Gamma-aminobutyric acid (GABA) receptor mediates suanzaorentang, a traditional Chinese herb remedy, -induced sleep alteration

Summary The sedative-hypnotic medications, including benzodiazepines and non-benzodiazepines, are the most common treatments for insomnia. However, concerns regarding patterns of inappropriate use, dependence and adverse effects have led to caution in prescribing those sedative-hypnotic medications. On the other hand, a traditional Chinese herb remedy, suanzaorentang, has been efficiently and widely used in clinic for insomnia relief without severe side effects in Asia. Although suanzaorentang has been reported to improve sleep disruption in insomniac patients, its mechanism is still unclear. The present study was designed to elucidate the effects of oral administration of suanzaorentang on physiological sleep-wake architectures and its underlying mechanism in rats. We found that oral administration of suanzaorentang at the beginning of the dark onset dose-dependently increased non-rapid eye movement sleep (NREMS) during the dark period, but had no significant effect on rapid eye movement sleep (REMS). Our results also indicated that intracerebroventricular (ICV) administration of γ-aminobutyric acid (GABA) receptor type A antagonist, bicuculline, significantly blocked suanzaorentang-induced enhancement in NREMS during the dark period, but GABA_B receptor antagonist, 2-hydroxysaclofen had no effect. These results implicated that this traditional Chinese herb remedy, suanzaorentang increases spontaneous sleep activity and its effects may be mediated through the GABA_A receptors, but not GABA_B receptors.     

鲢（Hypophthalmichthys molitrix）肿瘤坏死因子基因的克隆与表达分析

肿瘤坏死因子(TNF)是一种炎症细胞因子,在非特异性免疫系统中发挥着重要作用.TNF由单核细胞或巨噬细胞产生,能直接造成肿瘤细胞的死亡,并参与机体炎症和免疫应答的调节.用RACE(rapid amplification of cDNAends)-PCR方法,从鲢总RNA反转录产物中获得了1 254 bpTNF cDNA全序列.该序列包含394 bp的5'端非编码区,398 bp的3'端非编码区和462 bp的开放阅读框.鲢TNF的开放阅读框编码239个氨基酸,其中包含构成一对二硫键的2个保守半胱氨酸.同时利用RT-PCR技术,对该基因在鲢鱼体内不同组织之间的表达差异进行了分析研究,结果表明鲢TNF mRNA主要在脑、鳃和头肾中表达,肠和脾中有少量表达,而在肝中几乎没有表达.     

Spatio-temporal activation of cyclic AMP response element-binding protein, activity-regulated cytoskeletal-associated protein and brain-derived nerve growth factor: a mechanism for pontine-wave generator activation-dependent two-way active-avoidance memory processing in the rat

The present study explored possible physiological and molecular mechanisms of pontine-wave (P-wave) generator activation-dependent memory processing in the rat using a two-way active-avoidance learning paradigm. The results show that learning training increased rapid eye movement sleep and activated brainstem cells in the P-wave generator. During this period, there was a time-dependent increase in phosphorylation of cAMP response element-binding protein (CREB) in the dorsal hippocampus and amygdala and increased synthesis of activity-regulated cytoskeletal-associated protein (Arc) in the dorsal hippocampus, amygdala, frontal cortex and occipital cortex. Learning training also increased synthesis of brain-derived nerve growth factor (BDNF) in the occipital cortex, amygdala and dorsal hippocampus at different time intervals. During this time, the levels of nerve growth factor did not change. The results also show that the increase in rapid eye movement sleep P-wave density during the post-training 3-h recording session is positively correlated with the increased levels of phosphorylated CREB, BDNF and Arc in the dorsal hippocampus. These results suggest that memory processing of two-way active-avoidance learning may involve excitation of P-wave-generating cells in the brainstem and increased expression of phosphorylated CREB, Arc and BDNF in a time-dependent manner in the forebrain. These dynamic changes in cellular and molecular features provide considerable insight into the mechanisms of the P-wave generator activation-dependent memory consolidation process.     

