RESUMO
This study aimed to explore refugee women's experiences of interpreters in healthcare in Aotearoa, New Zealand (NZ). Semi-structured interviews were conducted with nine women who arrived in NZ as refugees. Analysis involved a 'text in context' approach. An iterative and interpretive process was employed by engaging with participant accounts and field notes. The various meanings behind participants' experiences were unpacked in relation to the literature and the broader socio-cultural contexts in which these experiences occurred. Findings highlighted issues with professional and informal interpreters. These issues included cost, discrepancies in dialect, translation outside appointments, and privacy. Findings indicate ethical and practical implications of using interpreters in healthcare for refugee women. A step to achieving equitable healthcare for refugee women in New Zealand entails putting in place accessible and robust communicative infrastructure.
Assuntos
Refugiados , Atenção à Saúde , Feminino , Humanos , Nova Zelândia , Pesquisa Qualitativa , TraduçãoRESUMO
PURPOSE: This study explored whether inhibitory control is associated with symptoms of orthorexia nervosa, a condition that involves substantial behavioral control in regard to healthy eating. METHOD: Participants (50 women, 13 men) completed the Eating Habits Questionnaire as a measure of orthorexia symptomatology, along with computerized versions of the Go/No-Go Task, Flanker Task, and Stroop Task. RESULTS: Orthorexia symptomatology did not correlate with either percent error or response time for any of the three tasks (all p's > 0.10). CONCLUSION: These results suggest that orthorexia is not associated with deficits or other differences in inhibitory control. LEVEL OF EVIDENCE: Level V, descriptive cross-sectional study.
Assuntos
Transtornos da Alimentação e da Ingestão de Alimentos , Comportamentos Relacionados com a Saúde , Estudos Transversais , Dieta Saudável , Comportamento Alimentar , Feminino , Humanos , Masculino , Inquéritos e QuestionáriosRESUMO
The kinetic aspects of lipolysis by pulmonary phospholipase A2 (ChPLA2-V), chicken intestinal phospholipase A2 (ChPLA2-IIA) and chicken pancreatic phospholipase A2 (ChPLA2-IB), from chicken have been compared using the monomolecular films technique, on short-chain phospholipids (with three different head groups) and on long-chain phospholipids. The main conclusions from our experimental data indicate that the maximum catalytic activities of ChPLA2-V on 1,2 phosphatidylcholine and 1,2 phosphatidylethanolamine reached 15.26 and 36.12 moles/cm2.min.mM, respectively, at a pressure of 15 and 35 dynes/cm, respectively. Whereas, those of ChPLA2-IB were 3.58 (at the pressure of 20 dynes/cm) and 4.9 moles/cm2.min.mM. However, hydrolysis of phosphatidylglycerol monolayers (C12PG), were very much higher compared with all the substrates tested with 122 moles/cm2.min. Surprisingly, the hydrolysis rate of ChPLA2-V on long-chain phosphatidylglycerol (C18PG) was very low (1.45 moles/cm2.min) compared with all tested substrates, even with the use of p-cyclodextrin. And thus, the fatty acid preference of ChPLA2-V was 2-decanoyl > 2-oleoyl with a PG head group. In order to gain significant correlations between enzyme's structures and their relative functions, we tried to examine the surface electrostatic potentials of the various secreted phospholipase 2 (sPLA2) from chicken. In the present study, we detailed that the substrate affinity, specificity and the hydrolysis rates of sPLA2 at each interface is governed by the surface electrostatic potentials and hydrophobic interactions operative at this surface.
Assuntos
Galinhas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácidos Graxos/metabolismo , Hidrólise , Intestinos/enzimologia , Cinética , Pâncreas/enzimologia , Pâncreas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMO
Bioleaching of heavy metals from industrial contaminated soil using metallotolerant fungi is the most efficient, cost-effective, and eco-friendly technique. In the current study, the contaminated soil samples from Hattar Industrial Estate revealed a total lead (Pb) and mercury (Hg) concentration of 170.90 mg L-1 and 26.66 mg L-1, respectively. Indigenous metallotolerant fungal strains including Aspergillus niger M1, Aspergillus fumigatus M3, Aspergillus terreus M6, and Aspergillus flavus M7 were isolated and identified by pheno- and genotyping. A. fumigatus and A. flavus of soil sample S1 showed higher efficiency for Pb removal (99.20% and 99.30%, respectively), in SDB medium. Likewise, A. niger and A. terreus of soil sample S2 showed higher efficiency for Hg removal (96% and 95.50%, respectively), in YPG medium. Furthermore, the maximum uptake efficiency for Pb removal (8.52 mg g-1) from soil sample S1 was noticed for A. fumigatus in YPG medium, while the highest uptake efficiency (4.23 mg g-1) of A. flavus M2 strain was observed with CYE medium. Similarly, the maximum uptake efficiency of 0.41 mg g-1 and 0.44 mg g-1 for Hg removal from soil sample S2 was found for A. niger and A. terreus strains, respectively, in CYE medium. Thus, in order to address the major issue of industrial waste pollution, indigenous fungal strains A. fumigatus (M1) and A. terreus (M7), isolated in this study, could be used (ex situ or in situ) to remediate soils contaminated with Pb and Hg.
