Expression and distribution of synaptotagmin family members in the zebrafish retina.

Synaptotagmins belong to a large family of proteins. Although various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1-10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR, qPCR, and in situhybridization, focusing on the family members whose products likely underlie Ca2+ -dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5, and 7). Most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5, and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ -dependent processes in different retinal neurons.

Synaptotagmin 1 Is Involved in Neuropathic Pain and Electroacupuncture-Mediated Analgesic Effect.

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral "Sanjinjiao" and "Zusanli" acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

Possible mechanism of Vitis vinifera L. flavones on neurotransmitters, synaptic transmission and related learning and memory in Alzheimer model rats.

BACKGROUND: This study explored the possible mechanism of flavones from Vitis vinifera L. (VTF) on neurotransmitters, synaptic transmission and related learning and memory in rats with Alzheimer disease (AD). METHODS: The researchers injected amyloid-ß(25-35) into the hippocampus to establish AD model rats. The Sprague-Dawley (SD) rats were divided into a control group, a donepezil group, an AD model group, a VTF low-dose group, a VTF medium-dose group and a VTF high-dose group. The researchers detected the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) according to kit instructions. The protein expression of brain-derived neurotrophic factor (BDNF), synaptotagmin-1 (SYT1) and cyclic adenosine monophosphate response element binding protein (CREB) in the rats' hippocampi was detected by immunohistochemistry and Western blot, and the gene expression of cAMP-regulated enhancer (CRE) was detected by real-time quantitative polymerase chain reaction (PCR). RESULTS: VTF may enhance the protein expression of p-CREB, BDNF and SYT1 in rat hippocampi, depending on dose. The messenger RNA (mRNA) level of CREB was significantly higher in the VTF high-dose group than in the model group, which was consistent with the results of Western blotting. VTF may reduce the activity of AChE and increase that of ChAT in rat hippocampi. Finally, VTF effectively improved the learning and memory abilities of AD rats. CONCLUSIONS: VTF can promote synaptic plasticity and indirectly affect the expression of cholinergic neurotransmitters, which may be one mechanism of VTF protection in AD rats.

Chronic acarbose treatment alleviates age-related behavioral and biochemical changes in SAMP8 mice.

The administration of maintaining the homeostasis of insulin/insulin-like growth factor 1 (IGF-1) signaling and/or glucose metabolism may reverse brain aging. In the present study, we investigated the effect of acarbose, an inhibitor of α-glucosidase, on age-related behavioral and biochemical changes. The SAMP8 mice were randomly divided into old control group and acarbose-treatment group. The mice in the acarbose group were administered acarbose (20 mg/kg/d, dissolved in drinking water) orally from 3 to 9 months of age when a new group of 3-month-old mice was added as young controls. The results showed that the aged controls exhibited declines in sensorimotor ability, open field anxiety, spatial and non-spatial memory abilities, decreased serum insulin levels, increased IGF-1 receptor and synaptotagmin 1 (Syt1) levels and decreased insulin receptor, brain-derived neurotrophic factor (BDNF) and syntaxin 1 (Stx1) levels in the hippocampal layers. The age-related behavioral deficits correlated with the serological and histochemical data. Chronic acarbose treatment relieved these age-related changes, especially with respect to learning and memory abilities. This protective effect of acarbose on age-related behavioral impairments might be related to changes in the insulin system and the levels of BDNF, IGF-1R, and the pre-synaptic proteins Syt1 and Stx1. In conclusion, long-term treatment with acarbose ameliorated the behavioral deficits and biochemical changes in old SAMP8 mice and promoted successful aging. This study provides insight into the potential of acarbose for the treatment of brain aging.

Molecular cloning and expression analysis of the synaptotagmin-1 gene in the hypothalamus and pituitary of Huoyan goose during different stages of the egg-laying cycle.

