824, July-Aug. 2016. tab, graf
Article in English | LILACS | ID: lil-794669

ABSTRACT

ABSTRACT Purpose: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. Materials and Methods: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. Results: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. Conclusion: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.


Subject(s)
Animals , Mice , Neoplastic Stem Cells/cytology , Urinary Bladder Neoplasms/pathology , Trypsin/pharmacology , Cell Adhesion/drug effects , Cell Separation/methods , Cell Culture Techniques/methods , Neoplastic Stem Cells/chemistry , Biomarkers, Tumor , Cell Differentiation , Culture Media, Serum-Free , Cancer Vaccines/immunology , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , Flow Cytometry , Mice, Nude
3.
Arq. bras. oftalmol ; 70(3): 407-411, maio-jun. 2007. ilus
Article in Portuguese | LILACS | ID: lil-459825

ABSTRACT

OBJETIVO: Avaliar as características morfológicas da membrana amniótica desepitelizada por diferentes técnicas. MÉTODOS: A membrana amniótica humana foi coletada no momento do parto, fixada em concentrações crescentes de glicerol (0-50 por cento em DMEM) e preservada a 80°C até a hora de ser usada. O estudo consistiu de 4 grupos: epitélio intacto (controle) e membranas desepitelizadas pela tripsina (2 mg/mL a 1:250), dispase (1,2 U/mL em solução salina balanceada de Hank livre de Mg2+ e Ca2+) e ácido etilenodiaminotetra-acético (EDTA), 0,02 por cento). As amostras foram submetidas à análise por microscopia eletrônica (de varredura e de transmissão). RESULTADOS: A microscopia eletrônica de varredura mostrou epitélio intacto no grupo controle e sua ausência nas membranas amnióticas desepitelizadas pela tripsina e pela dispase. Naquelas tratadas com o ácido etilenodiaminotetra-acético, havia áreas com e sem epitélio. Quando avaliadas pela microscopia eletrônica de transmissão, o epitélio estava intacto e firmemente aderido à membrana basal através de hemidesmossomos nos grupos controle e em parte do ácido etilenodiaminotetra-acético. Havia apenas fibras colágenas nas membranas tratadas com dispase e tripsina. CONCLUSÕES: O tratamento da membrana amniótica com tripsina e dispase pode causar completa retirada do epitélio e da membrana basal, ao passo que o ácido etileno- diaminotetra-acético pode preservar áreas com epitélio intacto e parcialmente destruir a membrana basal em outras.


PURPOSE: To evaluate the morphological features of the amniotic membrane denuded by different techniques. METHODS: Human amniotic membrane was collected at the time of delivery, fixed in increasing concentrations of glycerol (0-50 percent in DMEM) and preserved at -80°C until the time of use. The study consisted of 4 groups: intact epithelium (control) and denuded by trypsin (2 mg/mL at 1:250), dispase (1.2 U/mL in Mg2+ and Ca2+ free Hank's balanced salt solution) or ethylenediaminetetraacetic acid (EDTA), 0.02 percent. Specimens were submitted to electron (scanning and transmission) microscopy analysis. RESULTS: Scanning electron microscopy disclosed intact epithelium in the control group and its absence in the amniotic membranes denuded by trypsin and dispase. In those denuded by ethylenediaminetetraacetic acid there were areas with and without epithelium. When assessed by transmission electron microscopy, the epithelium was intact and firmly adhered to the basement membrane by hemidesmossomes in controls and in parts of ethylenediaminetetraacetic acid group. There were only collagen fibers in the dispase- and trypsin-treated groups. CONCLUSIONS: Trypsin and dispase treatment of the amniotic membrane may cause complete denuding of the epithelium and basement membrane whereas ethylenediaminetetraacetic acid may leave some intact epithelium-areas and partially destroy the basement membrane in others.


Subject(s)
Humans , Amnion/ultrastructure , Epithelial Cells/ultrastructure , Amnion/drug effects , Cell Culture Techniques , Edetic Acid/pharmacology , Endopeptidases/pharmacology , Microscopy, Electron , Trypsin/pharmacology
4.
Braz. j. biol ; 67(1): 105-109, Feb. 2007. ilus
Article in English | LILACS | ID: lil-449633

ABSTRACT

BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.


