Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

2-Amino-6-chloro-3,4-dihydroquinazoline: A novel 5-HT3 receptor antagonist with antidepressant character

2-Amino-6-chloro-3,4-dihydroquinazoline HCl (A6CDQ, 4) binds at 5-HT₃ serotonin receptors and displays antidepressant-like action in the mouse tail suspension test (TST). Empirically, 4 was demonstrated to be a 5-HT₃ receptor antagonist (two-electrode voltage clamp recordings using frog oocytes; IC₅₀ = 0.26 μM), and one that should readily penetrate the blood–brain barrier (log P = 1.86). 5-HT₃ receptor antagonists represent a potential approach to the development of new antidepressants, and 4 is an example of a structurally novel 5-HT₃ receptor antagonist that is active in a preclinical antidepressant model (i.e., the mouse TST).     

Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.     

Acute ghrelin administration reverses depressive-like behavior induced by bilateral olfactory bulbectomy in mice

This study aims to examine the antidepressant-like action of Ghrelin (Ghr), a hormone synthesized predominantly by gastrointestinal endocrine cells and released during periods of negative energy balance, in two behavioral models: tail suspension test (TST), a predictive model of antidepressant activity, and the olfactory bulbectomy (OB), an established animal model of depression. The reduction in the immobility time in the TST was the parameter used to assess antidepressant-like effect of Ghr. The depressive-like behavior in olfactory bulbectomized mice was inferred through the increase in the immobility time in the TST and the hyperlocomotor activity in the open-field test. Ghr produced antidepressant-like effect in TST (0.3 nmol/μl, i.c.v.), and reversed OB-induced depressive-like behavior. In conclusion, these results provide clear evidence that an acute administration of ghrelin produce antidepressant-like effect in the TST and OB.     

Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines

Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6 g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.     

Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs

Traditional reconstitution of membrane cytochromes P450 monooxygenase system requires efficient solubilization of both P450 heme enzymes and redox partner NADPH dependent reductase, CPR, either in mixed micellar solution or by incorporation in liposomes. Here we describe a simple alternative approach to assembly of soluble complexes of monomeric human hepatic cytochrome P450 CYP3A4 with CPR by co-incorporation into nanoscale POPC bilayer Nanodiscs. Stable and fully functional complexes with different CPR:CYP3A4 stoichiometric ratios are formed within several minutes after addition of the full-length CPR to the solution of CYP3A4 preassembled into POPC Nanodiscs at 37 °C. We find that the steady state rates of NADPH oxidation and testosterone hydroxylation strongly depend on CPR:CYP3A4 ratio and reach maximum at tenfold molar access of CPR. The binding of CPR to CYP3A4 in Nanodiscs is tight, such that complexes with different stoichiometry can be separated by size-exclusion chromatography. Reconstitution systems based on the co-incorporation of CPR into preformed Nanodiscs with different human cytochromes P450 are suitable for high-throughput screening of substrates and inhibitors and for drug-drug interaction studies.     

三种免疫学方法在矽肺合并肺结核诊断中的价值研究

目的:探讨T淋巴细胞酶联斑点实验(T-SPOT)、结核菌素皮肤试验(tuberculin test,TST)以及结核抗体(tuberculosis antibody)在矽肺合并肺结核(pulmonary silicosis complicated with tuberculosis)诊断中的应用价值。方法:收集2015年8月7日至2016年5月20日确诊为矽肺结核感染患者41例、非矽肺结核患者90例、健康体检学生对照组40例,对三组人群经以上免疫学方法检测的结果进行统计分析。结果:1)矽肺结核、非矽肺结核及健康人群中T-SPOT阳性率分别为73.17%、93.33%、15%,三者两两比较差异有统计学意义;矽肺结核、非矽肺结核及健康人群中TST阳性率分别为58.54%、88.89%、32.5%,非矽肺结核患者与健康人群及矽肺结核患者之间,差异均有统计学意义,但矽肺结核患者与健康人群之间,差异无统计学意义;矽肺结核、非矽肺结核及健康人群中结核抗体阳性率分别为36.58%、42.22%、52.5%,三者两两比较差异均无统计学意义。2)三种检测方法在矽肺结核患者中的敏感性分别为73.17%、58.54%、36.58%,差异有统计学意义,其中T-SPOT及TST的敏感性均高于结核抗体方法(P0.05);特异性分别为85%、67.5%、47.5%,其中T-SPOT的特异性高于结核抗体方法(P0.05);阳性预测值分别为83.33%、64.86%、41.67%,阴性预测值分别为75.56%、61.36%、42.22%,三种方法的阳性及阴性预测值存在差异,均有统计学意义。3)T-SPOT在I、II、III期矽肺结核检测的阳性率分别为52.38%、94.12%、100%,I期与II期或III期的阳性率之间差异有统计学意义,II期与III期之间阳性率差异无统计学意义。结论:T-SPOT方法具有较高的敏感性及特异性,对矽肺结核辅助诊断及临床分期具有较好的应用价值。     

Tobacco plants respond to the constitutive expression of the tospovirus movement protein NS(M) with a heat-reversible sealing of plasmodesmata that impairs development

Viral infection often results in typical symptoms, the biological background of which has remained elusive. We show that constitutive expression of the NSM viral movement protein (MP) of tomato spotted wilt virus in Nicotiana tabacum is sufficient to induce severe, infection-like symptoms, including pronounced deficiencies in root and shoot development. Leaves failed to expand and were arranged in a rosette due to the absence of internode elongation. Following the sink-source transition they accumulated excessive amounts of starch and developed fusing chlorotic patches in the mesophyll, resembling virus-induced chlorotic lesions. Eventually, the leaves became entirely white and brittle. With a combination of techniques, including photosystem II quantum-yield measurements, iontophoresis of symplasmic tracers, bombardment with pPVX.GFP and double immunolabelling it was shown that these symptoms correlated with the obstruction of NSM-targeted mesophyll plasmodesmata (Pd) in source tissues by depositions of 1,3-beta-D-glucan (GLU) or callose. Temperature-shift treatments (TST; 22-->32 degrees C), known to abolish chlorotic local lesions, also abolished the chlorotic 'superlesions' of transgenic plants and rescued plant development, by restoring the transport capacity of Pd through the action of 1,3-beta-D-glucanase (GLU-h) or callase. Return of these elongated, TST-recovered plants to 22 degrees C reintroduced superlesions and arrested shoot elongation, resulting in the formation of a rosette of clustered leaves at the shoot tip. Collectively, this indicates that the symptoms of NSM plants are self-inflicted and due to a basal defence response that counteracts prolonged interference of the MP with Pd functioning. This type of defence may also play a role in the formation of symptoms during viral infection.