BACKGROUND: Synaptotagmin-1 (Syt1) is an abundant, evolutionarily conserved integral membrane protein that plays essential roles in neurotransmitter release and hormone secretion. Neurotransmitters secreted by hypothalamic neurons can alter GnRH (gonadotropin-releasing hormones) neuronal activity by binding to and activating specific membrane receptors in pituitary cells and, in turn, control the release of gonadotropin hormones from the pituitary gland. To reveal the influence of Syt1 on the process of goose egg-laying, we cloned and characterized the cDNA of goose Syt1 originating from hypothalamus and pituitary tissues of Huoyan goose and investigated the mRNA expression profiles during different stages of the egg-laying cycle. METHODS: Hypothalamus and pituitary tissues were obtained from 36 Huoyan geese in the pre-laying period, early laying period, peak-laying period, and ceased period. The cDNA sequences of goose Syt1 were cloned and characterized from Huoyan goose tissues using 5'-RACE and 3'-RACE methods. Multiple alignments and phylogenetic analyses of the deduced Syt1 amino acid sequence were conducted using bioinformatics tools. The expression profiles of the Syt1 mRNA in the hypothalamus and pituitary during pre-laying, early laying, peak-laying and ceased period were examined using real-time PCR (qRT-PCR). RESULTS: The cDNA of Syt1 consisted of a 274 bp 5' UTR, a 1266 bp open reading frame (ORF) encoding 421 amino acids, and a 519 bp 3' UTR. The deduced amino acid sequence of goose Syt1 is highly conserved with the sequence from other species, especially with birds (more than 98%), and contains two protein kinase C2 conserved regions (C2 domain) from amino acids residue 157 to 259 and 288 to 402. The results of qRT-PCR demonstrated that the expression of Syt1 mRNA increased from the pre-laying period to the peak-laying period, reached its peak in the peak-laying period, and then decreased in the ceased period. CONCLUSIONS: To the best of our knowledge, this study is the first to obtain full-length cDNA sequences of the goose Syt1 gene, and the results of Syt1 mRNA expression profiling in the hypothalamus and pituitary tissues suggested that Syt1 may play an important role in regulating the secretion of hormones relevant to the reproduction and egg-laying of female geese.

Imbalance of leptin pathway and hypothalamus synaptic plasticity markers are associated with stress-induced depression in rats.

Increasing evidences have indicated that chronic stress is a contributing risk factor in the development of psychiatric illnesses including depression. The mechanisms of their psychopathology are multifaceted and include, besides others, alterations in neuroendocrine function and brain plasticity. In the present study, we investigated the behavior of stressed animals by the sucrose preference test, open field test (OFT), forced swimming test (FST), and tail-suspension test (TST). The response of hypothalamic-pituitary-adrenal (HPA) axis, leptin pathway, and synaptic plasticity markers in the hypothalamus were also detected. Our data demonstrated that chronic unpredictable mild stress (CUMS) could induce depression-like behavior in rat model, accompanied with the hyperactivity of HPA axis. The serum leptin level and hypothalamic mRNA expression of leptin receptor (LEPR) were both decreased. Results of Pearson test showed that the decreased serum leptin level was negatively related with the locomotion and rearing frequency in the open-field test, and the hypothalamic mRNA expression of LEPR was inversely related to serum CORT concentration. Moreover, our results showed that the mRNA expression of synaptotagmin I and synapsin I was both increased in the hypothalamus of CUMS rats, providing new evidence for the synaptic plasticity change in the hypothalamus of depressive rats. Furthermore, our results demonstrated that the mRNA expression of synaptotagmin I, but not synapsin I, was correlated with the depression-like behaviors and HPA axis hyperactivity in CUMS rats. Together with our previous results, the current findings suggested that a CUMS rat model could be effectively used to study molecular mechanisms underling the depressive symptomatology.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

BACKGROUND: Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. RESULTS: The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. CONCLUSIONS: Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Spatiotemporal pattern of concurrent spinal and supraspinal NF-κB expression after peripheral nerve injury.

