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To evaluate gemcitabine-cisplatin (GC) neoadjuvant cisplatin-based chemotherapy (NAC) for pathologic response (pR) and cancer-specific outcomes following radical cystectomy (RC) for muscle-invasive bladder cancer and identify clinical parameters associated with pR.We studied 150 consecutive cases of muscle-invasive bladder cancer that received GC NAC followed by open RC (2000-2013). A cohort of 121 patients treated by RC alone was used for comparison. Pathologic response and cancer-specific survival (CSS) were compared. We created the Johns Hopkins Hospital Dose Index to characterize chemotherapeutic dosing regimens and accurately assess sufficient neoadjuvant dosing regarding patient tolerance.No significant difference was noted in 5-year CSS between GC NAC (58%) and non-NAC cohorts (61%). The median follow-up was 19.6 months (GC NAC) and 106.5 months (non-NAC). Patients with residual non-muscle-invasive disease after GC NAC exhibit similar 5-year CSS relative to patients with no residual carcinoma (P = 0.99). NAC pR (≤ pT1) demonstrated improved 5-year CSS rates (90.6% vs. 27.1%, P0.01) and decreased nodal positivity rates (0% vs. 41.3%, P0.01) when compared with nonresponders (≥ pT2). Clinicopathologic outcomes were inferior in NAC pathologic nonresponders when compared with the entire RC-only-treated cohort. A lower pathologic nonresponder rate was seen in patients tolerating sufficient dosing of NAC as stratified by the Johns Hopkins Hospital Dose Index (P = 0.049), congruent with the National Comprehensive Cancer Network guidelines. A multivariate classification tree model demonstrated 60 years of age or younger and clinical stage cT2 as significant of NAC response (P0.05).Pathologic nonresponders fare worse than patients proceeding directly to RC alone do. Multiple predictive models incorporating clinical, histopathologic, and molecular features are currently being developed to identify patients who are most likely to benefit from GC NAC.

Meeting abstracts Vesigenurtacel-L is an allogeneic cell-based therapeutic cancer vaccine that was selected based on its expression of a range of tumor antigens (surviving, LAGE-1, MAGE-A3, MAGE-A11, PRAME and others) that are known to be expressed amongst a high proportion of primary bladder

The time required for a mechanical impulse to propagate from one end to the other was measured directly in frog sartorius muscles and in fiber bundles from the semitendinosus muscle. When the fibers were fully activated, the transmission velocity was 170 mm/ms. In resting fibers the transmission time was three to four times greater than in activated fibers. Control experiments indicated that the transmission time across the tendons was negligible. A muscle compliance of 55–80 Å per half sarcomere was estimated from these data. The "measurement time" of the method was calculated to be about 15 µs. This relatively short measurement time makes the method potentially useful for detecting changes in cross-bridge compliance.

Chaen et al. (1986. J. Biol. Chem. 261:13632-13636) showed that treatment of relaxed single muscle fibers with para-phenylenedimaleimide (pPDM) results in inhibition of a fiber's ability to generate active force and a diminished ATPase activity. They postulated that the inhibition of force production was due to pPDM's ability to prevent crossbridges from participating in the normal ATP hydrolysis cycle. We find that the crossbridges produced by pPDM treatment of relaxed muscle cannot bind strongly to the actin filaments in rigor, but do bind weakly to the actin filaments in the presence and also absence of ATP. After pPDM treatment, fiber stiffness, as measured using ramp stretches of varying duration, is ATP-insensitive and identical to that of untreated relaxed fibers (both at high [165 mM] and low [40 mM] ionic strength). These results suggest that the pPDM-treated crossbridges, in both the presence and absence of ATP, are locked in a state that resembles the weakly-binding myosin ATP state of normal crossbridges. Their resemblance to the ATP-crossbridges of relaxed untreated fibers is quite strong; both bind to actin about equally tightly and have similar attachment and detachment rate constants. We also found that crossbridges are locked in a weakly-binding state after treatment with N-phenylmaleimide (NPM). In muscle fibers, this method of producing weakly-binding crossbridges appears preferable to pPDM treatment because, unlike treatment with pPDM, it does not increase the fiber's resting tension and stiffness and it does not disrupt the titin band seen on SDS-PAGE.

The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-reacted crossbridge heads do not bind strongly to actin in rigor solution is not easily interpreted in terms of the solution studies of Xie and Schoenberg (Biochemistry 37 (1998) 8048) who found strong binding of NPM-reacted myosin subfragment-1 to actin in solutions devoid of MgATP. For this reason, the current work uses stiffness measurement to re-investigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bind strongly to actin in rigor solution providing one is extremely careful to reduce MgATP contamination to levels well below those that would have a detectable effect on unmodified fibers. The reason for this is that NPM-reacted crossbridge heads, which hydrolyze MgATP extremely slowly, are especially susceptible to contaminant MgATP. The new fiber results show a strong correlation with the solution results. A further manifestation of this correlation is that pPDM-reacted crossbridge heads are different from NPM-reacted ones in that, like in solution, they remain weakly binding to actin even at extremely low MgATP levels. The findings suggest that the covalent crosslinking of SH1 and SH2 by pPDM is likely playing a significant role in locking pPDM-reacted crossbridge heads in a weakly binding conformation.

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30–40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2–3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.

In vitro at low ionic strength (mu = 0.02 M) and 5 degrees C, myosin subfragment-1 shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca2+. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% of muscle length in 0.1 ms), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (mu = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 microns), the stiffness is less than 20% of the value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low ionic strength.

It is commonly believed, for both vertebrate striated and insect flight muscle, that when the ATP analogue adenyl-5'-yl imidodiphosphate (AMPPNP) is added to the muscle fiber in rigor, it causes the fiber to lengthen by 0.15%. This has been interpretated (Marston S.B., C.D. Roger, and R.T. Tregear. 1976. J. Mol. Biol. 104:263-267) as suggesting (a) that in rigor the crossbridge is fixed to, i.e., almost never detaches from the actin filament; (b), that the crossbridge remains fixed to the actin filament after AMPPNP addition; and (c) that the ability of AMPPNP to cause apparent lengthening of a muscle fiber is due to its ability to cause a conformational change in the myosin crossbridge that has an axial component of approximately 1.6 nm/half-sarcomere. The present study, done only on chemically-skinned rabbit psoas fibers, confirms that AMPPNP can cause muscle fibers to lengthen by 0.15% but only for a narrow set of experimental conditions. When experimental conditions are varied over a wider range, it becomes apparent that the extent of lengthening of a rigor muscle fiber upon AMPPNP addition depends almost entirely on the strain present in the rigor fiber before AMPPNP addition. Addition of AMPPNP to an unstrained rigor fiber (one supporting zero tension), induces zero length change while addition of AMPPNP to very highly strained rigor fibers induces length changes greater than 0.15%. The data thus do not support the hypotheses that the crossbridges remain fixed to the actin filament after AMPPNP addition and that the size of the apparent length change induced by AMPPNP is related to the size of the axial component of a conformational change. Instead, the data support the idea that the ability of AMPPNP to cause lengthening of a rigor muscle fiber is related to its ability to accelerate the rate at which strained crossbridges detach from actin and reattach in positions in lesser strain. The data do not rule out a conformational change upon AMPPNP binding, they simply make clear that any attempt to measure a force response conceivably due to a conformational change, would be more than obscured by the force changes due to crossbridges detaching and reattaching in positions of lesser strain.