INTRODUÇÃO: No tratamento do paciente grande queimado, onde se usa lâminas de epiderme cultivadas, o principal problema é o tempo necessário para sua produção. O isolamento tradicional de queratinócitos utiliza normalmente tripsina. No presente estudo, foi utilizada uma modificação do método de isolamento tradicional, que poderia produzir uma maior pureza e um maior número de colônias formadas a partir da população de células epidérmicas isoladas. OBJETIVO: Comparar a ação da tripsina e da termolisina no isolamento de queratinócitos usando pele de prepúcio de récem-nascidos. MÉTODOS: Essa metodologia utilizou a termolisina, que realiza a separação seletiva entre a epiderme e a derme. Após essa separação, a epiderme foi submetida à ação da tripsina para a obtenção da suspensão celular. RESULTADOS: Comparado ao método convencional, os experimentos mostraram que no grupo da termolisina mostrou facilidade para a separação entre a epiderme e derme, não houve contaminação por fibroblastos e produziu um maior número de colônias formadas, com diferença estatística significante. CONCLUSÃO: O número de colônias no grupo termolisina foi significantemente maior que no grupo tripsina.


Subject(s)
Humans , Infant, Newborn , Colony-Forming Units Assay , Cell Separation/methods , Keratinocytes/cytology , Thermolysin/pharmacology , Trypsin/pharmacology , Keratinocytes/transplantation , Tissue Engineering
5.
Article in English | WPRIM | ID: wpr-72709

ABSTRACT

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.


Subject(s)
Trypsin/pharmacology , Stem Cells/cytology , Rabbits , Limbus Corneae/cytology , Humans , Epithelium, Corneal/cytology , Edetic Acid/pharmacology , Cells, Cultured , Cell Culture Techniques , Cell Count , Animals
6.
Article in English | IMSEAR | ID: sea-34115

ABSTRACT

This study describes the development of Cryptosporidium parvum in MDCK, MA-104, Hep-2 and Vero cell lines. Differences in susceptibility, infectivity, and the methodology of excystation were determined. Various solutions were considered to determine the factors which enhanced the excystation (eg with and without sodium hypochlorite, trypsin or sodium taurocholate). It was shown that the sporozoites could be excysted in media either with or without trypsin and sodium taurocholate, but the number of sporozoites in the latter solution was less than the former one. Only oocysts digested by sodium hypochlorite and trypsin can enter the culture cells. Numerous meronts and oocysts were demonstrated and persisted for 9 days. Asexual stages were not observed in MA-104. Only few oocysts could be detected 1-3 days post-inoculation. There was a significant difference between the number of oocysts, which invaded MDCK, MA-104, and Hep-2 cells. MDCK gave the highest susceptibility to oocyst invasion among the three cell lines and asexual stages were also found. Among the 25 isolates, which had been cultivated, 23 isolates could infect MDCK and Hep-2. Only 2 isolates could not infect the MDCK cell. These 2 isolates could infect the Vero cell and yielded high numbers of trophozoites. Praziquantel (PZQ), doxycycline, and paromomycin (PRM) were tested on the infecting parasites. The drugs were added either with the inoculum or 24 hours after inoculation. None of them was effective, including PRM, which had been previously reported as effective.


Subject(s)
Acquired Immunodeficiency Syndrome/complications , Animals , Anthelmintics/administration & dosage , Anti-Bacterial Agents/administration & dosage , Cell Culture Techniques , Cell Line/drug effects , Cryptosporidiosis/complications , Cryptosporidium parvum/drug effects , Feces/parasitology , Humans , Oocysts/drug effects , Sodium Hypochlorite/pharmacology , Sporozoites/drug effects , Taurocholic Acid/pharmacology , Trypsin/pharmacology
7.
Indian J Exp Biol ; 2004 May; 42(5): 452-60
Article in English | IMSEAR | ID: sea-55694

ABSTRACT

A haemorrhagic protein toxin (SA-HT) was isolated and purified from the spine extract of the Indian venomous butterfish, S. argus Linn, by two step ion exchange chromatography. The toxin was homogeneous in native and SDS-PAGE gel. SDS-molecular weight of the toxin was found to be 18.1 +/- 0.09 kDa. SA-HT produced severe haemorrhage on stomach wall but devoid of cutaneous haemorrhage. UV, EDTA, trypsin, protease, cyproheptadine, indomethacin, acetylsalicylic acid and BW755C treatment significantly antagonized the haemorrhagic activity of SA-HT. The toxin produced dose and time dependent oedema on mice hind paw, which was significantly encountered by cyproheptadine, indomethacin and BW755C. SA-HT increased capillary permeability on guinea pig dorsal flank. On isolated guineapig ileum, rat fundus and uterus, SA-HT produced slow contraction which was completely antagonised by prostaglandin blocker SC19220. On isolated rat duodenum, SA-HT produced slow relaxation. SA-HT significantly increased plasma plasmin, serum MDA level and decreased serum SOD level indicating the possible involvement of cyclooxygenase and lipooxygenase pathway.


