Effects of Qigong Exercise on Physical and Cognitive Performance in Young Sedentary Females: A Quasi-Experimental Design, Placebo-Controlled Study.

Background: Qigong exercise represents one type of traditional Chinese exercise that might positively affect physical and psychological functioning, slow down disease development and improve quality of life. However, study findings are somewhat conflicting and mechanisms contributing to expected beneficial effects are rather poorly known. Objective: This study aims to evaluate the effects of qigong exercise training for 8 weeks on selected physical, cognitive, and biochemical outcomes in young sedentary females. Method/Design: Quasi-experimental design, placebo-controlled study. Setting: The study was performed at the Department of Physical Therapy, Faculty of Allied Health Science, Burapha University, Bangsean, Chonburi province, Thailand. Participants: Participants were 41 females with sedentary lifestyles. Interventions: 41 sedentary females were allocated to qigong exercise (QG, n = 20) or to the control group (CG, n = 21). Primary Outcome Measures: VO2 max predicted from step testing, aspects of cognitive functions (e.g., digit span forward, DSF, and digit span backward, DSB, task), hematological and biochemical parameters, and body composition were assessed in both groups before and after the 8-week training period. Results: Physical performance (estimated VO2 max) significantly increased after qigong training compared to the CG (P < .001). Working memory (DSB) increased after intervention only within the QG (P = .009) but changes did not reach significance between the groups. Changes in neutrophils (potential mediators of inflammation) tended to be improved in the QG in comparison to the CG (P = .075). Body composition remained unchanged. Conclusion: These findings indicate that 8 weeks of qigong training increased aerobic capacity and tended to improve working memory in otherwise sedentary young females. Neutrophils tended to decrease within the QG. Thus, it was speculated that enhanced oxygen supply to the brain and the decrease of neutrophils adhering to cortical capillaries might have contributed to improved cognitive function.

Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture.

The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally attributed to their ability to form transmembrane pores, causing lysis of target insect cells. Previously, the truncated tertiary structure of the chymotrypsin-treated Cry4Ba toxin lacking the N-terminal helices-α1 and α2 was reported. To elucidate a more complete functional structure, a 65-kDa trypsin-activated form of the Cry4Ba-R203Q mutant toxin was thus generated for X-ray crystallography by eliminating the Arg203-tryptic cleavage site. The 2.0 Å crystal structure of Cry4Ba-R203Q with R-factor of 21.5% and Rfree of 23.7.%, as subsequently improved with homology-based modeling and molecular dynamics (MD) simulations, revealed a wedge-shaped arrangement of three domains: a well-defined N-terminal domain of eight α-helices (α1, α2a, α2b, α3, α4, α5, α6 and α7) responsible for pore formation, a three-ß-sheet prism displaying two functional motifs and a C-terminal ß-sandwich domain. A full-atom structural model of the Cry4Ba pre-pore trimer constructed using a single-particle 3D-reconstructed template revealed that each toxin monomer forms the stable trimer by packing α3 and α4 together at the central interface. When MD simulations of a membrane-associated trimeric pore model comprising three α4-loop-α5 hairpins were performed, an stable open-pore structure at the membrane-water interface was clearly observed. Two conserved side-chains-Asn166 and Tyr170 in the α4-α5 loop were found to interact directly with phospholipid head groups, leading to pore opening and stability. Overall data provide the first complete view of the 3D structure of the Cry4Ba mosquito-active toxin and its trimeric pore architecture, underlining the importance of two critical loop residues-Asn166 and Tyr170.

Effects of Qigong exercise on muscle strengths and oxidative stress/antioxidant responses in young sedentary females: a quasi-experimental study.

Regular exercise is associated with the production of small amounts of oxidative stress which might promote individual antioxidant capacity contributing to favorable training effects potentially interrelated with skeletal muscle strength. Therefore, the present study was aimed at evaluating effects of an 8-week Qigong exercise training on muscle strengths associated with responses of oxidative stress and antioxidants in young sedentary females. A total of 41 sedentary women were allocated to the Qigong exercise group (QG, N=20) or to the control group (CG, N=21). After 8 weeks of Qigong training, back and leg strength was significantly improved compared to baseline and the CG (P<0.05). Plasma oxidative stress levels were reduced and total antioxidant capacity was enhanced in the QG compared to the CG (P<0.05). Correlation analyses revealed that improvements in muscle strength (including both groups) were associated with changes in the levels of oxidative stress (reduction) and antioxidants (elevation). The presented findings indicate that strength training effects seem at least partly to be interrelated with alterations of the oxidant-antioxidant balance generated by the 8-week Qigong training in young sedentary females.

Preferential modification of CyaA-hemolysin by CyaC-acyltransferase through the catalytic Ser30-His33 dyad in esterolysis of palmitoyl-donor substrate devoid of acyl carrier proteins.

