Light regulates Homer mRNA expression in the rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock.

Calmodulin-dependent activation of p38 and p42/44 mitogen-activated protein kinases contributes to c-fos expression by calcium in PC12 cells: modulation by nitric oxide.

Calcium and nitric oxide (NO) are important messengers for the activity-dependent immediate-early gene (IEG) expressions in neuronal cells. In the present study, we have investigated the roles of two mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase (p38 kinase) in calcium- and NO-induced c-fos expression in PC12 cells. Membrane depolarization-induced calcium increases activated both ERK and p38 kinase within 5 min. The activation of both ERK and p38 kinase by calcium was a calmodulin-dependent process since the pretreatment of W13 or calmidazolium, specific calmodulin antagonists, blocked calcium-induced activation of both MAP kinases. Calcium-induced c-fos expression was significantly reduced by the pretreatment of either MEK inhibitor (PD98059) or p38 kinase inhibitor (SB203580). This finding indicates that the calmodulin-dependent activation of ERK and p38 kinase is involved in calcium-induced c-fos expression. However, sodium nitroprusside and SIN-1, known to release NO, dose-dependently activated only ERK. NO-induced c-fos expression was partially inhibited by the PD98059. We also observed that NO dose-dependently potentiates not only calcium-induced c-fos expression but also calcium-induced ERK activation. In the presence of PD98059, the amplification of calcium-induced c-fos expression by NO was not observed. This result suggests that calcium- and NO-signals converge into the MEK/ERK pathway, thereby enhance IEG expressions in neuronal cells.

Expression, purification, characterization and crystallization of flap endonuclease-1 from Methanococcus jannaschii.

A gene coding for a protein homologous to the flap endonuclease-1 (FEN-1) was cloned from Methanococcus jannaschii, overexpressed, purified and characterized. The gene product from M. jannaschii shows 5' endo-/exonuclease and 5' pseudo-Y-endonuclease activities as observed in the FEN-1 in eukaryotes. In addition, Methanococcus jannaschii FEN-1 functions effectively at high concentrations of salt, unlike eukaryotic FEN-1. We have crystallized Methanococcus jannaschii FEN-1 and analyzed its preliminary character. The crystal belongs to the space group of P2(1) with unit cell dimensions of a = 58.93 A, b = 42.53 A, c = 62.62 A and beta = 92.250. A complete data set has been collected at 2.0 A resolution using a frozen crystal.

Postnatal development of detergent-insoluble properties of NMDA and AMPA receptor subunits in the rat brain synaptic membrane.

We investigated the developmental changes of detergent-insoluble characteristics of NMDA and AMPA receptor subunits in the synaptic membranes prepared from the rat cerebral cortex. At postnatal day (PND) 1, the majority of NMDAR1 and NMDAR2B subunits of NMDA receptors in the synaptic membranes were insoluble to the treatment of 1% Triton X-100. The detergent-insoluble properties of both subunits were not significantly changed during postnatal development. At PND 1, about 45% of GluR1 and 10% of GluR2/3 subunits of AMPA receptors in the synaptic membrane were insoluble to Triton X-100, whereas 70% of GluR1 and 56% of GluR2/3 subunits were insoluble at PND 22. These findings indicate that the postsynaptic clustering of NMDA and AMPA receptors during development seems to be differentially regulated in vivo.

Protein kinase A activity is required for depolarization-induced proline-rich tyrosine kinase 2 and mitogen-activated protein kinase activation in PC12 cells.

Protein kinase A (PKA) plays an essential role in the depolarization-induced c-fos expression in PC12 cells although the exact mechanism is unknown. Here we demonstrate that PKA is required for depolarization-induced activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase in PC12 cells. In addition, we have found that the depolarization-induced tyrosine phosphorylation of proline-rich tyrosine kinase (PYK) 2, a key calcium-sensitive upstream mediator of MAP kinase activation, is profoundly blocked by PKA inhibition. In contrast to the depolarization-induced signaling, the ERK and PYK2 activation by bradykinin (1 microM), a G-protein coupled receptor agonist, was not blocked by PKA inhibition. These findings suggest that PKA inhibition prevents depolarization-induced PYK2/MAP kinase pathway activation, thereby inhibiting the early gene expression.

The relationship of trigger finger and flexor tendon volar migration after carpal tunnel release.

It has been suggested that the increased frequency of trigger finger (TF) after carpal tunnel release (CTR) may be caused by the volar migration of the flexor tendons at the wrist altering the tendon biomechanics at the A1 pulley. This hypothesis has not been validated. We performed pre- and post-operative ultrasonography (USG) on the affected wrists of 92 patients who underwent CTR. Pre-operative USG was performed in neutral with no tendon loading; post-operative USG was performed in neutral unloaded and in various positions of wrist flexion whilst loading the flexor tendons with gripping. The mean volar migration of the flexor tendons after CTR was 2.2 (SD 0.4) mm in the unloaded neutral position. It was 1.8 (SD 0.4) mm in patients who did not develop TF and 2.5 (SD 0.5) mm in those who did (p = 0.0067). In loaded wrist flexion, the mean volar migration of flexor tendons after CTR in patients who did not develop TF and those who did was 2.1 and 3.0 mm in 0° flexion; 3.2 and 3.9 mm in 15° flexion; 4.3 and 5.1 mm in 30° flexion; and 4.9 and 5.8 mm in 45° flexion, respectively. There were significant differences between patients with and without TF at each flexion angle. Our data indicate that patients with greater volar migration of the flexor tendons after CTR are more likely to develop TF. This conclusion supports the hypothesis that the occurrence of TF after CTR may be caused by the bowstringing effects of the flexor tendons.

Use of the radial groove view intra-operatively to prevent damage to the extensor pollicis longus tendon by protruding screws during volar plating of a distal radial fracture.

The aims of this study were to assess the efficacy of a newly designed radiological technique (the radial groove view) for the detection of protrusion of screws in the groove for the extensor pollicis longus tendon (EPL) during plating of distal radial fractures. We also aimed to determine the optimum position of the forearm to obtain this view. We initially analysed the anatomy of the EPL groove by performing three-dimensional CT on 51 normal forearms. The mean horizontal angle of the groove was 17.8° (14° to 23°). We found that the ideal position of the fluoroscopic beam to obtain this view was 20° in the horizontal plane and 5° in the sagittal plane. We then intra-operatively assessed the use of the radial groove view for detecting protrusion of screws in the EPL groove in 93 fractures that were treated by volar plating. A total of 13 protruding screws were detected. They were changed to shorter screws and these patients underwent CT scans of the wrist immediately post-operatively. There remained one screw that was protruding. These findings suggest that the use of the radial groove view intra-operatively is a good method of assessing the possible protrusion of screws into the groove of EPL when plating a fracture of the distal radius.

Triterpenes from the spores of Ganoderma lucidum and their inhibitory activity against HIV-1 protease.

Two new lanostane-type triterpenes, lucidumol A and ganoderic acid beta, were isolated from the spores of Ganoderma (G.) lucidum, together with a new natural one and seven that were known. The structures of the new triterpenes were determined as (24S)-24,25-dihydroxylanost-8-ene-3,7-dione and 3 beta,7 beta-dihydroxy-11,15-dioxolanosta-8,24(E)-dien-26-oic acid, respectively, by chemical and spectroscopic means. The quantitative analyses of 5 fruiting bodies, antlered form and spores of G. lucidum were performed by high performance liquid chromatography and demonstrated that ganoderic alcohol and acid contents were quite high in the spore. Of the compound isolated, ganoderic acid beta, (24S)-lanosta-7,9(11)-diene-3 beta,24,25-triol (called lucidumol B), ganodermanondiol, ganodermanontriol and ganolucidic acid A showed significant anti-human immunodeficiency virus (anti-HIV)-1 protease activity with IC50 values of 20-90 microM.