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-κB activation

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.     

Hippocampal memory consolidation during sleep: a comparison of mammals and birds

The transition from wakefulness to sleep is marked by pronounced changes in brain activity. The brain rhythms that characterize the two main types of mammalian sleep, slow‐wave sleep (SWS) and rapid eye movement (REM) sleep, are thought to be involved in the functions of sleep. In particular, recent theories suggest that the synchronous slow‐oscillation of neocortical neuronal membrane potentials, the defining feature of SWS, is involved in processing information acquired during wakefulness. According to the Standard Model of memory consolidation, during wakefulness the hippocampus receives input from neocortical regions involved in the initial encoding of an experience and binds this information into a coherent memory trace that is then transferred to the neocortex during SWS where it is stored and integrated within preexisting memory traces. Evidence suggests that this process selectively involves direct connections from the hippocampus to the prefrontal cortex (PFC), a multimodal, high‐order association region implicated in coordinating the storage and recall of remote memories in the neocortex. The slow‐oscillation is thought to orchestrate the transfer of information from the hippocampus by temporally coupling hippocampal sharp‐wave/ripples (SWRs) and thalamocortical spindles. SWRs are synchronous bursts of hippocampal activity, during which waking neuronal firing patterns are reactivated in the hippocampus and neocortex in a coordinated manner. Thalamocortical spindles are brief 7–14 Hz oscillations that may facilitate the encoding of information reactivated during SWRs. By temporally coupling the readout of information from the hippocampus with conditions conducive to encoding in the neocortex, the slow‐oscillation is thought to mediate the transfer of information from the hippocampus to the neocortex. Although several lines of evidence are consistent with this function for mammalian SWS, it is unclear whether SWS serves a similar function in birds, the only taxonomic group other than mammals to exhibit SWS and REM sleep. Based on our review of research on avian sleep, neuroanatomy, and memory, although involved in some forms of memory consolidation, avian sleep does not appear to be involved in transferring hippocampal memories to other brain regions. Despite exhibiting the slow‐oscillation, SWRs and spindles have not been found in birds. Moreover, although birds independently evolved a brain region—the caudolateral nidopallium (NCL)—involved in performing high‐order cognitive functions similar to those performed by the PFC, direct connections between the NCL and hippocampus have not been found in birds, and evidence for the transfer of information from the hippocampus to the NCL or other extra‐hippocampal regions is lacking. Although based on the absence of evidence for various traits, collectively, these findings suggest that unlike mammalian SWS, avian SWS may not be involved in transferring memories from the hippocampus. Furthermore, it suggests that the slow‐oscillation, the defining feature of mammalian and avian SWS, may serve a more general function independent of that related to coordinating the transfer of information from the hippocampus to the PFC in mammals. Given that SWS is homeostatically regulated (a process intimately related to the slow‐oscillation) in mammals and birds, functional hypotheses linked to this process may apply to both taxonomic groups.     

Effects of tumour necrosis factor alpha (TNFalpha) on Mytilus haemocytes: role of stress-activated mitogen-activated protein kinases (MAPKs)

BACKGROUND INFORMATION: Many studies indicate that innate immunity in invertebrates can be modulated by a cytokine network like in vertebrates. In molluscs, the immune response is carried out by circulating haemocytes and soluble haemolymph factors. In the present study, the effects of heterologous TNFalpha (tumour necrosis factor alpha) on cell signalling and function in the haemocytes of the bivalve Mytilus galloprovincialis Lam. were investigated. RESULTS AND CONCLUSIONS: Addition of TNFalpha in the absence of haemolymph serum [in ASW (artificial sea water)] induced cellular stress, as indicated by lysosomal destabilization, and decreased phagocytosis; on the other hand, in the presence of serum, TNFalpha did not affect lysosomal stability and even stimulated phagocytosis. TNFalpha induced rapid phosphorylation of the stress-activated p38 and JNK (c-Jun N-terminal kinase) MAPKs (mitogen-activated protein kinases); both effects were persistent in ASW but transient in serum. Activation of p38 and JNKs in mediating the effects of TNFalpha was confirmed by the use of specific MAPK inhibitors. Moreover, flow cytometric analysis indicated that TNFalpha in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin V binding; in ASW, the cytokine resulted in a stable increase in the percentage of both annexin- and propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. The results indicate that TNFalpha can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated MAPKs and suggest that the haemocyte response to the cytokine is influenced by soluble haemolymph components.     

