An important role of tumor necrosis factor receptor-2 on natural killer T cells on the development of dsRNA-enhanced Th2 cell response to inhaled allergens.

BACKGROUND: Recent evidence indicates that TNF-α is a key mediator of the development of dsRNA-enhanced Th2 cell response to inhaled allergens. Natural killer T (NKT) cells may be a candidate source of Th2-polarizing cytokines. OBJECTIVE: The objective of this study was to evaluate the role of lung NKT cells on the development of TNF-α-mediated Th2 cell response. METHODS: A virus-associated asthma mouse model was generated by the administration of ovalbumin (OVA, 75 µg) and poly[I:C] (0.1 µg). Role of NKT and type I NKT cells was evaluated using CD1d- and Jα18-deficient mice. TNF-α receptors (TNFRs) were antagonized by using TNFR blocking peptides. RESULTS: The number of infiltrated NKT cells was increased in a virus-associated asthma mouse model. Increase in Th2 and Th17 cytokine levels in wild-type mice were abolished in both CD1d- and Jα18-deficient mice. In vitro co-culture experiments with alveolar macrophages and NKT cells showed that TNF-α produced by macrophages in the presence of poly[I:C] acts on NKT cells, inducing production of Th2-polarizing cytokines. Moreover, the induction of Th2-polarizing cytokines by poly[I:C] or recombinant TNF-α was impaired in both CD1d- and Jα18-deficient mice and that the above effect was reversed by a TNF-α receptor-2 (TNFR2) blocking peptide, but not by a TNFR1 blocker. CONCLUSIONS: These findings suggest that NKT cells play a key role in the development of Th2 cell response to inhaled allergens and that TNF-α produced by alveolar macrophages induces Th2 cell response, via TNFR2 on NKT cells.

Systemic Mycobacterium kansasii Infection in a Domestic Shorthair Cat.

A 1-year-old, female domestic shorthair cat was presented with anorexia, depression and weight loss, accompanied by multifocal nodules affecting the face, pinnae and periarticular tissue. Routine medical treatments were ineffective. The animal's physical condition continued to deteriorate and it finally died. Post-mortem examination revealed multifocal to coalescing firm nodules with occasional ulceration affecting the ears, peri-ocular areas, nasal planum, oral cavity and laryngopharyngeal region. Tan-coloured, firm, nodular lesions were also observed in the periarticular tissue, lungs and tracheobronchial and mediastinal lymph nodes. Impression smears of several of these lesions revealed a myriad of slender rod-shaped organisms, mainly in the cytoplasm of macrophages. Histopathological examination showed severe pyogranulomatous inflammation with or without necrosis in the nodules. Acid-fast staining revealed large numbers of acid-fast bacilli. Mycobacterium kansasii was detected in the tissues using multiplex polymerase chain reaction and DNA sequencing. No protozoal or fungal organisms were detected using special stains. On the basis of these results, the cat was diagnosed with systemic M. kansasii infection. To our knowledge, there have been few reports of M. kansasii infection, especially with systemic spread, in cats.

Modification to the accelerator of the NBI-1B ion source for improving the injection efficiency.

Minimizing power loss of a neutral beam imposes modification of the accelerator of the ion source for further improvement of the beam optics. The beam optics can be improved by focusing beamlets. The injection efficiencies by the steering of ion beamlets are investigated numerically to find the optimum modification of the accelerator design of the NBI-1B ion source. The beam power loss was reduced by aperture displacement of three edge beamlets arrays considering power loadings on the beamline components. Successful testing and operation of the ion source at 60 keV/84% of injection efficiency led to the possibility of enhancing the system capability to a 2.4 MW power level at 100 keV/1.9 µP.

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.

Region-specific expression of subunits of ionotropic glutamate receptors (AMPA-type, KA-type and NMDA receptors) in the rat spinal cord with special reference to nociception.

