An AgNP-deposited commercial electrochemistry test strip as a platform for urea detection.

We developed an inexpensive, portable platform for urea detection via electrochemistry by depositing silver nanoparticles (AgNPs) on a commercial glucose test strip. We modified this strip by first removing the enzymes from the surface, followed by electrodeposition of AgNPs on one channel (working electrode). The morphology of the modified test strip was characterized by Scanning Electron Microscopy (SEM), and its electrochemical performance was evaluated via Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). We evaluated the performance of the device for urea detection via measurements of the dependency of peak currents vs the analyte concentration and from the relationship between the peak current and the square root of the scan rates. The observed linear range is 1-8 mM (corresponding to the physiological range of urea concentration in human blood), and the limit of detection (LOD) is 0.14 mM. The selectivity, reproducibility, reusability, and storage stability of the modified test strips are also reported. Additional tests were performed to validate the ability to measure urea in the presence of confounding factors such as spiked plasma and milk. The results demonstrate the potential of this simple and portable EC platform to be used in applications such as medical diagnosis and food safety.

A microfluidic chamber to study the dynamics of muscle-contraction-specific molecular interactions.

In vitro motility and laser trap assays are commonly used for molecular mechanics measurements. However, chemicals cannot be added during these measurements, because they create flows that alter the molecular mechanics. Thus, we designed a microfluidic device that allows the addition of chemicals without creating bulk flows. Biocompatibility of the components of this device was tested. A microchannel chamber was created by photolithography with the patterns transferred to polydimethylsiloxane (PDMS). The PDMS chamber was bound to a polycarbonate membrane, which itself was bound to a molecular mechanics chamber. The microchannels ensured rapid distribution of the chemicals over the membrane, whereas the membrane ensured efficient delivery to the mechanics chamber while preventing bulk flow. The biocompatibility of the materials was tested by comparing the velocity (ν(max)) of propulsion by myosin of fluorescently labeled actin filaments to that of the conventional assay; no difference in ν(max) was observed. To estimate total chemical delivery time, labeled bovine serum albumin was injected in the channel chamber and TIRF was used to determine the time to reach the assay surface (2.7 ± 0.1 s). Furthermore, the standard distance of a trapped microsphere calculated during buffer diffusion using the microfluidic device (14.9 ± 3.2 nm) was not different from that calculated using the conventional assay (15.6 ± 5.3 nm, p = 0.922). Finally, ν(max) obtained by injecting adenosine triphosphate (ATP) in the microchannel chamber (2.37 ± 0.48 µm/s) was not different from that obtained when ATP was delivered directly to the mechanics chamber (2.52 ± 0.42 µm/s, p = 0.822). This microfluidic prototype validates the design for molecular mechanics measurements.

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin.

BACKGROUND: Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. METHODS: To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. RESULTS: Funb from unregulated actin (0.10±0.01pN) was significantly increased in the presence of CaD (0.17±0.02pN), tropomyosin (0.17±0.02pN) or both regulatory proteins (0.18±0.02pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06±0.01pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. CONCLUSIONS: CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. GENERAL SIGNIFICANCE: This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin.

BACKGROUND: Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. METHODS: To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. RESULTS: Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). CONCLUSIONS: This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. GENERAL SIGNIFICANCE: This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

Caffeine relaxes smooth muscle through actin depolymerization.

Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE. Although caffeine is an agonist of bitter taste receptors, which in turn mediate bronchodilation, its relaxant effect was not mimicked by quinine. After permeabilizing the membrane using ß-escin and depleting the internal Ca(2+) store using A23187, we found that 10 mM caffeine reversed tone evoked by direct application of Ca(2+), suggesting it functionally antagonizes the contractile apparatus. Using a variety of molecular techniques, we found that caffeine did not affect phosphorylation of myosin light chain (MLC) by MLC kinase, actin-filament motility catalyzed by MLC kinase, phosphorylation of CPI-17 by either protein kinase C or RhoA kinase, nor the activity of MLC-phosphatase. However, we did obtain evidence that caffeine decreased actin filament binding to phosphorylated myosin heads and increased the ratio of globular to filamentous actin in precontracted tissues. We conclude that, in addition to its other non-RyR targets, caffeine also interferes with actin function (decreased binding by myosin, possibly with depolymerization), an effect that should be borne in mind in studies using caffeine to probe excitation-contraction coupling in smooth muscle.

Acute response of airway muscle to extreme temperature includes disruption of actin-myosin interaction.

Despite the emerging use of bronchial thermoplasty in asthma therapy, the response of airway smooth muscle (ASM) to extreme temperatures is unknown. We investigated the immediate effects of exposing ASM to supraphysiologic temperatures. Isometric contractions were studied in bovine ASM before and after exposure to various thermal loads and/or pharmacologic interventions. Actin-myosin interactions were investigated using a standard in vitro motility assay. We found steep thermal sensitivity for isometric contractions evoked by acetylcholine, with threshold and complete inhibition at less than 50°C and greater than 55°C, respectively. Contractile responses to serotonin or KCl were similarly affected, whereas isometric relaxations evoked by the nitric oxide donor S-nitrosyl-N-acetylpenicillamine or the ß-agonist isoproterenol were unaffected. This thermal sensitivity developed within 15 minutes, but did not evolve further over the course of several days (such a rapid time-course rules out heat shock proteins, apoptosis, autophagy, and necrosis). Although heat-sensitive transient receptor potential (TRPV2) channels and the calmodulin-dependent (Cam) kinase-II-induced inactivation of myosin light chain kinase are both acutely thermally sensitive, with a temperature producing half-maximal effect (T(1/2)) of 52.5°C, the phenomenon we describe was not prevented by blockers of TRPV2 channels (e.g., ruthenium red, gadolinium, zero-Ca(2+) or zero-Na(+)/zero-Ca(2+) media, and cromakalim) or of Cam kinase-II (e.g., W7, trifluoperazine, and KN-93). However, direct measurements of actin-myosin interactions showed the same steep thermal profile. The functional changes preceded any histologic evidence of necrosis or apoptosis. We conclude that extreme temperatures (such as those used in bronchial thermoplasty) directly disrupt actin-myosin interactions, likely through a denaturation of the motor protein, leading to an immediate loss of ASM cell function.