Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.

Evaluation of post-repolarization refractoriness for conduction block in cardiac muscle: studies in an artificial isthmus in the canine right atrium.

Post-repolarization refractoriness (PRR) is an important factor in determining conduction block and is the difference between the effective refractory period (ERP) and the duration of the monophasic action potential (MAPD). In the present study, conduction block in an artificial isthmus in the canine atrium was evaluated and the coupling interval of a premature beat, which caused the block, was defined as the block coupling interval (BCI). The usefulness of this value was also evaluated. Radiofrequency linear ablation was performed on the right atrial surface parallel to the atrioventricular groove in 5 mongrel dogs, and an artificial isthmus (8-10mm wide and 25-30mm long) was created. Fourteen simultaneous unipolar recordings were performed in the isthmus with a resolution of 1.2 mm. Single extra-stimuli with basic drive train were delivered to induce conduction block in the isthmus and when it occurred, the coupling interval at the recording site just proximal to the site of the block was defined as the BCI. At the site of the block, the ERP and MAPD at each drive cycle length were measured. The PRR was calculated using 2 different formulae: (1) [ERP-MAPD], and (2) [BCI-MAPD]. It was found that each value was shortened in accordance with the shortening of the basic drive cycle length. In all basic drive trains, BCI>ERP>MAPD, and [ERP-MAPD] was always shorter than [BCI-MAPD]. In the shorter cycle length of basic drives, the difference between [ERP-MAPD] and [BCI-MAPD] was more prominent. In the artificial isthmus model in the canine atrium, BCI was always longer than the ERP measured at the same site as the block. Because the ERP may not directly reflect the block phenomenon, the electrophysiologic evaluation should use the BCI instead, as in the PRR evaluation.

Ovary-selective genes I: the generation and characterization of an ovary-selective complementary deoxyribonucleic acid library.

The importance of several ovary-selective/specific genes, i.e. genes preferentially or exclusively expressed in the ovary, has been established. Indeed, null mutant female mice for the c-mos, growth and differentiation factor-9, alpha-inhibin, and zona pellucida-3 genes proved sterile. A loss of function mutation of the human FSH receptor gene established its critical role in ovarian function. These data support the hypothesis that genes expressed selectively or specifically in the ovary are probably essential for the normal functioning of this organ system. We have used the differential screening technique suppression subtractive hybridization to systematically isolate and clone genes that are expressed in an ovary-selective/specific manner. The resultant target complementary DNA (cDNA) library has been exhaustively screened to a point at which additional sequencing was increasingly unlikely (< or = 4%) to yield additional previously unencountered cDNAs. In toto, 844 clones were sequenced and analyzed for homology to known genes using the Basic Local Alignment Tool (BLAST). Of those, 342 were determined to be independent (nonredundant). One hundred and fifty-nine independent clones proved identical to previously characterized genes, whereas an additional 100 independent clones proved significantly homologous (but not identical) to previously characterized genes. Yet 83 other independent clones did not display significant homology to previously characterized genes now listed in the publicly accessible nonredundant databases. As such, these latter genes were deemed novel. Of these 83 novel genes, a total of 36 displayed ovary-specific/selective expression, as determined by probing mouse multitissue Northern blots with 32P-labeled/PCR-amplified cDNA inserts. Under these circumstances, the false positive rate was minimal, as only one novel clone was expressed at a higher level in nonovarian tissues relative to ovary. Of the 36 ovary-specific/ selective novel genes, 22 proved subject to hormonal regulation during a simulated estrous cycle. In this communication we focus on 2 such novel ovary-specific/hormonally-dependent genes, the full-length sequences of which were isolated using rapid amplification of 3'-cDNA ends technology. Taken together, the present study accomplished systematic identification of those genes that are restricted in their expression to the ovary. These ovary-selective genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for the promotion of fertility or its control.