Implementation of Intelligent Physical Exercise Training at a Danish Hospital-A Qualitative Study of Employees' Barriers and Facilitators for Participation.

BACKGROUND: Exercise training at work has the potential to improve employees' productivity, health, and well-being. However, exercise interventions for healthcare workers in hospitals may be challenged by time pressure and the ongoing workflow with patient care. OBJECTIVE: The aim was to identify barriers and facilitators for participation in exercise training during work in a hospital department. METHODS: Eight semi-structured interviews of 13 individuals were conducted with hospital employees from different staff groups who participated in 12 weeks of exercise twice weekly. The data analysis was a thematic approach based on the Theoretical Domains Framework and the COM-B factors in the Behavior Change Wheel. RESULTS: Barriers and facilitators varied between different groups. Barriers included limited structure, busyness, and a discouraging culture. Facilitators included gaining a feeling of community and psychological and physical well-being. Seven contextual subthemes were vital for successful implementation of exercise in a hospital setting: sharing of knowledge and information; involvement; administration and structure; culture; individualization; purpose and objective; and incentives. CONCLUSIONS: The informants appreciated exercise training during work. Inpatient departments' informants found it difficult to participate in the intervention, whilst those with more administrative tasks found it easier. This study identified barriers and facilitators vital for a successful implementation of an exercise training intervention in a hospital department. The study explains how future interventions can improve reach, adoption, and implementation of exercise training interventions to hospital staffs.

Long Term Cardiovascular Health Effects of Intelligent Physical Exercise Training Among Office Workers-A 2 Year Follow up of a Randomized Controlled Trial.

OBJECTIVE: The aim of the study is to assess long-term effects of intelligent physical exercise training (IPET) on cardiorespiratory fitness (VO 2max ) and cardiometabolic measures. METHODS: Office workers were randomized to a control group (CG, n = 194) or a training group (TG, n = 193). The TG received 1-hour weekly IPET during paid working hours for 2 years and recommendations to perform 30-minute leisure time physical activity 6 d/wk (LPA). RESULTS: Training group compared with CG demonstrated a significantly larger increase in VO 2max of 0.13 ± 0.06 L/min and improved cardiometabolic measures at 1-year follow-up that were maintained at 2-year follow-up, with larger increases in VO 2max among high-adherence participants. CONCLUSIONS: Intelligent physical exercise training and LPA showed the potential for long-term improved VO 2max and cardiometabolic measures. These findings emphasize the effectiveness of integrating IPET during paid working hours, and the significance of adherence to training was underlined.

Exercise Training During Working Hours at a Hospital Department: A Pilot Study.

OBJECTIVES: This pilot study tested the use of an exercise offer to hospital employees during working hours and changes in work and health parameters. METHODS: Employees (n = 214) from a medical department on a Danish hospital were invited to 30 minutes' exercise training twice weekly for 12 weeks. Outcomes included health- and work-related parameters. RESULTS: Eighty employees (mean age, 44.4 [SD, 10.7] years; 81.3% women) completed the study. Intervention adherence was 36.3% (SD, 25.1%). Aerobic capacity increased from 34.6 (95% confidence interval [CI], 32.3 to 36.9) to 36.7 (95% CI, 34.1 to 39.4) mL O 2 /min per kilogram, P = 0.004. Blood pressure decreased from 120 (95% CI, 117 to 123)/79 (95% CI, 76 to 81) to 116 (95% CI, 112 to 120)/76 (95% CI, 74 to 79) mm Hg, P = 0.003. Waist circumference and musculoskeletal pain decreased. Well-being, social capital, and quality of life increased. CONCLUSIONS: Despite low training adherence, completers improved outcomes related to metabolic and self-rated health.

Attendance barriers experienced by female health care workers voluntarily participating in a multi-component health promotion programme at the workplace.

