Comparison of Peak Cardiopulmonary Performance Parameters on a Robotics-Assisted Tilt Table,a Cycle and a Treadmill

Robotics-assisted tilt table (RATT) technology provides body support, cyclicalstepping movement and physiological loading. This technology can potentially beused to facilitate the estimation of peak cardiopulmonary performance parametersin patients who have neurological or other problems that may preclude testing ona treadmill or cycle ergometer. The aim of the study was to compare themagnitude of peak cardiopulmonary performance parameters including peak oxygenuptake (VO_2peak) and peak heart rate (HR_peak) obtainedfrom a robotics-assisted tilt table (RATT), a cycle ergometer and a treadmill.The strength of correlations between the three devices, test-retest reliabilityand repeatability were also assessed. Eighteen healthy subjects performed sixmaximal exercise tests, with two tests on each of the three exercise modalities.Data from the second tests were used for the comparative and correlationanalyses. For nine subjects, test-retest reliability and repeatability ofVO_2peak and HR_peak were assessed. AbsoluteVO_2peak from the RATT, the cycle ergometer and the treadmill was(mean (SD)) 2.2 (0.56), 2.8 (0.80) and 3.2 (0.87) L/min, respectively (p< 0.001). HR_peak from the RATT, the cycle ergometer and thetreadmill was 168 (9.5), 179 (7.9) and 184 (6.9) beats/min, respectively (p< 0.001). VO_2peak and HR_peak from the RATT vs thecycle ergometer and the RATT vs the treadmill showed strong correlations.Test-retest reliability and repeatability were high for VO_2peak andHR_peak for all devices. The results demonstrate that the RATT isa valid and reliable device for exercise testing. There is potential for theRATT to be used in severely impaired subjects who cannot use the standardmodalities.     

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn²⁺ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M²⁺) cofactors, except Ca²⁺, for catalysis. We found that Zn²⁺ yielded the highest enzyme complex thermostability (T_m of 87 °C) and solvent tolerance. All AbHpaI•M²⁺ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn²⁺ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M²⁺ cofactors). For the aldol condensation reaction, AbHpaI•M²⁺ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M²⁺ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M²⁺ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn²⁺-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca²⁺ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn²⁺ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.