UNLABELLED: The expression of NF-κB in the spinal cord is associated with neuropathic pain. However, little is known about its expression beyond the spinal cord. Here we examined a spatial and temporal pattern of the NF-κB expression in both spinal and supraspinal regions. After chronic constriction injury (CCI) of the sciatic nerve, NF-κB (p65) expression was significantly increased in the ipsilateral spinal cord. In contrast, the NF-κB expression in the contralateral primary somatosensory cortex was decreased with no significant differences seen in the thalamus. In the contralateral anterior cingulate cortex, the NF-κB expression was increased significantly on day 14 as compared with the sham group. In the contralateral amygdala, the NF-κB expression showed a time-dependent downregulation after CCI, which became significant on day 14. MK-801 reduced nociceptive behaviors and reversed the direction of NF-κB expression. These results indicate that the CCI-induced expression of p65 NF-κB is both time-dependent and region-specific, in areas that process both sensory-discriminative and motivational-affective dimensions of pain. PERSPECTIVE: This article presents a spatiotemporal mapping of the NF-κB expression in spinal and supraspinal regions after peripheral nerve injury. These findings point to an involvement of NF-κB beyond the spinal cord in both the sensory discriminative and emotional affective aspects of neuropathic pain processing.

Suppressive effects of extracts from the aerial part of Coriandrum sativum L. on LPS-induced inflammatory responses in murine RAW 264.7 macrophages.

BACKGROUND: Coriandrum sativum is used not only as a spice to aid flavour and taste values in food, but also as a folk medicine in many countries. Since little is known about the anti-inflammatory ability of the aerial parts (stem and leaf) of C. sativum, the present study investigated the effect of aerial parts of C. sativum on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We further explored the molecular mechanism underlying these pharmacological properties of C. sativum. RESULTS: Ethanolic extracts from both stem and leaf of C. sativum (CSEE) significantly decreased LPS-induced nitric oxide and prostaglandin E(2) production as well as inducible nitric oxide synthase, cyclooxygenase-2, and pro-interleukin-1beta expression. Moreover, LPS-induced IkappaB-alpha phosphorylation and nuclear p65 protein expression as well as nuclear factor-kappaB (NF-kappaB) nuclear protein-DNA binding affinity and reporter gene activity were dramatically inhibited by aerial parts of CSEE. Exogenous addition of CSEE stem and leaf significantly reduced LPS-induced expression of phosphorylated mitogen-activated protein kinases (MAPKs). CONCLUSION: Our data demonstrated that aerial parts of CSEE have a strong anti-inflammatory property which inhibits pro-inflammatory mediator expression by suppressing NF-kappaB activation and MAPK signal transduction pathway in LPS-induced macrophages.

Purple sweet potato color repairs d-galactose-induced spatial learning and memory impairment by regulating the expression of synaptic proteins.

Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins used to color food (E163), has been reported to possess a variety of biological activities, including anti-oxidant, anti-tumor, and anti-inflammatory. The effect of PSPC on the spatial learning and memory of mice treated with d-galactose (d-gal) was evaluated by the Morris water maze; d-gal-treated mice had decreased performance compared with mice in the vehicle and PSPC groups, while the PSPC+d-gal group showed significantly shortened escape latency to platform, increased swimming speed, more target quadrant search time and more platform crossings as compared with the d-gal group. Brain functions, such as memory formation and recovery of function after injury, depend on proper regulation of the expression levels of the pre- and post-synaptic proteins. We investigated the expression of four pre-synaptic proteins (growth-associated protein-43, synapsin-I, synaptophysin, and synaptotagmin) and two post-synaptic proteins (post-synaptic density protein-95 and Ca(2+)/calmodulin-dependent protein kinase II) in the hippocampus and cerebral cortex, respectively, in response to different treatments. Western blotting analysis showed that there were significant decreases in the expression of these representative synaptic proteins in the hippocampus and cerebral cortex of d-gal-treated mice. Interestingly, these decreased expression levels of synaptic proteins could be reversed by PSPC. The levels of expression of these representative synaptic proteins in mice treated with PSPC alone were not significantly different from those in untreated mice. The results of this study suggested that memory impairment and synaptic protein loss in d-gal-treated mice may be improved by treatment with PSPC.

