Influence of activation frequency on cellular signalling pathways during fatiguing contractions in rat skeletal muscle

Activation frequency as a regulator of physiological responses in skeletal muscle, independent of contractile force, has received little attention. Here, the length-tension and force-frequency relationships were employed to keep active contractile force equal, despite a twofold difference in stimulation frequency (15 versus 30 Hz). Rat tibialis anterior muscles were tested in situ using 15 Hz stimulation at optimal length (15 Hz) and 30 Hz stimulation at shortened and lengthened positions (30 Hz(sub) and 30 Hz(supra)). Muscles were subjected to 1, 15, 30 and 80 Hz stimulation trains before and after 2 min of fatiguing stimulation. The principal findings were that the two 30 Hz protocols produced greater 38 kDa MAPK (p38) phosphorylation than the 15 Hz protocol (1.4- to 1.5-fold versus 1.1-fold), as well as greater fatigue (65-78 versus 43% decline in contraction force). In contrast, c-jun amino terminal kinase (JNK) phosphorylation appeared most responsive to total (active plus passive) tension such that the changes followed the pattern: 30 Hz(supra) > 15 Hz > 30 Hz(sub), while 44 and 42 kDa extracellular regulated kinase (ERK1/2) phosphorylation was not significantly increased in response to any of the protocols studied. Neither glycogen depletion nor myofibre damage accounted for any of the findings, but a decline in muscle excitation (m-wave) may have contributed to the greater fatigue seen at higher frequencies. These data suggest that neuromuscular activation frequency can influence certain signalling pathways in skeletal muscle, independent of force production.     

Acylguanidines as small-molecule beta-secretase inhibitors

BACE1 is an aspartyl protease responsible for cleaving amyloid precursor protein to liberate Abeta, which aggregates leading to plaque deposits implicated in Alzheimer's disease. We have identified small-molecule acylguanidine inhibitors of BACE1. Crystallographic studies show that these compounds form unique hydrogen-bonding interactions with the catalytic site aspartic acids and stabilize the protein in a flap-open conformation. Structure-based optimization led to the identification of potent analogs, such as 10d (BACE1 IC(50) = 110 nM).     

Therapeutic potential of TACE inhibitors in stroke

Stroke is the third leading cause of death and the leading cause of permanent disability in western countries and the incidence of stroke is expected to increase in the foreseeable future due to the ageing population. The effective treatment of stroke remains challenging due to the complexity and heterogenicity of the disease. Recombinant tissue plasminogen activator (rt-PA) is the only FDA-approved therapy for stroke during the first 3 hr after the disease onset. However the risk of hemorrhage and its narrow therapeutic window has limited its use in clinic. Inflammation has been known to play a crucial role in the induction and development of stroke and tumor necrosis factor-alpha (TNF-alpha) is a central player in the initiation of multiple inflammatory cascades. The recent success of three anti-TNF biologics in the clinic for the treatment of rheumatoid arthritis as well as other inflammatory diseases has further validated TNF159nflammation. TNF-alpha has also been shown to be associated with ischemic stroke. Anti-TNF biologics have been shown to be effective in reducing the disease symptoms in various pre-clinical stroke models. Small molecule TNF inhibitors are highly desirable due to the limitations of protein therapeutics. Tumor necrosis factor-alpha-converting enzyme (TACE) is the major sheddase of TNF-alpha and is essential for the generation of soluble, mature TNF-alpha. Thus TACE appears to be an attractive target for development of oral small molecule TNF-alpha inhibitors. This review summarizes the role of TNF-alpha in stroke and the effect of several TACE/MMP inhibitors in pre-clinical stroke models. The data strongly suggest that TACE/MMP inhibitors have great therapeutic potential and may be valuable alternatives in treating stroke in the clinic.     

Responses and limitations of the respiratory system to exercise

During maximal exercise, the gas exchange function of the lung is challenged because of the major cardiopulmonary changes that must occur to meet the increased metabolic demands imposed by exercise. In healthy untrained young adults, the respiratory system is able to meet these demands imposed on it during maximal exercise by implementing several key mechanisms. Nonetheless, there are several exceptional cases in which the lung is unable to accommodate the demands of exercise because of vascular or airway limitations.     

Effect of inspiratory muscle work on peripheral fatigue of locomotor muscles in healthy humans

The work of breathing required during maximal exercise compromises blood flow to limb locomotor muscles and reduces exercise performance. We asked if force output of the inspiratory muscles affected exercise-induced peripheral fatigue of locomotor muscles. Eight male cyclists exercised at ≥ 90% peak O₂ uptake to exhaustion (CTRL). On a separate occasion, subjects exercised for the same duration and power output as CTRL (13.2 ± 0.9 min, 292 W), but force output of the inspiratory muscles was reduced (−56% versus CTRL) using a proportional assist ventilator (PAV). Subjects also exercised to exhaustion (7.9 ± 0.6 min, 292 W) while force output of the inspiratory muscles was increased (+80% versus CTRL) via inspiratory resistive loads (IRLs), and again for the same duration and power output with breathing unimpeded (IRL-CTRL). Quadriceps twitch force ( Q _tw), in response to supramaximal paired magnetic stimuli of the femoral nerve (1–100 Hz), was assessed pre- and at 2.5 through to 70 min postexercise. Immediately after CTRL exercise, Q _tw was reduced −28 ± 5% below pre-exercise baseline and this reduction was attenuated following PAV exercise (−20 ± 5%; P < 0.05). Conversely, increasing the force output of the inspiratory muscles (IRL) exacerbated exercise-induced quadriceps muscle fatigue ( Q _tw=−12 ± 8% IRL-CTRL versus −20 ± 7% IRL; P < 0.05). Repeat studies between days showed that the effects of exercise per se , and of superimposed inspiratory muscle loading on quadriceps fatigue were highly reproducible. In conclusion, peripheral fatigue of locomotor muscles resulting from high-intensity sustained exercise is, in part, due to the accompanying high levels of respiratory muscle work.     

High-performance liquid chromatographic method for the assay of verapamil hydrochloride and related compounds in raw material

A modification of the USP HPLC method [ United States Pharmacopeia XXII, pp. 1444-1446] for the assay of the purity of verapamil hydrochloride has been evaluated for the determination of the drug content and related compounds in drug raw material. The method enables the resolution of 16 related compounds from the parent drug and, in most cases, from each other. The minimum quantifiable amount for most related compounds is less than 0.05%. Six drug raw material samples are analysed and the total impurities found to be 0.3% or less. All drug assay values were within the USP recommended limits of 99.0-100.5%.