Delta-opioid receptor endocytosis in spinal cord after dermenkephalin activation

Background

The delta(δ)-opioid receptors belong to the G protein-coupled receptors and in vitro studies have shown that δ-opioid receptors undergo an internalization process in response to agonist stimulation. The immediate consequence is the disappearance of receptors from the plasma membrane. This adaptation process reveals the cell's capacity to desensitize after a strong agonist stimulus. This process, if it occurs in vivo, could contribute to the tolerance phenomenon observed after opiate treatment. To study the mechanisms underlying regulation of the δ-opioid receptors in vivo, the effects of an application of the drug dermenkephalin, a potent and selective agonist of the δ-opioid receptor, were analysed in the rat spinal cord.

Results

Using immunocytochemistry and electron microscopy, we observed in control rats that membrane labelling was strictly localized at the interface between two neurites. Fifteen minutes after dermenkephalin stimulation, the plasma membrane labelling was associated with invaginated areas. Thirty minutes after stimulation, labelled vesicles were found in the cytoplasm confirming the internalization process.

Conclusions

The present findings support the view that δ-opioid receptors are internalized in response to prolonged exposure to dermenkephalin in vivo and confirm the presynaptic localization of δ-opioid receptors in the dorsal horn of the rat spinal cord.     

Measurement of the average γ-ray multiplicity and the mean γ-energy in the photon cascades following the reaction154Sm(12C,4n)162Er

By unfolding theγ-γ coincidence spectra from the reaction¹⁵⁴Sm(¹²C, 4n)¹⁶²Er at a beam energy of 64 MeV the mean energy of the continuousγ-ray spectrum feeding the ground state rotational band was determined as about 1.2 MeV. The averageγ-ray multiplicity \(\bar v\) including the rotational band transitions was measured for each of the levelsJ through which the cascade passed. The values for \(\bar v\) were found to increase regularly from \(\bar v = 8\) forJ=4 to \(\bar v = 14\) forJ=18.     

Corticospinal interaction during isometric compensation for modulated forces with different frequencies

Background

The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.

Results

We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.

Conclusions

The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.     

Computational framework for the prediction of transcription factor binding sites by multiple data integration

Background

Activation of extracellular signal-regulated protein kinase (ERK), a member of mitogen-activated protein kinase (MAPK) family, has been proposed to mediate neurite outgrowth-promoting effects of several neurotrophic factors in vitro. However, the precise activity of ERK during axonal regeneration in vivo remains unclear. Peripheral axotomy has been shown to activate ERK in the cell bodies of primary afferent neurons and associated satellite cells. Nevertheless, whether ERK is also activated in the axons and surrounded Schwann cells which also play a key role in the regeneration process has not been clarified.

Results

Phosphorylation of ERK in the sciatic nerve in several time-points after crush injury has been examined. Higher phosphorylation of ERK was observed in the proximal and distal nerve stumps compared to the contralateral intact nerve from one day to one month after crush. The activation of ERK was mainly localized in the axons of the proximal segments. In the distal segments, however, active ERK was predominantly found in Schwann cells forming Bungner's bands.

Conclusion

The findings indicate that ERK is activated in both the proximal and distal nerve stumps following nerve injury. The role of activated ERK in Wallerian degeneration and subsequent regeneration in vivo remains to be elucidated.     

E2 andE3 transition strengths in some rare-earth nuclei

Coulomb excitation byα-particles of vibrational-like states in even-mass rare-earth nuclei is used to determine the reduced transition probabilitiesB(E2; 0 _gs ⁺ →2 _γ ⁺ ),B(E2; 0 _gs ⁺ →2 _β ⁺ ),B(E2; 2 _gs ⁺ →0 _β ⁺ ) andB(E2; 0 _gs ⁺ →3 _oct ^? ) in¹⁵⁰Nd,^{152, 154}Sm,^{154, 158}Gd,¹⁶⁴Dy and¹⁶⁶Er. TheB(Eλ; 0 _gs ⁺ →I=λ)-values range from 2.4 to 6.5 single-particle units for transitions to the 2 _γ ⁺ -states, 0.8 single-particle units for the 2 _β ⁺ -states and from 14.1 to 21.7 single-particle units for the 3^?-states.     

