Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

PSA-based prostate cancer screening: the role of active surveillance and informed and shared decision making

Since the first publication describing the identification of prostate-specific antigen (PSA) in the 1960s, much progress has been made. The PSA test changed from being initially a monitoring tool to being also used as a diagnostic tool. Over time, the test has been heavily debated due to its lack of sensitivity and specificity. However, up to now the PSA test is still the only biomarker for the detection and monitoring of prostate cancer. PSA-based screening for prostate cancer is associated with a high proportion of unnecessary testing and overdiagnosis with subsequent overtreatment. In the early years of screening for prostate cancer, high rates of uptake were very important. However, over time the opinion on PSA-based screening has shifted towards the notion of informed choice. Nowadays, it is thought to be unethical to screen men without them being aware of the pros and cons of PSA testing, as well as the fact that an informed choice is related to better patient outcomes. Now, as the results of three major screening studies have been presented and the downsides of screening are becoming better understood, informed choice is becoming more relevant.     

Why choose myofibrils to study muscle myosin ATPase?

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction. In the absence of calcium, myofibrillar ATPase activity is basal, no shortening occurs and no active force is developed. In the presence of calcium, myofibrillar ATPase is activated and myofibrils either shorten with no external load (native myofibrils) or contract isometrically (cross-linked myofibrils). With this organised system, both chemical and mechanical studies can be carried out. For a decade, our laboratory has been using rabbit psoas myofibrils for exploring myosin ATPase activity. The first challenge was to successfully apply rapid kinetic approaches, such as rapid-flow-quench, to this organised system. Another challenge was to work with myofibrils in cryoenzymic conditions, i.e. in the presence of organic solvents and at sub-zero temperatures. In this overview, we highlight differences between the myosin ATPase in organised systems (myofibrils or fibres) and that of contractile proteins in solution (S1 or actoS1) that we observed using these approaches. We discuss the importance of these differences in terms of mechanochemical coupling. It is concluded that great care should be taken when extrapolating mechanochemical properties of the contractile proteins in solution to the whole muscle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rule-based versus probabilistic selection for active surveillance using three definitions of insignificant prostate cancer

Purpose

To study whether probabilistic selection by the use of a nomogram could improve patient selection for active surveillance (AS) compared to the various sets of rule-based AS inclusion criteria currently used.

Methods

We studied Dutch and Swedish patients participating in the European Randomized study of Screening for Prostate Cancer (ERSPC). We explored which men who were initially diagnosed with cT1-2, Gleason 6 (Gleason pattern ≤3 + 3) had histopathological indolent PCa at RP [defined as pT2, Gleason pattern ≤3 and tumour volume (TV) ≤0.5 or TV ≤ 1.3 ml, and TV no part of criteria (NoTV)]. Rule-based selection was according to the Prostate cancer Research International: Active Surveillance (PRIAS), Klotz, and Johns Hopkins criteria. An existing nomogram to define probability-based selection for AS was refitted for the TV1.3 and NoTV indolent PCa definitions.

Results

619 of 864 men undergoing RP had cT1-2, Gleason 6 disease at diagnosis and were analysed. Median follow-up was 8.9 years. 229 (37 %), 356 (58 %), and 410 (66 %) fulfilled the TV0.5, TV1.3, and NoTV indolent PCa criteria at RP. Discriminating between indolent and significant disease according to area under the curve (AUC) was: TV0.5: 0.658 (PRIAS), 0.523 (Klotz), 0.642 (Hopkins), 0.685 (nomogram). TV1.3: 0.630 (PRIAS), 0.550 (Klotz), 0.615 (Hopkins), 0.646 (nomogram). NoTV: 0.603 (PRIAS), 0.530 (Klotz), 0.589 (Hopkins), 0.608 (nomogram).

Conclusions

The performance of a nomogram, the Johns Hopkins, and PRIAS rule-based criteria are comparable. Because the nomogram allows individual trade-offs, it could be a good alternative to rigid rule-based criteria.

     

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M· ADP·Pi states predominate on the cross-bridge cycle

The ATPases (±Ca²⁺) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different E _a: −Ca²⁺, 78 and 60 kJ/mol and +Ca²⁺, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132–3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the E _a data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4°C. We used two methods: rapid flow quench (`cold ATP chase': titration of active sites, ATP binding kinetics, k <sub>cat</sub>; `P_i burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free P_i; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M·ADP·P_i. We propose a reaction pathway that includes several (A)M·ADP·P_i sub-states that are either `weak' or `strong', depending on the mechanical condition. This revised version was published online in July 2006 with corrections to the Cover Date.