Analysis of blood plasma proteins in patients with Alzheimer's disease by two-dimensional electrophoresis, sequence homology and immunodetection

Blood plasma proteins of patients with Alzheimer's disease (AD; senile dementia) and non-AD-type dementia were resolved by two-dimensional electrophoresis and identified by migration position in the electrophoresis pattern, sequence homology, and immunodetection by using antibodies. For the control experiments, blood plasma proteins of a healthy young individual and non-dementia patients were examined in a manner similar to that of the plasma samples of AD patients. In the plasma sample of the healthy young individual, more than 350 spots of silver-stained proteins were observed and among these spots, 73 spots were identified. Blood plasma proteins of the AD and non-AD-type dementia patients were compared with those of the control and non-dementia patients. In the blood plasma samples of five AD patients, three patients had apolipoprotein E4, and another patient showed apolipoprotein L and complement factor H. For the AD-related proteins apolipoprotein E, tau-1, and presenilin 2, proteins were examined by immunostaining with antibodies, in both AD and non-AD patients. Among the three samples of non-AD-type dementia patients, one was distinguishable by amyloid A proteins, and the other by haptoglobin isoforms.     

不稳定性心绞痛血瘀证的血浆蛋白质组学研究

为了寻找冠心病不稳定性心绞痛血瘀证血浆差异表达蛋白, 探索冠心病不稳定性心绞痛血瘀证的蛋白质组学特点. 采用差异凝胶双向电泳和质谱联用技术对12例冠心病不稳定性心绞痛血瘀证患者和12例健康人血浆进行比较研究. 初步发现了Fibrinogen β chain, Fibrinogen γ chain, α1-Antitrypsin, Haptoglobin β chain, Haptoglobin α2 chain在冠心病不稳定性心绞痛血瘀证患者中高表达, ApoA-IV, ApoA-I, Transthyretin, ApoJ在冠心病不稳定性心绞痛血瘀证患者中低表达. 差异表达蛋白根据功能可分为以下三类: (1)急性时相反应负相蛋白; (2)载脂蛋白; (3)凝血相关蛋白. 冠心病不稳定性心绞痛血瘀证可能与炎症反应、脂代谢紊乱以及凝血功能异常相关.     

Simultaneous determination of MHD and DMD in dog plasma by high-performance liquid chromatography with fluorescence detection and its application to pharmacokinetic studies

A rapid, reproducible high-performance liquid chromatographic (HPLC) method with fluorescence detection for the simultaneous determination of 3(or 8)-(1-methoxyethyl)-8(or 3)-(1-hydroxyethyl)-deuteroporphyrin IX (MHD) and 3,8-di-(1-methoxyethyl)-deuteroporphyrin IX (DMD) in dog plasma was described. Fluorescein was used as an internal standard. A simple extraction step with ethyl acetate was performed before chromatography on a Diamonsil C₁₈ column (5 μm, 4.6 mm×150 mm). The chromatography used 0.02 mol L⁻¹ sodium acetate/tetrahydrofuran (66:34 v/v). The analytical curve was linear over the concentration range 0.025– 2.5 μg mL⁻¹. For a 100 μL dog plasma sample, the limit of determination for both MHD and DMD was 0.025 μg mL⁻¹. The recoveries of MHD and DMD were more than 76% and 89%, respectively. The intra-assay (within-run) and interassay (between-run) coefficients of variation (precisions) for MHD and DMD were less than 15%. This method was found to be suitable for the analysis of biosamples and was successfully applied to pharmacokinetic studies of Deuxemether in dogs.     