BACKGROUND: Studies have shown that Workplace Health Promoting Programmes (WHPP) can facilitate healthier behaviour. Despite the benefits achieved from participating in a WHPP, a systematic review showed that only 10-50% of the employees participated and a challenge was lack of participation. Previous studies stress that understanding the barriers that prevent participants from attending WHPPs are important for designing highly effective interventions. To exploit the potential of a WHPP, it requires a deep insight into the attendance barriers experienced by the participants who voluntarily sign-up for a WHPP; and particularly those who want to stay in the programme but are prevented from participating in it regularly. Thus, the aim of this study was to identify and explore attendance barriers experienced by female Health Care Workers (HCWs) who voluntarily participated in a weekly one-hour multi-component training session, within a WHPP, over a one-year period. METHODS: This study was carried out within a RCT named FRIDOM (FRamed Intervention to Decrease Occupational Muscle pain) and was designed as a single-case study with an inductive approach for analysing the content of in-depth semi-structured qualitative interviews. Data was collected at two home care workplaces and two retirement homes in Denmark. Nine HCWs from the intervention group were selected as participants in the present study. RESULTS: The attendance barriers identified, consisted of three main themes and six related sub-themes: 1) organizational factors (work inflexibility, lack of support from team leaders), 2) intervention factors (training sessions organized outside normal work hours, incongruence between information received and reality, content and intensity of the program) and 3) individual factors (personal factors). CONCLUSION: Organizational and intervention factors are the two most important attendance barriers in future WHPPs. To overcome these barriers; training sessions should be organized within or in connection with work hours, support should be secured from team management and work shifts should be planned to enable attendance for all participants. Furthermore, the attendance barriers may be minimized by including participants in the decision-making process. This relates to both the content and intensity of the intervention, not only in the planning stage but throughout the intervention process. TRIAL REGISTRATION: ClinicalTrials.gov. NCT02843269 - 06.27.2016 - retrospectively registered.

Intelligent Physical Exercise Training in a Workplace Setting Improves Muscle Strength and Musculoskeletal Pain: A Randomized Controlled Trial.

PURPOSE: To assess effects of 1-year Intelligent Physical Exercise Training (IPET) on musculoskeletal health. METHODS: Office workers were randomized 1 : 1 to a training group, TG (N = 193), or a control group, CG (N = 194). TG received 1 h supervised high intensity IPET every week within working hours for 1 year and was recommended to perform 30 min of moderate intensity physical activity for 6 days a week during leisure. The IPET program was based on baseline health measures. RESULTS: No baseline differences were present. An intention-to-treat analysis showed significant between-group effect for muscle strength but not for musculoskeletal pain. However, a per-protocol analysis of those with an adherence of ≥70% demonstrated a significant between-group effect for neck pain during the past three months. Several significant within-group changes were present, where TG and TG ≥ 70% demonstrated clinically relevant pain reductions whereas minimal reductions were seen for CG. CONCLUSION: IPET and recommendations of moderate intensity physical activity demonstrated significant between-group effect on muscle strength. Interestingly, significant within-group reductions in musculoskeletal pain were seen not only in TG but also in CG. This may underlie the lack of such between-group effect and shows that a possible positive side effect of merely drawing attention can improve musculoskeletal health.

The Effect of Intelligent Physical Exercise Training on Sickness Presenteeism and Absenteeism Among Office Workers.

OBJECTIVE: The aim of this study was to investigate the effect of individually tailored intelligent physical exercise training (IPET) on presenteeism and absenteeism among office workers. METHODS: In a 1-year randomized controlled trial (RCT), employees were allocated to a training group TG (N = 193) or control group CG (N = 194). TG received 1-hour high-intensity IPET once a week within working hours, and was recommended to perform 30 minutes of moderate-intensity physical activity (PA) 6 days a week during leisure-time. RESULTS: An intention-to-treat analysis showed no effect on absenteeism, but a significant 4% increase in workability and 9% increase in general health in TG compared with CG. A per-protocol analysis [adherence of ≥70% (N = 89)] in addition showed a significant 6% increase in productivity and a 29% reduction in absenteeism compared with CG. CONCLUSION: IPET combined with recommendations of leisure-time PA significantly improved presenteeism and decreased absenteeism if following the protocol.

