Protective effects of naringin against gp120-induced injury mediated by P2X7 receptors in BV2 microglial cells.

This study was aimed at exploring the effects of P2X7 receptors on gp120-induced injury and naringin's protective effects against gp120-induced injury in BV2 microglia. BV2 microglia injury model was established by gp120 treatment and MTS assay was used to verify whether naringin has a cell-protective effect against gp120-induced injury. Changes in P2X7 receptor expression were assayed using RT-PCR, qPCR, and western blot. Results showed that the ODs of the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.91 ± 0.10, 0.71 ± 0.09, 0.83 ± 0.10, and 0.83 ± 0.10, respectively. Compared to the control group, the gp120 group showed a significantly decreased cell survival rate. Cell survival rates of the gp120+naringin group increased significantly compared to those of the gp120 group, while no difference was observed when compared to the gp120+BBG group. The relative P2X7 mRNA expression levels in the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.73 ± 0.06, 1.05 ± 0.06, 0.78 ± 0.05, and 0.81 ± 0.04, respectively. The corresponding P2X7 protein expression levels were 0.46 ± 0.04, 0.79 ± 0.04, 0.38 ± 0.07, and 0.42 ± 0.06. P2X7 mRNA and protein expression in the gp120 group increased significantly compared to those in the control group, and declined in the gp120+naringin group compared to those in the gp120 group. Therefore, P2X7 receptors might be involved in gp120-induced injury in BV2 microglia, and naringin might play a protective role by inhibiting the up-regulated expression of P2X7 receptors.

Characterization of chicken natural resistance-associated macrophage protein encoding genes (Nramp1 and Nramp2) and association with salmonellosis resistance.

Natural resistance-associated macrophage protein 1 and 2 encoding genes (Nramp1 and Nramp2) are related to many diseases. We cloned the cDNA of chicken Nramp1 and Nramp2 genes, characterized their expression and polymorphisms, and investigated the association of some SNPs with resistance to salmonellosis. The Nramp1 cDNA was 1746 bp long and the Nramp2 cDNA was 1938 bp long. These cDNAs are similar to previously reported cDNAs, varying by two and one amino acids, respectively. The chicken Nramp1 gene expressed predominantly in liver, thymus and spleen in both females and males. The Nramp2 gene expressed in almost all tissues, but predominantly in breast muscle, leg muscle, cerebrum, cerebellum, lung, kidney, and heart in both females and males. We identified 45 SNPs and 2 indels in the chicken Nramp1 gene; three of 13 SNPs in the exons were missense mutations (Arg223Gln, Ala273Glu and Arg497Gln). Association analysis indicated that A24101991G is significantly associated with chicken salmonellosis resistance. These results will be useful for functional investigation of chicken Nramp1 and Nramp2 genes.

The C-terminal cysteine of turbot Scophthalmus maximus translationally controlled tumour protein plays a key role in antioxidation and growth-promoting functions.

The translationally controlled tumour protein (TCTP) of turbot Scophthalmus maximus (SmTCTP) contains only one cysteine (Cys¹7°) at the C-terminal end. The biological role of this C-terminal Cys¹7° in the antioxidation and growth-promoting functions of SmTCTP was examined by site-directed mutation of C170A (Cys¹7° →Ala¹7°). It was found that C170A mutation not only obviously decreased the antioxidation capacity of the mutant-smtctp-transformed bacteria exposed to 0·22 mM hydrogen peroxide, but also significantly interrupted the normal growth and survival of the mutant-smtctp-transformed bacteria and flounder Paralichthys olivaceus gill (FG) cells, indicating a key role played by Cys¹7° in the antioxidation and growth-promoting functions of SmTCTP. This study also suggested that the self-dimerization or dimerization with other interacting proteins is critical to the growth-promoting function of SmTCTP.

