Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSB5) detected by PANT were present in neurons after as little as 1 mm of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TU-NEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB-and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis. Key Words: DNA damage-Single-strand breaks-Apoptosis-Cerebral ischemia.

Kinesin is a motor protein that transports organelles along a microtubule toward its plus end by using the energy of ATP hydrolysis. To clarify the nucleotide-dependent binding mode, we measured the unbinding force for one-headed kinesin heterodimers in addition to conventional two-headed kinesin homodimers under several nucleotide states. We found that both a weak and a strong binding state exist in each head of kinesin corresponding to a small and a large unbinding force, respectively; that is, weak for the ADP state and strong for the nucleotide-free and adenosine 5-[␤,␥-imido]triphosphate states. Model analysis showed that (i) the two binding modes in each head could be explained by a difference in the binding energy and (ii) the directional instability of binding, i.e., dependence of unbinding force on loading direction, could be explained by a difference in the characteristic distance for the kinesin-microtubule interaction during plus-and minus-end-directed loading. Both these factors must play an important role in the molecular mechanism of kinesin motility. Kinesin is a processive molecular motor that is essential for the transport of vesicles and organelles along a microtubule in various cells. Kinesin's processive movement has been explained by a mechanism that involves alternating between singleand double-headed bindings to a microtubule (1-5). Adjacent tubulin dimers of 8-nm length form consecutive binding sites (6), such that kinesin takes hundreds of 8-nm steps down a microtubule (7-10). Our recent single-molecule analysis of unbinding force (11) showed that conventional two-headed kinesin is involved in single-headed binding, both in the absence of nucleotides (nucleotide-free state) and in the coexistence of ADP and adenosine 5Ј-[␤,␥-imido]triphosphate (AMP-PNP) (ATP analogue), and double-headed binding in the presence of AMP-PNP (AMP-PNP state), which is consistent with the putative mechanism of kinesin motility.In the present study, we have measured the unbinding force of a single kinesin⅐microtubule complex under an optical microscope equipped with optical tweezers as was reported (11). To clarify the binding mode, we used one-headed kinesin heterodimers (12) in addition to conventional two-headed kinesin homodimers. Conventional two-headed homodimers or oneheaded heterodimers of kinesin molecules were attached to a polystyrene bead such that single kinesin binds to a single bead, and each bead was manipulated with optical tweezers on a microtubule that was adsorbed onto a coverslip (1, 9). An external load was imposed on the attached kinesin molecule by moving the bead toward the plus or the minus end of the microtubule. Here, we found that the two binding states exist in each head of kinesin depending on the nucleotide state. Also, we found that the dependence of the unbinding force on loading direction (where the unbinding force is smaller for the plus-end loading than for the minus-end loading) was independent of nucleotide states.We have analyzed the results for a weak and a strong bindi...

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Objective. To evaluate medial radial displacement (MRD) of the medial meniscus in osteoarthritic (OA) and normal knees, with and without weight bearing, using ultrasonography (US), and to prospectively evaluate the time course of changes in MRD in OA knees.Methods. The study subjects were 78 patients with OA of the knee (69% female; mean age 66.4 years) and 20 healthy, asymptomatic subjects (70% female; mean age 64.5 years) who served as a control group. The OA stage was determined according to the Kellgren/ Lawrence (K/L) radiographic grading system. US measurement of MRD was performed with subjects in the supine and standing positions. With the exception of subjects who dropped out, 58 OA knees (followup rate 74%) were evaluated at baseline and ϳ1 year later.Results. The medial meniscus was significantly displaced radially by weight bearing in control knees (P < 0.001) and in knees with K/L grades 1-3 OA (P < 0.01 for each comparison). MRD in either the supine or the standing position was not significantly different between the control knees and the K/L grade 1 knees, but significant differences were noted between the control knees and K/L grade 2 or more severe OA knees (P < 0.01 for each comparison). MRD of the medial meniscus had increased significantly on followup in all knees (P < 0.05 for each comparison) excluding K/L grade 4 knees in the standing position.Conclusion. MRD of the medial meniscus increased with weight bearing and during followup. These findings suggest a close association between extraarticular displacement of the medial meniscus and progression of OA.