RESUMO
The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Assuntos
Anti-Infecciosos , Metabolismo , Antígenos de Diferenciação de Linfócitos T , Genética , Brometo de Cianogênio , Farmacologia , Escherichia coli , Genética , Metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Genética , Corpos de Inclusão , Metabolismo , Estrutura Terciária de Proteína , Genética , Proteínas Recombinantes de Fusão , Genética , Tiorredoxinas , Genética , TransfecçãoRESUMO
A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.
Assuntos
Animais , Anticorpos Anti-Helmínticos/química , Antígenos/química , Antígenos de Helmintos/química , Bovinos , Brometo de Cianogênio/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/métodos , Fasciola/metabolismo , Fasciolíase/diagnóstico , Glicoproteínas/química , Cabras , Imunoglobulina G/química , Lymnaea , Coelhos , Sensibilidade e Especificidade , Sefarose/química , Fatores de Tempo , Infecções por Trematódeos/diagnósticoRESUMO
In search of the optimal preparation method for large-scale purification of human plasma fibronectin, we adopted affinity chromatography with gelatin and the Sepharose 4B activated with cyanogen bromide to purify fibronectin from type "C" plasma of healthy males, and scanned the best method under the conditions of different amount of plasma loading and different residence time in column. In a given column volume of gelatin, the absorbent was related with the plasma residence time in column and the total amount of plasma loaded. As a result, the optimal loading amount of plasma is 150 ml, and the residence time is 20 minutes. The preparation method, herein, has been proved to require small amount of plasma and yield large amount of fibronectin.
Assuntos
Humanos , Masculino , Cromatografia de Afinidade , Métodos , Brometo de Cianogênio , Química , Fibronectinas , Sangue , Gelatina , Sefarose , Química , CicatrizaçãoRESUMO
BACKGROUND: Photoaging skin is clinically characterized by wrinkled, dry, inelastic and irregularly pigmented skin, skin tumor and histologically by increased epidermal thickness, nurnerous fibroblasts, mast cells and inflammatory cells in the upper dermis, elastosis, degeneration of collagen fibers, increased proportion of type III collagen fibers in the dermis and increased numbers of keratinizing cysts in the lower dormis of hairless albino mouse skin. Chronic exposure to UVB induces photoaging skin and sunscreen agents are used to prevent photodamage to skin and reduce the incidence and extent of the chronic photoaging effects. OBJECTIVE: To evaluate the photoaging effects of UVB on the skin and to assess the ability of sunscreen agents to protect the skin from photoaging, we examined the clinical, histological and quantitative changes in collagen in the skin of Albino hairless Skh: HR-1 mice. MATERIALS AND METHODS: The experimental animals were male Albino hairless Skh: HR-1 mice, 12 weeks old. The control group was a chronologically aging group which had not been affected by UVB irradiation. The non-protected group was irradiated with UVB, a half of MED, 3 times weekly(Monday, Wednesday, Fr iday), for 12 weeks. To assess the photoprotective effects of sunscreen agents, the control group, the non-protected group and the sunscreen agent-applicated groups were compared to each other clinically, histologically, and by quantitative collagen analysis. An early increase in type III collagen during UVB irradiation was determined by cyanogen bromide digestion of the whole skin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. RESULTS: Clinically, the skin of the mice in the non-protected group showed mild changes caused by photoaging. Histoloically, the non-protected group showed increases in the size and number of keratinizing cysts in the lower dermis and increases in the mast cells in the upper dermis compared with the control group. However, no significant findings of increased epidermal thickness, inflammatory cell infiltration, elastic fiber change or degeneration of collagen fibers were shown. By the 12th week, four of the total of nine mice in the non-protected group developed at least one tumor. The sunscreen agent applicated groups showed slightly photoprotected effects clinically and histologically. In the collagen analysis, the proportion of type III collagen was significantly increased in the photoaging skin of mice in the non-protected group compared with the control group. Mice in the sunscreen agent applicated groups showed a significantly decreased proportion of type III collagen compared with the non-protected group. CONCLUSION: To summarize, UVB exposure of the skin induces photoaging. The number and size of keratinizing cysts increased in the photoaging skin of hairless albino mice. There was also a quantitative change in the collagen fibers. The use of sunscreen agents decreases the photoaging effects of UVB on the skin.
Assuntos
Animais , Humanos , Masculino , Camundongos , Envelhecimento , Colágeno , Colágeno Tipo III , Brometo de Cianogênio , Densitometria , Derme , Digestão , Tecido Elástico , Eletroforese , Fibroblastos , Incidência , Mastócitos , Pele , SódioRESUMO
BACKGROUND: Photoaging skin is clinically characterized by wrinkled, dry, inelastic and irregularly pigmented skin, skin tumor and histologically by increased epidermal thickness, nurnerous fibroblasts, mast cells and inflammatory cells in the upper dermis, elastosis, degeneration of collagen fibers, increased proportion of type III collagen fibers in the dermis and increased numbers of keratinizing cysts in the lower dormis of hairless albino mouse skin. Chronic exposure to UVB induces photoaging skin and sunscreen agents are used to prevent photodamage to skin and reduce the incidence and extent of the chronic photoaging effects. OBJECTIVE: To evaluate the photoaging effects of UVB on the skin and to assess the ability of sunscreen agents to protect the skin from photoaging, we examined the clinical, histological and quantitative changes in collagen in the skin of Albino hairless Skh: HR-1 mice. MATERIALS AND METHODS: The experimental animals were male Albino hairless Skh: HR-1 mice, 12 weeks old. The control group was a chronologically aging group which had not been affected by UVB irradiation. The non-protected group was irradiated with UVB, a half of MED, 3 times weekly(Monday, Wednesday, Fr iday), for 12 weeks. To assess the photoprotective effects of sunscreen agents, the control group, the non-protected group and the sunscreen agent-applicated groups were compared to each other clinically, histologically, and by quantitative collagen analysis. An early increase in type III collagen during UVB irradiation was determined by cyanogen bromide digestion of the whole skin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. RESULTS: Clinically, the skin of the mice in the non-protected group showed mild changes caused by photoaging. Histoloically, the non-protected group showed increases in the size and number of keratinizing cysts in the lower dermis and increases in the mast cells in the upper dermis compared with the control group. However, no significant findings of increased epidermal thickness, inflammatory cell infiltration, elastic fiber change or degeneration of collagen fibers were shown. By the 12th week, four of the total of nine mice in the non-protected group developed at least one tumor. The sunscreen agent applicated groups showed slightly photoprotected effects clinically and histologically. In the collagen analysis, the proportion of type III collagen was significantly increased in the photoaging skin of mice in the non-protected group compared with the control group. Mice in the sunscreen agent applicated groups showed a significantly decreased proportion of type III collagen compared with the non-protected group. CONCLUSION: To summarize, UVB exposure of the skin induces photoaging. The number and size of keratinizing cysts increased in the photoaging skin of hairless albino mice. There was also a quantitative change in the collagen fibers. The use of sunscreen agents decreases the photoaging effects of UVB on the skin.