The effects of morphine treatment on the NCAM and its signaling in the MLDS of rats.

Prolonged exposure to opiates induces a constellation of neuroadaptations, especially in the mesolimbic dopamine system (MLDS), which leads to alteration in the function of motivational circuitry. The neural cell adhesion molecule (NCAM) mediates cell-cell interactions and plays an important role in processes associated with neural plasticity. Moreover, it has been shown that NCAM were related to risk of alcoholism in human populations. Here, coimmunoprecipitation and western blotting were used to investigate whether morphine treatment induced alteration of the expression of NCAM or its signaling level in MLDS. The rats receiving escalating dose of morphine treatment were divided into three groups: morphine 1d, 3d and 5d group, which were injected subcutaneously with morphine hydrochloride for 1 day, 3 days and 5 days, respectively. Twelve hours after the last injection, animals were sacrificed and the tissues of ventral tegmental area (VTA), prefrontal cortex (PFC) and nucleus accumbens (NAc) were punched out to examine the expression of NCAM or its signaling level. The results showed that morphine treatment had no significant effect on the expression of NCAM, but downregulated the phosphorylation of NCAM-associated focal adhesion kinase (FAK) in the VTA and PFC of rats. In the NAc of rats, however, the expression of NCAM and its signaling were not altered significantly by morphine treatment. These results indicated that the downregulation of NCAM signaling in the VTA and PFC might be involved in the formation of morphine addiction.

NF-κB p65/p52 plays a role in GDNF up-regulating Bcl-2 and Bcl-w expression in 6-OHDA-induced apoptosis of MN9D cell.

Glial-cell-line-derived neurotrophic factor (GDNF) has been shown to protect dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA) toxicity. The mechanism underlying the antiapoptosis role of GDNF still needs further studies. We previously observed that nuclear factor-kappaB (NF-κB) signaling pathway, i.e. p65/p52, mediated the antiapoptosis role of GDNF in MN9D cells. Here, the DA cell line MN9D was used to explore the mechanisms underlying NF-κB p65/p52-mediated protection role of GDNF in DA neurons. The results showed that GDNF pretreatment blocked the apoptotic effects induced by 6-OHDA, with the upregulation of the antiapoptotic protein, Bcl-2 and Bcl-w, as well as the downregulation of the proapoptotic proteins, Bax and Bad. Furthermore, when sip100 plasmids were transfected into MN9D cells to inhibit the expression of p100, which was the precursor of p52, the effects of GDNF on upregulating Bcl-2 and Bcl-w were attenuated. These results indicated that GDNF could protect MN9D cells from apoptosis induced by 6-OHDA via upregulating Bcl-2 and Bcl-w expressions and downregulating Bax and Bad expressions. Moreover, NF-κB p65/p52 signaling mediated the effects of GDNF on Bcl-2 and Bcl-w expressions.

Repeated exposure to propofol in the neonatal period impairs hippocampal synaptic plasticity and the recognition function of rats in adulthood.

Anesthesia of neonates with propofol induces persistent behavioral abnormalities in adulthood. Although propofol-triggered apoptosis of neurons in the developing brain may contribute to the development of cognitive deficits, the mechanism of neurotoxicity induced by neonatal exposure to propofol remains unclear. In this study, the effects of neonatal propofol anesthesia on synaptic plasticity and neurocognitive function were investigated. Postnatal day 7 (PND-7) Sprague-Dawley rats were intraperitoneally injected with fat emulsion or 20, 40 or 60 mg/kg propofol for three consecutive days. The expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and postsynaptic density protein 95 (PSD-95) in the rat hippocampus at PND-10 and PND-12 was measured by Western blotting. The number of dendritic branches, total dendritic length and dendritic spine density were observed by Golgi-Cox staining 24 h and 72 h after the last propofol administration. Long-term potentiation (LTP) was measured electrophysiologically in hippocampus of PND-60 rats to evaluate the synaptic function. The learning and memory abilities of rats were evaluated by Morris water maze (MWM) experiments, Novel object recognition test (NORT) and Object location test (OLT) at PND-60. Our results showed that neonatal exposure to propofol significantly inhibited the expression of BDNF, TrkB and PSD-95 in the rat hippocampus. The number of dendritic branches, total dendritic length and dendritic spine density of neurons in the rat hippocampus were markedly reduced after neonatal propofol anesthesia. LTP was significantly diminished in hippocampus of PND-60 rats after repeated exposure to propofol in the neonatal period. Morris water maze experiments showed that repeated neonatal exposure to propofol significantly prolonged the escape latency and decreased the time spent in the target quadrant and the number of platform crossings. NORT and OLT showed that repeated neonatal exposure to propofol markedly reduced the Investigation Time for novel object or location. All of the results above indicate that repeated exposure to propofol in the neonatal period can impair hippocampal synaptic plasticity and the recognition function of rats in adulthood.