Granulocyte colony-stimulating factor improves neurological function and angiogenesis in intracerebral hemorrhage rats.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) plays a role in regulating phosphatidylinositol-3-kinase/serine/threonine kinase (PI3K/AKT) pathway, affecting cell proliferation and apoptosis, and inducing vascular endothelial growth factor (VEGF) expression. This study investigated the mechanism of G-CSF on angiogenesis and neural protection after intracerebral hemorrhage (ICH). MATERIALS AND METHODS: The rats were divided into four groups, including sham, ICH, ICH+G-CSF, and ICH+G-CSF+LY294002 (PI3K/AKT signaling pathway specific inhibitor). Cerebral neurological dysfunction was tested by Garcia scoring. Cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. Angiogenesis marker CD34 expression, PI3K/AKT signaling pathway, B-cell lymphoma-2 (Bcl-2), and VEGF expressions were compared by IHC. Rat cerebral nerve RN-c cells were divided into four groups, including control, oxygen-glucose deprivation (OGD), OGD+G-CSF, and OGD+G-CSF+LY294002. RESULTS: Neurological dysfunction was more evident; CD34+ cell number, VEGF expression, and cell apoptosis significantly increased; phosphorylated AKT (p-AKT) and Bcl-2 levels markedly reduced in ICH group compared with sham group. G-CSF apparently up-regulated p-AKT and Bcl-2 expressions, attenuated cell apoptosis, and elevated CD34+ cell number. LY294002 significantly decreased p-AKT, Bcl-2, and VEGF expressions, and alleviated the cell apoptosis protective and angiogenesis effect induced by G-CSF. OGD treatment induced RN-c cell apoptosis, down-regulated p-AKT and Bcl-2 expressions, and enhanced the tube capacity of vascular endothelial cells (VEC). G-CSF markedly elevated p-AKT and Bcl-2 contents in RN-c cells, declined cell apoptosis, increased p-AKT and VEGF levels in VEC, and enhanced tube capacity. CONCLUSIONS: G-CSF enhanced PI3K/AKT signaling pathway activity, promoted Bcl-2 and VEGF expression, reduced nerve cell apoptosis, and enhanced tube capacity of VECs, which may be the mechanism of G-CSF in improving neurological function and angiogenesis after ICH.

DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.

Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.

The involvement of P2X3 receptors of rat sympathetic ganglia in cardiac nociceptive transmission.

In this work we have examined the effects of P2X3 receptor antagonist A-317491 on P2X3 expression in superior cervical ganglion (SCG) from naive and myocardial ischemic rats to observe the effect of P2X3 receptors in cardiac nociceptive transmission. A-317491 improved nociceptive behavior. In the ganglia neurons of rats at 14 days after myocardial ischemic injury, the staining of P2X3 receptor in myocardial ischemic groups appeared to be more intense than those of naive rats detected by immunohistochemistry. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 immunoreactivity was lower than that in myocardial ischemic rats. The signals of P2X3 and its protein and mRNA in myocardial ischemic groups were higher than those in control group measured by western blotting and in situ hybridization. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 and its mRNA was lower than that in myocardial ischemic rats. These results suggest the involvement of P2X3 receptors in cardiac nociceptive transmission and A-317491 may inhibit the transmission mediated by P2X3 receptors in rat SCG after myocardial ischemia.

Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons.

Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (IATP) in rat DRG neurons. TMP (0.1-1 mM) concentration-dependently inhibited ATP (100 microM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 microM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose-response curves for IATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration-response curve of IATP downward markedly and the two EC50 values were very close (75 vs. 82 microM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on IATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on IATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.

A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5'-nucleotidase.