Implementing intelligent physical exercise training at the workplace: health effects among office workers-a randomized controlled trial.

PURPOSE: The aim was to assess 1-year cardiovascular health effects of Intelligent Physical Exercise Training, IPET. METHODS: Office workers from six companies were randomized 1:1 to a training group, TG (N = 194) or a control group, CG (N = 195). TG received 1-h supervised high intensity IPET every week within working hours for 1 year, and was recommended to perform 30-min of moderate intensity physical activity 6 days a week during leisure. The training program was based on baseline health check measures of cardiorespiratory fitness (CRF), body composition, blood pressure, blood profile, and musculoskeletal health. RESULTS: There were no baseline differences between groups. CRF assessed as VO2max in absolute values and relative to body weight was (mean ± SD): 3.0 ± 0.8 l/min and 35.4 ± 10.9 ml/min/kg for females, 3.9 ± 1.0 l/min and 37.9 ± 11.79 ml/min/kg for males. Intention to treat analysis demonstrated a significant almost 5 % increase in VO2max in TG compared with CG. A per protocol analysis of those with an adherence of ≥70 % demonstrated a significant increase in CRF of more than 10 % compared with CG, and a significant reduction in systolic blood pressure (-5.3 ± 13.7 mm Hg) compared with CG. CONCLUSION: High intensity IPET combined with the recommendations of moderate intensity physical activity demonstrated significant clinical relevant improvements in CRF and systolic blood pressure. This underlines the effectiveness of health promotion by implementing physical exercise training at the workplace.

Exercise is more than medicine: The working age population's well-being and productivity.

BACKGROUND: Physical activity (PA) includes muscle activity during exercise, manual work, and leisure time activities including sport. Conflicting results exist regarding health effects of PA that may deteriorate with manual work and elite sports, but improve when performed in moderation in accordance with international guidelines and may additionally enhance well-being and productivity. METHODS: In Denmark 15 randomized controlled trials have been conducted, introducing exercise at the workplace enrolling >3500 workers. The interventions lasted from 10 to 52 weeks and offered ~1 h weekly supervised exercise during working hours according to the concept of intelligent physical exercise training (IPET) that is based on evidenced sports sciences training principles and tailored to work exposure, employee health status, and physical capacity. Questionnaire surveys and health checks including blood and muscle sampling were performed at baseline and follow-up. The job groups included: office and computer workers, dentists, industrial technicians, cleaning personnel, health care workers, construction workers, and fighter/helicopter pilots. RESULTS: In all job groups significant improvements were documented regarding health outcomes. These were job group specific: neck pain was reduced among office and computer workers, dentists, industrial laboratory technicians, health care workers as well as fighter pilots. Cardio-respiratory fitness-a health risk indicator for cardio-metabolic diseases-was improved among office and computer workers, health care workers, and construction workers. Additionally, other improvements were evidenced such as increased muscle strength and balance control. Importantly, productivity increased with improved muscle strength and decreased body mass index. CONCLUSION: IPET does enhance health if an exercise program with evidenced efficacy is implemented by expert trainees with support of the employer. Accordingly, in every study group outcomes of improved health were documented and the effect sizes were of clinical relevance. Cost effectiveness estimates indicate acceptable cost relative to savings on health expenses and lost productivity.

Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

BACKGROUND: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7. RESULTS: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays. CONCLUSIONS: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

Mitochondrial localization of the OAS1 p46 isoform associated with a common single nucleotide polymorphism.

