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1.
Pharmeur Bio Sci Notes ; 2024: 12-26, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38533690

RESUMO

For more than 50 years, in vivo assays have been used for testing pharmaceutical product safety due to their assumed ability to broadly detect potential unidentified contaminants. As part of these in vivo tests, the animal tests for depressor substances and histamine have been described in the European Pharmacopoeia since its first edition in 1977. Both tests measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively. In 2024, the Histamine (2.6.10) general chapter is referenced in the Production section of four monographs and 10 monographs have variations of a sentence on designing the manufacturing process to eliminate or minimise substances lowering blood pressure in this same section, without referencing the chapter. The Depressor substances (2.6.11) chapter is referenced only in the Histamine (2.6.10) chapter as a next step if the histamine test is invalid. A historical search was performed and it has shown that the tests for histamine and for depressor substances were introduced by different groups of experts in an inconsistent way at different times, and for different reasons, leading to non-harmonised approaches across monographs. The control of histamine and other depressor substances has been the subject of numerous debates where their use was questioned. During these discussions, reports on positive cases or batches failing the test for histamine or depressor substances were anecdotal. In addition, in vivo tests can be considered non-specific, very variable, time-consuming, costly and ethically doubtful. More importantly, the majority of in vivo methods originate from a time when good manufacturing practice was not widely used and formal method validation requirements were not yet established. In view of the above, the removal of all references to animal tests for histamine or depressor substances from all texts still referring to them is proposed. Since the sentences in the Production section referring to the control of "substances lowering blood pressure", "vasoactive substances" or "hypotensive substances" appeared as remainders of the animal test without further guarantee of safety, it will also be proposed to remove all these sentences from the concerned monographs. Ultimately, the suppression of general chapters 2.6.10 and 2.6.11 from the Ph. Eur. is envisaged. Independently from the above, it is also envisaged to elaborate a new general chapter Histamine in active substances (2.5.47) to include physicochemical or immunochemical methods enabling the detection of histamine. This new text would aim at supporting manufacturers in their histamine control strategy following the inclusion of precaution statements in the general monograph on Products of fermentation (1468); these statements had been added in Ph. Eur. Supplements 9.6 and 10.4, following adverse events related to a GMP issue with gentamicin sulfate. This strategy has been endorsed by the European Pharmacopoeia Commission at its 177th Session in November 2023. The concerned monographs would be a subject of public consultation in Pharmeuropa 36.2 (April 2024).


Assuntos
Alternativas aos Testes com Animais , Histamina , Animais , Cobaias , Histamina/análise , Gatos
2.
Pharmeur Bio Sci Notes ; 2023: 60-68, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37711032

RESUMO

The manufacture of advanced therapy medicinal products (ATMPs) is critically impacted by the quality of the raw materials (RMs) used. Following the need expressed by stakeholders to establish a certification scheme for biological RMs used in the manufacture of ATMPs, the European Pharmacopoeia (Ph. Eur.) Cell Therapy Products Working Party (CTP WP) conducted an informal investigation with the aim of issuing a technical opinion on the feasibility of such a certification scheme. Seven RM Drug Master Files were reviewed for compliance of the RM with Ph. Eur. general chapter 5.2.12. Raw materials of biological origin for the production of cell-based and gene therapy medicinal products by members of the CTP WP who were representatives of competent authorities and experienced in the evaluation of RMs for ATMPs. This article presents the results of these case studies, including the potential benefits and concerns identified by the experts. It was concluded that it would be technically feasible, albeit challenging, to set up a certification system for RMs of biological origin against chapter 5.2.12. Although the establishment of such a scheme is currently perceived by some CTP WP members as premature, it could potentially be beneficial for all stakeholders (RM manufacturers, ATMP manufacturers and assessors).