Assuntos
Aspergillus/metabolismo , Chumbo/metabolismo , Mercúrio/metabolismo , Metais Pesados/metabolismo , Poluentes do Solo/metabolismo , Solo/química , Aspergillus/classificação , Aspergillus/genética , Monitoramento Ambiental , Recuperação e Remediação Ambiental , Genótipo , Resíduos Industriais , Chumbo/análise , Mercúrio/análise , Metais Pesados/análise , Fenótipo , Poluentes do Solo/análiseRESUMO
Remediation of heavy metals, other than microbial bioleaching method, is expensive and unsuitable for large contaminated areas. The current study was aimed to isolate, identify, and test the potential of indigenous fungal strains for heavy metal removal from contaminated soil. A total of three metallotolerant fungal strains, i.e., Aspergillus niger (M1DGR), Aspergillus fumigatus (M3Ai), and Penicillium rubens (M2Aii), were isolated and identified by phenotyping and genotyping from heavy metal-contaminated soil of Hattar Industrial Estate, Pakistan. A. niger was found to be the most successful strain for the removal of heavy metals from the contaminated soil with maximum bioaccumulation efficiency of 98% (Cd) and 43% (Cr). In contrast, A. fumigatus showed comparatively low but still considerable bioleaching potential, i.e., 79% and 69% for Cd and Cr removal, respectively. Maximum metal uptake efficiency, i.e., 0.580 mg g-1 and 0.152 mg g-1 by A. niger strain was noticed for Cd and Cr with Czapek yeast extract (CYE) and Sabouraud dextrose broth (SDB) media, respectively. A. fumigatus (M3Ai) exhibited the maximum bioleaching capacity (0.40 mg g-1) for Cr with CYE medium. The results reveal that A. niger M1DGR and A. fumigatus M3Ai could be used to develop new strategies to remediate soil contaminated with heavy metals (Cd and Cr) through either in situ or ex situ mycoremediation.
Assuntos
Aspergillus flavus/metabolismo , Aspergillus fumigatus/metabolismo , Cádmio/análise , Cromo/análise , Penicillium/metabolismo , Poluentes do Solo/análise , Biodegradação Ambiental , Monitoramento Ambiental , Poluição Ambiental/análise , Paquistão , Solo/químicaRESUMO
Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2-V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent.
Assuntos
Galinhas/genética , Fosfolipases A2/genética , Fosfolipases A2/farmacologia , Pichia/genética , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Cálcio/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Concentração de Íons de Hidrogênio , Coelhos , Ratos , Especificidade por Substrato , TemperaturaRESUMO
Leishmaniasis is a parasitic reticuloendotheliosis whose pathogen is a zooflagellate belonging to the genus Leishmania transmitted by the bite of an infected phlebotome. Recently, a unique secretory lipase from the human pathogen Leishmania donovani Ldlip3 has been identified and characterized. This lipase has a high identity with a putative triacylglycerol lipase of Leishmania major (Lmlip2). In the present study, Lmlip2 was expressed in the eukaryotic heterologous expression system Pichia pastoris as tagged enzyme of 308 amino acids. Maximal protein production was reached after 2 days of fermentation. Optimal Lmlip2 lipase activity was measured using the pH stat technique at pH 8â¯at 26⯰C using vinyl esters and triacylglycerols (true lipids) as substrates. Moreover, biochemical characterization of Lmlip2 contained in culture supernatant, illustrates that L. major secreted lipase is active and stable at low temperatures especially 26°and prefer neutral pH; concerning substrate specificityLmlip2 presents a preference for short chains lipid substrates vinyl esters such as VC2, VC3 and VC4 likewise, it is capable to hydrolyze long chain triacylglycerols like olive oil. Metal ions and surfactants tested in this study decrease Lmlip2 activity. Further studies are needed to clarify the relation between the lipase activity and the virulence. Thus, it could lead to the identification of novel targets to block cutaneous Leishmaniasis in human hosts.