BACKGROUND: The expression of 2'-5'-Oligoadenylate synthetases (OASs) is induced by type 1 Interferons (IFNs) in response to viral infection. The OAS proteins have a unique ability to produce 2'-5' Oligoadenylates, which bind and activate the ribonuclease RNase L. The RNase L degrades cellular RNAs which in turn inhibits protein translation and induces apoptosis. Several single nucleotide polymorphisms (SNPs) in the OAS1 gene have been associated with disease. We have investigated the functional effect of two common SNPs in the OAS1 gene. The SNP rs10774671 affects splicing to one of the exons in the OAS1 gene giving rise to differential expression of the OAS1 isoforms, and the SNP rs1131454 (former rs3741981) resides in exon 3 giving rise to OAS1 isoforms with either a Glycine or a Serine at position 162 in the core OAS unit. RESULTS: We have used three human cell lines with different genotypes in the OAS1 SNP rs10774671, HeLa cells with the AA genotype, HT1080 cells with AG, and Daudi cells with GG. The main OAS1 isoform expressed in Daudi and HT1080 cells was p46, and the main OAS1 isoform expressed in HeLa cells was p42. In addition, low levels of the OAS1 p52 mRNA was detected in HeLa cells and p48 mRNA in Daudi cells, and trace amounts of p44a mRNA were detected in the three cell lines treated with type 1 interferon. We show that the OAS1 p46 isoform was localized in the mitochondria in Daudi cells, whereas the OAS1 isoforms in HeLa cells were primarily localized in cytoplasmic vacuoles/lysosomes. By using recombinantly expressed OAS1 mutant proteins, we found that the OAS1 SNP rs1131454 (former rs3741981) did not affect the enzymatic OAS1 activity. CONCLUSIONS: The SNP rs10774671 determines differential expression of the OAS1 isoforms. In Daudi and HT1080 cells the p46 isoform is the most abundantly expressed isoform associated with the G allele, whereas in HeLa cells the most abundantly expressed isoform is p42 associated with the A allele. The SNP rs1131454 (former rs3741981) does not interfere with OAS1 enzyme activity. The OAS1 p46 isoform localizes to the mitochondria, therefore a full 2-5A system can now be found in the mitochondria.

A conceptual model for worksite intelligent physical exercise training--IPET--intervention for decreasing life style health risk indicators among employees: a randomized controlled trial.

BACKGROUND: Health promotion at the work site in terms of physical activity has proven positive effects but optimization of relevant exercise training protocols and implementation for high adherence are still scanty. METHODS/DESIGN: The aim of this paper is to present a study protocol with a conceptual model for planning the optimal individually tailored physical exercise training for each worker based on individual health check, existing guidelines and state of the art sports science training recommendations in the broad categories of cardiorespiratory fitness, muscle strength in specific body parts, and functional training including balance training. The hypotheses of this research are that individually tailored worksite-based intelligent physical exercise training, IPET, among workers with inactive job categories will: 1) Improve cardiorespiratory fitness and/or individual health risk indicators, 2) Improve muscle strength and decrease musculoskeletal disorders, 3) Succeed in regular adherence to worksite and leisure physical activity training, and 3) Reduce sickness absence and productivity losses (presenteeism) in office workers. The present RCT study enrolled almost 400 employees with sedentary jobs in the private as well as public sectors. The training interventions last 2 years with measures at baseline as well as one and two years follow-up. DISCUSSION: If proven effective, the intelligent physical exercise training scheduled as well as the information for its practical implementation can provide meaningful scientifically based information for public health policy. TRIAL REGISTRATION: ClinicalTrials.gov, number: NCT01366950.

Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals.

Characterization of human phosphodiesterase 12 and identification of a novel 2'-5' oligoadenylate nuclease - The ectonucleotide pyrophosphatase/phosphodiesterase 1.

The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5' oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5'-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2'-5'-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V(max) and K(m) kinetic parameters for the enzyme. We have also identified a novel 2'-5'-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process.

Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover.

The vertebrate 2-5A system is part of the innate immune system and central to cellular antiviral defense. Upon activation by viral double-stranded RNA, 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2'-5'-oligoadenylate synthetases. These unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5' bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that 2'-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA like canonical cytoplasmic deadenylases. Furthermore, we document a marked negative association between 2'-PDE and mitochondrial mRNA levels following siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The results indicate that 2'-PDE, apart from playing a role in the cellular immune system, may also function in mitochondrial RNA turnover.