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Nascimento Prematuro , Feminino , Humanos , Certificação , Comércio , Processos Grupais
3.
ESMO Open ; 8(4): 101610, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37536254

RESUMO

BACKGROUND: Investigation of the disparities in the access to experimental treatment in early-phase clinical trials is lacking. The objective of the EGALICAN-2 study was to identify the factors underpinning such inequalities. METHODS: A national prospective survey was conducted in 11 early-phase clinical trial centers (CLIP2) certified by the French National Cancer Institute. Sociodemographic, socioeconomic and medical data were collected. Univariate logistic regression models were carried out to estimate odds ratios and 90% confidence intervals associated with the effect of each study variable. A multivariate logistic regression model was built to explore the independent factors associated with the administration of the experimental treatment (C1D1). A post hoc analysis was carried out excluding female cancer patients. RESULTS: Between 2015 and 2016, 1355 patients referred from 11 CLIP2 centers in France were included in the study. Eight hundred and forty-eight patients received C1D1 (73%) and 320 patients (27%) were screening failure. Median age was 58 years (range 17-97 years) and 667 patients (54%) were female. Most patients had a metastatic disease (n = 751, 87%). In the multivariate logistic regression analysis, the significant independent factors associated with C1D1 were male sex, initial care received in a hospital with an early-phase unit and living in wealthy metropolitan areas (P values <0.05). In the post hoc analysis, the sex factor was no longer significant [odds ratio = 1.21 (95% confidence interval 0.86-1.70), P value = 0.271]. CONCLUSIONS: This study investigated the factors producing social inequalities in the context of early-phase clinical trials in oncology. Our research highlights factors of sex, care pathway and geographic location. Gynecological cancer was found to impact C1D1 significantly, unlike breast cancer. The results of this study should contribute to improve patient access to early-phase clinical trials.


Assuntos
Neoplasias da Mama , Humanos , Masculino , Feminino , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Estudos Prospectivos , França/epidemiologia , Neoplasias da Mama/diagnóstico
4.
ESMO Open ; 7(3): 100468, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35533427

RESUMO

BACKGROUND: Access to clinical trials and especially early-phase trials (ECT) is an important issue in geriatric oncology. As cancer can be considered an age-related disease because the incidence of most cancers increases with age, new drugs should also be evaluated in older patients to assess their safety and efficacy. The EGALICAN-2 study was primarily designed to identify social and/or regional inequalities regarding access to ECT. We focused on the factors of inequalities in access to ECT in older patients. PATIENTS AND METHODS: During a 1-year period (2015-2016), a survey was conducted in 11 early-phase units certified by the French National Cancer Institute. RESULTS: A total of 1319 patients were included in the analyses: 1086 patients (82.3%) were <70 years and 233 patients (17.7%) were >70 years. The most common tumor types at referral in older patients were gastrointestinal (19.3%), hematological (19.3%), and thoracic tumors (18.0%). Most patients referred to the phase I unit had signed informed consent and the rate was similar across age (92.7% in younger patients versus 90.6% in older patients; P = 0.266). The rate of screening failure was also similar across age (28.5% in younger patients versus 24.3% in older patients; P = 0.219). Finally, in older patients, univariate analyses showed that initial care received in the hospital having a phase I unit was statistically associated with first study drug administration (odds ratio 0.49, 90% confidence interval 0.27-0.88; P = 0.045). CONCLUSIONS: Older patients are underrepresented in early clinical trials with 17.7% of patients aged ≥70 years compared with the number of new cases of cancer in France (50%). However, when invited to participate, older patients were prone to sign informed consent.


Assuntos
Neoplasias , Idoso , Ensaios Clínicos como Assunto , França/epidemiologia , Humanos , Incidência , Neoplasias/tratamento farmacológico , Neoplasias/terapia
5.
Comput Methods Programs Biomed ; 214: 106537, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34879326