Assuntos
Leishmania major/enzimologia , Leishmania major/genética , Lipase/genética , Lipase/metabolismo , Pichia/genética , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Lipase/antagonistas & inibidores , Lipase/química , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , TemperaturaRESUMO
A Staphylococcus aureus strain, isolated from an Algerian biotope, secretes a non-induced lipase in the culture medium. The S. aureus lipase (SAL) was purified to homogeneity. Pure SAL is a monomeric protein (43 kDa). The 20 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. SAL presents specific activities of about 1600 and 555 U mg-1 using tributyrin and olive oil emulsion as substrates, respectively. In contrast to other staphylococcal lipases previously characterized, SAL was stable at a pH range from 6 to 9 after 1 h incubation, and retained 50% of its activity after 10 min incubation at 50 °C. The purified enzyme was also characterized using monolayer technique. Lipase activity can be measured only when the surface pressure exceeds 15 mN m-1 . The critical surface pressure (πc ) measured with egg-PC films was estimated at 33 mN m-1 . SAL showed a preference for the distal ester groups of the diglyceride isomers at low surface pressure, for the adjacent ester groups at high surface pressure and a preference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. Cloned and sequenced gene part, encoding the mature lipase shows, in comparison with S. aureus lipase 3 (SAL3), a deletion of three residues (LKA) at the N-terminal extremity and a substitution of glycine 208 and isoleucine 226 with an arginine and leucine, respectively.
Assuntos
Lipase/genética , Lipase/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Clonagem Molecular , Meios de Cultura/química , Emulsões , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Lipase/química , Azeite de Oliva/metabolismo , Pressão , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Estereoisomerismo , Especificidade por Substrato , Tensoativos/química , Tensoativos/isolamento & purificação , Triglicerídeos/metabolismoRESUMO
Hypertension in paediatric age group is commonly secondary to a known cause. It is crucial to identify the cause of hypertension and treat it before development of any associated complications to prevent morbidity and mortality. Paediatric Hypertension is one of the important clinical finding in a child with certain clinical syndrome. We are presenting a case of a 10 month old child presenting with hypertension and hypokalaemia, after excluding all identifiable causes and her positive response to therapy, that is amiloride, along with supportive biochemical data she was diagnosed as a case of monogenic type of hypertension known as Liddle's syndrome.
Assuntos
Síndrome de Liddle/diagnóstico , Amilorida/uso terapêutico , Bloqueadores do Canal de Sódio Epitelial/uso terapêutico , Feminino , Humanos , Lactente , Síndrome de Liddle/tratamento farmacológicoRESUMO
High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZαA with the Saccharomyces cerevisiae α-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6-tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75 mg/L), a high level secretion of rFSL (310 mg/L) and tFSL (240 mg/L) was achieved providing an important improvement in enzyme production. Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4mM) and calcium ions enhanced lipase activity. During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection.
Assuntos
Fusarium/genética , Expressão Gênica , Lipase/genética , Lipase/metabolismo , Pichia/genética , Proteínas Recombinantes , Ácidos e Sais Biliares/farmacologia , Cálcio/farmacologia , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Fusarium/enzimologia , Lipase/química , Lipase/isolamento & purificaçãoRESUMO
Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols.