Evolution of the 2'-5'-oligoadenylate synthetase family in eukaryotes and bacteria.

The 2'-5'-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins in bacteria that contains the five OAS-specific motifs. This indicates a specific relationship to OAS. The wide distribution of the OAS genes has made it possible to suggest how the OAS1 gene could have evolved from a common ancestor to choanoflagellates and metazoans. Furthermore, we suggest that the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may differ from its function in the interferon system, and that this original function of OAS is preserved even in OAS genes that code for proteins, which do not have 2'-5'-oligoadenylate synthetase activity.

In vitro effects of metal pollution on Mediterranean sponges: species-specific inhibition of 2',5'-oligoadenylate synthetase.

Heavy metals are among the main pollutants of the Mediterranean coastal waters where they can harm sublittoral biota. Filter-feeder, long-living invertebrates that remain fixed to the rocky bottom, such as sponges, are good targets to metal contamination studies since they may be exposed to potential low levels of contamination for years. Several molecular and biochemical mechanisms are developed by sponges to counteract the effects of noxious metals. As a result, some of the normal cell functions can be altered. Here we show that the main heavy metals that can be found in marine sublittoral waters (i.e. copper, iron, zinc and manganese) may alter the immune system of sponges by inhibiting the activity of the sponge 2',5'-oligoadenylate synthetase (2-5A synthetase), which is an enzyme involved in the immune system of vertebrates. We selected the widespread Mediterranean sponges Geodia cydonium, Crella elegans and Chondrosia reniformis for the study. They exerted a high 2-5A synthetase activity and gave a unique profile of 2',5'-oligoadenylate product production. Several metals alter the 2-5A synthetase activity differently, in a species-specific manner. 2-5A synthetases from G. cydonium and C. elegans were inhibited by all the metal ions assayed. However, in C. reniformis, 2-5A synthetase was either activated or inhibited by the same ions depending on their final concentrations. Like in humans, metal contamination may have an effect on the OAS activity and thus it might alter the sponge immune system. However, since the effects are species-specific, 2-5A synthetase cannot be used as general biomarker of metal pollutions.

2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera].

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2',5'-oligoadenylate synthetase (OAS). The components of the antiviral 2',5'-oligoadenylate (2-5A) system (OAS, 2'-Phosphodiesterase (2'-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2'-PDE activity, which highlights the probable existence of a premature 2-5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2-5A degrading activity. Upon this finding, two out of three elements forming the 2-5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family.

2',5'-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family.

Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium.

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.

The human 2'-5'oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2'-5' instead of 3'-5' phosphodiester bond formation.

The demonstration by Kerr and colleagues that double-stranded (ds) RNA inhibits drastically protein synthesis in cell-free systems prepared from interferon-treated cells, suggested the existence of an interferon-induced enzyme, which is dependent on dsRNA. Consequently, two distinct dsRNA-dependent enzymes were discovered: a serine/threonine protein kinase that nowadays is referred to as PKR and a 2'-5'oligoadenylate synthetase (2'-5'OAS) that polymerizes ATP to 2'-5'-linked oligomers of adenosine with the general formula pppA(2'p5'A)(n), n>or=1. The product is pppG2'p5'G when GTP is used as a substrate. Three distinct forms of 2'-5'OAS exist in human cells, small, medium, and large, which contain one, two, and three OAS units, respectively, and are encoded by distinct genes clustered on the 2'-5'OAS locus on human chromosome 12. OASL is an OAS like IFN-induced protein encoded by a gene located about 8 Mb telomeric from the 2'-5'OAS locus. OASL is composed of one OAS unit fused at its C-terminus with two ubiquitin-like repeats. The human OASL is devoid of the typical 2'-5'OAS catalytic activity. In addition to these structural differences between the various OAS proteins, the three forms of 2'-5'OAS are characterized by different subcellular locations and enzymatic parameters. These findings illustrate the apparent structural and functional complexity of the human 2'-5'OAS family, and suggest that these proteins may have distinct roles in the cell.