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1.
Braz. j. biol ; 83: e240015, 2023. tab, graf
Artigo em Inglês | LILACS-Express | MEDLINE, LILACSEXPRESS | ID: biblio-1285624

RESUMO

Abstract Zinc is an essential micronutrient that is required for optimum plant growth. It is present in soil in insoluble forms. Bacterial solubilization of soil unavailable form of Zn into available form, is an emerging approach to alleviate the Zn deficiency for plants and human beings. Zinc solubilizing bacteria (ZSB) could be a substitute for chemical Zn fertilizer. The present study aimed to isolate and characterize bacterial species from the contaminated soil and evaluate their Zn solubilizing potential. Zn resistant bacteria were isolated and evaluated for their MIC against Zn. Among the 13 isolated bacterial strains ZSB13 showed maximum MIC value upto 30mM/L. The bacterial strain with the highest resistance against Zn was selected for further analysis. Molecular characterization of ZSB13 was performed by 16S rRNA gene amplification which confirmed it as Pseudomonas oleovorans. Zn solubilization was determined through plate assay and broth medium. Four insoluble salts (zinc oxide (ZnO), zinc carbonate (ZnCO3), zinc sulphite (ZnS) and zinc phosphate (Zn3(PO4)2) were used for solubilization assay. Our results shows 11 mm clear halo zone on agar plates amended with ZnO. Likewise, ZSB13 showed significant release of Zn in broth amended with ZnCO3 (17 and 16.8 ppm) and ZnO (18.2 ppm). Furthermore, Zn resistance genes czcD was also enriched in ZSB13. In our study, bacterial strain comprising Zn solubilization potential has been isolated that could be further used for the growth enhancement of crops.


Resumo O zinco é um micronutriente essencial necessário para o crescimento ideal das plantas. Ele está presente no solo em formas insolúveis. A solubilização bacteriana da forma indisponível de Zn no solo para a forma disponível é uma abordagem emergente para aliviar a deficiência de Zn em plantas e seres humanos. Bactérias solubilizadoras de zinco (ZSB) podem ser um substituto para fertilizantes químicos de Zn. O presente estudo teve como objetivo isolar e caracterizar espécies bacterianas de solo contaminado e avaliar seu potencial de solubilização de Zn. Bactérias resistentes ao Zn foram isoladas e avaliadas quanto ao seu MIC contra o Zn. Entre as 13 cepas bacterianas isoladas, ZSB13 apresentou valor máximo de MIC de até 30 mM/L. A cepa bacteriana com maior resistência ao Zn foi selecionada para análise posterior. A caracterização molecular de ZSB13 foi realizada por amplificação do gene 16S rRNA que o confirmou como Pseudomonas oleovorans. A solubilização do Zn foi determinada através de ensaio em placa e meio caldo. Quatro sais insolúveis (óxido de zinco (ZnO), carbonato de zinco (ZnCO3), sulfito de zinco (ZnS) e fosfato de zinco (Zn3 (PO4) 2) foram usados ​​para o ensaio de solubilização. Nossos resultados mostram uma zona de halo clara de 11 mm em placas de ágar corrigidas com ZnO. Da mesma forma, ZSB13 mostrou liberação significativa de Zn em caldo alterado com ZnCO3 (17 e 16,8 ppm) e ZnO (18,2 ppm). Além disso, os genes de resistência ao Zn czcD também foram enriquecidos em ZSB13. Em nosso estudo, a cepa bacteriana compreendendo potencial de solubilização de Zn foi isolada e poderia ser usada posteriormente para o aumento do crescimento de safras.

2.
Braz. j. biol ; 82: e239991, 2022. tab, graf
Artigo em Inglês | LILACS-Express | MEDLINE, LILACSEXPRESS | ID: biblio-1278503

RESUMO

Abstract High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-1) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.