Direct interaction of SNARE complex binding protein synaphin/complexin with calcium sensor synaptotagmin 1.

Although the binding of synaphin (also called complexin) to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is critical for synaptic vesicle exocytosis, the exact role of synaphin remains unclear. Here, we show that synaphin directly binds to synaptotagmin 1, a major Ca(2+) sensor for fast neurotransmitter release, in a 1:1 stoichiometry. Mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the C-terminal region is essential for the interaction between these two proteins. Binding was sensitive to ionic strength, suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven consecutive glutamic acid residues (residues 108-114) at the C-terminal region of synaphin to alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. Furthermore, a peptide from the C-terminus of synaphin (residues 91-124) blocked the binding of synaptotagmin 1 to synaphin, an effect that was abolished by mutating the consecutive glutamic acid residues to alanine. Immunoprecipitation experiments with brain membrane extracts showed the presence of a complex consisting of synaphin, synaptotagmin 1, and SNAREs. We propose that synaphin recruits synaptotagmin 1 to the SNARE-based fusion complex and synergistically functions with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis.

Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis.

Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) play a key role in the pathogenesis of osteoarthritis (OA). Anti-inflammatory agents capable of suppressing the production and catabolic actions of these cytokines may have therapeutic potential in the treatment of OA and a range of other osteoarticular disorders. The purpose of this study was to examine the effects of curcumin (diferuloylmethane), a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in human articular chondrocytes maintained in vitro. The effects of curcumin were studied in cultures of human articular chondrocytes treated with IL-1beta and TNF-alpha for up to 72h. Expression of collagen type II, integrin beta1, cyclo-oxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) was monitored by western blotting. The effects of curcumin on the expression, phosphorylation and nuclear translocation of protein components of the NF-kappaB system were studied by western blotting and immunofluorescence, respectively. Treatment of chondrocytes with curcumin suppressed IL-1beta-induced NF-kappaB activation via inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation and p65 nuclear translocation. Curcumin inhibited the IL-1beta-induced stimulation of up-stream protein kinase B Akt. These events correlated with down-regulation of NF-kappaB targets including COX-2 and MMP-9. Similar results were obtained in chondrocytes stimulated with TNF-alpha. Curcumin also reversed the IL-1beta-induced down-regulation of collagen type II and beta1-integrin receptor expression. These results indicate that curcumin has nutritional potential as a naturally occurring anti-inflammatory agent for treating OA through suppression of NF-kappaB mediated IL-1beta/TNF-alpha catabolic signalling pathways in chondrocytes.

Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice.

OBJECTIVE: We investigated the antiinflammatory properties of a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. MATERIAL AND METHODS: Zymosan-induced arthritis and pleurisy in Swiss and C57/Bl6 mice (n = 10 per group). Western blot analysis was performed to analyze nuclear factor-kappaB (NFkappaB) translocation in mice peritoneal macrophages stimulated in vitro with zymosan (500 microg/ml). ELISA was performed to evaluate cytokine levels in knee joints. Values of p

[Effect of ginkgolide B on the function of rat aorta smooth cells and U937 cells stimulated by oxLDL].

AIM: To investigate the effect of ginkgolide B on the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL or PAF. In addition, to analyze whether the effect of oxLDL is mediated through PAF receptor. METHODS: Using 3H-Tdr incorporation assay, the proliferation of VSMC was measured. The protein and mRNA level of MCP-1 and IL-8 in U937 cells were determined by RT-PCR and ELISA. Using Western blotting the p65 and IkappaB was quantified. The binding of oxLDL to U937 cell was measured by a radio-ligand binding assay of 3H-PAF. RESULTS: Ginkgolide B inhibited, in dose-dependent manner, the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL, and inhibited the oxLDL-induced p65 activation and depletion of IKappaB. oxLDL inhibited PAF binding to U937 cells. CONCLUSION: Ginkgolide B, as a PAF antagonist, possesses the effect of inhibiting the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL in vitro. The effect of oxLDL is, at least in part, mediated through PAF receptor.