Membrane oxidation assay--a novel lipoxygenase-based evaluation of membrane oxidizability

A membrane oxidation assay is presented which uses isolated erythrocyte membranes ("ghosts") and lipoxygenase as a selective catalyst for the transfer of oxygen to cis - cis -1,4-pentadiene-moieties. The latter are, for instance, present in linoleic and arachidonic acids, both of which are integral parts of membranes. These non-conjugated double bonds represent energetically favorable sites for oxidative attack and therefore, may be rearranged and partially consumed during oxidative stress. Consequently, the measurement of oxygen consumption in the course of the lipoxygenase-mediated oxidation provides a tool for the quick and reliable determination of such double bonds. Significant inter-individual differences have been noted in 11 subjects, which also correlate to the total radical antioxidant parameter (TRAP) values obtained. This assay will be helpful in the assessment of oxidizable structures in erythrocyte membranes that may be diminished as a consequence of oxidative damage suffered by an individual. In conclusion, a simple and rapid assay for the assessment of the oxidizability of erythrocyte membranes is presented complementing the TRAP assay for plasma antioxidative status.     

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( •OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 μM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of •OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced •OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with •OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular •OH in damage associated with intracerebral iron injection.     

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P700A1 by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz

The radical-pair state of the primary electron donor and the secondary electron acceptor (P₇₀₀⁺A₁⁻) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 Å resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C₂ symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.     

血清CEA、G-17、MK、ProGRP与胃癌根治术患者术后复发风险的关系研究

摘要 目的：探讨血清癌胚抗原（CEA）、胃泌素17（G-17）、中期因子（MK）、胃泌素释放肽前体（ProGRP）与胃癌根治术（RG）患者术后复发风险的关系。方法：选取2018年1月～2020年8月新疆医科大学第一附属医院普外科收治的156例胃癌患者为胃癌组,根据RG后是否复发分为复发组和无复发组,另选取同期我院52名健康体检志愿者为对照组。收集胃癌患者临床资料,采用酶联免疫吸附法检测血清CEA、G-17、MK、ProGRP水平。通过单因素和多因素Logistic回归分析RG患者术后复发的影响因素,受试者工作特征（ROC）曲线分析血清CEA、G-17、MK、ProGRP水平对RG患者术后复发的预测价值。结果：与对照组比较,胃癌组血清CEA、G-17、MK、ProGRP水平升高（P＜0.05）。随访2年,失访2例,154例RG患者术后复发率为28.57%（44/154）。多因素Logistic回归分析显示,CEA、G-17、MK、ProGRP升高、TNM分期Ⅲ期、分化程度为低分化、淋巴结转移为RG患者术后复发的独立危险因素（P＜0.05）。ROC曲线分析显示,血清CEA、G-17、MK、ProGRP水平联合预测RG患者术后复发的曲线下面积（AUC）大于CEA、G-17、MK、ProGRP单独预测。结论：血清CEA、G-17、MK、ProGRP水平升高与RG患者术后复发密切相关,可能成为RG患者术后复发的辅助预测指标,且血清CEA、G-17、MK、ProGRP联合预测RG患者术后复发的价值较高。     

胶原生物膜在耳内镜下中耳胆脂瘤乳突根治术中的应用效果分析

摘要 目的：探讨胶原生物膜在耳内镜下乳突根治术中的应用效果。方法：选取徐州医科大学附属医院2021年4月至2022年 2月收治的51例中耳胆脂瘤患者进行回顾性分析,其中研究组27例患者予以胶原生物膜修复皮肤缺损,对照组予以颞肌筋膜修复术腔皮肤缺损,观察两组患者术后临床症状,手术时长,术腔完全上皮化时间、干耳时间及术前术后听力改变。结果：研究组术后患者因外耳道进水,存在感染及肉芽生长者1例,予以清理后未再次生长;对照组术后发生1例外耳道口狭窄的情况,予以橡胶扩张管进行扩张并后并定期清理术腔肉芽、脱落痂皮,患者外耳道恢复良好。两组术前耳闷、耳痛、耳鸣及术后耳痛VAS评分无明显差异（P>0.05）;研究组术后耳闷及耳鸣VAS评分较对照组降低（P<0.05）。研究组平均手术时长、术后术腔完全上皮化时间及平均干耳时间短于对照组（P<0.05）。两组术前术后气骨导差（ABG）、平均气导听阈（AC）比较差异均无统计学意义（P＞0.05）。结论：作为术区移植物,胶原生物膜应用于耳内镜下中耳胆脂瘤乳突根治术可加快创面术腔的修复,减少局部创伤与操作步骤,改善临床症状,缩短手术时间、术后术腔完全上皮化时间及获得干耳时间,可作为临床上有效的修复材料。     