RESUMO

BACKGROUND AND OBJECTIVE: Longitudinal analysis of patient-reported outcome (PRO) data remains challenging, as no standardization of statistical methods has been proposed, making comparison of PRO results between clinical trials difficult. In this context, the time to deterioration approach has recently been proposed and is regularly used as a modality of longitudinal PRO analysis in oncology. METHODS: Two new SAS macro programs were developed, %TTD and %TUDD, which implement longitudinal analysis of PRO data according to the time to deterioration approach. These programs implement the recommended deterioration definitions. We described the programs with their different functionalities. RESULTS: The %TTD macro calculates the time to first or transient deterioration, and the %TUDD macro calculates the time until definitive deterioration. These macros allow to obtain the survival variables from the time to deterioration approach. We illustrate our programs by presenting different applications on the randomized phase II AFUGEM GERCOR clinical trial. CONCLUSION: The implementation of the deterioration definitions in SAS software allows the dissemination of this approach, in order to move toward the goal of standardization of longitudinal PRO analysis in oncology clinical trials.


Assuntos
Neoplasias , Qualidade de Vida , Humanos , Oncologia , Medidas de Resultados Relatados pelo Paciente , Software
6.
BMC Med Res Methodol ; 20(1): 223, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883216

RESUMO

BACKGROUND: Health-related quality of life (HRQoL) has become a major endpoint to assess the clinical benefit of new therapeutic strategies in oncology clinical trials. Typically, HRQoL outcomes are analyzed using linear mixed models (LMMs). However, longitudinal analysis of HRQoL in the presence of missing data remains complex and unstandardized. Our objective was to compare the modeling alternatives that account for informative dropout. METHODS: We investigated three alternative methods-the selection model (SM), pattern-mixture model (PMM), and shared-parameters model (SPM)-in relation to the LMM. We first compared them on the basis of methodological arguments highlighting their advantages and drawbacks. Then, we applied them to data from a randomized clinical trial that included 267 patients with advanced esophageal cancer for the analysis of four HRQoL dimensions evaluated using the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire. RESULTS: We highlighted differences in terms of outputs, interpretation, and underlying modeling assumptions; this methodological comparison could guide the choice of method according to the context. In the application, none of the four models detected a significant difference between the two treatment arms. The estimated effect of time on HRQoL varied according to the method: for all analyzed dimensions, the PMM estimated an effect that contrasted with those estimated by the SM and SPM; the LMM estimated effects were confirmed by the SM (on two of four HRQoL dimensions) and SPM (on three of four HRQoL dimensions). CONCLUSIONS: The PMM, SM, or SPM should be used to confirm or invalidate the results of LMM analysis when informative dropout is suspected. Of these three alternative methods, the SPM appears to be the most interesting from both theoretical and practical viewpoints. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov , number NCT00861094 .


Assuntos
Neoplasias Esofágicas , Qualidade de Vida , Neoplasias Esofágicas/tratamento farmacológico , Humanos , Estudos Longitudinais , Oncologia , Inquéritos e Questionários
7.
Qual Life Res ; 29(4): 867-878, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31776827

RESUMO

PURPOSE: The time to deterioration (TTD) approach has been proposed as a modality of longitudinal analysis of patient-reported outcomes (PROs) in cancer randomized clinical trials (RCTs). The objective of this study was to perform a systematic review of how the TTD approach has been used in phase III RCTs to analyze longitudinal PRO data. METHODS: A systematic literature search was conducted in PubMed/MEDLINE, the Cochrane Library and through manual search to identify studies published between January 2014 and June 2018. All phase III cancer RCTs including a PRO endpoint using the TTD approach were considered. We collected general information about the study, PRO assessment and the TTD approach, such as the event definition, the choice of reference score and whether the deterioration was definitive or not. RESULTS: A total of 1549 articles were screened, and 39 studies were finally identified as relevant according to predefined criteria. Among these 39 studies, 36 (92.3%) were in advanced and/or metastatic cancer. Several different deterioration definitions were used in RCTs, 10 studies (25.6%) defined the deterioration as "definitive", corresponding to a deterioration maintained over time until the last PRO assessment available for each patient. The baseline score was explicitly stated as the reference score to qualify the deterioration for most studies (n = 31, 79.5%). CONCLUSION: This review highlights the lack of standardization of the TTD approach for the analysis of PRO data in RCTs. Special attention should be paid to the definition of "deterioration", and this should be based on the specific cancer setting.


Assuntos
Deterioração Clínica , Neoplasias/patologia , Neoplasias/terapia , Medidas de Resultados Relatados pelo Paciente , Humanos , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto
8.
Pharmeur Bio Sci Notes ; 2018: 12-36, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29845933

RESUMO

Since the opening for signature of the European Convention for the Protection of Animals Used for Experimental and Other Scientific Purposes in 1986, the European Pharmacopoeia Commission and its experts have carried out a programme of work committed to Replacing, Reducing and Refining (3Rs) the use of animals for test purposes. While updates on achievements in the field of the 3Rs are regularly provided, this article summarises the activities of the Ph. Eur. Commission in this field within the last decade.


Assuntos
Alternativas aos Testes com Animais/normas , Bem-Estar do Animal/normas , Farmacopeias como Assunto/normas , Vacinas/normas , Comitês Consultivos , Alternativas aos Testes com Animais/legislação & jurisprudência , Animais , Europa (Continente) , Humanos , Testes de Toxicidade/normas
9.
Pharmeur Bio Sci Notes ; 2016: 129-134, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28279254

RESUMO

For more than twenty years, the European Pharmacopoeia (Ph. Eur.) monographs for biotherapeutic proteins have been elaborated using the multisource approach (Procedure 1), which has led to robust quality standards for many of the first-generation biotherapeutics. In 2008, the Ph. Eur. opened up the way towards an alternative mechanism for the elaboration of monographs (Procedure 4-BIO pilot phase), which is applied to substances still under patent protection, based on a close collaboration with the Innovator company, to ensure a harmonised global standard and strengthen the quality of the upcoming products. This article describes the lessons learned during the P4-BIO pilot phase and addresses the current thinking on monograph elaboration in the field of biotherapeutics. Case studies are described to illustrate the standardisation challenges associated with the complexity of biotherapeutics and of analytical procedures, as well as the approaches that help ensure expectations are met when setting monograph specifications and allow for compatibility with the development of biosimilars. Emphasis is put on monograph flexibility, notably by including tests that measure process-dependent microheterogeneity (e.g. glycosylation) in the Production section of the monograph. The European Pharmacopoeia successfully concluded the pilot phase of the P4-BIO during its 156th session on 22-23 November 2016.


Assuntos
Medicamentos Biossimilares/análise , Fator IX/análise , Fator VIIa/análise , Farmacopeias como Assunto/normas , Terapia Biológica/métodos , Terapia Biológica/tendências , Medicamentos Biossimilares/uso terapêutico , Europa (Continente) , Fator IX/uso terapêutico , Fator VIIa/uso terapêutico , Humanos , Projetos Piloto
10.
Haemophilia ; 21(4): 543-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25623631

RESUMO

A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed.


Assuntos
Fator IX/análise , Fator VIII/análise , Testes de Coagulação Sanguínea/normas , Calibragem , Cuidadores/psicologia , Compostos Cromogênicos/química , Compostos Cromogênicos/metabolismo , Fator IX/normas , Fator VIII/normas , Humanos , Cooperação Internacional , Laboratórios , Tempo de Tromboplastina Parcial , Rotulagem de Produtos , Proteínas Recombinantes/análise , Proteínas Recombinantes/normas
11.
Pharmeur Bio Sci Notes ; 2010(2): 68-85, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21144490

RESUMO

Due to the depletion in stocks of the World Health Organization (WHO) 2nd International Standard (IS) for nystatin, an international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish a replacement batch. Seventeen laboratories participated in the collaborative study, performing the microbiological diffusion assay to estimate the potency of the candidate 3rd International Standard for nystatin. The 2nd International Standard for nystatin was used as a standard to ensure the continuity of the unitage. Follow-up accelerated degradation studies demonstrated that the IS is stable when at the customary storage temperature of - 20 °C. The 3rd IS for nystatin was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in 2006 with an assigned potency of 5710 International Units per mg (IU/mg). The 3rd IS for nystatin is available from the EDQM.