Subject(s)
4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Capillaries , Chromatography, Ion Exchange , Cyproheptadine/pharmacology , Dose-Response Relationship, Drug , Edema/chemically induced , Edetic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Female , Fish Proteins/chemistry , Fish Venoms/chemistry , Gastrointestinal Agents/pharmacology , Guinea Pigs , Indomethacin/pharmacology , Lipoxygenase/metabolism , Mice , Muscle, Smooth/drug effects , Perciformes , Permeability , Rats , Spine/metabolism , Superoxide Dismutase/metabolism , Time Factors , Trypsin/pharmacology , Ultraviolet Rays , Uterus/drug effects
8.
J Health Popul Nutr ; 2003 Jun; 21(2): 90-5
Article in English | IMSEAR | ID: sea-834

ABSTRACT

Forty-five strains of Shigella were screened for haemagglutinin production and broad-spectrum haemagglutination reaction. Mannose-sensitive haemagglutinin (MSHA) was found in 22 strains [Shigella flexneri (7), S. dysenteriae (7), S. sonnei (3), and S. boydii (5)]. Eighteen strains harboured mannose-resistant haemagglutinin (MRHA), and 8 strains were observed to be non-haemagglutinating to guinea pig erythrocyte. With the exception of human erythrocytes (O, A, B, and AB), the observed MSHA and MRHA also agglutinated the erythrocytes of rabbit, sheep, rat, chicken, and horse, suggesting a broad-spectrum haemagglutinating property. Haemagglutinins of S. flexneri and S. dysenteriae elicited a relatively stronger haemagglutinating activity with agglutinability to chicken and rabbit erythrocytes enhanced by trypsinization. Haemagglutination reaction with guinea pig erythrocyte was generally inhibited by sialic acid, while simple sugars, such as D-glucose, D-galactose, N-acetylgalactosamine, N-acetylglucosamine, and D-rhamnose, elicited no inhibitory effect. The results of the study revealed broad-spectrum haemagglutinin expression by circulating Shigella strains in Nigeria.


Subject(s)
Animals , Bacterial Adhesion , Chickens , Erythrocytes/metabolism , Guinea Pigs , Hemagglutination/drug effects , Hemagglutinins/metabolism , Horses , Humans , Nigeria , Rabbits , Rats , Sheep , Shigella/physiology , Trypsin/pharmacology
9.
Indian J Biochem Biophys ; 2001 Dec; 38(6): 361-7
Article in English | IMSEAR | ID: sea-27937

ABSTRACT

Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by very low concentrations of trypsin at 37 degrees C. PEP+Mg2+ and several other effectors of PEP carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition (in presence of PEP, Mg2+ and HCO3) although yielded a truncated subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic condition, only malate sensitivity was appreciably affected. Significant protection of the enzyme activity against trypsin during catalytic phase could be either due to a conformational change induced on substrate binding. Several lines of evidence indicate that the inactivation caused by a cleavage at a highly conserved C-terminal end of the subunit.


Subject(s)
Bicarbonates/pharmacology , Fluorescence , Glucose-6-Phosphate/pharmacology , Glycine/pharmacology , Kinetics , Magnesium/pharmacology , Malates/pharmacology , Phosphoenolpyruvate Carboxylase/antagonists & inhibitors , Phosphorylation , Protein Conformation , Sulfhydryl Compounds/chemistry , Trypsin/pharmacology , Zea mays/enzymology
10.
Mem. Inst. Oswaldo Cruz ; 95(5): 693-700, Sept.-Oct. 2000. tab, ilus
Article in English | LILACS | ID: lil-267896

ABSTRACT

Bacillus thuringiensis produces d-endotoxins that require proteolytic processing to become active. The activation of the B. thuringiensis subsp. medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin of B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity. Amino terminal sequence of the C. quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys31 and Asp32, with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu29 and Arg30, and for the chymotrypsin-resistant fragment between Arg30 and Lys31.


Subject(s)
Animals , Bacillus thuringiensis/metabolism , Bacterial Proteins/metabolism , Endotoxins/metabolism , In Vitro Techniques , Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Chymotrypsin/pharmacology , Culex , Endotoxins/chemistry , Erythrocytes/metabolism , Hydrogen-Ion Concentration , Sequence Analysis, Protein , Sheep , Trypsin/pharmacology
11.
Mem. Inst. Oswaldo Cruz ; 95(5): 701-5, Sept.-Oct. 2000. graf
Article in English | LILACS | ID: lil-267897

ABSTRACT

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80§C for 10 min or at 55§C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.


