DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Summary The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a β-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 × 10⁵ cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

Fingerprinting complex pectins by chromatographic separation combined with ELISA detection

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-α-l-arabinan and (1,4)-β-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Identification and comparative analysis of the large subunit mitochondrial ribosomal proteins of Neurospora crassa

The mitochondrial ribosome (mitoribosome) has highly evolved from its putative prokaryotic ancestor and varies considerably from one organism to another. To gain further insights into its structural and evolutionary characteristics, we have purified and identified individual mitochondrial ribosomal proteins of Neurospora crassa by mass spectrometry and compared them with those of the budding yeast Saccharomyces cerevisiae. Most of the mitochondrial ribosomal proteins of the two fungi are well conserved with each other, although the degree of conservation varies to a large extent. One of the N. crassa mitochondrial ribosomal proteins was found to be homologous to yeast Mhr1p that is involved in homologous DNA recombination and genome maintenance in yeast mitochondria.     

Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products.     

MHR、血清ESM-1、sST2与急性ST段抬高型心肌梗死患者直接PCI术中慢血流/无复流的关系及对近期预后的预测价值

摘要 目的：探讨单核细胞与高密度脂蛋白胆固醇比值（MHR）、血清内皮细胞特异性分子-1（ESM-1）、可溶性致癌抑制因子2（sST2）与急性ST段抬高型心肌梗死（STEMI）患者直接经皮冠状动脉介入治疗（PPCI）术中慢血流/无复流（SRF/NRF）的关系及对其近期预后的预测价值。方法：选取2019年1月～2022年4月首都医科大学附属北京朝阳医院心内科收治的187例接受PPCI术的急性STEMI患者为急性STEMI组,根据PPCI术中心肌梗死溶栓治疗（TIMI）血流分级分为SRF/NRF组47例和无SRF/NRF组140例,随访6个月,根据是否发生主要不良心血管事件分为预后不良组和预后良好组,另选取同期56名体检健康志愿者为对照组。收集急性STEMI患者临床资料,计算MHR并检测血清ESM-1、sST2水平。采用Spearman相关性分析SRF/NRF患者MHR和血清ESM-1、sST2水平与TIMI血流分级的相关性,多因素Logistic回归分析急性STEMI患者PPCI后近期预后不良的影响因素,受试者工作特征（ROC）曲线分析MHR和血清ESM-1、sST2水平对急性STEMI患者PPCI后近期预后不良的预测价值。结果：急性STEMI组MHR和血清ESM-1、sST2水平高于对照组（P＜0.05）。SRF/NRF组MHR和血清ESM-1、sST2水平高于无SRF/NRF组（P＜0.05）。SRF/NRF患者MHR和血清ESM-1、sST2水平与TIMI血流分级呈负相关（P＜0.05）。左心室射血分数升高为急性STEMI患者PPCI后近期预后不良的独立保护因素,年龄增加、SRF/NRF和MHR、ESM-1、sST2升高则为独立危险因素（P＜0.05）。ROC曲线分析显示,MHR和血清ESM-1、sST2水平联合预测急性STEMI患者PPCI后近期预后不良的曲线下面积（AUC）大于MHR、ESM-1、sST2单独预测。结论：急性STEMI患者MHR和血清ESM-1、sST2水平升高与PPCI术中SRF/NRF和近期预后不良密切相关,三者联合预测急性STEMI患者近期预后不良的价值较高。