Resumo A alta resistência aos antimicrobianos está associada à formação de biofilme responsável por micróbios infecciosos para suportar condições severas. Portanto, novas alternativas são necessárias como inibidores de biofilme para controlar infecções. Neste estudo, as atividades antimicrobiana e antibiofilme dos extratos de Fagonia indica foram avaliadas contra isolados clínicos MDR. O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica tem efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e valor de concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados clínicos multirresistentes (MDR). O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica teve efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados MDR. Os efeitos inibitórios máximos do extrato de clorofórmio Fagonia indica na formação de biofilme foi observada em Staphylococcus aureus (71,84%), seguido por Klebsiella pneumoniae (70,83%) após 48 horas, mostrando que a inibição também é dependente do tempo. Nossos resultados sobre extravasamento de proteínas de células bacterianas indicaram que isolados MDR tratados com extrato clorofórmico de Fagonia indica apresentaram vazamento máximo de proteínas de K. pneumoniae (59,14 µg mL-1), seguido por S. aureus (56,7 µg mL-1). Ensaios de fixação de células indicaram que o extrato de clorofórmio resultou em uma inibição de 43,5-53,5% da aderência das células a uma superfície de poliestireno. Nossos resultados revelaram que extratos de Fagonia indica inibiram significativamente a formação de biofilme entre isolados clínicos MDR, portanto, poderiam ser aplicados como agentes antimicrobianos e inibidores de biofilme de baixo custo contra esses isolados MDR.

3.
Braz J Biol ; 83: e240015, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34320047

RESUMO

Zinc is an essential micronutrient that is required for optimum plant growth. It is present in soil in insoluble forms. Bacterial solubilization of soil unavailable form of Zn into available form, is an emerging approach to alleviate the Zn deficiency for plants and human beings. Zinc solubilizing bacteria (ZSB) could be a substitute for chemical Zn fertilizer. The present study aimed to isolate and characterize bacterial species from the contaminated soil and evaluate their Zn solubilizing potential. Zn resistant bacteria were isolated and evaluated for their MIC against Zn. Among the 13 isolated bacterial strains ZSB13 showed maximum MIC value upto 30mM/L. The bacterial strain with the highest resistance against Zn was selected for further analysis. Molecular characterization of ZSB13 was performed by 16S rRNA gene amplification which confirmed it as Pseudomonas oleovorans. Zn solubilization was determined through plate assay and broth medium. Four insoluble salts (zinc oxide (ZnO), zinc carbonate (ZnCO3), zinc sulphite (ZnS) and zinc phosphate (Zn3(PO4)2) were used for solubilization assay. Our results shows 11 mm clear halo zone on agar plates amended with ZnO. Likewise, ZSB13 showed significant release of Zn in broth amended with ZnCO3 (17 and 16.8 ppm) and ZnO (18.2 ppm). Furthermore, Zn resistance genes czcD was also enriched in ZSB13. In our study, bacterial strain comprising Zn solubilization potential has been isolated that could be further used for the growth enhancement of crops.


Assuntos
Pseudomonas oleovorans , Poluentes do Solo , Humanos , RNA Ribossômico 16S/genética , Solo , Microbiologia do Solo , Zinco
4.
Braz J Biol ; 82: e239991, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34190801

RESUMO

High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-1) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.


Assuntos
Extratos Vegetais , Staphylococcus aureus , Bactérias , Biofilmes , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Extratos Vegetais/farmacologia
5.
Braz. J. Biol. ; 81(2): 398-405, Mar.-May 2021. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: vti-762746

RESUMO

Continuous occurrence of heavy metals is a major cause of environmental pollution due to its toxic effects. At minimum concentrations, these metals are highly reactive and can gather in the food chains and food web, causing major dangers to public health concerns. Soil samples were collected from Paharang drain, Faisalabad. Cadmium tolerant bacteria were isolated and evaluated for their MIC against Cd. The isolated bacterial strain GCFSD01 showed MIC value upto 30 mM/L. The bacterial strain with the highest resistance against Cd was selected for further study. Molecular characterization of bacterial isolate GCFSD01 was performed by 16S rRNA which confirmed it as Bacillus cereus. Optimum growth conditions of bacterial strain were also evaluated. Strain GCFSD01 showed optimum growth at pH 7 and 37 °C temperature. Our result revealed that B. cereus strain GCFSD01 reduced 61.3% Cd after 48 hrs. Multiple metal tolerance and Cd reduction by B. cereus indicate its potential for further use for decontamination of polluted soil.(AU)