Assuntos
Pancrelipase/metabolismo , Fosfolipases/metabolismo , Proteínas Recombinantes , Animais , Catálise , Ativação Enzimática , Hidrólise , Cinética , Lipólise , Modelos Moleculares , Conformação Molecular , Pancrelipase/química , Pancrelipase/genética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Especificidade por Substrato , PerusRESUMO
INTRODUCTION: Stress among medical students induced by academic pressures is on the rise among the student population in Pakistan and other parts of the world. Our study examined the relationship between two different systems employed to assess academic performance and the levels of stress among students at two different medical schools in Karachi, Pakistan. METHODS: A sample consisting of 387 medical students enrolled in pre-clinical years was taken from two universities, one employing the semester examination system with grade point average (GPA) scores (a tiered system) and the other employing an annual examination system with only pass/fail grading. A pre-designed, self-administered questionnaire was distributed. Test anxiety levels were assessed by The Westside Test Anxiety Scale (WTAS). Overall stress was evaluated using the Perceived Stress Scale (PSS). RESULTS: There were 82 males and 301 females while four did not respond to the gender question. The mean age of the entire cohort was 19.7 ± 1.0 years. A total of 98 participants were from the pass/fail assessment system while 289 were from the GPA system. There was a higher proportion of females in the GPA system (85% vs. 59%; p < 0.01). Students in the pass/fail assessment system had a lower score on the WTAS (2.4 ± 0.8 vs. 2.8 ± 0.7; p = 0.01) and the PSS (17.0 ± 6.7 vs. 20.3 ± 6.8; p < 0.01), indicating lower levels of test anxiety and overall stress than in students enrolled in the GPA assessment system. More students in the pass/fail system were satisfied with their performance than those in the GPA system. CONCLUSION: Based on the present study, we suggest governing bodies to revise and employ a uniform assessment system for all the medical colleges to improve student academic performance and at the same time reduce stress levels. Our results indicate that the pass/fail assessment system accomplishes these objectives.
Assuntos
Avaliação Educacional/métodos , Estresse Psicológico/epidemiologia , Estudantes de Medicina/psicologia , Adolescente , Ansiedade/psicologia , Estudos Transversais , Feminino , Humanos , Masculino , Paquistão/epidemiologia , Fatores Sexuais , Fatores Socioeconômicos , Estresse Psicológico/psicologia , Adulto JovemRESUMO
Starting from total uropygial glands mRNAs, chicken uropygial carboxylesterase (cuCES) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the cuCES is compared to that of human liver carboxylesterase 1 (hCES1). Given the high amino acid sequence homology between the two enzymes, a 3-D structure model of the chicken carboxylesterase was built using the structure of hCES1 as template. By following this model and utilizing molecular dynamics (MD) simulations, the resistance of the chicken carboxylesterase at high temperatures could be explained. The docking of substrate analogs into the cuCES active site was used to explain the fact that the chicken carboxylesterase cannot hydrolyze efficiently large substrate molecules.
Assuntos
Proteínas Aviárias/química , Carboxilesterase/química , Clonagem Molecular , Glândulas Perianais/química , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Sequência de Bases , Carboxilesterase/genética , Domínio Catalítico , Galinhas , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Humanos , Isoenzimas/química , Isoenzimas/genética , Cinética , Fígado/química , Fígado/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Glândulas Perianais/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.
Assuntos
Proteínas Aviárias/química , Lipase/química , Pichia/metabolismo , Proteínas Aviárias/biossíntese , Proteínas Aviárias/genética , Diglicerídeos/química , Expressão Gênica , Hidrólise , Cinética , Lipase/biossíntese , Lipase/genética , Pâncreas/enzimologia , Pressão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato , TurquiaRESUMO
The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as "classical lipase", was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA1 and PLA2 activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.
Assuntos
Lipase/química , Fosfolipases A1/química , Fosfolipases A1/genética , Animais , Humanos , Cinética , Lipase/genética , Lipase/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipases A1/metabolismo , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Ligação Proteica , Conformação Proteica , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , SuínosRESUMO
The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin-water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.
Assuntos
Colipases/metabolismo , Lipase/química , Lipase/metabolismo , Pâncreas/enzimologia , Triglicerídeos/metabolismo , Perus/metabolismo , Animais , Células Clonais , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Vetores Genéticos/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lipase/isolamento & purificação , Oligopeptídeos/metabolismo , Pichia/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Temperatura , Fatores de Tempo , Transformação Genética , Trioleína/metabolismoRESUMO
The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group II sPLA2, has been amplified. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3 mg per litre of pure refolded fully active enzyme to be obtained. Recombinant expression of chPLA2-IIA in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme.