We previously demonstrated that the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was acylated at Lys983 and thus activated its hemolytic activity. Here, attempts were made to provide greater insights into such toxin activation via fatty-acyl modification by CyaC-acyltransferase. Non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaC were separately expressed in E. coli and subsequently purified by FPLC to near homogeneity. When effects of acyl-chain length were comparatively evaluated through CyaC-esterolysis using various p-nitrophenyl (pNP) derivatives, Michaelis-Menten steady-state kinetic parameters (KM and kcat) of CyaC-acyltransferase revealed a marked preference for myristoyl (C14:0) and palmitoyl (C16:0) substrates of which catalytic efficiencies (kcat/KM) were roughly the same (~1.5 × 103 s-1mM-1). However, pNP-palmitate (pNPP) gave the highest hemolytic activity of NA/CyaA-Hly after being acylated in vitro with a range of acyl-donor substrates. LC-MS/MS analysis confirmed such CyaC-mediated palmitoylation of CyaA-Hly occurring at Lys983, denoting no requirement of an acyl carrier protein (ACP). A homology-based CyaC structure inferred a role of a potential catalytic dyad of conserved Ser30 and His33 residues in substrate esterolysis. CyaC-ligand binding analysis via molecular docking corroborated high-affinity binding of palmitate with its carboxyl group oriented toward such a dyad. Ala-substitutions of each residue (S30A or H33A) caused a drastic decrease in kcat/KM of CyaC toward pNPP, and hence its catalytic malfunction through palmitoylation-dependent activation of NA/CyaA-Hly. Altogether, our present data evidently provide such preferential palmitoylation of CyaA-Hly by CyaC-acyltransferase through the enzyme Ser30-His33 nucleophile-activation dyad in esterolysis of palmitoyl-donor substrate, particularly devoid of a natural acyl-ACP donor.

Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation.

Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met482-Arg1706) from Bordetella pertussis was palmitoylated at Lys983 when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met482-Leu750) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala751-Arg1706) containing the Lys983-acylation region (AR, Ala751-Gln1000), but lacking HP, was co-produced with CyaC in E. coli. Hemolysis assay indicated that CyaA-RTX showed no hemolytic activity. Additionally, MALDI-TOF/MS and LC-MS/MS analyses confirmed that CyaA-RTX was non-acylated, although the co-expressed CyaC-acyltransferase was able to hydrolyze its chromogenic substrate-p-nitrophenyl palmitate and acylate CyaA-Hly to become hemolytically active. Unlike CyaA-RTX, the ∼70-kDa His-tagged CyaA-HP/BI fragment which is hemolytically inactive and contains both HP and AR was constantly co-eluted with CyaC during IMAC-purification as the presence of CyaC was verified by Western blotting. Such potential interactions between the two proteins were also revealed by semi-native PAGE. Moreover, structural analysis via electrostatic potential calculations and molecular docking suggested that CyaA-HP comprising α1-α5 (Leu500-Val698) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic surfaces. Overall, our results demonstrated that the HP region of CyaA-Hly is conceivably required for not only membrane-pore formation but also functional association with CyaC-acyltransferase, and hence effective palmitoylation at Lys983.

Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation.

Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly(530), Gly(533), Gly(537) and Gly(544)) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly(530)_Gly(533)_Gly(537) cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix-helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly(530)XXGly(533)XXXGly(537) could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly(530)_Gly(533)_Gly(537) cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.

Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.

Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.

Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation.

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity. Here, a 100-kDa RTX (Repeat-in-ToXin) subcloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated. The recombinant CyaA-RTX protein, although expressed as a soluble form in a protease-deficient E. coli strain BL21(DE3)pLysS, was found to be highly sensitive to proteolytic degradation. Interestingly, the addition of calcium ions in a millimolar range into the CyaA-RTX preparation significantly prevented the degradation. Moreover, levels of proteolytic degradation were dependent on calcium concentrations, implying an important role for calcium-binding sites in the RTX subdomain for structural stability. Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel ß-roll structure with calcium ions acting as a structural stabilizing bridge.

Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis.

To obtain a complete structure of the Bacillus thuringiensis Cry4Ba mosquito-larvicidal protein, a 65 kDa functional form of the Cry4Ba-R203Q mutant toxin was generated for crystallization by eliminating the tryptic cleavage site at Arg203. The 65 kDa trypsin-resistant fragment was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 184.62, c = 187.36 A. Diffraction data were collected to at least 2.07 A resolution using synchrotron radiation and gave a data set with an overall R(merge) of 9.1% and a completeness of 99.9%. Preliminary analysis indicated that the asymmetric unit contained one molecule of the active full-length mutant, with a V(M) coefficient and solvent content of 4.33 A(3) Da(-1) and 71%, respectively.

Esterase activity of Bordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanism in vivo.

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.