Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of ≈20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 ± 24 s. We define the latter component to be the "super relaxed state." The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate. metabolic rate | thick filament | phosphorylation | fluorescent nucleotides T he metabolic activity of resting skeletal muscle is of interest because it plays a significant role in the whole-body restingenergy expenditure (1). Muscle metabolism is involved in coldinduced thermogenesis, in consumption of calories from excess food intake, and is a major regulator of blood-sugar levels (1-5). The mechanism of muscle thermogenesis and its regulation remain an active area of investigation. Here we identify another mechanism that plays a role in thermogenesis of resting skeletal muscle, the modulation of myosin ATPase activity by the structure of the thick filament.Our study also addresses a long-standing discrepancy concerning myosin kinetics. Ferenczi et al. (6) noted an inconsistency between the ATP turnover rate of purified frog myosin and the rate of oxygen consumption of living, resting frog muscle. The low metabolic activity of living, resting muscle (6, 7) sets an upper limit on the myosin ATPase activity, and this limit was less than one-fifth of the rate observed for myosin in vitro. A similar difference is seen in mammalian fibers (8, 9). The ATP turnover rate of rabbit myosin [0.16 s −1 at in vivo temperatures (6, 9) would also have to be inhibited by more than a factor of 5 to be compatible with the resting-energy consumption of rabbit muscle [0.7 J × L −1 s × −1 (8, 9)]. These observations show unambiguously that myosin in living, resting vertebrate muscle is inhibited by a large factor relative to purified myosin measured in vitro.Although more than 30 years have passed since the original observation, the mechanism of myosin inhibition, unambiguously required to...

Although good evidence suggests that many agents are effective in preventing osteoporotic fractures, the data are insufficient to determine the relative efficacy or safety of these agents.

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Purpose: A new humanized anti-CD20 monoclonal antibody (MAb), IMMU-106, was evaluated to elucidate its action as an antilymphoma therapeutic, as a single agent, and in combination with the anti-CD22 MAb, epratuzumab.Experimental Design: Antiproliferative effects, apoptotic effects, and the ability of IMMU-106 to mediate complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity on a panel of non-Hodgkin's lymphoma (NHL) cell lines were compared with the chimeric anti-CD20 MAb, rituximab, and evaluated in light of the various levels of antigen expression by the cell lines. In vivo therapy studies were performed in SCID mice bearing disseminated Raji lymphoma.Results: The mechanisms of cytotoxicity of IMMU-106 were found to be similar to rituximab, and include direct apoptosis, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity. IMMU-106 was also found to be very similar to rituximab in terms of antigenbinding specificity, binding avidity, and dissociation constant. Treatment of Raji-bearing SCID mice with IMMU-106 yielded median survival increases of up to 4.2-fold compared with control mice. Survival in mice treated with IMMU-106 plus epratuzumab was compared with IMMU-106 treatment alone. Although the combined treatment did not improve median survival, an increased proportion of long-term survivors was observed. An enhanced antiproliferative effect was also observed in vitro in SU-DHL-6 cells when IMMU-106 was combined with epratuzumab. These findings are consistent with the up-regulation of CD22 expression observed after pretreatment of NHL cells in vitro with CD20 MAb (IMMU-106).Conclusions: It is expected that in humans IMMU-106 should be at least as effective as rituximab and, due to its human framework construction, it may exhibit different pharmacokinetic, toxicity, and therapy profiles. In addition, it may be possible to enhance efficacy by combination therapy comprised of anti-CD20 and other B-cell lineage targeting MAbs, such as epratuzumab. The current results emphasize that in vitro as well as in vivo studies with many of the NHL cell lines were generally predictive of the known activity of anti-CD20 MAbs in NHL patients, as well as the enhanced efficacy of epratuzumab combined with rituximab observed in early clinical trials.

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