RESUMO
We have isolated a lipolytic halotolerant bacterium, designated as CJ3, that was identified as a Staphylococcus sp. Culture conditions were optimized and the highest extracellular lipase production amounting to 5 U/ml was achieved after 24 h of cultivation. The extracellular lipase was puriï¬ed 24-fold by ammonium sulfate precipitation and a Sephacryl S-200 chromatography, and its molecular mass was found to be around 38 kDa, as revealed by SDS-PAGE and gel filtration. The lipase substrate specificity was investigated using short (tributyrin) and long (olive oil) chain triglyceride substrates. The lipase was inhibited by submicellar concentrations of Triton X-100, and maximum specific activities were found to be 802 U/mg on tributyrin and 260 U/mg on olive oil at pH 8.0 and 45 °C. The lipase was fairly stable in the pH range from 6.0 to 9.0, and about 69% of its activity was retained after incubation at 45 °C for 60 min. The enzyme showed a high tolerance to a wide range of salt concentration and a good stability in organic solvents, especially in long-chain alcohols.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Lipase/química , Lipase/isolamento & purificação , Staphylococcus/enzimologia , Triglicerídeos/química , Cromatografia em Gel , Peso Molecular , Especificidade por Substrato/fisiologiaRESUMO
BACKGROUND: Diabetes has become a serious health problem and a major risk factor associated with troublesome health complications, such as metabolism disorders and liver-kidney dysfunctions. The inadequacies associated with conventional medicines have led to a determined search for alternative natural therapeutic agents. The present study aimed to investigate and compare the hypoglycemic and antilipidemic effects of kombucha and black tea, two natural drinks commonly consumed around the world, in surviving diabetic rats. METHODS: Alloxan diabetic rats were orally supplied with kombucha and black tea at a dose of 5 mL/kg body weight per day for 30 days, fasted overnight, and sacrificed on the 31st day of the experiment. Their bloods were collected and submitted to various biochemical measurements, including blood glucose, cholesterol, triglcerides, urea, creatinine, transaminases, transpeptidase, lipase, and amylase activities. Their pancreases were isolated and processed to measure lipase and α-amylase activities and to perform histological analysis. RESULTS: The findings revealed that, compared to black tea, kombucha tea was a better inhibitor of α-amylase and lipase activities in the plasma and pancreas and a better suppressor of increased blood glucose levels. Interestingly, kombucha was noted to induce a marked delay in the absorption of LDL-cholesterol and triglycerides and a significant increase in HDL-cholesterol. Histological analyses also showed that it exerted an ameliorative action on the pancreases and efficiently protected the liver-kidney functions of diabetic rats, evidenced by significant decreases in aspartate transaminase, alanine transaminase, and gamma-glytamyl transpeptidase activities in the plasma, as well as in the creatinine and urea contents. CONCLUSIONS: The findings revealed that kombucha tea administration induced attractive curative effects on diabetic rats, particularly in terms of liver-kidney functions. Kombucha tea can, therefore, be considered as a potential strong candidate for future application as a functional supplement for the treatment and prevention of diabetes.
Assuntos
Camellia sinensis , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Hipolipemiantes/uso terapêutico , Fitoterapia , Preparações de Plantas/uso terapêutico , Chá , Animais , Glicemia/metabolismo , Camellia sinensis/microbiologia , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Creatinina/sangue , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Enzimas/sangue , Hipoglicemiantes/farmacologia , Hipolipemiantes/farmacologia , Rim/efeitos dos fármacos , Lipase/antagonistas & inibidores , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Preparações de Plantas/farmacologia , Ratos , Ratos Wistar , Chá/microbiologia , Triglicerídeos/sangue , Ureia/sangue , alfa-Amilases/antagonistas & inibidoresRESUMO
BACKGROUND: Pancreatic colipase is a required co-factor for pancreatic lipase, being necessary for its activity during hydrolysis of dietary triglycerides in the presence of bile salts. In the intestine, colipase is cleaved from a precursor molecule, procolipase, through the action of trypsin. This cleavage yields a peptide called enterostatin knoswn, being produced in equimolar proportions to colipase. RESULTS: In this study, colipase from the common stingray Dasyatis pastinaca (CoSPL) was purified to homogeneity. The purified colipase is not glycosylated and has an apparent molecular mass of around 10 kDa. The NH2-terminal sequencing of purified CoSPL exhibits more than 55% identity with those of mammalian, bird or marine colipases. CoSPL was found to be less effective activator of bird and mammal pancreatic lipases than for the lipase from the same specie. The apparent dissociation constant (Kd) of the colipase/lipase complex and the apparent Vmax of the colipase-activated lipase values were deduced from the linear curves of the Scatchard plots. We concluded that Stingray Pancreatic Lipase (SPL) has higher ability to interact with colipase from the same species than with the mammal or bird ones. CONCLUSION: The fact that colipase is a universal lipase cofactor might thus be explained by a conservation of the colipase-lipase interaction site. The results obtained in the study may improve our knowledge of marine lipase/colipase.