No attempt has been made so far to classify the subtypes of presynaptic inhibitory adenosine receptors located in the myenteric plexus and to localize ecto-ATPase and 5'-nucleotidase in the intestine. The release of [3H]acetylcholine and smooth muscle responses to acetylcholine were measured and the effect of selective adenosine receptor ligands was studied using field-stimulated isolated longitudinal muscle strips of guinea-pig ileum. Release of ATP and its hydrolysis rate were also measured using the luciferin-luciferase technique. A histochemical method combined with electron microscopy was used for localization of ecto-ATPase and 5'-nucleotidase, enzymes responsible for destruction of extracellular ATP, ADP and AMP. Subtype-selective A1-receptor agonists and antagonists inhibited and enhanced, respectively, the release of acetylcholine associated with neuronal activity. A significant amount of ATP was released in response to electrical stimulation and administration of carbamylcholine. The release of ATP was inhibited by atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3-receptor antagonist. Hydrolysis of ATP was rapid and resulted in an accumulation of extracellular adenosine involved in presynaptic A1-receptor-mediated inhibition of acetylcholine release. While the inhibitory effect of adenosine and ATP was significantly potentiated by dipyridamol, an adenosine uptake blocker, that of 2-ms ATP was not. The effect of ATP was not competitively antagonized by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1-receptor antagonist. In conclusion, axon terminals of cholinergic interneurons are equipped with inhibitory A1- and P2 gamma-receptors. Therefore, both adenosine and ATP control the release of acetylcholine through these receptors. ATP is mainly released from the smooth muscle in response to stimulation of M3-muscarinic receptors by endogenous acetylcholine (cascade transmission [Vizi E. S. et al. (1992) Neuroscience 50, 455-465]) and is rapidly hydrolysed by ecto-ATPase localized on the surface of the smooth muscle and axon terminals producing ADP and AMP, and by 5'-nucleotidase present only on the surface of smooth muscle cells producing adenosine.

Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP.

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca2+]o-dependent manner. The Na+ channel blocker, tetrodotoxin (1 microM) inhibited the stimulation-evoked release of endogenous ATP and of [3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz. when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 microM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A1-adenosine receptors. When the temperature was reduced to 7 degrees C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9 +/- 5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the Km (apparent Michaelis constant) and Vmax (apparent maximal velocity) were 475 +/- 24 microM and 3.50 +/- 0.18 nmol/min per mg for ectoATPase and 1550 +/- 120 microM and 14.5 +/- 0.9 nmol/min per mg tissue for 5'-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca2+]o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue. The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.

Prolactin plays a stimulatory role in ovarian follicular development and egg laying in chicken hens.

The aim of this study was to show a stimulatory role in ovarian follicle development by prolactin (PRL) in chicken hens. In experiment 1, anti-PRL antibodies were generated in hen plasma by intramuscular administrations of recombinant PRL antigen. Egg laying remained at levels lower (P < 0.05) in the PRL-immunized group than in the BSA-immunized group of hens, whereas development of incubation was depressed in the former but not the latter group. Throughout the experiment, plasma PRL concentrations were lower in the PRL-immunized hens than in non-incubating control hens; LH concentrations were similar between the PRL- and BSA-immunized hens until the end of the experiment when LH was lower in the BSA-immunized hens (P < 0.05). In experiment 2, anti-PRL receptor (PRLR) antibodies were raised in hens with the use of immunizations against recombinant PRLR extracellular domain. Immunization against PRLR initially increased the egg-laying rate when measured under the short photoperiod (12 h) but blocked the laying rate increase that occurred in the BSA-immunized control hens when the photoperiod was extended from 12 to 16 h. The development of incubation behavior was not affected by immunization against PRLR nor was plasma PRL or LH concentration. In experiment 3, when the egg-laying rate was depressed in PRL immunization hens, developmental speed of large white follicles was found to be slower than in the BSA-immunized control hens (P < 0.05). These results indicate that immunization against PRL slows down ovarian follicular development and reduces hen egg-laying performance, suggesting that PRL plays a stimulatory role in ovarian follicular development in chicken hens.

Effects of immunisation against leptin on feed intake, weight gain, fat deposition and laying performance in chickens.

1. Three experiments were conducted to study the effects of leptin on weight gain and body composition in laying hens. 2. The effects of immunisation against chicken leptin on feed intake (FI), fat deposition and laying rate were observed in laying Guangdong yellow-feathered hens. Ten hens were inoculated with leptin immunogen on d 3, 31, 63 and 84, together with 10 control hens immunised with bovine serum albumin (BSA). In the 100-d experiment, immunisation against leptin increased blood anti-leptin antibody titres, slightly reduced plasma T3 concentrations, slightly decreased FI and increased live weight; however, laying rate was significantly depressed and abdominal fat mass was increased by the end of the 100-d experiment. 3. Passive immunisation of 50-d-old pullets with yolk extract containing anti-leptin antibody IgY significantly increased FI within 6 h of treatment compared with physiological saline treated controls. 4. In growing 70-d-old pullets, inoculation with 0.5 (group 1) or 1 (group 2) ml leptin immunogen on d 1 and 28 of the experiment slightly increased FI and significantly increased daily gain compared with BSA-immunised control pullets. Abdominal fat mass on d 49 increased from 48+/-4.5 g in controls to 66+/-3.5 and 80+/-3.1 g in groups 1 and 2, respectively. 5. It was suggested that immunisation against leptin mimicked loss of leptin bioactivity and might become a novel technique to stimulate fat growth in certain types of animal production.