The present study attempted to explore the gene expression of the subunits (GluR1-4) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor, subunit (GluR5) of kainic acid (KA)-type receptor, NR1 [a subunit of N-methyl-D-aspartate (NMDA) receptors] and the possible glutamate-binding subunit of an NMDA receptor complex in the dorsal horn of the rat spinal cords using in situ hybridization histochemistry. These results were compared with those of the spinal motor neurons. Expression of the subunits of the AMPA-type receptor was also examined at the protein level using immunocytochemistry, with reference to the motor neurons. Although all the four subunits of the AMPA-type receptor were expressed throughout the dorsal horn, the pattern of expression was different according to the dorsal horn region and to the subunits. GluR2 showed the strongest expression in the dorsal horn. Huge numbers of strongly labelled cells formed a dense collection in lamina II and superficial parts of lamina III. Many neurons in lamina II and superficial parts of lamina III expressed GluR1 moderately. Scattered neurons moderately expressing GluR3 were also seen in these regions, while the expression of GluR4 was very low. Labelling of the dorsal horn neurons by the GluR5 probe was low, and NR1 probe and a glutamate-binding subunit of an NMDA receptor complex probe labelled them diffusely with low to moderate intensity. These findings show a close relationship between the glutamergic nociceptive primary afferent system and AMPA-type receptors in which GluR2 is especially highly expressed. The present study further showed that the expression pattern of the glutamate receptors in the spinal sensory neurons differs considerably from that of spinal motor neurons. Motor neurons very strongly express GluR3 and 4, while the expression of GluR2 and GluR1 is moderate and low, respectively. Expression of GluR5 is also low in the motor neurons. However, expression of NR1 and the glutamate-binding subunit of an NMDA receptor complex is very strong. These findings indicate that the subunit composition of the AMPA-type receptors regulating motor neurons is different from that of the AMPA-type receptors in the spinal sensory neurons, and that there are at least two kinds of glutamergic systems which regulate motor neurons: via AMPA-type receptors and via NMDA receptors.

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.

AMPA, KA and NMDA receptors are expressed in the rat DRG neurones.

The localization of AMPA receptor subunits (GluR1-4), a KA receptor subunit (GluR5) and NMDA receptor subunits (NR1 and NRgbs; the glutamate binding subunits of an NMDA receptor complex) was investigated using immunohistochemistry and in situ hybridization histochemistry in the rat dorsal root ganglion. Small neurones expressed GluR1-, GluR2/3-like immunoreactivities and GluR5, NR1, NRgbs mRNAs, while large neurones expressed GluR2/3-like immunoreactivity and NR1 and NRgbs mRNAs. These data suggest that the glutamatergic system plays an important role in the primary sensory afferent systems and that the composition of glutamate receptors differs according to the cell size.

Distributional characteristics of the mRNA for retinoid Z receptor beta (RZR beta), a putative nuclear melatonin receptor, in the rat brain and spinal cord.

We investigated the distribution of retinoid Z receptor beta (RZR beta), a putative nuclear melatonin receptor, mRNA in the rat brain and spinal cord using in situ hybridization. The RZR beta riboprobe uniquely hybridized with the neurons in several sensory pathways including the dorsal horn of the spinal cord, whereas the regions involved in the motor control were generally unlabeled. RZR beta mRNA was also highly expressed in the dorsomedial portion of the suprachiasmatic nucleus, pars tuberalis and some limbic areas. This result suggests that RZR beta plays a specific role as a transcription factor in the sensory, neuroendocrine and limbic systems.

Profile of Fos-like immunoreactivity induction by light stimuli in the intergeniculate leaflet is different from that of the suprachiasmatic nucleus.

Light stimuli induce Fos-like immunoreactivity (FLI) in suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL). Short pulses of light stimuli that synchronize the circadian rhythms induce FLI in SCN. The characteristics of light induction of FLI in the IGL were studied using immunohistochemistry. In the IGL, at least 2 h of sustained light stimuli were necessary to show an increase of FLI. This FLI persisted while the light was turned on. FLI induction in the IGL by light stimuli was not circadian time specific response. These findings imply that the functional significance of Fos activation on circadian rhythms and mechanism of FLI induction in IGL would be different from that in SCN.

Calcitonin gene-related peptide-like immunoreactive (CGRPI) elements in the circadian system of the mouse: an immunohistochemistry combined with retrograde transport study.

This study was based on immunohistochemical detection of calcitonin gene-related peptide-like immunoreactive (CGRPI) neurons and fibers in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) of the mouse. CGRPI neurons and fibers were found within the ventrolateral part of the SCN, in the whole extent of IGL and sparsely distributed in ventral lateral geniculate body. The presence of CGRPI structures in the SCN and IGL of the mouse further supports the hypothesis of differences in the content of neuroactive substances in the circadian clock between mammalian species. Fluorogold retrograde transport combined with CGRP immunofluorescence demonstrates that CGRPI neurons in the IGL constitute a part of IGL reciprocal connections.

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.

Enhancement of phospholipase D activity by overexpression of amyloid precursor protein in P19 mouse embryonic carcinoma cells.

