Respiratory endurance training by means of a Spirotiger in extending intermittent claudication distance — a case study

According to Fontaine’s classification, intermittent claudication is a symptom of the 2nd stage of peripheralarterial occlusive disease. Intermittent claudication is described as muscle pain that occurs during walking. Patientsexperiencing it observe major reduction of exercise tolerance. Rehabilitation of patients with peripheral arterialocclusive disease uses many different training programs which lead to extension of intermittent claudicationdistance. Respiratory training is one of them and it is the training of the respiratory muscles that improves theirendurance, force, speed, coordination, and elasticity. Respiration therapy causes delayed metaboreflex, whichin turn the blood flow in lower limbs increases..We present a case of a patient who underwent percutaneous balloon angioplasty of iliac artery and then conductedrespiratory training by means of Spirotiger what resulted in further increase of claudication distance

Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 å resolution

AbstractBackground: Aureolysin is an extracellular zinc-dependent metalloproteinase from the pathogenic bacterium Staphylococcus aureus. This enzyme exhibits in vitro activity against several molecules of biological significance for the host, indicating that it is involved in the pathology of staphylococcal diseases.Results: Here we report the amino-acid sequence and inhibitor-free X-ray crystal structure of aureolysin, a member of the thermolysin family of zinc-dependent metalloproteinases. This enzyme, which binds one zinc and three calcium ions, comprises a single chain of 301 amino acids that consists of a β-strand-rich upper domain and an α-helix-rich lower domain.Conclusions: The overall structure of aureolysin is very similar to that of the other three members of this family whose structures are known – thermolysin (TLN) from Bacillus thermoproteolyticus, neutral protease (NP) from Bacillus cereus and elastase (PAE) from Pseudomonas aeruginosa. But an important difference has been encountered: in contrast to what has been observed in the other three members of this family (TLN, NP and PAE), inhibitor-free aureolysin displays a ‘closed’ active site cleft conformation. This new structure therefore raises questions about the universality of the hinge-bending motion model for the neutral metalloproteinases

Applied methods of exercise based therapy for the extension of walking distance in patients with intermittent claudication

Intermittent claudication, according to the Fontaine Classification Scale is a symptom of 2nd degree atherosclerosis of the arteries of the lower limbs. The process of atherosclerosis involves increased narrowing of the blood vessel lumina and their eventual closure. Patients with atherosclerosis often suffer bouts of muscular pain while walking which eventually leads to restricted mobility. The treatment of those affected by furring up of the arteries of the lower limbs includes intravascular procedures, insertion of balloon devices and stents and in severe cases of atherosclerosis surgical intervention is required. The more conservative areas of treatment involve pharmacotherapy, patient participation in educational training sessions, lifestyle changes and appropriate physiotherapy referrals. If applied early on, lifestyle changes such as smoking cessation, an improved diet, as well as targeted training can help avoid the need for surgical intervention. At the moment, the goal of mainstream physiotherapy in the treatment of peripheral artery disease is to determine the most appropriate forms of exercise which can increase the walking distance of patients with intermittent claudication, improve blood-flow in particular to the lower extremities as well as improve patients’ overall quality of life. The purpose of this study is to gather and analyse exercise strategies that result in increased walking distance in intermittent claudication. The best results have been observed in groups who teamed up ambulatory therapy with strength training; Nordic walking therapy along with strength training and finally walking training with upper body aerobic training on the cross-trainer. The variety of training combinations gives us the ability to cater for and accommodate individual patient needs

Identification and Characterization of Prokaryotic Dipeptidyl-Peptidase 5 from Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is one of the major causative organisms of chronic periodontitis. The bacterium utilizes amino acids as energy and carbon sources, and incorporates them mainly as dipeptides. Therefore, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. A DPP7-like activity still remained in a dpp4-7-11 disrupted P. gingivalis ATCC 33277. PGN_0756, currently annotated as a prolyl oligopeptidase, possessed an activity indistinguishable from that of the triple dpp gene-disrupted strain, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with an apparent molecular weight of 66 kDa, while it preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated kcat/Km values for Gly-Phe-MCA and Lys-Ala-MCA of 13.01 and 11.02 μM-1 s-1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of dpp- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also distributed in prokaryotes

Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate

Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria

Establishment of potent and specific synthetic substrate for dipeptidyl-peptidase 7

Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40?±?0.83?μM?1?s?1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06?3.77?μM?1?s?1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides

Exopeptidases including dipeptidyl- and tripeptidyl-peptidase are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tri-peptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser615 and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9-family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, though sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, as AOP scarcely released an N-acyl-amino acid as compared to di- and tri-peptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys- Met-4-methycoumaryl-7-amide, the most potential substrate, was 123.3 ± 17.3 μM-1sec-1, optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, while equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met16-Glu101). The three dimensional modeling revealed the three domain structures: residues Met16-Ala126, which has no similar homologue with known structure, residues Leu127-Met495 (β-propeller domain) and residues Ala496-Phe736 (α/β hydrolase domain), and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides

Discrimination based on Gly and Arg/Ser at position 673 between dipeptidyl-peptidase (DPP) 7 and DPP11, widely distributed DPPs in pathogenic and environmental gram-negative bacteria

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium, expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al. (2011) J. Biol. Chem. 286, 38115–38127), which has been categorized as a member of the S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate specificities of the five clusters and an unassigned group were determined with recombinant DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens. Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity, and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser673 exhibited Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with Arg673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg673/Ser673 were indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was mediated by Gly673. Taken together with the distribution of the two genes, all 264 members of the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A more compelling phylogenic tree based on the conserved C-terminal region suggested two gene duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the genus Bacteroides