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

Background

Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drug's clinical value in acute stroke setting.

Results

Here, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.

Conclusion

The present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.     

Localization in the ground state of the ising model with a random transverse field

We study the zero-temperature behavior of the Ising model in the presence of a random transverse field. The Hamiltonian is given by $$H = - J\sum\limits_{\left\langle {x,y} \right\rangle } {\sigma _3 (x)\sigma _3 (y) - \sum\limits_x {h(x)\sigma _1 (x)} } $$ whereJ>0,x,y∈Z ^d, σ₁, σ₃ are the usual Pauli spin 1/2 matrices, andh={h(x),x∈Z ^d} are independent identically distributed random variables. We consider the ground state correlation function 〈σ₃(x)σ₃(y)〉 and prove:

1. Letd be arbitrary. For anym>0 andJ sufficiently small we have, for almost every choice of the random transverse fieldh and everyx∈Z ^d, that $$\left\langle {\sigma _3 (x)\sigma _3 (y)} \right\rangle \leqq C_{x,h} e^{ - m\left| {x - y} \right|} $$ for ally∈Z ^d withC _{x h} <∞.
2. Letd≧2. IfJ is sufficiently large, then, for almost every choice of the random transverse fieldh, the model exhibits long range order, i.e., $$\mathop {\overline {\lim } }\limits_{\left| y \right| \to \infty } \left\langle {\sigma _3 (x)\sigma _3 (y)} \right\rangle > 0$$ for anyx∈Z ^d.

     

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

Background

As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.

Methods

Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.

Results

By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells.

Conclusion

Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.     

Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation

Background

Cigarette smoking enhances the risk of stroke. However, the underlying molecular mechanisms are largely unknown. The present study established an in vivo rat secondhand cigarette smoking (SHS) model and examined the hypothesis that SHS upregulates endothelin receptors with increased cerebrovascular contraction via the Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway.

Results

Rats were exposed to SHS for up to 8 weeks. The cerebral artery vasoconstriction was recorded by a sensitive myograph. The mRNA and protein expressions for endothelin receptors in cerebral arteries were studied by real-time PCR and Western blot. Compared to fresh air exposed rats, cerebral arteries from SHS rats exhibited stronger contractile responses (P < 0.05) mediated by endothelin type A (ET_A) receptors. The expressions of mRNA and protein for ET_A receptors in the cerebral arteries from SHS rats were higher (P < 0.05) than that in control. SHS did not affect endothelin type B (ET_B) receptor-mediated contractions, mRNA or protein levels. The results suggest that SHS upregulates ET_A, but not ET_B receptors in vivo. After SHS exposure, the mRNA levels of Raf-1 and ERK1/2, the protein expression of phosphorylated (p)-Raf-1 and p-ERK1/2 were increased (P < 0.05). Raf-1 inhibitor, GW5074 suppressed the enhanced ET_A receptor-mediated contraction, mRNA and protein levels induced by SHS. In addition, GW5074 inhibited the SHS-caused increased mRNA and phosphorylated protein levels of Raf-1 and ERK1/2, suggesting that SHS induces activation of the Raf/ERK/MAPK pathway.

Conclusions

SHS upregulates cerebrovascular ET_A receptors via the Raf/ERK/MAPK pathway, which provides novel understanding of mechanisms involved in SHS-associated stroke.     

Angular correlation measurements in154Gd

γ-γ angular correlation measurements were performed to determine the mixing parameters δ of some γ-transitions in¹⁵⁴Gd. High energy compton background could easily be eliminated using a two dimensional data aquisition system. The relativeB (E2)-values of (K=2→K=0) interband transitions were determined and compared with collective model predictions. Radioactivity:¹⁵⁴Eu[from¹⁵³Eu(n, γ)], enriched target. \(^{154} Eu\xrightarrow{{\beta ^{ --} }}^{154} Gd.\)      

Enhanced effect of microdystrophin gene transfection by HSV-VP22 mediated intercellular protein transport

Background

Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo.

Results

Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice.

Conclusion

These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD.     