A ocorrência contínua de metais pesados é uma das principais causas de poluição ambiental devido aos seus efeitos tóxicos. A contaminação por metais pesados representa um grande risco para todas as formas de vida encontradas no meio ambiente. Em concentrações mínimas, esses metais são altamente reativos e podem se acumular nas cadeias alimentares e na cadeia alimentar, causando grandes perigos às preocupações com a saúde pública. Amostras de solo foram coletadas no esgoto de Paharang, Faisalabad. Bactérias tolerantes ao cádmio foram isoladas da amostra coletada pelo método da placa de ágar. As colônias separadas individuais selecionadas foram avaliadas quanto às suas concentrações inibitórias mínimas contra Cd. A cepa bacteriana isolada GCFSD01 apresentou valores de CIM de 30 mM/L. A colônia bacteriana que apresentou maior resistência contra o Cd foi selecionada para identificação. Após seleção da maior colônia bacteriana resistente ao Cd, coloração de Gram e diferentes testes bioquímicos foram realizados para a caracterização da bactéria isolada. A caracterização molecular do isolado bacteriano GCFSD01 foi realizada por PCR 16S rRNA confirmando a presença de Bacillus cereus. Após a identificação molecular, as condições ótimas de crescimento da cepa bacteriana também foram verificadas. A cepa GCFSD01 apresentou crescimento ótimo em pH 7 e temperatura de 37 °C. Nosso resultado revelou que a cepa de B. cereus GCFSD01 reduziu 61,3% de Cd após 48 horas. A tolerância a múltiplos metais e a redução de Cd por B. cereus indicam seu potencial para uso posterior na descontaminação do solo poluído.(AU)


Assuntos
Bacillus cereus/efeitos dos fármacos , Bacillus cereus/isolamento & purificação , Intoxicação por Cádmio , Análise do Solo
6.
Braz. j. biol ; 81(2): 398-405, Jan.-May 2021. tab, graf
Artigo em Inglês | LILACS-Express | LILACS | ID: biblio-1153348

RESUMO

Abstract Continuous occurrence of heavy metals is a major cause of environmental pollution due to its toxic effects. At minimum concentrations, these metals are highly reactive and can gather in the food chains and food web, causing major dangers to public health concerns. Soil samples were collected from Paharang drain, Faisalabad. Cadmium tolerant bacteria were isolated and evaluated for their MIC against Cd. The isolated bacterial strain GCFSD01 showed MIC value upto 30 mM/L. The bacterial strain with the highest resistance against Cd was selected for further study. Molecular characterization of bacterial isolate GCFSD01 was performed by 16S rRNA which confirmed it as Bacillus cereus. Optimum growth conditions of bacterial strain were also evaluated. Strain GCFSD01 showed optimum growth at pH 7 and 37 °C temperature. Our result revealed that B. cereus strain GCFSD01 reduced 61.3% Cd after 48 hrs. Multiple metal tolerance and Cd reduction by B. cereus indicate its potential for further use for decontamination of polluted soil.


Resumo A ocorrência contínua de metais pesados é uma das principais causas de poluição ambiental devido aos seus efeitos tóxicos. A contaminação por metais pesados representa um grande risco para todas as formas de vida encontradas no meio ambiente. Em concentrações mínimas, esses metais são altamente reativos e podem se acumular nas cadeias alimentares e na cadeia alimentar, causando grandes perigos às preocupações com a saúde pública. Amostras de solo foram coletadas no esgoto de Paharang, Faisalabad. Bactérias tolerantes ao cádmio foram isoladas da amostra coletada pelo método da placa de ágar. As colônias separadas individuais selecionadas foram avaliadas quanto às suas concentrações inibitórias mínimas contra Cd. A cepa bacteriana isolada GCFSD01 apresentou valores de CIM de 30 mM/L. A colônia bacteriana que apresentou maior resistência contra o Cd foi selecionada para identificação. Após seleção da maior colônia bacteriana resistente ao Cd, coloração de Gram e diferentes testes bioquímicos foram realizados para a caracterização da bactéria isolada. A caracterização molecular do isolado bacteriano GCFSD01 foi realizada por PCR 16S rRNA confirmando a presença de Bacillus cereus. Após a identificação molecular, as condições ótimas de crescimento da cepa bacteriana também foram verificadas. A cepa GCFSD01 apresentou crescimento ótimo em pH 7 e temperatura de 37 °C. Nosso resultado revelou que a cepa de B. cereus GCFSD01 reduziu 61,3% de Cd após 48 horas. A tolerância a múltiplos metais e a redução de Cd por B. cereus indicam seu potencial para uso posterior na descontaminação do solo poluído.