Endotoxaemia, production of tumour necrosis factor alpha and polymorphonuclear neutrophil activation following strenuous exercise in humans

To examine whether endotoxaemia accompanying long-term, strenuous physical exercise is involved in exercise-induced increase in plasma tumour necrosis factor alpha (TNF-alpha) concentration and polymorphonuclear neutrophil (PMN) activation, 14 male recreational athletes [mean age 28 (SEM 1) years] were studied. Exercise consisted of a 1.5-km river swim, a 40-km bicycle race, and a 10-km road race. Mean time to complete the race was 149.8 (SEM 4.8) min. The plasma concentrations of granulocyte myeloperoxidase (MPO) and TNF-alpha were significantly higher than baseline values immediately and 1 h after exercise (P<0.001). Both variables returned to pre-race levels the day after exercise. Marked, transient decreases in plasma concentrations of anti-lipopolysaccharide (LPS) immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies directed against a panel of selected smooth gram-negative LPS were observed after the race, reaching in most cases minimal values in the blood sample drawn immediately following the completion of the triathlon. There was no significant correlation between the magnitude of PMN activation, as assessed by the increase in plasma concentrations of MPO, and the humoral markers of endotoxaemia and TNF-alpha. An inverse, highly significant relationship between the increase in plasma TNF-alpha concentrations and the changes in circulating anti-LPS IgM antibodies concentrations was observed (r = -0.7; P<0.01). These findings suggest that exercise-induced endotoxaemia was involved in the release of TNF-alpha, that the magnitude of the TNF-alpha response to exercise was down-regulated by anti-LPS antibodies of the IgM class, and that the production of TNF-alpha and endotoxaemia did not seem to play a role in the activation of circulating PMN in the exercising subjects.     

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.     

Fumonisin B(1) alters sphingolipid metabolism and tumor necrosis factor alpha expression in heart and lung of mice

Fumonisin B(1) (FB(1)), produced by Fusarium verticillioides, is a common contaminant in foods and feeds. Increase in tissue free sphingoid bases resulting from the inhibition of ceramide synthase is a biomarker of fumonisin exposure. Tumor necrosis factor alpha (TNFalpha) is induced in liver in response to FB(1) treatment. This study determined whether fumonisin B(1) caused increases in free sphingoid bases and altered the expression of TNFalpha in heart and lung, organs that are not targets of FB(1) toxicity, of male and female mice treated with 5-daily subcutaneous injection of 2.25 mg/kg FB(1). A significant increase in free sphingoid bases was observed in both heart and lung of FB(1)-exposed mice. The magnitude of increases in free sphingoid bases in both organs of female mice was much higher than that in males. The expression of TNFalpha was increased by FB(1) treatment in the lung of male mice and in the heart of female mice, whereas the expression of interferon gamma was unaltered. Results suggest that both sphingolipid accumulation and TNFalpha induction are observed in the tissues of mice that are not associated with FB(1) toxicity.     

Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.

Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.     

Changes in the expression of tumor necrosis factor (TNF) alpha,TNFalpha receptor (TNFR) 2, and TNFR-associated factor 2 in granulosa cells during atresia in pig ovaries

Tumor necrosis factor (TNF) alpha can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFalpha-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFalpha and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFalpha and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.