Calculation of Oxygen Uptake during Ambulatory Cardiac Rehabilitation.

Background: Cardiopulmonary exercise testing is not used routinely. The goal of this study was to determine whether accurate estimates of VO2 values can be made at the beginning and at the end of a rehabilitation program. Methods: A total of 91 cardiac rehabilitation patients were included. Each participant had to complete cardiopulmonary exercise testing at the beginning and at the end of a rehabilitation program. Measured VO2 values were compared with estimates based on three different equations. Results: Analyses of the means of the differences in the peak values showed very good agreement between the results obtained with the FRIEND equation or those obtained with a combination of rules of thumb and the results of the measurements. This agreement was confirmed with the ICCs and with the standard errors of the measurements. The ACSM equation performed worse. The same tendency was seen when considering the VO2 values at percentage-derived work rates. Conclusions: The FRIEND equation and the more easily applicable combination of rules of thumb are suitable for estimating the peak VO2 and the VO2 at a percentage-derived work rate in cardiac patients both at the beginning and at the end of a cardiac rehabilitation program.

The Impact of Medical Face Masks and Rehabilitation Duration on the Performance Output and Outcomes of Cardiologic Rehabilitants.

During the COVID-19 pandemic, wearing a medical face mask became mandatory in daily life and also in cardiological rehabilitation. In order to investigate whether the performance and outcomes of cardiological rehabilitation were affected by face masks, we compared data from patients who underwent rehabilitation with face masks with data from patients without face masks. In total, 114 patients from an ambulant rehabilitation center were included. Of them, 60 patients completed rehabilitation without a face mask (NFM). In contrast, 54 patients (with a face mask, WFM) completed their rehabilitation during the COVID-19 pandemic and had to wear medical face masks for the entire day and also during ergometer training or other interventions. Subgroups were formed with patients who accepted to extend rehabilitation for one week (4 WG); the other patients only completed 3-week rehabilitation (3 WG). We analyzed the performance and outcomes of all groups (NFM; WFM, 3 WG and 4 WG). At baseline, no group differences were detected. All groups significantly improved their power output and heart rate recovery, without any group differences. We conclude that face masks and also an additional rehabilitation week do not affect the exercise performance or outcomes of out-house cardiological rehabilitation.

Neuroinflammatory markers in patients with haemophilia and healthy controls: Where are the differences?

INTRODUCTION: People with haemophilia (PwH) suffer from haemophilic arthropathy which is accompanied by acute and chronic inflammation. The aim of this study was to examine the neuroinflammatory network operative in PwH and to compare it to healthy controls. MATERIAL AND METHODS: Blood samples were collected from 41 PwH (age 54.7 ± 11.7 years) and 33 healthy controls (age 50.9 ± 10.5 years) and the levels of 13 neuroinflammatory markers were analyzed by applying an antibody-based detection kit in a flow cytometer. RESULTS: From 13 analyzed markers, three-ß-nerve growth factor (ß-NGF), soluble receptor for advanced glycation endproducts (sRAGE) and Interleukin-18 (IL-18) differed significantly between the groups (ß-NGF p = .045; sRAGE p = .003; IL-18 p = .007). While ß-NGF was downregulated in PwH, sRAGE and IL-18 were upregulated. None of the analyzed markers corelated to the joint status of PwH while CCL2 (C-C motif ligand 2 chemokine) correlated to HIV infections in PwH (r = .313, p = .007). Correlation analyses of the markers studied also revealed many differences between PwH and controls suggesting a number of deregulations in PwH. CONCLUSION: The altered levels of sRAGE and ß-NGF in PwH, which have not been analyzed in PwH before, may help to understand the neuroinflammatory network operative in PwH. The general inflammatory processes in PwH and the involved biomarkers in PwH remain poorly understood. PwH could benefit from new therapies against neuroinflammation which may help to reduce inflammation or also chronic pain.

Endothelial dysfunction and atherosclerosis related miRNA-expression in patients with haemophilia.