Evaluation of the influence of mirabilite on the absorption and pharmacokinetics of the ingredients in Dahuang‐mudan decoction by a validated UPLC/QTOF–MS/MS method

Dahuang‐mudan decoction (DMD) has been widely used for disease treatment in China for 1700 years. The formula consists of Rhubarb, moutan bark, Prunus persica, wax gourd kernel and mirabilite, which have been well studied by multidisciplinary approaches. However, the role of the mineral mirabilite in DMD is unclear. The objective of this study was to investigate the effects of mirabilite on the absorption and pharmacokinetics of the ingredients in DMD. The constituents were identified in DMD extract and the plasma of mirabilite–DMD (MDMD, 50 g kg^?1) treated rats and nonmirabilite–DMD (NMDMD, 50 g kg^?1) treated rats. The plasma was also used to investigate the effects of mirabilite on the pharmacokinetics of active ingredients in DMD using a new validated UPLC–MS/MS method. The results showed that 63 compounds were identified in the extract of DMD, 27 and 22 of which were found in the plasmas of MDMD‐ and NMDMD‐treated rats, respectively. Furthermore, the results of a pharmacokinetic study suggested that mirabilite influenced the absorption of the five constituents by decreasing the absorption of emodin and rhein while increasing the absorption of aloe‐emodin, paeoniflorin and amygdalin; the pharmacokinetic parameters, including the T_max, C_max, AUC_0–t, MRT_0–t, CLz and t_1/2 of five constituents, significantly changed in MDMD‐treated rats compared with the NMDMD. The method validation for selectivity, precision, accuracy, matrix effect, recovery and stability met the acceptance criteria. These findings uncover the roles of mirabilite in DMD and demonstrate the application of scientific principles to the study of DMD in human health care.     

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t_1/2, 0.09–0.14 h; DMD: t_1/2, 0.07–0.11 h) and a relatively slow elimination phase (MHD: t_1/2β, 2.03–3.20 h; DMD: t_1/2β, 2.51–3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75–1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36–89.99% for MHD; 45.43–76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50–50.0 μg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.     

Dystrophin Protein Quantification as a Duchenne Muscular Dystrophy Diagnostic Biomarker in Dried Blood Spots Using Multiple Reaction Monitoring Tandem Mass Spectrometry: A Preliminary Study

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle loss, leading to difficulties in movement. Mutations in the DMD gene that code for the protein dystrophin are responsible for the development of DMD disorder, where the synthesis of this protein is completely halted. Therefore, circulating dystrophin protein could be a promising biomarker of DMD disease. Current methods for diagnosing DMD have sensitivity, specificity, and reproducibility limitations. Herein, a quantitative liquid chromatography–tandem spectrometry (LC–MS/MS) technique in multiple reaction monitoring (MRM) mode was designed and validated for accurate dystrophin protein measurement in a dried blood spot (DBS). The method was successfully validated on the basis of international guidelines regarding calibration curves, precision, and accuracy. In addition, patients and healthy controls were used to test the amount of dystrophin protein circulating in DBS samples as a potential biomarker for DMD disorders. DMD patients were found to have considerably lower levels than controls. To the best of our knowledge, this is the first study to report dystrophin levels in DBS through LC–MS/MS as a diagnostic marker for DMD to the proposed MRM method, providing a highly specific and sensitive approach to dystrophin quantification in a DBS that can be applied in DMD screening.     

Utilization of magnetic nanobeads for analyzing haptoglobin in human plasma as a marker of Alzheimer’s disease by capillary electrophoretic immunoassay with laser-induced fluorescence detection

Alzheimer’s disease (AD) is a neurodegenerative disorder resulting from an impaired cholinergic function with loss of cognitive activity in the brain. Haptoglobin is a useful biomarker for AD analysis. Compared to the conventional enzyme-linked immunosorbent assay for haptoglobin analysis, the proposed immunoassay procedure reduces sample analysis time by approximately 55 min. Therefore, immunoassay was coupled with capillary electrophoresis (CE) to determine haptoglobin concentrations indirectly by using magnetic nanobeads (MBs) as a support and laser-induced fluorescence detection. In human plasma sample, the haptoglobin was immobilized on the MBs and reacted with the purified anti-haptoglobin antibody. The optimum separation time for the analyte was shorter than 6 min at 25 °C with a fused-silica capillary column of 40.2 cm × 50 μm ID (effective length 30 cm) and a run buffer containing 25 mM phosphate (pH 8.0) with 0.01% poly(ethylene oxide) (PEO). When using Atto 495 NHS ester as an internal standard (IS) (250.0 ng mL⁻¹), the linear range of the proposed method for indirect determination of haptoglobin was 0.2–3.0 mg mL⁻¹. The method was further used to monitor the course of AD in patients with behavioral and psychological symptoms of dementia (BPSD).     