7.
Trop Biomed ; 38(1): 106-110, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33797532

RESUMO

Syphilis is a sexually transmitted disease and its actual prevalence among Pakistani blood donors is currently unknown. A cross sectional study was conducted at different district healthcare hospitals of Punjab, Pakistan with an aim to evaluate the prevalence and risk factors associated with syphilis in blood donors using immunochromatographic test (ICT) and enzyme linked immunosorbent assay (ELISA). A total (n=1200) blood samples were collected from donors aged 18-65 years. All the information regarding personal data, demographic data and risk factors was collected via structured questionnaire. On the basis of ICT and ELISA, the overall prevalence of syphilis was 3.91% among blood donors. The demographic factors positively linked with syphilis were age (P= 0.000; Odds ratio, OR= 7.18; 95% confidence interval CI= 2.816-18.295) and education status (P= 0.000; Odds ratio, OR= 12.33; 95% confidence interval CI= 3.469-43.849) of donors. Similarly among the risk factors analyzed, marital status (P= 0.012; Odds ratio OR= 2.251; 95% confidence interval CI= 1.206- 4.202) and blood transfusion history (P= 0.030; Odds ratio OR= 1.981; 95% confidence interval CI= 1.083-3.623) were also strongly associated with syphilis. We emphasized the importance of promoting preventive measures for syphilis. The syphilis diagnosis should not be based on a single test. The present study indicates that higher prevalence is alarming for blood donors in Pakistan. Stringent donor screening is highly recommended to ensure maximum safe blood transfusion.

8.
Braz J Biol ; 81(2): 398-405, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32696847

RESUMO

Continuous occurrence of heavy metals is a major cause of environmental pollution due to its toxic effects. At minimum concentrations, these metals are highly reactive and can gather in the food chains and food web, causing major dangers to public health concerns. Soil samples were collected from Paharang drain, Faisalabad. Cadmium tolerant bacteria were isolated and evaluated for their MIC against Cd. The isolated bacterial strain GCFSD01 showed MIC value upto 30 mM/L. The bacterial strain with the highest resistance against Cd was selected for further study. Molecular characterization of bacterial isolate GCFSD01 was performed by 16S rRNA which confirmed it as Bacillus cereus. Optimum growth conditions of bacterial strain were also evaluated. Strain GCFSD01 showed optimum growth at pH 7 and 37 °C temperature. Our result revealed that B. cereus strain GCFSD01 reduced 61.3% Cd after 48 hrs. Multiple metal tolerance and Cd reduction by B. cereus indicate its potential for further use for decontamination of polluted soil.


Assuntos
Metais Pesados , Poluentes do Solo , Bacillus cereus/genética , Biodegradação Ambiental , Cádmio/toxicidade , Metais Pesados/análise , RNA Ribossômico 16S/genética , Solo , Microbiologia do Solo , Poluentes do Solo/toxicidade
9.
Artigo em Inglês | VETINDEXEXPRESS | ID: vti-746049

RESUMO

Abstract Continuous occurrence of heavy metals is a major cause of environmental pollution due to its toxic effects. At minimum concentrations, these metals are highly reactive and can gather in the food chains and food web, causing major dangers to public health concerns. Soil samples were collected from Paharang drain, Faisalabad. Cadmium tolerant bacteria were isolated and evaluated for their MIC against Cd. The isolated bacterial strain GCFSD01 showed MIC value upto 30 mM/L. The bacterial strain with the highest resistance against Cd was selected for further study. Molecular characterization of bacterial isolate GCFSD01 was performed by 16S rRNA which confirmed it as Bacillus cereus. Optimum growth conditions of bacterial strain were also evaluated. Strain GCFSD01 showed optimum growth at pH 7 and 37 °C temperature. Our result revealed that B. cereus strain GCFSD01 reduced 61.3% Cd after 48 hrs. Multiple metal tolerance and Cd reduction by B. cereus indicate its potential for further use for decontamination of polluted soil.