It has been shown that phospholipase D (PLD) activity is stimulated by the beta-amyloid protein in neuronal cells. The aim of this study was to determine whether overexpression of the amyloid precursor protein (APP) affects the activity and the level of PLD expression in P19 embryonic carcinoma cells. We observed that the unstimulated basal PLD activity was higher in wild-type APP(695)-transfected cells than in non-transfected control cells. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), has been shown to activate PLD. PMA-stimulated PLD activity was 3-fold higher in the APP overexpressing cells than in the control cells. P19 cells express two distinct PLD isozymes, PLD1 and PLD2. The level of PLD2 expression was increased by APP overexpression. Although the PKC inhibitor, GF109203X, inhibited PMA-stimulated PLD activity, it did not affect the high basal PLD activity induced by APP overexpression. Neuronal differentiation of the P19 cells by retinoic acid did not affect the basal or PMA stimulated-PLD activity. Interestingly, APP overexpression in the differentiated P19 cells also led to an increase in PLD activity. The PLD activity of the P19 cells is apparently regulated by amyloid protein through both PKC-dependent and -independent mechanisms.

Developmental expression of 'RZR beta, a putative nuclear-melatonin receptor' mRNA in the suprachiasmatic nucleus of the rat.

RZR beta is a member of the retinoid Z receptor (RZR) family of orphan receptors, and its expression is brain-specific. Recently, it was reported that the distribution of RZR beta mRNA is partially coincident with melatonin binding sites and that RZR beta is a putative nuclear receptor of melatonin. Using in situ hybridization, we investigated the developmental expression of RZR beta mRNA in the suprachiasmatic nucleus (SCN) of the rat, which is considered as circadian pacemaker. The RZR beta mRNA was first found in the dorsomedial portion of the SCN at embryonic day 20, and RZR beta mRNA expression in the dorsomedial SCN continued until postnatal day 60. This result suggests that RZR beta plays specific roles as a transcription factor in the dorsomedial SCN but not in the ventrolateral SCN during development and throughout postnatal life.

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.

Production of env-deficient rous sarcoma virus (RSV) early after infection.

Production of defective virus particles during the early stage of Rous sarcoma virus (RSV) infection of chicken embryo fibroblasts (CEF) was examined. RSV harvested 2 days post infection (2pi) had 10 to 30 times lower specific infectivity (focus forming units/unit reverse transcriptase activity) than 5pi harvest. Virus particles produced on day 2 contained less env proteins than particles harvested on day 5. The amount of other viral proteins was equal in particles harvested on day 2 and day 5. Analysis of infected cells revealed that these cells synthesized less env proteins on day 2 than on day 5. RSV RNA in infected cells was spliced normally on day 2. Infection at a low multiplicity of infection (moi) prolonged the production of defective particles. When infection was initiated by a low moi (0.01), particles harvested on day 5 had the same characteristics as 2pi particles after infection with a high moi (1.0). We conclude that the low infectivity of early harvest is due to the reduced amount of env proteins in virus particles, which is a consequence of the reduced env protein synthesis.

Production of defective virus by terminally differentiated myotubes infected with Rous sarcoma virus.

The generally accepted concept that the replication of Rous sarcoma virus (RSV) is dependent on host cell DNA synthesis was reexamined. As the host we used terminally differentiated myotubes (MT), in which no cellular DNA synthesis is observed. As an extension of our previous study which indicated that RSV-infected MT produce various virus components, we examined viral particles produced by infected MT. Electron microscopy showed presence of viral particles released from infected MT. Immunoprecipitation analysis revealed that these particles contained an equal amount of the gag but a decreased amount of the env proteins as compared with the particles from infected chicken embryo fibroblasts (CEF). Consequently, viral particles from infected MT had an infectivity only 6% of that of particles from infected CEF cells. In a parallel experiment, we microinjected molecularly cloned RSV DNA into MT. In contrast to the infection mediated by viral particles, both MT and CEF cells produced the same amount of infectious particles when microinjected with viral DNA. We conclude that RSV replicates in the complete absence of host DNA synthesis, though infectivity of the progeny virus depends on the initial condition of the infection.

Characteristic travelling patterns of non-recurrent laryngeal nerves.

BACKGROUND: The non-recurrent laryngeal nerve is subject to potential injury during thyroid surgery. Intra-operative identification and preservation of this nerve can be challenging. Its presence is associated with an aberrant subclavian artery and the developmental absence of the brachiocephalic trunk. This study aimed to evaluate the incidence of non-recurrent laryngeal nerves and present a new classification system for the course of these nerves. METHODS: Non-recurrent laryngeal nerves were identified on the right side in 15 patients who underwent thyroidectomy. The incidence of non-recurrent laryngeal nerves (during thyroidectomy) and aberrant subclavian arteries (using neck computed tomography) was evaluated, and the course of the nerves was classified according to their travelling patterns. RESULTS: The overall incidence of non-recurrent laryngeal nerves was 0.68 per cent. The travelling patterns of the nerves could be classified as: descending (33 per cent), vertical (27 per cent), ascending (20 per cent) or V-shaped (20 per cent). CONCLUSION: Clinicians need to be aware of these variations to avoid non-recurrent laryngeal nerve damage. A retroesophageal subclavian artery (on neck computed tomography) virtually assures a non-recurrent laryngeal nerve. This information is important for preventing vocal fold paralysis. Following a review of non-recurrent laryngeal nerve travelling patterns, a new classification was devised.

