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Overexpression of human glucocerebrosidase containing different-sized leaders.

ABSTRACT: Gaucher disease results from impaired activity of the lysosomal enzyme glucocerebrosidase. Aiming at overexpressing the human glucocerebrosidase and testing the efficiency of the two in-frame ATGs of its gene in directing synthesis of an active enzyme, it was coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). cDNAs containing either one or both ATGs of the glucocerebrosidase mRNA were linked to the T7 polymerase promoter. Recombinant viruses were produced and used for infecting human cells in tissue culture. The results demonstrated that both ATGs directed translation of active glucocerebrosidase, resulting in a 10-fold increase in enzymic activity. Most of the protein remained sensitive to endoglycosidase H. The active enzyme represented a small fraction of the expressed glucocerebrosidase. The recombinant enzyme had the same Km and optimal pH towards the artificial substrate 4-methylumbelliferyl glucopyranoside as the authentic endogenous human enzyme. Measurements of intracellular enzymic activity directed by the cDNAs with either one or both ATGs in cells loaded with a fluorescent glucosylceramide demonstrated a 30% increase in activity directed by the cDNAs containing the first ATG over that containing the second ATG. This indicates that the protein synthesized from the first ATG, with a 38 amino acid leader, is translocated through the endoplasmic reticulum more readily than its counterpart directed by the second ATG, with a 19 amino acid leader. The elevation in glucocerebrosidase activity and the reproducibility of the data leads us to propose the use of the vaccinia virus-derived expression system as a tool for studying glucocerebrosidase mutants in Gaucher disease.

SUBMITTER: Pasmanik-Chor M

PROVIDER: S-EPMC1217489 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundOccupational health interventions for leaders are underrepresented in small and medium-sized enterprises (SMEs). When creating and developing effective occupational health interventions, identification of the specific needs of the target group is regarded as an essential step before planning an intervention. Therefore, the aim of this study was (1) to examine the subjectively experienced work-related stressors of leaders in small and medium-sized IT and technological services enterprises, (2) to explore coping behaviors leaders use to deal with the experienced work-related stressors, (3) to investigate resources supporting the coping process and (4) to identify potentially self-perceived consequences resulting from the experienced stressors.MethodsTen semi-structured interviews with leaders in small and medium-sized IT and technological services enterprises were conducted. The interviews were transcribed and analyzed with content-structuring qualitative content analysis in accordance to Kuckartz.ResultsLeaders in small and medium-sized IT and technological services enterprises experience various stressors caused by work organization as well as industry-related stressors and other work-related stressors. To address the experienced stressors, leaders apply problem focused coping behaviors (e.g. performing changes on structural and personal level), emotional focused coping behaviors (e.g. balancing activities, cognitive restructuring) as well as the utilization of social support. Helpful resources for the coping process include organizational, social and personal resources. As a result of the experienced work-related stressors, interviewees stated to experience different health impairments, negative effects on work quality as well as neglect of leisure activities and lack of time for family and friends.ConclusionThe identified experienced work-related stressors, applied coping behaviors, utilized resources and emerging consequences underpin the urgent need for the development and performance of health-oriented leadership interventions for leaders in small and medium- sized IT and technological services. The results of this study can be used when designing a target-oriented intervention for the examined target group.

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Overexpression of human glucocerebrosidase containing different-sized leaders.

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