Cingulate seizure-like activity reveals neuronal avalanche regulated by network excitability and thalamic inputs

Background

Cortical neurons display network-level dynamics with unique spatiotemporal patterns that construct the backbone of processing information signals and contribute to higher functions. Recent years have seen a wealth of research on the characteristics of neuronal networks that are sufficient conditions to activate or cease network functions. Local field potentials (LFPs) exhibit a scale-free and unique event size distribution (i.e., a neuronal avalanche) that has been proven in the cortex across species, including mice, rats, and humans, and may be used as an index of cortical excitability. In the present study, we induced seizure activity in the anterior cingulate cortex (ACC) with medial thalamic inputs and evaluated the impact of cortical excitability and thalamic inputs on network-level dynamics. We measured LFPs from multi-electrode recordings in mouse cortical slices and isoflurane-anesthetized rats.

Results

The ACC activity exhibited a neuronal avalanche with regard to avalanche size distribution, and the slope of the power-law distribution of the neuronal avalanche reflected network excitability in vitro and in vivo. We found that the slope of the neuronal avalanche in seizure-like activity significantly correlated with cortical excitability induced by γ-aminobutyric acid system manipulation. The thalamic inputs desynchronized cingulate seizures and affected the level of cortical excitability, the modulation of which could be determined by the slope of the avalanche size.

Conclusions

We propose that the neuronal avalanche may be a tool for analyzing cortical activity through LFPs to determine alterations in network dynamics.     

Formulation of a killed whole cell pneumococcus vaccine - effect of aluminum adjuvants on the antibody and IL-17 response

Background

Streptococcus pneumoniae causes widespread morbidity and mortality. Current vaccines contain free polysaccharides or protein-polysaccharide conjugates, and do not induce protection against serotypes that are not included in the vaccines. An affordable and broadly protective vaccine is very desirable. The goal of this study was to determine the optimal formulation of a killed whole cell pneumococcal vaccine with aluminum-containing adjuvants for intramuscular injection.

Methods

Four aluminium-containing adjuvants were prepared with different levels of surface phosphate groups resulting in different adsorptive capacities and affinities for the vaccine antigens. Mice were immunized three times and the antigen-specific antibody titers and IL-17 responses in blood were analyzed.

Results

Although all adjuvants induced significantly higher antibody titers than antigen without adjuvant, the vaccine containing aluminum phosphate adjuvant (AP) produced the highest antibody response when low doses of antigen were used. Aluminum hydroxide adjuvant (AH) induced an equal or better antibody response at high doses compared with AP. Vaccines formulated with AH, but not with AP, induced an IL-17 response. The vaccine formulated with AH was stable and retained full immunogenicity when stored at 4°C for 4 months.

Conclusions

Antibodies are important for protection against systemic streptococcal disease and IL-17 is critical in the prevention of nasopharyngeal colonization by S. pneumoniae in the mouse model. The formulation of the whole killed bacterial cells with AH resulted in a stable vaccine that induced both antibodies and an IL-17 response. These experiments underscore the importance of formulation studies with aluminium containing adjuvants for the development of stable and effective vaccines.     

Strong decays ofΨ(4.03) andΨ(4.16) as radial excitations of charmonium

Considering the signals detected at 4.03 and 4.16 GeV as radial excitations of charmonium, we study their relative decay rates intoD \(\bar D\) ,D \(\bar D^* \) ,D ^* \(\bar D\) ,D ^* \(\bar D^* \) . We point out that one can understand these two peaks as ac \(\bar c\) 3S?2D wave state system with a large mixing angle in a Coulomb+linear interquark potential. We also examine the possibility that these two signals are respectively 3S and 4S wave excitations by studying a logarithmic charmonium potential model. We show that both these interpretations lead to drastically different predictions for the Ψ (4.16) decay rates (eitherD \(\bar D^* \) +D ^* \(\bar D\) orD \(\bar D\) mode is strongly suppressed) which would be very instructive to test experimentally.     

