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Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.

Affinity of hemoglobin (Hb) for O2 determines in part the rate of O2 diffusion from capillaries to myocytes by altering capillary PO2. We hypothesized that a decrease in Hb O2 affinity (increased P50) would increase capillary and tissue PO2 (PtiO2) and improve O2 consumption during ischemia. To test this hypothesis, blood flow to the pump-perfused left hindlimb of 18 anesthetized and paralyzed dogs was progressively decreased over 90 min while hindlimb O2 consumption and O2 delivery (QO2) and PtiO2 were measured at the muscle surface. Arterial PO2 was maintained at 150 +/- 10 Torr in all dogs. We increased P50 by 12.3 +/- 0.9 (SE) Torr in nine dogs with RSR-13, an allosteric modifier of Hb. This decreased arterial O2 saturation to 90-92% but increased mean PtiO2 from 35.5 +/- 11.6 to 44.1 +/- 15.2 (SD) Torr (P < 0.05) with no change in controls (n = 9). O2 extraction ratio at critical QO2 was 74 +/- 2% in controls and 79 +/- 1% in RSR-13-treated dogs (P = not significant). PtiO2 was 30-40% higher in the RSR-13-treated group at any QO2 above critical but did not differ between groups below critical QO2. Perfusion heterogeneity and convergence of the dissociation curves near critical QO2 may have mitigated any effect of increased P50 on O2 diffusion. Still, increasing P50 by 12 Torr with RSR-13 significantly increased PtiO2 at QO2 values above critical.

Nitric oxide synthase (NOS) inhibition has been used to increase blood pressure in humans with septic shock despite a lack of data regarding its effects on O2 delivery (QO2). We studied the effects of NG-nitro-L-arginine methyl ester (L-NAME) on systemic, gut, and hindlimb circulations of endotoxic dogs. Twelve dogs were infused with 2 mg/kg of LPS over 1 h followed by 60 mL/kg of 6% dextran over 2 h. Six dogs also received 20 mg/kg of L-NAME, LPS caused mean arterial pressure (MAP), flow and QO2 to whole body, hindlimb and gut to decrease, but O2 uptake (VO2) did not change. Dextran resuscitation alone produced a hyperdynamic state with increased blood flow to or above baseline. With L-NAME, systemic and regional resistances increased twofold and MAP returned to near baseline. Late in the study, these dogs had significantly lower blood flow and QO2 to the gut but maintained VO2 by increasing oxygen extraction to near critical levels. These data suggest that in acute endotoxicosis, L-NAME may significantly improve blood pressure but may markedly encroach on O2 transport reserves to the gut.

The symposium, "Mechanisms Which Control VO2 Near VO2max," led to a general agreement that there is a variable impediment to the movement of O2 from the interior of the red cell to the interior of the mitochondrion. By changing the variables associated with O2 delivery or by altering the conditions for muscle contractions, the effective O2 diffusing capacity for muscle can be altered as well. Because it is often measured as the ratio of VO2/PvO2, it was suggested that this be referred to as O2 conductance rather than diffusing capacity. In contrast to the wide range of O2 conductance values found by the symposium contributors, a very narrow range of O2 extraction values was found when VO2 was graphed against O2 delivery. The only experimental values that departed from this relationship to any degree were those where hemoglobin function was altered or if blood flow was forced to extraordinary high levels by a pump. The limits for VO2 in contracting isolated muscle are set not only by O2 supply but by O2 demand associated with stimulus patterns. Other intriguing and perhaps useful questions are: 1) What is the relative contribution of such factors as diffusional shunting, flow heterogeneity, red cell transit time, etc., to the apparent O2 conductance? 2) How is blood flow to contracting muscle controlled? 3) How is contractile force adjusted to energy supply?

We investigated the effect of hypoxia on acetylcholine (ACh) stimulated, endothelium-derived relaxing factor/nitric oxide (EDRF/NO)-dependent relaxation, and on basal tension in rat aortic rings. ACh (10(-9)-10(-6) M)-mediated relaxation at high [95%, Emax -76.2 +/- 4.5% of phenylephrine (PE)-induced constriction] and normal (20%, Emax -81.2 +/- 3.6%) O2 levels was inhibited by hypoxia (5%, Emax -36.2 +/- 7.2%); residual hypoxic relaxation was blocked by the K+ channel antagonist glibenclamide. To address whether O2 influenced EDRF/NO and K+ channel contributions to basal tone, the effect of stepwise reduction of available O2 (95, 20, 5, and 0%) was studied in intact and endothelial cell (EC)-denuded rings. The effects in these rings were compared with results of the same progressive reduction in O2 in the presence of the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) or glibenclamide (10(-4) M). EC-intact and EC-denuded rings constricted to 0.80 +/- 0.10 and 1.41 +/- 0.15 g, respectively. Reducing O2 to 20% had no significant effect on vascular tension, but 5% caused constriction (p < 0.05) in EC-intact rings (0.90 +/- 0.15 g). This hypoxic vasoconstriction was blocked by L-NAME, but not by glibenclamide, suggesting that hypoxic vasoconstriction was mediated by withdrawal of EDRF/NO. In contrast, EC-denuded rings showed a significant relaxant response at 5% O2. When O2 was then reduced further (95% N2/5% CO2), both EC-intact and EC-denuded rings relaxed, and this relaxation reached baseline tension (0.10 +/- 0.1 g).(ABSTRACT TRUNCATED AT 250 WORDS)