Involvement of NCAM in the effects of GDNF on the neurite outgrowth in the dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) exerts its biological effects via a multi-component receptor system including the ligand binding receptor--GDNF family receptor-alpha1 (GFRalpha1) and the signaling receptor--RET tyrosine kinase. Recently, the neural cell adhesion molecule (NCAM) has been identified as an alternative signaling receptor for GDNF. The purpose of this study was to investigate whether NCAM could mediate the protective effect of GDNF on injured dopamine (DA) neurons and to determine which cytoplasmic signal molecule associated with NCAM was activated while GDNF performing this effect. The results showed that the phosphorylation of NCAM-associated Fyn was upregulated with GDNF treatment, and this upregulation was inhibited by pre-treatment with the NCAM function-blocking antibody. Moreover, pre-treatment with the antibody could abolish the effect of GDNF on promoting the neurite outgrowth of these DA neurons, except for the effect of GDNF on promoting the expression of tyrosine hydroxylase (TH) in these DA neurons. These results suggest that NCAM is involved in the promotive effect of GDNF on the neurite outgrowth in lesioned DA neurons, but not involved in the promotive effect of GDNF on TH expression in these neurons.

Calbindin-D28K expression induced by glial cell line-derived neurotrophic factor in substantia nigra neurons dependent on PI3K/Akt/NF-kappaB signaling pathway.

Calbindin-D28K is a calcium-binding protein in neuronal cytoplasm, which has the capability to protect neurons from degeneration. It was reported that glial cell line-derived neurotrophic factor (GDNF) increased calbindin-D28K expression in dopaminergic neurons in vitro. It was observed in our research that GDNF also enhanced the expression of calbindin-D28K in adult rat substantia nigra neurons in vivo. To investigate the intracellular signaling pathways underlying the calbindin-D28K expression induced by GDNF, immunoblot and immunoprecipitation analyses were performed in our present study. Our results showed that injection of GDNF alone into substantia nigra of an adult rat brain increased the calbindin-D28K expression; meanwhile, the phosphorylation level of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) increased. However, the calbindin-D28K expression induced by GDNF was specifically blocked by the inhibitor of phosphatidylinositol 3-kinase (PI3K), but the inhibitor of ERK1/2 did not block the calbindin-D28K expression. Furthermore, GDNF administration also caused the nuclear factor kappaB (NF-kappaB/p65), to translocate from cytoplasm into the nucleus, and the inhibitor of PI3K effectively blocked the translocation. Immunoprecipitation assay results further demonstrated that it was the p65/p52 complex of NF-kappaB, rather than the p65/p50 complex that translocated into the neuronal nucleus. The calbindin-D28K expression induced by GDNF was also inhibited when the NF-kappaB signaling pathway was blocked by Helenalin. These results described a novel mechanism by which the activation of PI3K/Akt-->NF-kappaB (p65/p52) signaling pathway could play a role in the calbindin-D28K expression induced by GDNF.

Necrostatin-1 protection of dopaminergic neurons.

Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson's disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range (5-30 µM) elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson's disease.

The distributions and signaling directions of the cerebrospinal fluid contacting neurons in the parenchyma of a rat brain.