INTRODUCTION: Elevated markers of endothelial dysfunction and inflammation indicate worse endothelial function in the aging haemophilia population. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Several miRNAs have been shown to be involved in the process of endothelial dysfunction and atherosclerosis. AIM: The aim of this study was to determine the underlying molecular pathways of endothelial dysfunction and inflammation in haemophilia patients. METHODS: A total of 25 patients with severe or moderate haemophilia A (20 patients) or B (5 patients), 14 controls and 18 patients with coronary artery disease (CAD) after myocardial infarction were included in this study. Expression of miRNA-126, -155, -222, -1, -let7a, -21 and -197 were analysed using a real time polymerase chain reaction. Network-based visualisation and analysis of the miRNA-target interactions were performed using the MicroRNA ENrichment TURned NETwork (MIENTURNET). RESULTS: Expression of miRNA-126 (p < .05) and miRNA-let7a (p < .05) were significantly higher in CAD patients compared to haemophilia patients and controls. MiRNA-21 (p < .05) was significantly elevated in CAD patients compared to controls. MiRNA-155 (p < .05), miRNA-1 (p < .05) and miRNA-197 (p < .05) were significantly higher expressed in CAD and haemophilia patients compared to controls and showed a strong correlation with increased levels of interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1). The network analysis revealed interactions in the cytokine signalling, focal adhesion and VEGFA-VEGFR2 pathway (Vascular endothelial growth factor, -receptor). CONCLUSION: This study characterises miRNA expression in haemophilia patients in comparison to CAD patients and healthy controls. The results imply comparable biological processes in CAD and haemophilia patients.

Effects of high-intensity interval training in a three-week cardiovascular rehabilitation: a randomized controlled trial.

OBJECTIVE: The aim of this study was to assess the benefit of using high-intensity interval training for cardiovascular patients undergoing outpatient rehabilitation in a standard short-term (three-week) program in Germany. DESIGN: This is a randomized controlled trial (RCT). SETTING: This study was conducted at Cardiowell (Wuppertal, Germany), an outpatient rehabilitation center. INTERVENTION: Patients underwent the typical three-week German outpatient rehabilitation program using either moderate continuous training (i.e. the standard training program) or high-intensity interval training. MAIN MEASURES: A total of 50 patients of an outpatient rehabilitation center were randomized into two groups. The control group underwent the standard rehabilitation protocol that applied moderate continuous training, and the intervention group trained according to a high-intensity interval protocol. Patients trained on a bicycle ergometer. Peak power output, oxygen uptake parameters, heart frequencies, and blood pressure were compared at the beginning and at the end of the rehabilitation program. RESULTS: After three weeks, the intervention group had improved to a significantly greater extent in maximal performance parameters than the control group: the peak power output (20.9 (±14.1) W; control 8.8 (±10.4) W), maximum oxygen uptake (0.33 (±0.33) L/min; control 0.05 (±0.29) L/min)), relative maximum oxygen uptake (3.4 (±4.2) mL/kg/min; control 0.9 (±3.1) mL/kg/min), and O2 pulse (1.8 (±2.2) mL/heart beat; control 0.35 (±1.7) mL/heart beat). CONCLUSION: The implementation of high-intensity interval training during a typical three-week German cardiac rehabilitation has the power to increase the outcome for the patients.

Acute Response of Circulating Vascular Regulating MicroRNAs during and after High-Intensity and High-Volume Cycling in Children.

AIM: The aim of the present study was to analyze the response of vascular circulating microRNAs (miRNAs; miR-16, miR-21, miR-126) and the VEGF mRNA following an acute bout of HIIT and HVT in children. METHODS: Twelve healthy competitive young male cyclists (14.4 ± 0.8 years; 57.9 ± 9.4 ml·min(-1)·kg(-1) peak oxygen uptake) performed one session of high intensity 4 × 4 min intervals (HIIT) at 90-95% peak power output (PPO), each interval separated by 3 min of active recovery, and one high volume session (HVT) consisting of a constant load exercise for 90 min at 60% PPO. Capillary blood from the earlobe was collected under resting conditions, during exercise (d1 = 20 min, d2 = 30 min, d3 = 60 min), and 0, 30, 60, 180 min after the exercise to determine miR-16, -21, -126, and VEGF mRNA. RESULTS: HVT significantly increased miR-16 and miR-126 during and after the exercise compared to pre-values, whereas HIIT showed no significant influence on the miRNAs compared to pre-values. VEGF mRNA significantly increased during and after HIIT (d1, 30', 60', 180') and HVT (d3, 0', 60'). CONCLUSION: RESULTS of the present investigation suggest a volume dependent exercise regulation of vascular regulating miRNAs (miR-16, miR-21, miR-126) in children. In line with previous data, our data show that acute exercise can alter circulating miRNAs profiles that might be used as novel biomarkers to monitor acute and chronic changes due to exercise in various tissues.

