Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide-Alkyne Cycloaddition.

We investigate the effect of buffer identity, ionic strength, pH, and organic cosolvents on the rate of strain-promoted azide-alkyne cycloaddition with the widely used DIBAC cyclooctyne. The rate of reaction between DIBAC and a hydrophilic azide is highly tolerant to changes in buffer conditions but is impacted by organic cosolvents. Thus, bioconjugation reactions using DIBAC can be carried out in the buffer that is most compatible with the biomolecules being labeled, but the use of organic cosolvents should be carefully considered.

Acute hypertensive pulmonary edema: a new paradigm.

Although acute hypertensive pulmonary edema is sometimes regarded as the most severe form of heart failure, at the peak of symptoms, hearts perform well above resting levels and cannot be said to be failing. Another characteristic of the condition, the rapidity of its onset and reversal when properly treated, suggests positive feedback as a causal mechanism. It is proposed that the syndrome results from a feedback loop with increased sympathetic tone as the efferent output, increased pulmonary vascular pressure as the stimulus to increased sympathetic tone, and positive feedback occurring because elevated sympathetic tone constricts systemic veins, thereby transferring blood from peripheral veins to the pulmonary vasculature.

Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries.

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.

Mechanism and significance of early, rapid shortening in sensitized airway smooth muscle.

It has been reported that sensitization of animals to allergens increases both early shortening velocity and myosin light-chain kinase of their airway smooth muscle without increasing force generated by these muscles. Since early shortening sets muscle length for the duration of a contraction, these responses might be expected to produce greater airway obstruction. Here, it is explained how the more rapid early shortening without increased force production is predicted by the 2-stage process of activation followed by contraction posited by the crossbridge theory of contraction when the rate, but not the extent, of activation is increased.

The extensive length-force relationship of porcine airway smooth muscle.

The full functional length range of trachealis muscle was measured to identify a precise reference length and to assess the length changes that the myofilament lattice can accommodate. The initial reference length (L(10%)) was that where rest tension equaled 10% of total force (passive tension plus active force). Total force at this length served as a force reference (F(ref) = 219 +/- 12 kPa, N = 7).

Inhibition of myosin light-chain phosphorylation inverts the birefringence response of porcine airway smooth muscle.

Muscle birefringence, caused mainly by parallel thick filaments, increases in smooth muscle during stimulation, signalling thick filament formation upon activation. The reverse occurs in skeletal muscle, where a decrease in birefringence has been correlated with crossbridge movement away from the thick filaments. When force generation by trachealis muscle was inhibited with wortmannin, which inhibits myosin light-chain phosphorylation and thick-filament formation, but not the calcium increase caused by stimulation, the birefringence response inverted, suggesting crossbridge movement similar to that of skeletal muscle.

Plasticity in airway smooth muscle: an update.

At a similar meeting 10 years ago, we proposed (i) that the long functional range of some smooth muscles is accommodated by plastic alterations that place more myofilaments in series at longer lengths, (ii) that this plasticity is facilitated by myosin filament evanescence, with filaments dissociating partially during relaxation and reforming upon activation, and (iii) that filament lengthening during the rise of activation would cause velocity to fall. Since that meeting, we have accumulated a substantial body of evidence to support these proposals, as follows: (i) muscles develop nearly the same force when adapted to a range of lengths that can vary by 3-fold; (ii) other physiological parameters including shortening velocity, maximum power, compliance, ATPase rate, and thick-filament mass increase by about 2/3 for a doubling of muscle length; (iii) thick-filament density increases substantially during the rise of activation; and (iv) velocity falls as force rises during the rise of tetanic force, and when correction is made for differences in activation, velocity and force vary exactly in inverse proportion. This review explains the rationale for the different experimental measurements and their interpretation.

Length-dependent filament formation assessed from birefringence increases during activation of porcine tracheal muscle.

Birefringence and force produced by pig tracheal smooth muscles were recorded every 100 ms during electrically stimulated tetani at muscle lengths that varied 1.5-fold and at the peak of acetylcholine contractures at the same lengths. Isometric force was nearly the same at all lengths.

On the terminology for describing the length-force relationship and its changes in airway smooth muscle.

The observation that the length-force relationship in airway smooth muscle can be shifted along the length axis by accommodating the muscle at different lengths has stimulated great interest. In light of the recent understanding of the dynamic nature of length-force relationship, many of our concepts regarding smooth muscle mechanical properties, including the notion that the muscle possesses a unique optimal length that correlates to maximal force generation, are likely to be incorrect. To facilitate accurate and efficient communication among scientists interested in the function of airway smooth muscle, a revised and collectively accepted nomenclature describing the adaptive and dynamic nature of the length-force relationship will be invaluable.

Computed tomography demonstration of lipomatous metaplasia of the left ventricle following myocardial infarction.

Replacement of myocardium by fat, particularly of the right ventricle, is often diagnosed as arrhythmogenic right ventricular dysplasia. At autopsy, however, 68% of scars associated with chronic ischemic heart disease have shown fatty metaplasia in the scar. Four patients with a past history of previous myocardial infarctions and computed tomography demonstration of fatty change in left ventricular regions of hypokinesis and infarction are presented.

Force and myosin light chain phosphorylation in dog airway smooth muscle activated in different ways.

To assess activation mechanisms of dog trachealis muscle and test whether isometric force generation could be separated from myosin light-chain (MLC) phosphorylation, force and phosphorylation were measured in the presence of wortmannin (a light-chain kinase inhibitor) or Y-27632 (a rho-kinase inhibitor) during electrically stimulated tetani and sustained contractures induced by acetylcholine, KCl, or calyculin A, a light-chain phosphatase inhibitor which caused irreversible contractures and both di- and mono-phosphorylation of light chain. Phosphorylation was not much more than half under any circumstances. A nearly constant proportionality between steady force and phosphorylation existed over a 9-fold force range during contractures and 25-sec tetani, except that force correlated best with the di-phosphorylated light chain produced by calyculin A.

Structure-function correlation in airway smooth muscle adapted to different lengths.

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.

Differing effects of inotropic agents on length change deactivation of isolated rat myocardium.

Background: A rapid change in length of cardiac muscle during isometric contraction is followed by developed force that is less than appropriate for the new length because of deactivation of the contractile system. Length change deactivation may have favorable or unfavorable effects on cardiac function, depending on the circumstances under which it is produced.

Methods: Left ventricular papillary muscles from male Sprague-Dawley rats were arranged for recording of isometric force.