Assuntos
Antibacterianos/normas , Nistatina/normas , Cromatografia Líquida , Comportamento Cooperativo , Estabilidade de Medicamentos , Cooperação Internacional , Laboratórios/normas , Nistatina/química , Nistatina/farmacologia , Padrões de Referência , Organização Mundial da Saúde
12.
Pharmeuropa Bio ; 2003(2): 1-8, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14960259

RESUMO

Collaborative studies were initiated by the European Directorate for the Quality of Medicines (EDQM) to assign potency values for candidate European Pharmacopoeia Chemical Reference Substances (Ph. Eur. CRSs) used for the microbiological assay of antibiotics. The candidates were assayed against their respective International Standard (IS), using the methods by diffusion or turbidimetry. Potencies were assigned to all the antibiotics concerned, which were adopted by the European Pharmacopoeia Commission.


Assuntos
Antibacterianos/análise , Antibacterianos/normas , Europa (Continente) , Cooperação Internacional , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Farmacopeias como Assunto/normas , Padrões de Referência
13.
Pharmeuropa Bio ; 2004(1): 35-45, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15659284

RESUMO

Human Growth hormone (hGH, somatotrophin) is a 22 kDa, 191 amino-acid single chain protein produced by somatroph cells of the anterior pituitary gland. It is the major physiological regulator of growth, and deficiencies in growth hormone levels have long been recognized as the underlying cause of growth disorders (dwarfism). The ability of exogenous hGH to restore normal rates of growth in both human and animal models of growth retardation has long been recognized and the use of hGH in therapy goes back several decades. Initial preparations were prepared by extraction and purification from cadaveric pituitary tissue, and since 1984, hGH has been prepared by recombinant Deoxyribosenucleic acid (rDNA) technology. As is usually the case with "biologicals", characterization of the drug substance depended on a combination of physico-chemical and biological methods, and the hGH molecule became well characterized fairly early in its life as a drug. Indeed, by 1980 the major degradation forms and structural variants of the hGH molecule had been described and reviewed. Little satisfactory progress had been made in refining biological assays for hGH, and, although in vitro assays were described, potency-defining assays remained dependant on the whole body growth response in rats, and were both invasive and imprecise. In the early 1990's a series of collaborative studies on analysis of recombinant hGH (somatropin) established that available bioassays were much less selective that physico-chemical methods in detecting and quantifying structural degradation, and 1994 saw an international consensus to replace the bioassays with physico-chemical analytical methods for the routine batch release of somatropin. During the last decade in most markets somatropin has, unusually for a protein, been subject to batch release and control dependent entirely on physico-chemical analysis, without the routine use of any form of bioassay. During that time there has been a continuous development and refinement of methods, and the identification of a range of structural variants and degradation products of the molecule. The present review sets out to summarise the current knowledge on physico-chemical analytical methods for somatropin, and the structural variants that have been identified and characterized. A survey of available biological analytical methods is beyond the scope of this review, as is consideration of the earlier pituitary preparations and the recombinant 192 amino-acid methionyl form of the molecule (somatrem), although it is likely that many of the methods and variants described would be equally applicable to somatrem.


Assuntos
Hormônio do Crescimento Humano/química , Cromatografia Líquida de Alta Pressão/normas , Clonagem Molecular/métodos , Eletroforese Capilar , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/normas , Humanos , Focalização Isoelétrica , Mapeamento de Peptídeos , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/normas , Transcrição Gênica
14.
Pharmeuropa Bio ; 2004(1): 47-58, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15659285

RESUMO

In the European Pharmacopoeia, the monographs on somatropin, somatropin bulk solution and somatropin for injection prescribe a number of tests including "related substances", "dimer and related substances of higher molecular weight" and "isoform distribution" which are intended to control the levels of impurities in the substance. The aim of this study was to verify the robustness of a new method by capillary electrophoresis and to compare its performance with that of the existing test for "isoform distribution" by isoelectric focusing. It was demonstrated that the capillary electrophoresis method was superior to the method of isoelectric focusing. The interest of the CZE method consists in the resolution of related impurities that might be process specific and/or generated by the expression system.