Subject(s)
Animals , Chitinases/metabolism , Encephalitozoon/enzymology , Cellulase/metabolism , Chitinases/antagonists & inhibitors , Spores/enzymology , Trypsin/pharmacology
12.
Mem. Inst. Oswaldo Cruz ; 95(4): 495-501, July-Aug. 2000.
Article in English | LILACS | ID: lil-264230

ABSTRACT

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.


Subject(s)
Humans , Animals , Erythrocytes/metabolism , Glycophorins/metabolism , Merozoite Surface Protein 1/chemistry , Plasmodium falciparum/metabolism , Amino Acid Sequence , Chymotrypsin/pharmacology , Erythrocytes/drug effects , Erythrocytes/parasitology , Glycophorins/biosynthesis , Merozoite Surface Protein 1/metabolism , N-Acetylneuraminic Acid/metabolism , Plasmodium falciparum/drug effects , Sequence Analysis, Protein , Trypsin/pharmacology
13.
Article in English | WPRIM | ID: wpr-159772

ABSTRACT

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.


Subject(s)
Animals , Astrocytes/enzymology , Blotting, Western , Brain/enzymology , Calcium/metabolism , Chromatography, Ion Exchange , Endopeptidases/pharmacology , Enzyme Stability , Ethanol/pharmacology , Glioma , Immunoblotting , Immunohistochemistry , Male , Molecular Weight , Neurons/enzymology , PC12 Cells , Transglutaminases/isolation & purification , Transglutaminases/immunology , Transglutaminases/chemistry , Rats , Rats, Sprague-Dawley , Trypsin/pharmacology , Tumor Cells, Cultured
14.
Article in English | WPRIM | ID: wpr-159769

ABSTRACT

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.


Subject(s)
Animals , B-Lymphocytes/physiology , B-Lymphocytes/drug effects , Bone Marrow/metabolism , Cell Division/physiology , Chromatography, High Pressure Liquid , Chymotrypsin/pharmacology , Dose-Response Relationship, Drug , Growth Substances/pharmacology , Growth Substances/chemistry , Hot Temperature , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Weight , Polymyxin B/pharmacology , Protein Denaturation , Spleen/metabolism , Thymus Gland/metabolism , Trypsin/pharmacology
15.
Rev. farm. bioquim. Univ. Säo Paulo ; 29(2): 89-95, jul.-dez. 1993. ilus, tab
Article in Portuguese | LILACS | ID: lil-135293

ABSTRACT

Foi realizada avaliacao da replicacao "in vitro" das cepas do virus influenza A/SP/1/80 (H3N2), A/SP/3/84 (H1N1) e B/SP/1/83 em culturas de celulas MDCK (Madin-Darby Canine Kidney). Sob a influencia da tripsina observou-se aumento de multiplicacao viral do que sem a mesma. Atraves dos testes de hemaglutinacao (HA) e de infecciosidade em ovos (DIO50) e em celulas (DICC50) foram obtidos titulos medios hemaglutinantes de: 46 e 84,66 (p<0,05); de infecciosidade em ovos: 5,76 e 6,52 e em celulas: 5,0 e 6,75 (p<0,01) para a cepa A/SP/1/80(H3N2) e os titulos medios hemaglutinantes de 40 e 74,66 (p<0,05) e de infecciosidade em ovos: 4,93 e 5,90 e em celulas: 5,10 e 6,18 (p<0,01) para cepa A/SP/1/84 (H1N1), quando o cultivo do virus se realizou na ausencia e na presenca da tripsina, respectivamente. Dentro do mesmo parametro de acao da tripsina sobre o cultivo viral, a cepa B/SP/1/83 apresentou os seguintes titulos medios hemaglutinantes: 4,75 e 4,45 (p<0,2) e de infecciosidade em ovos: 62,6 e 44 (p<0,2) e em celulas: 3,97 e 3,56 (p<0,1) quando a cepa foi cultivada em presenca ou nao da tripsina, respectivamente. Os resultados obtidos, alem de sugerirem que a tripsina promove aumento da replicacao viral "in vitro", sugerem, tambem, que existem diferencas na resposta a acao enzimatica entre as cepas de virus influenza tipo A e B. O presente estudo teve como perspectiva avaliar a acao da tripsina na multiplicacao do virus influenza "in vitro", visando o preparo de antigenos para reagentes imunologicos e de vacinas


Subject(s)
Animals , Chick Embryo , Culture Media , Influenza, Human/immunology , Virus Replication , Trypsin/pharmacology , Antigens, Viral , Influenza Vaccines/immunology
16.
Braz. j. med. biol. res ; 26(1): 81-91, Jan. 1993. ilus, tab
Article in English | LILACS | ID: lil-148677