Resumo A ocorrência contínua de metais pesados é uma das principais causas de poluição ambiental devido aos seus efeitos tóxicos. A contaminação por metais pesados representa um grande risco para todas as formas de vida encontradas no meio ambiente. Em concentrações mínimas, esses metais são altamente reativos e podem se acumular nas cadeias alimentares e na cadeia alimentar, causando grandes perigos às preocupações com a saúde pública. Amostras de solo foram coletadas no esgoto de Paharang, Faisalabad. Bactérias tolerantes ao cádmio foram isoladas da amostra coletada pelo método da placa de ágar. As colônias separadas individuais selecionadas foram avaliadas quanto às suas concentrações inibitórias mínimas contra Cd. A cepa bacteriana isolada GCFSD01 apresentou valores de CIM de 30 mM/L. A colônia bacteriana que apresentou maior resistência contra o Cd foi selecionada para identificação. Após seleção da maior colônia bacteriana resistente ao Cd, coloração de Gram e diferentes testes bioquímicos foram realizados para a caracterização da bactéria isolada. A caracterização molecular do isolado bacteriano GCFSD01 foi realizada por PCR 16S rRNA confirmando a presença de Bacillus cereus. Após a identificação molecular, as condições ótimas de crescimento da cepa bacteriana também foram verificadas. A cepa GCFSD01 apresentou crescimento ótimo em pH 7 e temperatura de 37 °C. Nosso resultado revelou que a cepa de B. cereus GCFSD01 reduziu 61,3% de Cd após 48 horas. A tolerância a múltiplos metais e a redução de Cd por B. cereus indicam seu potencial para uso posterior na descontaminação do solo poluído.

10.
J Int Med Res ; 35(6): 930-2, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18035005

RESUMO

Ophthalmoplegia is an unusual finding in diffuse large B cell lymphoma. This report describes the case of a 72-year old male patient who presented with ophthalmoplegia but no gross neurological manifestations and an isolated mass in the left upper thigh. His ophthalmoplegia improved from his first cycle of systemic and intrathecal chemotherapy, his thigh mass effusion spontaneously resolved, and his laboratory tests also showed improvement. The patient remained well, though weak, for several months with no other neurological signs. He subsequently died from clinically diagnosed pulmonary embolism.


Assuntos
Linfoma Difuso de Grandes Células B/complicações , Oftalmoplegia/etiologia , Idoso , Diagnóstico Diferencial , Evolução Fatal , Humanos , Masculino , Embolia Pulmonar
11.
J Virol ; 81(10): 5144-54, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17360759

RESUMO

Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency (K(i) = 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (-TDeltaS = -14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (DeltaH = -0.7 kcal/mol, 25 degrees C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.


Assuntos
Farmacorresistência Viral/genética , Inibidores da Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Mutação , Piridinas/metabolismo , Pironas/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Piridinas/farmacologia , Pironas/farmacologia , Sulfonamidas
12.
Biochim Biophys Acta ; 1481(2): 229-36, 2000 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-11018713

RESUMO

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.