Assuntos
DNA Complementar/normas , Eletroforese Capilar/normas , Hormônio do Crescimento Humano/normas , Europa (Continente) , Humanos , Reprodutibilidade dos Testes
15.
Dev Biol (Basel) ; 111: 79-88, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12678227

RESUMO

In vivo bioassays for peptide and protein hormones have, until recently, represented a major use of laboratory animals. Advances in physico-chemical methodology, particularly HPLC, have moved most of these substances into the "non-biological" category. Newer soluble regulators (interferons, cytokines, growth factors), will probably be controlled by in vitro bioassays, obviating the need for validation of alternatives. Validation of in vitro alternatives for glycoproteins remains problematic, and is the subject of current research and development.


Assuntos
Alternativas aos Testes com Animais , Bioensaio/métodos , Hormônios/química , Hormônios/metabolismo , Animais , Humanos , Técnicas In Vitro , Farmacopeias como Assunto
18.
Biochim Biophys Acta ; 1127(2): 191-8, 1992 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-1643106

RESUMO

Geotrichum candidum CMICC 335426 was previously shown to produce two lipases termed lipase A and lipase B, lipase B being highly specific for hydrolysis of esters of cis-delta 9 fatty acids. We now describe the isolation of polyclonal antibodies specific for lipase A and lipase B. These antibodies were used in Western blotting techniques to detect the appearance of the lipases during the course of the fermentation of G. candidum CMICC 335426. A and B were found to be produced simultaneously in the extracellular medium at the start of the growth phase. The two lipases were always present at similar levels in the medium. The specific antibodies were then used to detect the presence of A- and B-like lipases in crude lipase samples from other strains of G. candidum. The lipases were found at different levels in all these samples, and the specificities of the crude lipases varied significantly from one strain to another. Differences in specificity could therefore be explained by different levels of specific (B-type) and non-specific (A-type) lipases in the medium. This was verified by purifying A- and B-type lipases from the G. candidum strain ATCC 34614.


Assuntos
Geotrichum/enzimologia , Lipase/análise , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Western Blotting , Reações Cruzadas , Fermentação , Geotrichum/ultraestrutura , Lipase/imunologia , Lipase/metabolismo , Especificidade por Substrato
19.
Biochim Biophys Acta ; 1123(1): 59-64, 1992 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-1730047

RESUMO

The mould Geotrichum candidum produces extracellular lipases with different substrate specificities according to strains. We purified two lipases - termed lipase A and lipase B - from Geotrichum candidum CMICC 335426. The specificity of the two lipases was investigated using hydrolysis assays on emulsions of pure acylglycerols and a wide range of fatty acid esters. Lipase B was very highly specific for hydrolysis of esters of cis-delta 9-fatty acids. Lipase A did not show such strict specificity, because it hydrolysed a wider variety of fatty acid esters, in particular those of palmitic acid and isomers of oleic acid. We think that differences in specificity previously observed for crude lipases from various strains of G. candidum can be explained by the presence of different levels of specific (lipase B) and non-specific (lipase A) lipases. As lipases A and B are structurally related proteins, a minor variation in structure may be responsible for the differing specificities.


Assuntos
Geotrichum/enzimologia , Isoenzimas/metabolismo , Lipase/metabolismo , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Ésteres/metabolismo , Ácidos Graxos/metabolismo , Glicerídeos/metabolismo , Hidrólise , Isoenzimas/isolamento & purificação , Lipase/isolamento & purificação , Especificidade por Substrato
20.
Eur J Biochem ; 202(2): 485-91, 1991 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-1761048

RESUMO

We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively. Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo-beta-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.


Assuntos
Geotrichum/enzimologia , Isoenzimas/metabolismo , Lipase/metabolismo , Western Blotting , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/metabolismo , Glicosilação , Focalização Isoelétrica , Isoenzimas/isolamento & purificação , Lipase/isolamento & purificação , Peso Molecular , Especificidade por Substrato
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