The photomagneton\hat B^{\left( 3 \right)} and electrodynamic conservation lawsand electrodynamic conservation laws

It is shown that the basic electrodynamical conservation laws are unaffected by the presence in free space of the photomagneton of light, $\hat B^{\left( 3 \right)} = B^{\left( 0 \right)} \hat J/\rlap{--} h$ , the fundamental photon property responsible for magnetization by light. The expectation value $B^{\left( 3 \right)} = \left\langle {\hat B^{\left( 3 \right)} } \right\rangle $ does not affect the Poynting vector, so that it does not contribute to electromagnetic flux density. The electromagnetic energy density can be expressed in terms ofB ⁽³⁾ through the equation $$\rlap{--} h\omega = \frac{1}{{\mu _0 }}\smallint B^{\left( 3 \right)} \cdot B^{\left( 3 \right) * } dV.$$ When light magnetizes matter, the unitB ⁽³⁾ of magnetic flux density per photon is transferred from light to matter. This is equivalent to an elastic transfer of angular momentum. Experimental indications for the existence ofB ⁽³⁾ are discussed.     

Covariant partition temperature model ande + e − annihilation

The rapidity distributions of inclusive \(e^ + e^ - \to h\bar h + \cdot \cdot \cdot\) of PEP and DESY experiments are analyzed in terms of the covariant partition temperatureT _p model. The estimates ofT _p ^* in the fireball system are comparable to the conventional temperature, the energy dependence follows approximately Stefan's law, the radius of the specific volume ralative to the energy density being ～1.18 fm. In the c.m.s. of collision, \(T_p = AW^a (W = \sqrt s in GeV)\) witha=0.60±0.05 andA=0.256±0.006, it is found \(T_p \cong {W \mathord{\left/ {\vphantom {W {\tfrac{3}{2}\left\langle {n_ \pm } \right\rangle }}} \right. \kern-0em} {\tfrac{3}{2}\left\langle {n_ \pm } \right\rangle }}\) . These properties hold also for \(\bar pp\) collision, but not forpp→π^?+...     

Inclusive production ofK *(892) andΣ(1385) in\bar p p interactions at 3.6 GeV/c and a test of factorization hypothesis

We present a study of the inclusive production ofK ^*(892) and ∑^t+-(1385)+cc at 3.6 GeV/c from \(\bar p\) p interactions. The sensitivity of the exposure is 35.4 events/μb. Longitudinal and transverse momentum distributions are presented. The indirect production ofK _s ⁰ from parentK ^* and that of Λ's from parent Σ(1385) are studied. The shape of thex distribution of Λ's for \(p\xrightarrow{{\bar p}}\Lambda \) are calculated from \(p\xrightarrow{p}\Lambda \) and \(p\xrightarrow{{\pi ^ - }}\Lambda \) and compared with the experimental distributions. The difference of antiparticle production cross-section ofK _s ⁰ in the central region is compared with the expectation from Mueller-Regge formalism.     

Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

Background

Domoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo.

Results

Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation.

Conclusion

In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.     

{^6_{\Lambda} \rm{H}} Modeled as {^4_{\Lambda} \rm{H}} + n + n

A three-body calculation for the \({^4_{\Lambda} \rm{He}}\) and \({^6_{\Lambda}{\rm H}}\) hypernuclei has been undertaken. The respective cores are \({^4_{\Lambda}{\rm H}}\) . The interactions in the \({^6_{\Lambda}{\rm He}}\) system, modeled as \({^4_{\Lambda} {\rm H+p+n}}\) , are reasonably well known. For example, the p n interaction is well determined by the p n scattering data, the \({^4_{\Lambda}{\rm H}}\) –p interaction can be fitted to the \({^5_{\Lambda}{\rm He}}\) binding energy. The \({^4_{\Lambda}{\rm He}}\) –n interaction can be fitted to α–n scattering data. For the ⁴He–n system the s-wave can be modeled alternatively as a repulsive potential or as an attractive potential with a forbidden bound state. We explore these alternatives in ⁶He, because the interaction comes into play in modeling \({^6_{\Lambda}{\rm He}}\) as well as in our \({^4_{\Lambda}{\rm H}}\) + n + n model of \({^6_{\Lambda}{\rm H}}\) , where the valence neutrons are Pauli blocked from the s-shell of the core nucleus.