Genotyping of exons 1 to 20 in Duchenne muscular dystrophy by universal multiplex PCR and short‐end capillary electrophoresis

One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1–20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short‐end injection CE (short‐end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9 min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1× TBE buffer at 6 kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation‐dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.     

Sequential changes of the cardiomyopathy in Duchenne muscular dystrophy by 201Tl myocardial SPECT

201Tl myocardial SPECT was performed to evaluate cardiomyopathy in Duchenne type of progressive muscular dystrophy (DMD). Follow up SPECT images of the same patients were also obtained about 1 year after the first scan. Cases subjected to study were 10 DMD. At the first study the hypoperfusion area of the left ventricular muscle was observed in 6 cases (60%) out of 10. At the second study the hypoperfusion areas became wider and lower in 4 out of 6 cases (66.7%). The new hypoperfusion area which was not demonstrated at the first study was observed at the second study in one case of these cases. These results suggested that the positive rate of cardiomyopathy in DMD by 201Tl myocardial SPECT was high, and 201Tl myocardial SPECT is a useful examination to detect the change of myocardial damage in DMD.     

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

There are three types of human Haptoglobin, Hp 1‐1, Hp 2‐1, and Hp 2‐2, each characterized by a distinct combination of the two α chains of the holoprotein. A modified Coomassie Brilliant Blue R 250 (mCBB‐R250) staining method for detecting the phenotypes of human blood haptoglobin molecules is presented. Addition of excess hemoglobin to the sample allowed specific formation of different haptoglobin‐hemoglobin complexes, which, in turn can be separated into distinctive migration pattern on native‐PAGE and stained by CBB‐R250. The typing results are consistent with that using Western blotting. In comparison to the existing methods for haptoglobin typing, our method is comparable in accuracy, and is easier to carry out. The results on typing of 29 plasma samples from Taipei Blood Center were also presented.     

肌营养不良症患者红细胞膜磷脂组成的高效液相色谱分析

采用高效液相色谱－紫外检测法对肌营养不良症患者红细胞膜上磷脂的组成进行了分析测定,并将定量结果与正常人的正常对比,发现肌营养不良症患者红细胞膜中的卵磷脂、脑磷脂、磷脂酰丝氨酸均在正常值范围内,而神经鞘磷脂明显低于正常人的     

Improvement in reflective-emissive dual-mode properties of electrochemical displays by electrode modification

We studied the electrochromic (EC) and electrochemiluminescent (ECL) properties of a novel dual-mode display (DMD) cell that was enabled for reflective and emissive modes of representation by introducing both EC and ECL materials into an electrochemical cell. We fabricated EC, ECL, and DMD cells based on a simple-mixture solution or modified electrodes and compared their properties to clarify the advantage of a DMD system based on modified electrodes. Both the solution- and modified electrode-based DMDs showed EC properties in the reflective mode under dc bias application and ECL properties in the emissive mode under ac bias application. Although the solution-based DMD cell featured a very simple structure, some improvements related to side reaction and quenching reaction were required. The modified electrode-based DMD cell was fabricated to improve these aspects. The advantage of the DMD model based on the modified electrodes was certainly suggested by comparisons of the results with those of EC, ECL, and DMD cells based on a simple-mixture solution.     