ABSTRACT

1. The effects of Phneutria nigriventer venom (PNV) on rabbit vascular smooth muscle have been investigated. De-endothelialized vascular strips were superfused in a cascade system with oxygenated (95 per cent O2 + 5 per cent CO2) Krebs solution at 37 degrees C. 2. Phoneutria nigriventer venom (0.3-30 micrograms) produced dose-dependent and short-lived contractions of both venous (cava, mesenteric and jugular veins) and arterial (pulmonary and mesenteric arteries) tissues. 3. Methysergide (5.0 microM) did not significantly affect PNV-induced contractions in venous tissues (cava and mesenteric veins) or pulmonary artery, indicating that serotonin is not involved in the contraction. This was confirmed when PNV was dialyzed (24-48 h) since the contracting activity was still observed on the above tissues. In addition, the spasmogenic activity induced by dialyzed PNV was greatly reduced by incubating the venom with trypsin. 4. Neither tetrodotoxin (3.0 microM) nor phenoxybenzamine (0.05 microM) significantly affected PNV-induced contractions, suggesting that voltage-dependent sodium channel activation or endogenous catecholamine release from autonomic nerve endings on the vascular walls do not play a role in the response to PNV. 5. Our results demonstrate that PNV contains non-dialyzable components, probably peptides, that are responsible for the contractile activity on rabbit veins and pulmonary artery strips


Subject(s)
Animals , Male , Guinea Pigs , Rabbits , Muscle, Smooth, Vascular , Spider Venoms/pharmacology , Spider Venoms/antagonists & inhibitors , Spider Venoms/chemistry , Time Factors , Trypsin/pharmacology
17.
Biol. Res ; 26(1/2): 141-50, 1993. graf
Article in English | LILACS | ID: lil-228601

ABSTRACT

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...


Subject(s)
Animals , Humans , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Calmodulin/pharmacology , Homeostasis , Intracellular Membranes/enzymology , Leishmania mexicana/enzymology , Calcium-Transporting ATPases/antagonists & inhibitors , Cell Membrane/enzymology , Enzyme Activation , Erythrocyte Membrane/enzymology , Trypsin/pharmacology
18.
Ciênc. cult. (Säo Paulo) ; 42(7): 453-7, jul. 1990. ilus
Article in English | LILACS | ID: lil-96123

ABSTRACT

O extrato de Mandevilla velutina inibe as contraçöes induzidas pela bradicinina e cininogenases (calicreína, tripsina e tonina) no útero de rata. As contraçöes por angiotensina II e mesmo por cloreto de bário também foram inibidas, quando o íleo de cobaia foi usado, a contraçäo evocada pela histamina foi inibida. Esses experimentos sugerem que a inibiçäo causada pelo extrato de Mandevilla velutina näo é seletiva para bradicinina


Subject(s)
Guinea Pigs , Rats , Animals , Female , Uterine Contraction , Ileum , Plant Extracts/pharmacology , Angiotensin II/pharmacology , Barium/pharmacology , Bradykinin/pharmacology , Kallikreins/pharmacology , Histamine/pharmacology , Peptidyl-Dipeptidase A/pharmacology , Trypsin/pharmacology
19.
Yonsei Medical Journal ; : 103-109, 1990.
Article in English | WPRIM | ID: wpr-38653

ABSTRACT

There is evidence from other studies that some degree of cartilage healing may take place after the initiation of an inflammatory response. It is postulated that the induction of the platelet-cartilage interaction may eventuate in cartilage repair. The treatment of fresh articular cartilage with proteolytic enzymes rendered the tissue active as a platelet aggregant. During platelet aggregation a host of active substances are released which are known to play a role in the inflammatory response (Thompson 1975). This study was undertaken to evaluate the effects of trypsin on the surface injury of rabbit hyaline cartilage. The results were as follows: 1) Hyaline cell regeneration was observed only in the group treated with trypsin and blood; 2) Hyaline cartilage regeneration did not occur in the group treated with a single injection of trypsin or blood; 3) There was no significant damage to the healthy articular cartilage by the single injection of trypsin or blood, or both; and 4) Platelets do not adhere to cartilage and superficial damaged cartilage does not induce platelet aggregation.


Subject(s)
Animals , Cartilage, Articular/drug effects , Cell Division , Mitosis/physiology , Platelet Aggregation/drug effects , Rabbits , Regeneration , Trypsin/pharmacology , Wound Healing/drug effects
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