Assuntos
Álcoois/química , Quimotripsinogênio/química , Sais/química , 1-Propanol/química , Naftalenossulfonato de Anilina , Cloratos/química , Dicroísmo Circular , Etanol/química , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Cloreto de Potássio/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Sulfatos/química , Trifluoretanol/química
13.
Biochim Biophys Acta ; 1479(1-2): 103-13, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11004533

RESUMO

The role of salt bridge(s) (between epsilon-NH(2) groups of lysine residues of human serum albumin (HSA) and carboxyl groups of bilirubin) in the binding and photoconversion of bilirubin bound to high affinity site on HSA was investigated by covalent modification of approximately 20% internal (buried) lysine residues of HSA with acetic anhydride, succinic anhydride and O-methylisourea and white light irradiation of their complexes with bilirubin. The different HSA derivatives, namely, acetylated HSA (aHSA), succinylated HSA (sHSA) and guanidinated HSA (gHSA), thus obtained, were found to be homogeneous with respect to charge and size and characterized in detail in terms of mean residue ellipticity, Stokes radius, tryptophan fluorescence, bilirubin binding and the photochemistry of their complexes with bilirubin. All the three derivatives retained helical contents and molecular size (Stokes radius) similar to HSA except for sHSA which showed a slight increase in the Stokes radius from 3.56 to 3.64 nm. Further, fluorescence properties of aHSA and sHSA were also found to be different from HSA and gHSA. Based on difference spectral change, fluorescence quenching and fluorescence enhancement results of bilirubin bound to HSA and its derivatives, nearly 46 and 48% reduction in bilirubin binding was observed in the case of aHSA and sHSA, respectively. Both aHSA and sHSA showed a decrease of 8- and 10-fold, respectively, in association constant compared to native HSA. Although the bisignate circular dichroism (CD) spectra of an equimolar (1:1) bilirubin-HSA complex was retained by all three HSA derivatives, the intensity of both positive and negative CD Cotton effects decreased significantly in both aHSA and sHSA. gHSA which retained net charge identical to native HSA, showed little decrease in bilirubin binding and the intensity of bisignate CD Cotton effects. The photochemical reaction of bilirubin bound to aHSA and sHSA produced opposite results to those observed with HSA and gHSA. A brief (2 min) irradiation of an equimolar complex of bilirubin with both aHSA and sHSA accompanied a rapid shift (14-15 nm) in the absorption spectrum of the bound pigment towards the blue region and almost complete elimination of negative CD Cotton effects while only moderately affecting the magnitude of positive CD Cotton effects. On the other hand, similar treatment of the complexes of bilirubin with HSA and gHSA did not show any change in the absorption spectrum, only a slight decrease in the intensity of both positive and negative CD Cotton effects was observed. The fluorescence intensity of bilirubin bound to HSA and gHSA was increased upon irradiation with white light and after 30 min it was nearly twice the value observed at 0 min irradiation. Interestingly, no change in the fluorescence intensity of bilirubin bound either to aHSA or sHSA was observed upon irradiation, even on increasing the duration of irradiation to 1 h. Taken together, the results on fluorescence quenching, fluorescence enhancement, CD spectral changes and visible absorption spectroscopy suggest that salt bridge(s) of the type (-COO(-).(+)H(3)N-) in which the epsilon-NH(2) group(s) contributed by lysine residues, are not only involved in the enantioselective binding of bilirubin but also in the stereospecific photoisomerization of bilirubin bound to a high affinity site on HSA.


Assuntos
Bilirrubina/metabolismo , Sais/metabolismo , Albumina Sérica/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Fotoquímica , Ligação Proteica , Conformação Proteica , Sais/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
14.
Biochimie ; 82(3): 203-9, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10863003