De novo mutations in sporadic deletional Duchenne muscular dystrophy (DMD) cases

Dinucleotide repeat polymorphism based genetic analysis is a powerful approach to gain insight into rare genetic events like germline mosaicism and de novo mutations. The loss of heterozygosity of polymorphic dinucleotide loci at "deletional hotspot" of dystrophin gene can provide direct evidence of carrier status in female relatives of affected DMD patients with overlapped exonic deletions. We have used 4 STR loci of the central deletional hotspot of the dystrophin gene for genetic analysis in sporadic unrelated DMD families. Twenty-nine mothers of sporadic deletional cases were analysed and their carrier status was determined. Eighteen of them showed heterozygosity in the deleted loci suggesting the occurrence of de novo mutations. In 9 cases, the carrier status was indeterminate while 2 showed germline mosaicism. Our observations reiterated the importance of STR analysis in determining the status of mothers of sporadic deletional DMD cases in order to provide proper genetic counselling.     

胰岛素联合沙格列汀降糖疗效及安全性的研究

目的评估胰岛素联合沙格列汀治疗2型糖尿病的有效性和安全性。方法回顾性对照研究50例2型糖尿病患者胰岛素加用沙格列汀或阿卡波糖治疗(48±8)周后血糖水平和血糖波动等指标的变化。结果治疗(48±8)周后,两组血糖、Hb A1c、MAGE均明显下降(P均≤0.001),其中沙格列汀组MAGE的降幅明显大于阿卡波糖组(P=0.024),胃肠道不良反应发生率显著低于阿卡波糖组。结论沙格列汀联合胰岛素具有更强的控制血糖波动的疗效,胃肠道不良反应发生率更低。     

Change in serum proteome during allogeneic hematopoietic stem cell transplantation and clinical significance of serum C-reactive protein and haptoglobin

Successful hematopoietic stem cell transplantation (HSCT) involves the restoration of hematopoietic function after engraftment, arising from the differentiation and proliferation of hematopoietic stem cells. Several factors could influence the course of allogeneic-HSCT (allo-HSCT). Therefore, knowledge of serum proteome changes during the allo-HSCT period might increase the efficacy of diagnosis and disease prevention efforts. This study conducted proteomic analyses to find proteins that were significantly altered in response to allo-HSCT. Sera from five representative patients who underwent allo-HSCT were analyzed by 2-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, and were measured on a weekly basis before and after allo-HSCT in additional 78 patients. Fourteen protein spots showing changes in expression were further examined, and most proteins were identified as acute phase proteins (APPs). Studies of 78 additional patients confirmed that C-reactive protein (CRP) and haptoglobin undergo expression changes during allo-HSCT and thus may have the potential to serve as representative markers of clinical events after allo-HSCT. Maximal CRP level affected the development of major transplant-related complications (MTCs) and other problems such as fever of unknown origin. Particularly, an increase in CRP level 21 days after allo-HSCT was found to be an independent risk factor for MTC. Maximal haptoglobin and haptoglobin level 14 days after allo-HSCT were predictive of relapses in underlying hematologic disease. Our results indicated that CRP and haptoglobin were significantly expressed during allo-HSCT, and suggest that their level can be monitored after allo-HSCT to assess the risks of early transplant-related complications and relapse.     

Capillary electrophoresis for analysis of deletion and duplication in exon 44–55 of Duchenne muscular dystrophy gene