Positive feedback modulation of acetylcholine release from isolated rat superior cervical ganglion.

The effects of selective nicotinic acetylcholine (ACh) receptor (nAChR) agonists and antagonists on the stimulation-evoked release of [3H]ACh were studied in rat isolated superior cervical ganglion loaded with [3H]choline and superfused in a 2-ml chamber. Nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), but not cytisine, increased the stimulation (2 Hz)-evoked release of [3H]ACh in a concentration-dependent manner. The rank order of potency to increase stimulation-evoked release for the nAChR agonists (nicotine > DMPP >> cytisine) suggests that the beta4 subunit of nAChRs is not involved in the release. The finding that alpha-bungarotoxin was effective in preventing the effect of DMPP and itself significantly reduced the release indicates that the alpha7 subunit is located presynaptically and may be involved in the positive feedback modulation. Hexamethonium inhibited the effect of DMPP with an apparent dissociation constant (Kd) of 11.5 +/- 1.5 microM. Hexamethonium and other nAChR antagonists, i.e., (+)-tubocurarine (100 microM), mecamylamine (3 microM), dihydro-beta-erythroidine (3 microM), pancuronium (10 microM) and alpha-bungarotoxin (2 microM), also decreased the stimulation-evoked release of [3H]ACh. The effect of hexamethonium was independent of stimulation frequency (2, 10 and 30 Hz) applied. Atropine enhanced the stimulation-evoked release of ACh, indicating that there is negative feedback modulation of ACh release associated with neuronal activity. In contrast, when the nicotinic positive feedback was prevented by hexamethonium, atropine failed to enhance the release. These findings indicate that muscarinic receptor-mediated inhibition of ACh release functions in cases in which the release is enhanced by ACh via stimulation of presynaptic nAChRs. A similar interaction was found between A1 receptor-mediated reduction and nAChR-mediated positive feedback modulation of [3H]ACh release. The results suggest the presence of positive feedback modulation of ACh release via presynaptic nAChRs in rat superior cervical ganglion.

Presynaptic release of ATP from superior cervical ganglion of rats modulated by various receptors.

AIM: To determine whether adenosine 5'-triphosphate (ATP) is released from the superior cervical ganglion (SCG) of rats and whether the release is regulated by presynaptic mechanism. METHODS: Using the luciferin-luciferase technique. RESULTS: Electric stimulation evoked the release of ATP from the rat SCG. Adenosine (100 mumol.L-1), P1(A1) purinoceptor agonist N6-cyclopentyladenosine (0.1 mumol.L-1), the muscarinic agonist oxotremorine (1 mumol.L-1), and 5-hydroxytryptamine (100 mumol.L-1) decreased the evoked release of ATP from the rat SCG. On the contrary, P1(A1) purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 nmol.L-1), P2 purinoceptor antagonist pyridoxal-5-phosphate-6-azophenyl-2',4-disulphonic acid (10 mumol.L-1), muscarinic antagonist atropine (1 mumol.L-1), alpha 2 adrenoceptor antagonist yohimbine (3 mumol.L-1), D2 dopamine receptor antagonist sulpiride (20 mumol.L-1), and histamine (100 mumol.L-1) increased the evoked release of ATP from the rat SCG. CONCLUSION: ATP is released from the rat SCG and the release of ATP can be presynaptically modulated by P1(A1), P2, muscarinic, alpha adrenergic, D2, 5-HT, and H1 receptor agonists and antagonists.