RESUMO

Chloroform-induced conformational changes of bilirubin (BR) bound to different serum albumins were studied by circular dichroism (CD) and fluorescence spectroscopy. Addition of a small amount of chloroform ( approximately 20 mM) to a solution containing 20 microM albumin and 15 microM BR changed the sign order and magnitude of the characteristic CD spectra of all BR-albumin complexes except BR-PSA complex which showed abnormal behavior. Monosignate negative CD Cotton effects (CDCEs) of BR complexed with SSA, GSA and BuSA were transformed into bisignate CDCEs in presence of chloroform akin to those exhibited by chloroform free solution of BR-HSA complex, indicating that the pigment acquired right handed plus (P) chirality when chloroform was added to these complexes. Bisignate CD spectra of BR complexed with HSA and BSA showed complete inversion upon addition of chloroform corroborating earlier findings. On the other hand, changes observed with BR-RSA complex were slightly different showing an additional CD band of weak intensity centered around 390 nm though inversion of CDCEs was similar to that of BR-HSA complex. Monosignate CD spectra of BR-PSA complex also showed three CD bands occurring at 409, 470 and 514 nm after chloroform addition. These results indicated significant but different effects of chloroform on the conformation of bound BR in BR-albumin complexes which can be ascribed to the changes in the exciton chirality of bilirubin probably due to altered hydrophobic microenvironment induced by the binding of chloroform at or near the ligand binding site. Chloroform severely quenched the intrinsic tryptophan fluorescence of the protein and shifted the emission maxima towards blue region in all the albumins except PSA. However, quantitative differences in both quenching and blue shift were noted in different serum albumins. This suggests that chloroform probably binds in the close vicinity of tryptophan residue(s) located in subdomain(s) IIA or IB and II both. The fluorescence of BR-albumin complexes was also found to be sensitive to the presence of a small amount of chloroform. But the changes observed in the fluorescence of the bound pigment in presence of chloroform were less marked as compared to the changes in the intrinsic fluorescence of protein per se. Taken together, these results suggest that there is at least one conserved site for chloroform binding in all these albumins which is at or near the BR binding site.


Assuntos
Bilirrubina/química , Clorofórmio/química , Albumina Sérica/química , Dicroísmo Circular , Conformação Proteica , Espectrometria de Fluorescência , Triptofano/química
15.
Proteins ; 40(1): 29-38, 2000 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10813828

RESUMO

The unfolding of human serum albumin (HSA), a multidomain protein, by urea was followed by far-UV circular dichroism (CD), intrinsic fluorescence, and ANS fluorescence measurements. The urea-induced transition, which otherwise was a two-step process with a stable intermediate at around 4.8 M urea concentration as monitored by far-UV CD and intrinsic fluorescence, underwent a single-step cooperative transition in the presence of 1.0 M KCl. The free energy of stabilization (DeltaDelta G(H2O)D) in the presence of 1 M KCl was found to be 1,090 and 1,200 cal/mol as determined by CD and fluorescence, respectively. The salt stabilization occurred in the first transition (0-5.0 M urea), which corresponded to the formation of intermediate (I) state from the native (N) state, whereas the second transition, corresponding to the unfolding of I state to denatured (D) state, remained unaffected. Urea denaturation of HSA as monitored by tryptophan fluorescence of the lone tryptophan residue (Trp(214)) residing in domain II of the protein, followed a single-step transition suggesting that domain(s) I and/or III is (are) involved in the intermediate formation. This was also confirmed by the acrylamide quenching of tryptophan fluorescence at 5 M urea, which exhibited little change in the value of Stern-Volmer constant. ANS fluorescence data also showed single-step transition reflecting the absence of accumulation of hydrophobic patches. The stabilizing potential of various salts studied by far-UV CD and intrinsic fluorescence was found to follow the order: NaClO(4) > NaSCN >Na(2)SO(4) >KBr >KCl >KF. A comparison of the effects of various potassium salts revealed that anions were chiefly responsible in stabilizing HSA. The above series was found similar to the electroselectivity series of anions towards the anion-exchange resins and reverse of the Hofmeister series, suggesting that preferential binding of anions to HSA rather than hydration, was primarily responsible for stabilization. Further, single-step transition observed with GdnHCl can be ascribed to its ionic character as the free energy change associated with urea denaturation in the presence of 1.0 M KCl (5,980 cal/mol) was similar to that obtained with GdnHCl (5,870 cal/mol).


Assuntos
Albumina Sérica/química , Ureia , Resinas de Troca Aniônica , Ânions/química , Dicroísmo Circular , Humanos , Indicadores e Reagentes , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína
16.
Biochim Biophys Acta ; 1476(1): 139-48, 2000 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-10606775