Acute Effects of Different Exercise Protocols on the Circulating Vascular microRNAs -16, -21, and -126 in Trained Subjects.

Aim: mircoRNAs (miRNAs), small non-coding RNAs regulating gene expression, are stably secreted into the blood and circulating miRNAs (c-miRNAs) may play an important role in cell-cell communication. Furthermore, c-miRNAs might serve as novel biomarkers of the current vascular cell status. Here, we examined how the levels of three vascular c-miRNAs (c-miR-16, c-miR-21, c-miR-126) are acutely affected by different exercise intensities and volumes. Methods: 12 subjects performed 3 different endurance exercise protocols: 1. High-Volume Training (HVT; 130 min at 55% peak power output (PPO); 2. High-Intensity Training (HIT; 4 × 4 min at 95% PPO); 3. Sprint-Interval Training (SIT; 4 × 30 s all-out). c-miRNAs were quantified using quantitative real-time PCR with TaqMan probes at time points pre, 0', 30', 60', and 180' after each intervention. The expression of miR-126 and miR-21 was analyzed in vitro, in human coronary artery endothelial cells, human THP-1 monocytes, human platelets, human endothelial microparticles (EMPs) and human vascular smooth muscle cells (VSMCs). To investigate the transfer of miRNAs via EMPs, VSMCs were incubated with EMPs. Results: HVT and SIT revealed large increases on c-miR-21 [1.9-fold by HVT (cohen's d = 0.85); 1.5-fold by SIT (cohen's d = 0.85)] and c-miR-126 [2.2-fold by SIT (cohen's d = 1.06); 1.9-fold by HVT (cohen's d = 0.85)] post-exercise compared to pre-values, while HIT revealed only small to moderate changes on c-miRs-21 (cohen's d = -0.28) and c-miR-126 (cohen's d = 0.53). c-miR-16 was only slightly affected by SIT (1.4-fold; cohen's d = 0.57), HVT (1.3-fold; cohen's d = 0.61) or HIT (1.1-fold; cohen's d = 0.2). Further in vitro experiments revealed that miR-126 and miR-21 are mainly of endothelial origin. Importantly, under conditions of endothelial apoptosis, miR-126 and miR-21 are packed from endothelial cells into endothelial microparticles, which were shown to transfer miR-126 into target vascular smooth muscle cells. Conclusion: Taken together, we found that HVT and SIT are associated with the release of endothelial miRNAs into the circulation, which can function as intercellular communication devices regulating vascular biology.

Comparative proteome analysis of Actinoplanes sp. SE50/110 grown with maltose or glucose shows minor differences for acarbose biosynthesis proteins but major differences for saccharide transporters.

Actinoplanes sp. SE50/110 is known for the production of the α-glucosidase inhibitor and anti-diabetic drug acarbose. Acarbose (acarviosyl-maltose) is produced as the major product when the bacterium is grown in medium with maltose, while acarviosyl-glucose is the major product when glucose is the sole carbon source in the medium. In this study, a state-of-the-art proteomics approach was applied combining subcellular fractionation, in vivo metabolic labeling and shotgun mass spectrometry to analyze differences in the proteome of Actinoplanes sp. SE50/110 cultures grown in minimal medium containing either maltose or glucose as the sole carbon source. To study proteins in distinct subcellular locations, a cytosolic, an enriched membrane, a membrane shaving and an extracellular fraction were included in the analysis. Altogether, quantitative proteome data was obtained for 2497 proteins representing about 30% of the ca. 8270 predicted proteins of Actinoplanes sp. SE50/110. When comparing protein quantities of maltose- to glucose-grown cultures, differences were observed for saccharide transport and metabolism proteins, whereas differences for acarbose biosynthesis gene cluster proteins were almost absent. The maltose-inducible α-glucosidase/maltase MalL as well as the ABC-type saccharide transporters AglEFG, MalEFG and MstEAF had significantly higher quantities in the maltose growth condition. The only highly abundant saccharide transporter in the glucose condition was the monosaccharide transporter MstEAF, which may indicate that MstEAF is the major glucose importer. Taken all findings together, the previously observed formation of acarviosyl-maltose and acarviosyl-glucose is more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. BIOLOGICAL SIGNIFICANCE: Diabetes is a global pandemic accounting for about 11% of the worldwide healthcare expenditures (>600 billion US dollars) and is projected to affect 592 million people by 2035 (www.idf.org). Whether Actinoplanes sp. SE50/110 produces type 2 diabetes drug acarbose (acarviosyl-maltose) or another acarviose metabolite such as acarviosyl-glucose as the major product depends on the offered carbon source. The differences observed in this proteome in this study suggest that the differences in the formation of acarviosyl-maltose and acarviosyl-glucose are more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. In addition, the present study provides a comprehensive overview of the proteome of Actinoplanes sp. SE50/110.

Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. BIOLOGICAL SIGNIFICANCE: Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species.

Carbon source dependent biosynthesis of acarviose metabolites in Actinoplanes sp. SE50/110.

In this work the biosynthesis of the type 2 diabetes mellitus therapeutic acarviosyl-maltose (acarbose) and related acarviose metabolites produced by Actinoplanes sp. SE50/110 was studied in liquid minimal medium supplemented with the defined carbon sources maltose, glucose, galactose or mixtures of maltose/glucose and maltose/galactose. Quantifying acarviosyl-maltose by HPLC and UV detection revealed that only cultures grown in maltose-containing minimal media produced acarviosyl-maltose in significant amounts. A qualitative analysis of the cytosolic and extracellular proteome for the presence of proteins from the acarbose biosynthesis gene cluster showed that these were not only synthesized in maltose-containing media, but also in media with glucose or galactose as the sole carbon source. A LC-MS-based detection method was applied to test the hypothesis that different acarviose metabolites are produced in media with maltose, glucose or galactose. The analysis revealed that a spectrum of acarviose metabolites (acarviose with 1-4 glucose equivalent units) was formed under all tested conditions. As expected, in maltose-containing minimal media acarviosyl-maltose was produced as the major component exceeding the remaining minor components by 2-3 orders of magnitude. In minimal medium supplemented with glucose acarviosyl-glucose was the major component, while in minimal medium with galactose no major component was present. Based on the results presented, a model for the intracellular biosynthesis of major and minor acarviose metabolites was developed.

Capillary earlobe blood may be used for RNA isolation, gene expression assays and microRNA quantification.

An increasing number of studies examining gene expression associated with diseases in children is likely, in the near future, to provide simple and easy to use methods for the isolation of RNA for gene expression profiling. Prerequisites for such studies are likely to encompass the use of small amounts of blood, as well as less invasive blood collection methods. In the current study, RNA was isolated from 20 µl capillary blood samples from the earlobes of 10 adults for quantitative PCR experiments. The results were compared with RNA isolated from venipuncture samples of the 10 samples. The expression of 4 mRNAs and 1 microRNA (miRNA), miRNA­126, was measured. The quantitative PCR results obtained with the capillary blood probes were similar to results using venous blood samples. The few differences observed may result from a variation in the blood cell composition. The use of capillary blood samples from the earlobe for gene expression analysis is likely to allow this method to be used in newborns, babies and children. In addition, such a method, using microliters of blood samples, may also be useful for other medical studies e.g., in cases where repetitive blood sampling is necessary or in patients with bleeding disorders.

The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism.

The pseudotetrasaccharide acarbose is a medically relevant secondary metabolite produced by strains of the genera Actinoplanes and Streptomyces. In this study gene products involved in acarbose metabolism were identified by analyzing the cytosolic and extracellular proteome of Actinoplanes sp. SE50/110 cultures grown in a high-maltose minimal medium. The analysis by 2D protein gel electrophoresis of cytosolic proteins of Actinoplanes sp. SE50/110 resulted in 318 protein spots and 162 identified proteins. Nine of those were acarbose cluster proteins (Acb-proteins), namely AcbB, AcbD, AcbE, AcbK, AcbL, AcbN, AcbR, AcbV and AcbZ. The analysis of proteins in the extracellular space of Actinoplanes sp. SE50/110 cultures resulted in about 100 protein spots and 22 identified proteins. The identifications included the three acarbose gene cluster proteins AcbD, AcbE and AcbZ. After their identification, proteins were classified into functional groups. The dominant functional groups were the carbohydrate binding, carbohydrate cleavage and carbohydrate transport proteins. The other functional groups included protein cleavage, amino acid degradation, nucleic acid cleavage and a number of functionally uncharacterized proteins. In addition, signal peptide structures of extracellularly found proteins were analyzed. Of the 22 detected proteins 19 contained signal peptides, while 2 had N-terminal transmembrane helices explaining their localization. The only protein having neither of them was enolase. Under the conditions applied, the secretome of Actinoplanes sp. SE50/110 was dominated by seven proteins involved in carbohydrate metabolism (PulA, AcbE, AcbD, MalE, AglE, CbpA and Cgt). Of special interest were the identified extracellular pullulanase PulA and the two solute-binding proteins MalE and AglE. The identifications suggest that Actinoplanes sp. SE50/110 has two maltose/maltodextrin import systems. We postulate the identified MalEFG transport system of Actinoplanes sp. SE50/100 as the missing acarbose-metabolite importer and present a model of acarbose metabolism that is extended by the newly identified gene products.