Many studies have been made on the distributions of CSF contacting neurons (CSF-CNs) in the parenchyma of the brain with horseradish peroxidase (HRP) or autoradiographics. A significant amount of data has shown that both HRP and autoradiographical substances could pass freely through the spaces of ependyma into the parenchyma of the brain. It is therefore possible that the results were not exact. We found that CB-HRP was a dependable tracer to CSF-CNs and studied the distributions and the signaling directions of cerebrospinal fluid contacting neurons (CSF-CNs) in the parenchyma of the brain with the cholera toxin subunit B with horseradish peroxidase (CB-HRP) tracing combined with transmission electron microscopy. The results were as follows: (1) CSF contacting tanycytes existed not only in the wall of the third ventricle (3V), but also in the walls of the lateral ventricle (LV), the fourth ventricle (4V) and the central canal (CC) of the spinal cord. (2) Some CSF contacting glia cells were observed in the lateral septal nucleus (LS). (3)The distal CSF-CNs in the parenchyma were found in LS, the anterodorsal thalamic nucleus (AD), the supramammillary nucleus (SuM), the dorsal raphe nucleus (DR), the floor of 4V and the lateral superior olive (LSO), but they were mainly found in DR and divided into groups A and B. (4) Axon terminals labeled by CB-HRP were found in the cavity of the brain ventricle. (5) The synaptic relationships between the neurons were labeled by CB-HRP in DR and no-labeled by CB-HRP in the parenchyma. Both synapses Gray I and II were found. It was significant that the presynaptic elements were formed by the neurons no-labeled CB-HRP and the postsynaptic elements labeled CB-HRP. Our results suggested firstly that the signaling directions of CSF-CNs in DR were only from the parenchyma to CSF.

[Short-term curative efficacy of autologous cytokine induced killer cells combined with low-dose IL-2 regimen containing immune enhancement by thymic peptide in elderly patients with B-cell chronic lymphocytic leukemia].

This study was purposed to evaluate the safety and curative effect of autologous cytokine induced killer cells (CIK) combined with low-dose IL-2 regimen containing immune enhancement of thymic peptide on elderly patients with B-cell chronic lymphocytic leukemia (B-CLL). Thymic peptide α1 was subcutaneously given as the immunoenhancement agent at a dose of 1.6 mg/d, 14 days as one cycle. Peripheral blood mononuclear cells (PBMNC) from 5 patients with B-CLL were isolated once a week to induce ex vivo CIK cells through culture in the context of interferon (IFN)-γ, interleukin (IL)-2 and anti-CD3 monoclonal antibody. The PBMNC were separated from patients before and after 14 days as one cycle of thymic peptide α1 administration. Parameters of amplification ability, effector cells quantity, lymphocyte subgroups percentage and antitumor cytotoxicity were compared before and after thymic peptide administration. The 5 patients were treated with CIK cells combined with low-dose IL-2 regimen immediately after injection of thymic peptide α1. The CIK cells plus low-dose IL-2 regimen containing thymic peptide enhancement was defined as: thymic peptide α1 1.6 mg/d was subcutaneously administered once every other day; (4 - 6) ×10(9) of CIK cells were transfused followed by IL-2 subcutaneous administration of 1 mU/d on days 1-10, 28 days as one cycle. Clinical evaluation parameters including cellular immunity function, CLL related biomarkers, disease state and infectious frequency and degree were investigated before and after CIK cells infusion puls IL-2. The results showed that the amount of amplified CIK cells, the percentage and amplification times of effector cells and antitumor cytotoxicity more significantly increased after thymic peptide α1 treatment than before its use (P < 0.05). The total 46 cycles of CIK cells infusion plus IL-2 were completed in the 5 CLL patients. No adverse reaction was observed. After treatment of CIK cells plus IL-2, the general conditions of 5 CLL patients were to different extent improved. Simultaneously, percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD56(+) cells in peripheral blood remarked by raised (P < 0.05), the serum level of ß2 microglobulin was significantly declined (P < 0.05), and the frequency and degree of infection was also decreased (P < 0.05). Following CIK cells plus IL-2 therapy, the transformation of disease state from partial remission (PR) to complete remission was seen in 3 patients, from stable disease (SD) to PR in 1 patient, and from progress of disease to SD in 1 patient. It is concluded that the regimen of autologous CIK cells combined with low-dose IL-2 containing immune enhancement of thymic peptide is safety and effective for the treatment of elderly patients with B-CLL.

[Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype avian influenza virus possessing H9N2 internal genes].

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008 (Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live bird market in East China during our epidemiological surveillance. To investigate the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with the characterization of the HPAIV. According to the newest unified nomenclature system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M) displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08 might be a natural reassortant virus. The phylogenetic analysis further indicated that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern China might provide the internal genes for Dk/ SD/009/08.