RESUMO

We studied the effect of various anions (of acids and salts) on the acid denatured state of HSA by near-UV circular dichroism (CD), far-UV CD, 1-anilinonaphthalene-8-sulfonate (ANS) binding, tryptophan fluorescence and thermal transition. Addition of different acids and salts caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity (MRE) value at 222 nm and loss of ANS binding sites exhibited by the decrease in the ANS fluorescence intensity at 480 nm. However, the concentration range of acids/salts required to bring about the transition varied greatly among different acids and salts. Among various acids/salts tested, K(3)Fe(CN)(6) was found to be most effective whereas HCl and KCl were least effective in inducing the properties close to native structure. Further, they followed the electroselectivity series. The near-UV CD spectra showed an increase in MRE towards the native state, whereas the tryptophan fluorescence emission spectra produced a red shift of about 6 nm on addition of KClO(4). The temperature-induced transition in the presence of 40 mM KClO(4) monitored by ellipticity measurements at 222 nm was characterized by the presence of an intermediate state in the temperature range 30-50 degrees C having abundant secondary structure. These results suggest that human serum albumin at low pH and in the presence of acids or salts exists in a partially folded state characterized by native-like secondary structure and tertiary folds.


Assuntos
Albumina Sérica/química , Naftalenossulfonato de Anilina , Ânions/química , Dicroísmo Circular , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
17.
Int J Biol Macromol ; 26(2-3): 173-80, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10517526

RESUMO

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in alpha-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.


Assuntos
Fármacos Cardiovasculares/metabolismo , Indometacina/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Anidridos Acéticos/química , Sítios de Ligação , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Lisina/química , Compostos de Metilureia/química , Concentração Osmolar , Conformação Proteica , Anidridos Succínicos/química
18.
Eur J Biochem ; 266(1): 26-32, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10542047

RESUMO

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.


Assuntos
Concentração de Íons de Hidrogênio , Conformação Proteica , Albumina Sérica/química , Naftalenossulfonato de Anilina , Dicroísmo Circular , Temperatura Alta , Humanos , Isomerismo , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Viscosidade
19.
Biochem Mol Biol Int ; 46(5): 943-50, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9861448

RESUMO

Interactions of tetracycline with bovine serum albumin (BSA) were studied by fluorescence quenching and circular dichroism (CD) analysis. The binding isotherm exhibited at least 13 tetracycline binding sites on the albumin molecule. Amongst these, four were found to be high affinity sites and the remainder were loose sites. The Scatchard analysis demonstrated the binding constant and capacity of BSA to be 4.6 x 10(6) liters/mole and 3.6, respectively. The CD data revealed a significant decrease in the mean residue ellipticity (MRE), indicating alterations in the protein helicity. A reduction of 20% in the alpha-helical content of the albumin was noted at higher levels of tetracycline in the presence of Cu (II) ions. Thus the strong in vitro interactions of tetracycline with albumin resulted in conformational changes in its globular structure and insinuate potential health risk due to possible macromolecular damage, under physiological conditions, from the formation of tetracycline/Cu(II) complexes.


Assuntos
Albumina Sérica/metabolismo , Tetraciclina/metabolismo , Animais , Sítios de Ligação , Cátions/metabolismo , Bovinos , Dicroísmo Circular , Cobre/metabolismo , Cobre/farmacologia , Corantes Fluorescentes , Luz , Ligação Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos , Albumina Sérica/química , Tetraciclina/farmacologia
20.
J Biochem Biophys Methods ; 37(1-2): 47-52, 1998 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-9825299

RESUMO

Serum albumin, when incubated with bilirubin prior to electrophoresis, was visualized as a yellow-colored band during the electrophoretic run and did not require any staining. Furthermore, free bilirubin served as a tracking dye. A minimum of 20 micrograms of protein was detected very well by this method. The formation of a bilirubin-albumin complex did not affect the electrophoretic mobility as the protein complexed with bilirubin as well as free albumin moved with the same mobility. Only a single protein band was visualized by this method after electrophoresis of human plasma. The method is simple, less time-consuming and may be used in identifying bilirubin-binding proteins in various biological samples.


Assuntos
Bilirrubina , Eletroforese das Proteínas Sanguíneas/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Albumina Sérica/isolamento & purificação , Animais , Proteínas de Transporte/sangue , Proteínas de Transporte/isolamento & purificação , Bovinos , Estudos de Avaliação como Assunto , Humanos , Ligantes , Soroalbumina Bovina/isolamento & purificação
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