Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice.

Human epidermal growth factor receptor2/Neu, which is overexpressed in about 30% of human breast cancers, transduces growth signals in large part via the Ras-Raf-MEK-ERK pathway. Nevertheless, it is a matter of controversy whether high ERK activity in breast cancer tissues correlates with better or worse prognosis, leaving the role of ERK activity in the progression of breast cancers unresolved. To address this issue, we live-imaged ERK activity in mammary tumors developed in mouse mammary tumor virus-Neu transgenic mice, which had been crossed with transgenic mice expressing a Förster resonance energy transfer biosensor for ERK. Observation of the tumor by two-photon microscopy revealed significant heterogeneity in ERK activity among the mammary tumor cells. The level of ERK activity in each cell was stable up to several hours, implying a robust mechanism that maintained the ERK activity within a limited range. By sorting the mammary tumor cells on the basis of their ERK activity, we found that ERK(high) cells less efficiently generated tumorspheres in vitro and tumors in vivo than did ERK(low) cells. In agreement with this finding, the expressions of the cancer stem cell markers CD49f, CD24 and CD61 were decreased in ERK(high) cells. These observations suggest that high ERK activity may suppress the self-renewal of mammary cancer stem cells.

Familial cortical myoclonic tremor as a unique form of cortical reflex myoclonus.

Previously it was reported that some patients with tremor had the same electrophysiological findings as those seen in patients with cortical reflex myoclonus, and consequently the tremor was named "cortical tremor." In the present study, we examined six patients from three families with cortical tremor of relatively late onset. The inheritance pattern of cortical tremor was compatible with autosomal dominant trait. Those patients had relatively rhythmic involuntary movements (tremor) in the distal upper and lower limbs, especially during posture and/or fine movements. There was no cerebellar ataxia or dementia, and fits of loss of consciousness occurred only infrequently. Electrophysiologically, they had generalized spikes on electroencephalogram (EEG), giant cortical components of somatosensory evoked potential, an enhanced long-loop reflex (C-reflex), and cortical spikes preceding the rhythmic jerk demonstrable by the jerk-locked back averaging method, thus fulfilling the criteria of cortical reflex myoclonus. Furthermore, they had slow negative EEG shift starting 1-2 s prior to voluntary movements, suggesting that, as opposed to the conventional form of progressive myoclonus epilepsy (PME), the cerebellar efferent input to the motor cortices was normal. These clinical and electrophysiological pictures are distinct from those of familial essential tremor, familial essential myoclonus, or the conventional form of PME, and the term "familial cortical myoclonic tremor" will represent the clinical and electrophysiological features of this unique entity most appropriately.

Reduction of cerebral blood flow in chronic schizophrenia: relation to age.

The effect of age on resting-state cerebral blood flow (CBF) was studied in 40 medicated patients with chronic schizophrenia aged 20 to 57 and 32 age- and sex-matched normal controls, using the regional CBF with the xenon-133 inhalation technique. Global CBF (average of 16 cerebral regions) and the left prefrontal flow ratio (left prefrontal/global CBF) were significantly lower in schizophrenics aged 20-29 than in age-matched controls. Both global CBF and the left prefrontal flow ratio correlated with age in controls, but neither of such correlations was found in schizophrenics. Therefore, the CBF deficits in chronic schizophrenia appear to be unaffected by the aging process.

Lumbar trapezoid plate for lumbar spondylolisthesis. A clinical study on preoperative and postoperative instability.

The authors studied the effects of a "lumbar trapezoid plate" (spinal plate and pedicle screwing), performed for lumbar spondylolisthesis, observing the effect on the remaining adjacent discs with regard to preoperative and postoperative instability. The authors examined changes in preoperative and postoperative lumbar ROM (range of motion), displacement of motor unit levels, and occurrence of instability in the remaining discs, such as horizontal and rotational displacement, in 26 patients who were followed up for a mean of 29 postoperative months; 13 patients had spondylolytic spondylolisthesis and 13 patients degenerative spondylolisthesis. The authors studied the effects of the fused vertebral angle and reduction of spondylolisthesis on the remaining upper and lower adjacent discs and the preoperative and postoperative fused disc heights. Intervertebral fusion must affect the remaining adjacent discs, but compensatory function of the remaining motor unit level was more influenced by the fused intervertebral angle than by repositioning of the spondylolisthesis. Fusion at a physiologically lordotic position is quite necessary. For this purpose, it is considered important to prevent grafted bones of the posterior lumbar interbody fusion (PLIF) from collapse and to maintain the achieved alignment of the lumbar spine.

Nucleotide sequence of cDNA and primary structure for hard tail growth hormone.

Full-length cDNA for hard tail growth hormone (htGH) has been cloned, and the nucleotide and deduced amino acid sequences have been analyzed. htGH is composed of 188 amino acid residues, and it shows 79, 74, 72, 59, 56, 37, 33 and 30% identity of amino acid with yellow tail, tuna, sea bream, flounder, salmon, blue shark, bullfrog and human GHs, respectively.

Postoperative instability of cervical OPLL and cervical radiculomyelopathy.

The presence of cervical spine instability with respect to preoperative and postoperative changes in angular, horizontal, and rotational displacement of the vertebral body were studied. With the anterior approach, the instability in the remaining unfused segments, and their relation to the kyphotic or lordotic fused segment were studied. With the posterior approach, postoperative ROM (range of motion) could be better maintained, and horizontal displacement was improved in more cases by laminoplasty compared with laminectomy. With the anterior approach, the compensatory function for the loss of motion of the segments resulting from fusion was most remarkable at the levels of C2-3 and C6-7. In the alignment of the anterior fused segments, it appears important that the physiologic lordotic position be maintained.

[Composite tissue (bone and ligaments) allografts in the reconstruction of the posterior element of the canine spine (preliminary report)].

Reconstructions of the posterior element of the spine with allograft transplantation in adult mongrel dogs were studied histologically. The bone and ligament composite allografts were frozen at -80 degrees for reduction of the antigenicity. Within four weeks, a new bone formation was observed, and the bony union was completed at 8 weeks after the grafting. The transplanted lamina was replaced by the newly formed bone by creeping substitution within 32 weeks. However a part of the spinous process was absorbed, and was not replaced by new bone. The supraspinal ligament and the ligamentum flavum were gradually replaced by connective tissue resembling a ligament. The scar tissue did not invade into the spinal canal, and symptoms of myelopathy did not appear throughout the observation. Although a biomechanical investigation was not performed, these results indicated reconstruction of the posterior element of the spine with allograft transplantation is possible for clinical application.

Generation of free radical and hydrogen peroxide from 2-hydroxyemodin, a direct-acting mutagen, and DNA strand breaks by active oxygen.

Among several hydroxylated metabolites of emodin, a fungal anthraquinone and constituent of rhubarb, 2-hydroxyemodin was a direct-acting mutagen showing a large electron-spin resonance (ESR) signal in the presence of DNA, especially at alkaline pH. Coupled with generation of free radical, hydrogen peroxide but not superoxide was formed. The active oxygen produced from 2-hydroxyemodin induced strand breaks in phi X 174 replicative form I DNA (supercoiled covalently closed circular duplex DNA). These results suggest a possible role of active oxygen in the process of mutagenesis.