Comparative RNA-sequencing of the acarbose producer Actinoplanes sp. SE50/110 cultivated in different growth media.

Actinoplanes sp. SE50/110 is known as the producer of the alpha-glucosidase inhibitor acarbose, a potent drug in the treatment of type-2 diabetes mellitus. We conducted the first whole transcriptome analysis of Actinoplanes sp. SE50/110, using RNA-sequencing technology for comparative gene expression studies between cells grown in maltose minimal medium, maltose minimal medium with trace elements, and glucose complex medium. We first studied the behavior of Actinoplanes sp. SE50/110 cultivations in these three media and found that the different media had significant impact on growth rate and in particular on acarbose production. It was demonstrated that Actinoplanes sp. SE50/110 grew well in all three media, but acarbose biosynthesis was only observed in cultures grown in maltose minimal medium with and without trace elements. When comparing the expression profiles between the maltose minimal media with and without trace elements, only few significantly differentially expressed genes were found, which mainly code for uptake systems of metal ions provided in the trace element solution. In contrast, the comparison of expression profiles from maltose minimal medium and glucose complex medium revealed a large number of differentially expressed genes, of which the most conspicuous genes account for iron storage and uptake. Furthermore, the acarbose gene cluster was found to be highly expressed in maltose-containing media and almost silent in the glucose-containing medium. In addition, a putative antibiotic biosynthesis gene cluster was found to be similarly expressed as the acarbose cluster.

Endurance training modifies exercise-induced activation of blood coagulation: RCT.

Randomized controlled trials (RCTs) on the topic of physical training and blood coagulation are rare and the effects are unclear. The aim of this study was to investigate whether endurance training adjusts blood coagulation and fibrinolysis at rest and after exercise. The study included 50 healthy untrained non-smokers randomized into training (TR 49 ± 6 years) or control group (CO 48 ± 6 years). Each subject performed an exercise test adjusted at 80 % individual anaerobic threshold (IAT) for 60 min before and after 12 weeks (80 % IAT: before TR 123 ± 20, CO 125 ± 26 W; after TR 148 ± 23 W, CO 127 ± 25 W; mean ± SD). Blood was taken at rest and after exercise to determine coagulation (e.g., aPTT, thrombin potential, TAT, F1+2, several coagulation factors) and fibrinolytic (e.g., tPA, PAI) parameters. The training intervention induced an elevation of physical capacity in TR by 17 % (rel. VO2max) that led to a statistical relevant prolongation of aPTT at rest. Although absolute power output during the second exercise test was 20 % higher in TR, we detected an attenuated exercise-induced decrease of aPTT and attenuated increase of F1+2 after training. Resting levels of tPA- and PAI-Ag decreased slightly but not significantly after training. Exercise-induced changes were comparable after training in spite of higher power output in TR. Although the effects are small in healthy men, training modifies exercise-induced blood coagulation positively. The fact that exercise-induced changes in blood coagulation and fibrinolysis are rather attenuated or unchanged in the training group, in spite of a 20 % higher absolute power output during exercise, substantiates the adjusting effect of endurance training and the importance of physical fitness in primary prevention.

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110.

BACKGROUND: Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. RESULTS: Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. CONCLUSIONS: The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.

Crystal structures of the bacterial solute receptor AcbH displaying an exclusive substrate preference for ß-D-galactopyranose.

Solute receptors (binding proteins) are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high-affinity monosaccharide-binding protein with a dissociation constant (K(d)) for ß-d-galactopyranose of 9.8±1.0 nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including d-galacturonic acid, l-arabinose, d-xylose and l-rhamnose competed with [(1)(4)C]galactose for binding to AcbH. Moreover, AcbH does not bind d-glucose, which is a common property of all but one d-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose-binding proteins rather than to glucose-binding proteins. Two helices are inserted in the substrate-binding pocket, which reduces the cavity size and allows the exclusive binding of monosaccharides, specifically ß-d-galactopyranose, in the (4)C(1) conformation. Site-directed mutagenesis of three residues from the binding pocket (Arg82, Asp361 and Arg362) that interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analysis indicated that these residues are crucial for galactose binding. To our knowledge, this is the first report of the tertiary structure of a solute receptor with exclusive affinity for ß-d-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed.