Expressions and possible roles of GDNF receptors in the developing dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) has an essential role in the survival and maturation of the dopaminergic (DA) neurons in the substantia nigra (SN) of mammalian embryonic brain. In addition to Ret, cell adhesion molecules (CAMs) were also proposed to function as transmembrane signaling receptors of GDNF. The present study was to investigate whether these transmembrane receptors of GDNF were correlated with the tyrosine hydroxylase (TH) expression of SN DA neurons during early developmental stage. RT-PCR and Western blot were performed to detect TH expression in SN of perinatal rats at mRNA and protein level respectively; meanwhile, Western blot was performed to detect the expressions of the transmembrane proteins including Ret, neural cell adhesion molecule-140 (NCAM-140), integrin ß1 and N-cadherin. The results showed that TH mRNA expression was positively correlated with both Ret and N-cadherin protein, while there was no correlation with NCAM-140 and integrin ß1; TH protein expression was correlated with all of these transmembrane molecules. These data suggested that the expression of either TH mRNA or TH protein was subject to the mediation of different transmembrane receptor combinations of GDNF.

The involvement of NF-kappaB p65/p52 in the effects of GDNF on DA neurons in early PD rats.

Glial cell line-derived neurotrophic factor (GDNF) can exert neuroprotective effects on the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons that are undergoing degeneration in Parkinson's disease (PD). In an attempt to investigate the molecular signaling mechanisms underlying GDNF protection the DA neurons from degeneration, we established early PD rat models in which the DA neurons in SNc were degenerating. Whether the cytoplasmic NF-kappaB signaling pathway was involved in the protection of GDNF on the degenerating DA neurons was examined in the present study. The results showed that the nuclear NF-kappaB p65 levels in the DA neurons increased when GDNF was injected into SNc of early PD rat models. Immunoprecipitation assays showed that the nuclear NF-kappaB p65/p52 complex levels increased after GDNF administration, while the p65/p50 complex levels decreased. These results indicated that GDNF could activate the NF-kappaB signaling pathway in the degenerating DA neurons. And it was the noncanonical NF-kappaB signaling pathway, which contained the NF-kappaB p65/p52 complex that was involved in the effects of GDNF on DA neurons.

Integrin beta1 is involved in the signaling of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the substantia nigra (SN) dopamine (DA) neurons. The transmembrane signaling of GDNF is mediated by a unique receptor system, including the ligand binding receptor GDNF family receptor alpha (GFRalpha) and the transmembrane signaling receptor Ret or neural cell adhesion molecule-140 (NCAM-140). Here, we found that another transmembrane cell adhesion molecule, integrin, a heterodimer consisting of alpha and beta subunits, also mediates the transmembrane signaling of GDNF. The results showed that the level of phosphorylated Src homology 2 domain containing (Shc), which was associated with the cytoplasmic domain of integrin beta1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin beta1 could form a complex with GFRalphal. The simulation of molecular modeling showed that four H-bonds were formed between integrin beta1 and GFRalpha. These data indicate that integrin beta1 is involved in the transmembrane signaling of GDNF and suggest that integrin beta1 may be an alternative signaling receptor for GDNF.

Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells.

AIM: To explore the intracellular mechanisms underlying the survival/differentiation effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine (DA) cells. METHODS: Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting. RESULTS: GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76+/-0.75; GDNF group, 18.63+/-0.95. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: control group, 3.65+/-0.88 and 2.49+/-0.42; GDNF group, 6.01+/-0.43 and 4.89+/-0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98+/-0.58. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Akt pathway-inhibited group, 3.79+/-0.62 and 2.50+/-0.25, respectively). CONCLUSION: The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.

Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement.

A visual gene-detecting technique using nanoparticle-supported gene probes is described. With the aid of gold nanoparticle-supported 3'-end-mercapto-derivatized oligonucleotide serving as detection probe, and 5'-end -amino-derivatized oligonucleotide immobilized on glass surface acting as capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nano-amplification" and silver staining. Different genotypes of Hepatitis B and C viruses in the serum samples from infected patients were detected using home-made HBV, HCV, and HBV/HCV gene chips by the gold/silver nanoparticle staining amplification method. The present visual gene-detecting technique may avoid limitations with the reported methods, for its high sensitivity, good specificity, simplicity, speed, and cheapness. This technique has potential applications in many fields, especially in multi-gene detection gene chips coupled with the detection will find applications in clinic. Additionally, resonance Rayleigh light scattering (RLS) spectroscopy is used, for the first time, to judge and monitor the immobilization of gene probes on gold nanoparticle surfaces.