In vitro muscle contraction force measurements on isolated and entire rat diaphragms

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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Tubular Repair of the Median Nerve in the Human Forearm

Journal of Hand Surgery (European Volume) ◽

10.1016/0266-7681(94)90068-x ◽

1994 ◽

Vol 19 (3) ◽

pp. 273-276 ◽

Cited By ~ 91

Author(s):

G. LUNDBORG ◽

B. ROSÉN ◽

S. O. ABRAHAMSON ◽

L. DAHLIN ◽

N. DANIELSEN

Keyword(s):

Muscle Contraction ◽

Median Nerve ◽

Hand Function ◽

Nerve Trunk ◽

Contraction Force ◽

Silicone Tube ◽

Human Forearm ◽

Normal Hand ◽

Male Patients ◽

Chamber Technique

Transected median nerves in the forearm of two male patients, 12 and 21 years of age, were treated with a chamber technique leaving a 3 to 5 mm gap between the nerve ends. The nerve ends were enclosed in a silicone tube of such a dimension that would not cause compression of the nerve. Post-operative examination including sensory evaluation and assessment of muscle contraction force was carried out after 3 years. In both cases there was excellent motor recovery of the thenar muscles. Outgrowth of sensory fibres was remarkably fast, resulting ultimately in functional sensibility allowing almost normal hand function. 2PD was ⩽ 6 mm (12year-old patient) and 8 to 10 mm (21-year-old patient) respectively. In one case the silicone tube was re-explored because of minor local discomfort 2 years after the repair. The former gap was bridged by a smooth continuous nerve-like structure of the same diameter as the adjacent nerve trunk and with no signs of nenroma formation or compression of the nerve.

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Myogenic mechanism for peristalsis in the cat esophagus

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.277.2.g306 ◽

1999 ◽

Vol 277 (2) ◽

pp. G306-G313 ◽

Cited By ~ 6

Author(s):

Harold G. Preiksaitis ◽

Nicholas E. Diamant

Keyword(s):

Muscle Contraction ◽

Slow Wave ◽

Circular Muscle ◽

Smooth Muscle Contraction ◽

Slow Waves ◽

Mechanical Activity ◽

Control Mechanisms ◽

Slow Wave Oscillations

A myogenic control system (MCS) is a fundamental determinant of peristalsis in the stomach, small bowel, and colon. In the esophagus, attention has focused on neuronal control, the potential for a MCS receiving less attention. The myogenic properties of the cat esophagus were studied in vitro with and without nerves blocked by 1 μM TTX. Muscle contraction was recorded, while electrical activity was monitored by suction electrodes. Spontaneous, nonperistaltic, electrical, and mechanical activity was seen in the longitudinal muscle and persisted after TTX. Spontaneous circular muscle activity was minimal, and peristalsis was not observed without pharmacological activation. Direct electrical stimulation (ES) in the presence of bethanechol or tetraethylammonium chloride (TEA) produced slow-wave oscillations and spike potentials accompanying smooth muscle contraction that progressed along the esophagus. Increased concentrations of either drug in the presence of TTX produced slow waves and spike discharges, accompanied by peristalsis in 5 of 8 TEA- and 2 of 11 bethanechol-stimulated preparations without ES. Depolarization of the muscle by increasing K+ concentration also produced slow waves but no peristalsis. We conclude that the MCS in the esophagus requires specific activation and is manifest by slow-wave oscillations of the membrane potential, which appear to be necessary, but are not sufficient for myogenic peristalsis. In vivo, additional control mechanisms are likely supplied by nerves.

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Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00430.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. G327-G335 ◽

Cited By ~ 6

Author(s):

Karnam S. Murthy ◽

Wimolpak Sriwai

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Okadaic Acid ◽

Protein Phosphatase ◽

Dependent Inactivation ◽

Rhythmic Cycles ◽

Phosphatase 2A ◽

Contraction And Relaxation ◽

Camp Levels

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Gαq minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

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