Crystal structures of the solute receptor GacH of Streptomyces glaucescens in complex with acarbose and an acarbose homolog: comparison with the acarbose-loaded maltose-binding protein of Salmonella typhimurium.

GacH is the solute binding protein (receptor) of the putative oligosaccharide ATP-binding cassette transporter GacFG, encoded in the acarbose biosynthetic gene cluster (gac) from Streptomyces glaucescens GLA.O. In the context of the proposed function of acarbose (acarviosyl-1,4-maltose) as a 'carbophor,' the transporter, in complex with a yet to be identified ATPase subunit, is supposed to mediate the uptake of longer acarbose homologs and acarbose for recycling purposes. Binding assays using isothermal titration calorimetry identified GacH as a maltose/maltodextrin-binding protein with a low affinity for acarbose but with considerable binding activity for its homolog, component 5C (acarviosyl-1,4-maltose-1,4-glucose-1,1-glucose). In contrast, the maltose-binding protein of Salmonella typhimurium (MalE) displays high-affinity acarbose binding. We determined the crystal structures of GacH in complex with acarbose, component 5C, and maltotetraose, as well as in unliganded form. As found for other solute receptors, the polypeptide chain of GacH is folded into two distinct domains (lobes) connected by a hinge, with the interface between the lobes forming the substrate-binding pocket. GacH does not specifically bind the acarviosyl group, but displays specificity for binding of the maltose moiety in the inner part of its binding pocket. The crystal structure of acarbose-loaded MalE showed that two glucose units of acarbose are bound at the same region and position as maltose. A comparative analysis revealed that in GacH, acarbose is buried deeper into the binding pocket than in MalE by exactly one glucose ring shift, resulting in a total of 18 hydrogen-bond interactions versus 21 hydrogen-bond interactions for MalE(acarbose). Since the substrate specificity of ATP-binding cassette import systems is determined by the cognate binding protein, our results provide the first biochemical and structural evidence for the proposed role of GacHFG in acarbose metabolism.

Enzymology of aminoglycoside biosynthesis-deduction from gene clusters.

The classical aminoglycosides are, with very few exceptions, typically actinobacterial secondary metabolites with antimicrobial activities all mediated by inhibiting translation on the 30S subunit of the bacterial ribosome. Some chemically related natural products inhibit glucosidases by mimicking oligo-alpha-1,4-glucosides. The biochemistry of the aminoglycoside biosynthetic pathways is still a developing field since none of the pathways has been analyzed to completeness as yet. In this chapter we treat the enzymology of aminoglycoside biosyntheses as far as it becomes apparent from recent investigations based on the availability of DNA sequence data of biosynthetic gene clusters for all major structural classes of these bacterial metabolites. We give a more general overview of the field, including descriptions of some key enzymes in various aminoglycoside pathways, whereas in Chapter 20 provides a detailed account of the better-studied enzymology thus far known for the neomycin and butirosin pathways.

The gac-gene cluster for the production of acarbose from Streptomyces glaucescens GLA.O: identification, isolation and characterization.

The C7N-cyclitol containing alpha-glucosidase inhibitor acarbose is commercially produced using developed strains of Actinoplanes and is used in the treatment of patients suffering from diabetes type II. We have identified a second acarbose production cluster using a genomic cosmid gene bank from Streptomyces glaucescens GLA.O and sequenced a region (42658bp; accession AM409314) which clearly contained a gene cluster (gac-cluster) for the synthesis of acarbose or acarbose related endproducts. The gac-cluster exhibited large similarities to the acb-gene cluster from Actinoplanes. However, remarkable differences are found in the biosynthesis of the C7N-cyclitol in the two acarbose biosynthesis pathways. We show the expression of selected genes using RT-PCR approaches, we were able to detect small amounts of acarbose or acarbose related metabolites and we have characterized the GacK protein, an acarbose kinase, which specifically phosphorylates acarbose and acarbose homologs. All these data in combination with the postulated functions of the encoded Gac proteins clearly indicate that also in S. glaucescens a recycling mechanism for acarbose ("carbophor") which had been described for the first time for acarbose cluster from Actinoplanes, is also realised.