Nicotine-Induced Neuroprotection Against Ischemic Injury Involves Activation of Endocannabinoid System in Rats

Nicotine has been reported to exert certain protective effect in the Parkinson’s and Alzheimer’s diseases. Whether it has a similar action in focal cerebral ischemia was unclear. In the present study, rats received either an injection of (?)-nicotine hydrogen tartrate salt (1.2 mg/kg, i.p.) or the vehicle 2 h before the 120 min middle cerebral artery occlusion. Neurological deficits and histological injury were assessed at 24 h after reperfusion. The content of endocannabinoids and the expression of cannabinoid receptor CB1 in brain tissues were determined at different time points after nicotine administration. Results showed that nicotine administration ameliorated neurological deficits and reduced infarct volume induced by cerebral ischemia in the rats. The neuroprotective effect was partially reversed by CB1 blockage. The content of the endocannabinoids N-arachidonylethanolamine and 2-arachidonoylglycerol, as well as the expression of cannabinoid receptor CB1 were up-regulated in brain tissues after nicotine delivery. These results suggest that endogenous cannabinoid system is involved in the nicotine-induced neuroprotection against transient focal cerebral ischemia.     

Effects of Sevoflurane on Self-Renewal Capacity and Differentiation of Cultured Neural Stem Cells

Sevoflurane anesthesia in infant rats can result in long-term cognitive impairment, possibly by inhibiting neurogenesis. The hippocampus is critical for memory consolidation and is one of only two mammalian brain regions where neural stem cells (NSCs) are renewed continuously throughout life. To elucidate the pathogenesis of sevoflurane-induced cognitive dysfunction, we measured the effects of clinical sevoflurane doses on the survival, proliferation, and differentiation of hippocampal NSCs. Neural stem cells were isolated from Sprague–Dawley rat embryos, expanded in vitro, and exposed to sevoflurane at 0.5, 1, or 1.5 minimal alveolar concentration (MAC) for 1 or 6 h. Two days after treatment, cell viability, cytotoxicity, and apoptosis rate were estimated by WST-1 assay, lactate dehydrogenase (LDH) activity, and TdT-mediated dUTP-biotin nick end labeling (TUNEL), respectively, while proliferation rate was assessed by 5-ethynyl-2′-deoxyuridine (BrdU) incorporation and Ki67 staining. Differentiation was assayed 7 days after treatment by immunocytochemistry and Western blots of neuron and glial markers. The phosphorylation level of p44/42 extracellular regulated kinases (ERK1/2) was measured in the proliferation and differentiation phases respectively. Sevoflurane at 1 MAC or 1.5 MAC for 1 h increased viable cell number whereas a 6 h exposure at these same concentrations suppressed proliferation and promoted apoptotic death (P < 0.01). Sevoflurane had no effect on NSC differentiation, and a sub-clinical concentration (0.5 MAC) altered neither proliferation nor viability. The phosphorylation level of ERK1/2 increased after 1 h of 1 MAC or 1.5 MAC of sevoflurane exposure in the proliferation phase, but not in the differentiation phase. Brief (1 h) exposure to sevoflurane at clinical concentrations enhanced proliferation of cultured NSCs possibly mediated by ERK1/2, but a 6 h exposure suppressed proliferation and induced apoptosis. Prolonged sevoflurane exposure may decrease the self-renewal capacity of hippocampal NSCs, resulting in cognitive deficits.     

Novel SLC20A2 mutations identified in southern Chinese patients with idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (IBGC) is a rare neuropsychiatric disorder characterized by bilateral and symmetric cerebral calcifications. Recently, SLC20A2 was identified as a causative gene for familial IBGC, and three mutations were reported in a northern Chinese population. Here, we aimed to explore the mutation spectrum of SLC20A2 in a southern Chinese population. Sanger sequencing was employed to screen mutations within SLC20A2 in two IBGC families and 14 sporadic IBGC cases from a southern Han Chinese population. Four novel mutations (c.82G > A p.D28N, c.185T > C p.L62P, c.1470_1478delGCAGGTCCT p.Q491_L493del and c.935-1G > A) were identified in two families and two sporadic cases, respectively; none were detected in 200 unrelated controls. No mutation was found in the remaining 12 patients. Different mutations may result in varied phenotypes, including brain calcification and clinical manifestations. Our study supports the hypothesis that SLC20A2 is a causative gene of IBGC and expands the mutation spectrum of SLC20A2, which facilitates the understanding of the genotype–phenotype correlation of IBGC.     