In this study, a genotyping CGE method was established for analysis of Duchenne muscular dystrophy (DMD) gene deletions and duplications in exon 44–55. A total of 12 DMD exons (exon 44–55) and 2 internal standard gene fragments were simultaneously amplified by using a universal multiplex PCR (UMPCR) and determined by CGE. The conditions of UMPCR and CGE were optimized, including the kinds of polymerase, temperatures in UMPCR, separation matrix, separation temperature, and voltage. Finally, the separation was performed by 1.2% poly(ethylene oxide) in 1× TBE buffer at ?6 kV and 25°C. After validation, our results showed the peak patterns for differentiation of genetic deletion or duplication in 27 DMD patients and normal subjects, according to the peak height ratios by comparison of two internal standard peaks. Among the 27 subjects, 23 cases are deletion type and four are duplication type. The data of two patients analyzed by this CGE‐PCR method were different from that of multiplex ligation dependent probe amplification method, and the sequencing results demonstrated that our results were correct. This UMPCR‐CGE method was considered better than the multiplex ligation dependent probe amplification method. Furthermore, this method can be used for eugenics in clinical applications.     

Chemo- and diastereoselectivity in the dimethyldioxirane oxidation of 2,3-dihydro-4H-1-benzothiopyran-4-ones and 4H-1-benzothiopyran-4-ones. Unusual reactivity of 4H-1-benzothiopyran-4-one 1-oxides

The oxidation of the 1-thiochromanones 1-3 by dimethyldioxirane (DMD) produced the corresponding sulfoxides 4-6 or sulfones 7-9; their relative amounts depended on the amount of oxidant used. A low diastereoselectivity was observed in the sulfoxidation of the 2-substituted 1-thiochromanones 2 and 3, due to the small steric differentiation during the DMD attack. An unusual reactivity pattern was found in the DMD oxidation of the 1-thiochromones 10-12, in that the sulfoxides 13-15 were more reactive toward the electrophilic oxidizing agent than the corresponding sulfides. The observed anomaly may be explained in terms of transannular stabilization of the transition structure (TS) for the sulfone formation, promoted through favorable conformational effects in the sulfoxide. Higher sulfoxide/sulfone ratios were found in solvents of greater hydrogen bond donor capacity, which is in accordance with the postulated stabilizing effect.     

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).     

First isolation of eclipsed vic-disulfoxide: 7,8-dithiabicyclo[4.2.1]nona-2,4-diene 7-exo,8-exo-dioxide

[reaction: see text] The oxidation of 7,8-dithiabicyclo[4.2.1]nona-2,4-diene 7-exo-oxide with dimethyldioxirane (DMD) provided the 7-exo-8-exo-dioxide, the structure of which was determined by X-ray crystallography [S-S 2.341(2) A and 90 degree angle O-S-S-O 4.1(3) degrees ]. The exo attack of DMD to give the exo,exo-dioxide was kinetically more favorable than the endo attack to give the endo,exo-dioxide. DFT calculations showed that the exo,exo-dioxide is thermodynamically more stable than the other stereoisomers.     

A robust combination of dysprosium vanadate/halloysite nanotubes: the electrochemical system for dimetridazole detection

Dimetridazole (DMD) is one of the significant antibiotic drugs of nitroimidazoles derivates that have attracted increasing attention in the medical field due to its pharmacological and toxicological activity. The development of high-performance sensors for continuous monitoring of DMD in food and environments is receiving increasing attention. Herein, an electrochemical platform was designed based on a dysprosium vanadate/halloysite nanotubes (DyV/HNTs) nanocomposite for the detection of DMD. The DyV/HNTs nanocomposite was examined by various spectroscopic and analytical techniques. The DyV/HNTs based electrochemical sensor reveals a distinctly higher electrocatalytic response to the reduction of DMD due to the good physiochemical properties compared to other electrodes. The DyV/HNTs based electrochemical sensor for DMD covered two linear ranges of 0.001–0.54 and 0.54–188 μM with a detection limit of 0.9 nm through the amperometric method, which is better than those previously reported. Furthermore, selectivity, stability, repeatability, and reproducibility studies were performed. Moreover, the fabricated DyV/HNTs sensor was successfully applied for the reliable discrimination of DMD in biological and water samples with satisfactory recovery values. The results indicated that this DyV/HNTs nanocomposite may be a promising electrochemical sensing platform for the determination of DMD.