Mitochondrial swelling and microtubule depolymerization are associated with energy depletion in axon degeneration.

Although mitochondrial dysfunction is intimately related to axonal degeneration following nerve injury, the molecular mechanisms of mitochondrial swelling and its mechanistic relation to axonal degeneration are largely unknown. Previous studies have demonstrated that axonal degeneration in the injured peripheral nerves shows two morphologically distinct phases: (1) A latency period (∼24h), in which the morphology of axonal cytoskeletons seems unchanged, followed by (2) an execution period (36-48h), which shows a catastrophic granular degeneration of most axonal structures in rodent axons. In the present study, we found that, in the sciatic nerve axotomy model, energy failure and microtubule depolymerization occurred during the latency period whereas mitochondrial swelling and neurofilament degradation started in the execution period. The energy repletion with NAD or an NAD/pyruvate mixture inhibited microtubule depolymerization, mitochondrial swelling and axonal degeneration in transected sciatic nerve axons. In addition, microtubule perturbing agents enhanced axonal degeneration and mitochondrial swelling. Extracellular calcium chelation did not affect energy failure, microtubule depolymerization or mitochondrial swelling, but it did prevent neurofilament degradation. These findings suggest that an early disturbance in energy dynamics regardless of mitochondrial swelling might be a key trigger for the initiation of axonal degeneration and that extracellular calcium influx is a late effector for neurofilament degradation.

First neutral beam injection experiments on KSTAR tokamak.

The first neutral beam (NB) injection system of the Korea Superconducting Tokamak Advanced Research (KSTAR) tokamak was partially completed in 2010 with only 1∕3 of its full design capability, and NB heating experiments were carried out during the 2010 KSTAR operation campaign. The ion source is composed of a JAEA bucket plasma generator and a KAERI large multi-aperture accelerator assembly, which is designed to deliver a 1.5 MW, NB power of deuterium at 95 keV. Before the beam injection experiments, discharge, and beam extraction characteristics of the ion source were investigated. The ion source has good beam optics in a broad range of beam perveance. The optimum perveance is 1.1-1.3 µP, and the minimum beam divergence angle measured by the Doppler shift spectroscopy is 0.8°. The ion species ratio is D(+):D(2)(+):D(3)(+) = 75:20:5 at beam current density of 85 mA/cm(2). The arc efficiency is more than 1.0 A∕kW. In the 2010 KSTAR campaign, a deuterium NB power of 0.7-1.5 MW was successfully injected into the KSTAR plasma with a beam energy of 70-90 keV. L-H transitions were observed within a wide range of beam powers relative to a threshold value. The edge pedestal formation in the T(i) and T(e) profiles was verified through CES and electron cyclotron emission diagnostics. In every deuterium NB injection, a burst of D-D neutrons was recorded, and increases in the ion temperature and plasma stored energy were found.

Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins.

We studied Rous sarcoma virus (RSV) protein synthesis in RSV-infected, terminally differentiated chicken myotubes ('late-infected' myotubes), in which no viral DNA integration takes place but all three viral mRNAs (38S, 28S and 21S) are transcribed normally. With the use of specific anti-RSV protein antisera, we found that only the viral gag and pol proteins were synthesized at levels similar to those synthesized in RSV-transformed fibroblasts; the synthesis of env and v-src proteins was significantly reduced in these infected myotubes. We concluded that the viral RNA transcribed from the unintegrated RSV DNA was functional but that genes at the 3' end of the RSV genome were translated at a lower level. By contrast, when mononucleated replicating chicken myoblasts were infected with a mutant (tsNY68) carrying a temperature-sensitive v-src gene and maintained at the non-permissive temperature for this gene, they developed into myotubes with viral DNA integrated in their chromosomal DNA. These 'early-infected' myotubes expressed all four viral genes (gag, pol, env and v-src) at a level similar to that in infected fibroblasts. This result ruled out the possible presence of specific factor(s) in myotubes that preferentially inhibit the 3' genes of RSV, and suggested other translational control(s) of viral gene expression in late-infected myotubes.