白念珠菌铁通透酶基因CaFTH1的功能研究

[目的]白念珠菌CaFTH1是一种铁通透酶编码基因.为了研究CaFTH1对胞内铁代谢和液泡功能的影响,构建fth1△/△单基因缺失菌株和fth1△/△fet33△/△双基因缺失菌株.[方法]利用生物信息学软件对CaFTH1进行序列比对和分析;通过实时荧光定量PCR技术研究铁离子丰度对CaFTH1表达的影响;利用PCR介导的同源重组方法构建基因缺失菌株;利用原子吸收光谱方法测定基因缺失菌株胞内铁含量的变化,并对基因缺失菌株在缺铁条件和菌丝诱导条件下的生长状况进行研究;通过代谢转换实验,研究CaFTH1对细胞液泡功能的影响.[结果]序列比对结果表明白念珠菌CaFth1蛋白属于铁通透酶Ftr1超家族,与酿酒酵母液泡膜蛋白ScFth1具有最高的同源性.铁匮乏条件会诱导CaFTH1的表达,而富铁条件则会抑制其表达.白念珠菌CaFTH1的缺失会导致胞内铁含量的降低,fth1△/△突变菌株基础上CaFET33的缺失则会进一步降低胞内铁含量.在缺铁条件下,fth1△/△fet33△/△双基因缺失菌株在一定程度上表现出代谢转换能力的缺陷.另外,在某些固体菌丝诱导培养条件下,fth1△/△fet33△/△缺失菌株菌落表面形成褶皱能力显著增强;而在液体菌丝诱导条件下,则表现为增强的菌丝聚集能力.[结论]CaFTH1是一种低铁应答基因,在维持白念珠菌胞内铁离子稳态及液泡功能方面具有重要作用.CaFTH1和CaFET33基因的双缺失会对白念珠菌的菌落形态和菌丝聚集产生影响.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.     

Study of PE and iPP orientations on the surface of carbon nanotubes by using molecular dynamic simulations

In this study, the early stage of interfacial crystallisation behaviour of low molecular weight polyethylene (PE) and isotactic polypropylene (iPP) oligomer on the surface of carbon nanotubes (CNTs) with different diameters, chiralities and topography structures was studied using MD simulations. We started to simulate the effect of CNTs chirality and diameter on PE molecular chain orientation, and then the effect of CNTs topography structure on PE and iPP molecular chain orientation was investigated. Finally, some experiments were carried out to prove the simulated results. Our study shows that for CNTs with a diameter comparable with the radius of gyration (R_g) of a polymer chain, an easy orientation of PE chains along CNTs axis is observed for all the systems of the CNTs with different chiralities due to a geometric confinement effect. For CNTs with a much larger diameter, multiple orientation of PE chains is induced on its surface due to the lattice matching between graphite lattice and PE molecular chains. In this case, the chirality of CNTs dominates the orientation of graphite lattice, which determines the orientation of PE chains arrangement on CNTs surface. More importantly, it was found that the groove structure formed by CNT bundles is very useful for the stabilisation of polymer chain, and thus facilitates the orientation of molecular chain along the long axis of CNTs. As a result, a novel nanohybrid shish–kebab (NHSK) structure with CNTs acting as central shish while polymer lamellae as kebab can be successfully obtained for both PE with zigzag conformation and iPP with helical conformation. This simulation result was well supported by the experimental observation. Our study could provide not only a deep understanding of the origin of the polymer chain orientation on CNTs surface but also the guidance for the preparation of polymer/CNTs nanocomposites with novel NHSK structure.     

Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and oxLDL through LOX-1 pathway

Vein grafts interposed into arteries are susceptible to the development of atherosclerosis due to rapid increases in blood pressure. This process is accelerated in patients with hyperlipidemia. The molecular mechanism underlying this process is unknown. In this study, quiescent rat vascular smooth muscle cells (VSMCs) were treated in vitro with mechanical stretch stress (10% elongation) with and without oxLDL (25 μg/ml) in the presence and absence of simvastatin (2.5 μmol/L). The results demonstrate that stretch stress and oxLDL can each induce activation of ERK1/2 and Ki-67 expression in VSMCs, but the peak levels of ERK activation and Ki-67 expression were observed in groups subjected to both stretch stress and oxLDL. Simvastatin was found to inhibit increased ERK activation and Ki-67 expression in VSMCs subjected to stretch stress with or without oxLDL. Mechanically, simvastatin was also found to inhibit increased expression of LOX-1 (a receptor of oxLDL) in VSMCs subjected to stretch stress with or without oxLDL. Knockdown of LOX-1 via small interfering RNAs (siRNA-LOX-1) resulted in obvious inhibition of ERK activation in VSMCs subjected to stretch stress with and without oxLDL. These results suggest that combined stretch stress and oxLDL can additively promote the activation of ERK1/2 leading to accelerated proliferation of VSMCs (e.g. increased Ki-67 expression) via LOX-1 signal pathway. This was found to be partially inhibited by simvastatin. These results may provide important data for the treatment and prevention of hypertension with or without hyperlipidemia.     

Molecular Cloning and Expression Analysis of Nine ThTrx Genes in Tamarix hispida

Thioredoxins are small conserved proteins that play key roles in the oxidative stress response. In this study, nine Trx genes, including five Trxhs, three Trxms, and one Trx-like gene, were cloned from Tamarix hispida. The roles of these ThTrx genes were investigated under various abiotic stress conditions. Expression profiles of the nine ThTrx genes in response to different abiotic stresses in leaf and root tissues were constructed using quantitative real time-polymerase chain reaction. Differential expression of all nine ThTrx genes was observed (>2-fold) in response to NaCl, PEG, or CdCl₂ stress in at least one tissue, indicating that all of these genes act in abiotic stress responses. All ThTrx genes were induced (>2-fold) by abscisic acid (ABA) treatment in the leaves and especially in the roots, suggesting that ABA-dependent signaling pathways regulate ThTrxs. These results demonstrate that ThTrx expression constitutes an adaptive response to abiotic stress in T. hispida and plays an important role in abiotic stress tolerance.     

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry

Key message

The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

Abstract

Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6–10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic ‘Aurora’ and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.