Effects of multi-joint muscular fatigue on biomechanics of slips

The objective of the present study was to investigate the effects of multi-joint muscular fatigue on biomechanics of slips. Both lower-limb fatigue and upper-limb fatigue were examined, and the fatiguing exercises involved multi-joint movements to replicate muscular fatigue in realistic scenarios. Sixty healthy young adults participated in the study, and were evenly categorized into three groups: no fatigue, lower-limb fatigue, and upper-limb fatigue. These participants were instructed to walk on a linear walkway, and slips were induced unexpectedly during walking. The results showed that multi-joint muscular fatigue affects biomechanics of slips in all three phases of slips (i.e. initiation, detection, and recovery). In particular, adaptive safer postural control strategies were adopted with the application of both lower-limb fatigue and upper-limb fatigue to maintain the likelihood of slip initiation as in the no fatigue condition. In the phases of detection and recovery, lower-limb fatigue was found to compromise biomechanics of slips while upper-limb fatigue did not show any effects. Based on these findings, minimizing exposures to lower-limb fatigue should be given higher priority compared to upper-limb fatigue when developing interventions to prevent slip-induced falls. In addition, these findings also suggest that interventions aimed at enhancing proprioceptive acuity and increasing muscular strength in the lower limb could also be effective in slip-induced fall prevention.     

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.     

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.     

DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection

We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.     

Plasticity in the Rat Prefrontal Cortex: Linking Gene Expression and an Operant Learning with a Computational Theory

The plasticity in the medial Prefrontal Cortex (mPFC) of rodents or lateral prefrontal cortex in non human primates (lPFC), plays a key role neural circuits involved in learning and memory. Several genes, like brain-derived neurotrophic factor (BDNF), cAMP response element binding (CREB), Synapsin I, Calcium/calmodulin-dependent protein kinase II (CamKII), activity-regulated cytoskeleton-associated protein (Arc), c-jun and c-fos have been related to plasticity processes. We analysed differential expression of related plasticity genes and immediate early genes in the mPFC of rats during learning an operant conditioning task. Incompletely and completely trained animals were studied because of the distinct events predicted by our computational model at different learning stages. During learning an operant conditioning task, we measured changes in the mRNA levels by Real-Time RT-PCR during learning; expression of these markers associated to plasticity was incremented while learning and such increments began to decline when the task was learned. The plasticity changes in the lPFC during learning predicted by the model matched up with those of the representative gene BDNF. Herein, we showed for the first time that plasticity in the mPFC in rats during learning of an operant conditioning is higher while learning than when the task is learned, using an integrative approach of a computational model and gene expression.     

Chronic exposure to lead causes persistent alterations in the electric membrane properties of neurons in cell culture

The effects of chronic lead (Pb) exposure on neuronal electric membrane properties (EMP) were determined using neural cell cultures of adult mouse dorsal root ganglia (DRG). Cultures were exposed to Pb concentrations ranging from 0 to 100 microM for 12 days (8 DIV to 20 DIV). EMP were determined in Pb-free medium either immediately after withdrawal (IWD), or 6 days after withdrawal (6WD) from Pb. For IWD, regression analysis indicated that a number of EMP varied significantly with increasing Pb concentration. The largest such change occurred for electrical excitability which decreased significantly with increasing Pb (P = 0.000), being reduced by approximately two-thirds for neurons exposed to 100 microM Pb; resting membrane potential increased with Pb (P = 0.000); membrane time constant decreased with Pb (P = 0.007); action potential afterhyperpolarization decreased with Pb (P = 0.023). There was also evidence that the time course of action potentials was accelerated with increasing Pb concentrations, the rate of fall of neurons with biphasic falling phases being particularly increased (P = 0.047). This general pattern of altered EMP was observed for the 6WD condition also, indicating that chronic exposure to Pb caused persistent abnormalities in neuronal membranes even after 6 days of cultivation in Pb-free medium. The patterns of alterations in EMP suggested that chronic Pb exposure caused a prolonged increase in potassium permeability. It was proposed that the latter was mediated through a Pb-induced increase in intracellular ionic calcium and the associated disruption of calcium homeostasis.