乳腺癌根治术患者术前焦虑的影响因素分析及其对术后恢复、细胞免疫功能和生命质量的影响

摘要 目的：分析乳腺癌根治术患者术前焦虑的影响因素,并探讨术前焦虑对患者术后恢复、细胞免疫功能和生命质量的影响。方法：选择我院2020年3月~2021年12月期间收治的拟行乳腺癌根治术的120例患者作为研究对象,术前1 d采用焦虑自评量表（SAS）评估所有患者的焦虑状况,根据是否存在焦虑分为焦虑组和无焦虑组,乳腺癌根治术患者术前焦虑的影响因素采用多因素Logistic回归分析。对比焦虑组和无焦虑组的术后恢复、细胞免疫功能和生命质量情况。结果：120例乳腺癌根治术患者中,有31例患者无术前焦虑,89例患者存在焦虑症状,根据是否存在术前焦虑分为焦虑组（n=89）和无焦虑组（n=31）。乳腺癌根治术患者术前焦虑与年龄、文化程度、家庭人均月收入、付费方式、婚姻状况、家庭支持、既往有无全麻史、术前住院时长、定期体检有关（P<0.05）。多因素Logistic回归分析结果表明：年龄＜60岁、文化程度为小学及其以下、家庭人均月收入＜3000元、婚姻状况为未婚、无家庭支持、既往无全麻史、术前住院时长>1 d是乳腺癌根治术患者术前焦虑的危险因素（P<0.05）。焦虑组的术后首次肛门排气时间、首次下床活动时间、术后住院时间均长于无焦虑组（P<0.05）。两组术后1个月CD3⁺、CD4⁺、CD4⁺/CD8⁺水平升高,且无焦虑组高于焦虑组（P<0.05）,两组术后1个月CD8⁺水平下降,且无焦虑组低于焦虑组（P<0.05）。两组术后1个月生理状况、情感状况、社会/家庭状况、功能状况、附加关注评分和总分下降,且无焦虑组低于焦虑组（P<0.05）。结论：乳腺癌根治术患者术前焦虑发生率较高,其发生受到年龄、文化程度、家庭人均月收入、婚姻状况、家庭支持、既往有无全麻史、术前住院时长等多种因素的影响,可导致患者术后恢复时间延长,细胞免疫功能和生命质量降低。     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications

Summary We have obtained a revised estimate of the pattern of point mutation by considering more pseudogene sequences. Compared with our previous estimate, it agrees better with expectations based on the double-strand structure of DNA. The revised pattern, like the previous one, indicates that mutation occurs nonrandomly among the four nucleotides. In particular, the proportion of transitional mutations (59%) is almost twice as high as the value (33%) expected under random mutation. The same high proportion of transitions is observed in synonymous substitutions in genes. The proportion of transitional changes observed among electrophoretic variants of human hemoglobin is about the same as that predicted by the revised pattern of mutation. We also show that nonrandom mutation increases, by about 15%, the proportion of synonymous mutations due to single-nucleotide changes in the codon table, and increases, from 10% to 50%, the rate of synonymous mutation in the seven genes studied. However, nonrandom mutation reduces (by about 10%) the proportion of polar changes among nonsynonymous mutations in a gene. As far as single-nucleotide changes (in the codon table) are concerned, nonrandom mutation only slightly favors relatively conservative amino acid interchanges, and has virtually no effect on the proportions of radical changes and nonsense mutations.     

Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers

Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, ³P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 10⁷s^?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 10⁸s^?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of ³P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some ³P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed.