北京市方庄地区儿童全血微量营养素含量检测结果分析

目的:通过分析北京市方庄地区儿童全血微量营养素的含量及影响因素,研究探讨微量营养素缺乏的防治措施。方法:2011年1月至12月北京中医药大学东方医院儿科门诊1887例健康体检儿童,收集末梢血,采用原子吸收光谱仪检测全血5种微量营养素铜、锌、钙、镁、铁的含量,并从性别、年龄、季节(月份)方面进行比较分析。结果:全血5种微量营养素含量无性别差异(P0.05),但存在季节差异,4-6月铜、锌含量降低;7-9月钙、镁含量降低;10-12月铁含量降低(P0.05)。全血锌、铁含量存在年龄差异,婴幼儿最低(P0.05)。1887例儿童中,缺乏较多的元素依次为铁(38.8%)、钙(22.3%)、锌(12.8%),其中,4-7岁(17.0%)、7-12岁(21.5%)年龄段组以锌缺乏居多。1岁以内(51.2%)、1-3岁(44.6%)年龄段组以铁缺乏居多。全血铜、锌、钙、铁缺乏无性别差异(P0.05)。全血5种微量营养素缺乏存在季节差异,1-3月镁缺乏明显,4-6月铜、锌缺乏明显,10-12月钙、铁缺乏明显(P0.05)。结论:北京市方庄地区儿童主要存在锌、钙、铁的缺乏,以秋冬季及初春明显。其中婴幼儿以铁缺乏为主,学龄前期及学龄期儿童以锌缺乏为主。应加强对此地区儿童的营养宣教和饮食指导。     

马麝全血和被毛矿物元素测定

检测了马麝主要树叶饲料和健康马麝全血、被毛中6种微量元素(Cu、Mn、Fe、Zn、Mo、Se)及2种常量元素(Ca、Mg)的含量,并与当地健康山羊的全血和被毛值进行了比较分析。结果表明,马麝的10种树叶饲料为低Se饲料,被毛中Cu、Mn、Zn、Ca、Mg含量明显高于全血水平,而Fe则反之。马麝全血和毛样中的各元素分布比较集中,离散度均很小。     

重组人血清白蛋白生产工艺研究进展

人血清白蛋白是一种重要的临床药物 ,人源血浆白蛋白可能会传染病原体将逐步被重组白蛋白所代替。对用巴斯德毕赤酵母生产重组人血清白蛋白的表达系统、发酵方法、分离纯化技术以及重组白蛋白的理化及生理特性进行了综述。     

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.High-resolution structures of proteins are essential for understanding cellular processes. Determining protein structures, however, is difficult: protein stability, purity, quantity, and solubility critically affect success. Nuclear magnetic resonance (NMR)¹ spectroscopy can only be applied to proteins of limited size, whereas x-ray crystallography necessitates prior crystallization of the protein. These conditions make structure determination challenging for many proteins of biological relevance. This includes especially proteins that contain intrinsically unstructured or long coiled-coil regions, proteins associated to a membrane (1, 2) or parts of multi-protein complexes (3). New developments to overcome some of these restrictions include x-ray free electron lasers (XFEL) (4), which only require microcrystals, new detectors in cryo-electron microscopy (5) and in-cell NMR (6), which analyzes the structure of small proteins in a cellular context. Further advancements that assist with protein structure determination have included the development of being able to use sparse NMR data, for example using backbone only data (7), and the understanding of evolutionary constraints for predicting protein structure (8).We present a novel approach to obtain structural details of proteins by mass spectrometry. This can be accomplished through cross-linking and mass spectrometry (CLMS) (9–11). Cross-links establish covalent bonds between residue pairs close in space but not necessarily in sequence. This conserves structural information throughout an analysis that follows the standard proteomics workflow. Typically, a bi-functional chemical reagent, the cross-linker, is incubated with a protein of interest. The cross-linker reacts with two residues—often involving the side-chain amine of lysine—that are near each other in the folded protein. A protease such as trypsin is used to degrade the protein. The resulting mix of cross-linked peptides is then analyzed by mass spectrometry and database searching akin to other shotgun proteomics approaches (12). The pairs of cross-linked residues are identified from the mass spectrometric data and provide information on which residues are near each other in the folded protein. This information is represented in the form of distance constraints, deducible from the length of the cross-linking agent.CLMS data has been used to study large multi-protein complexes (13), networks (14) and proteins in whole cells (15). The distance constraints obtained are sparse but complement other structural data in integrated structural biology well (10). Cross-link data allow placing high-resolution structures of individual sub-units in the electron microscopy structure of an assembled multi-protein complex to obtain its quasi-atomic resolution structure, e.g. the proteasome (16). In an alternative approach, genetic site-directed positioning of a photo-reactive group, azide, as part of a phenylalanine analog, was recently used to derive proximity information that allowed modeling of receptor CRF1R bound to its native ligand (17). Young et al. used 15 cross-links to identify the correct fold of bovine basic fibroblast growth factor using threading and homology modeling (18). In a similar study, Singh et al. used eight cross-links to build a monomer homology model of the major capsid protein E of bacteriophage lambda and to derive a pseudoatomic model of the lambda procapsid shell (19). In both of the aforementioned cases, the cross-link information was merely used to verify structural models by threading and homology modeling, and did not significantly impact model building. Prior attempts to leverage cross-linking data in structure determination delivered improvements, however, without leading to high-resolution models (20).Here, we increase the spatial resolution of information obtained through cross-linking by using a highly reactive chemical as a cross-linking agent. This broadens the specificity of cross-linking and thus increases the spatial resolution in conjunction with mass spectrometry. We employ the heterobifunctional chemical cross-linker sulfosuccinimidyl 4,4′-azipentanoate, sulfo-SDA (21), to chemically cross-link a protein, human serum albumin (HSA).We combine the distance constraints provided by cross-linking and mass spectrometry with computational, conformational space search. This approach allows us to generate structural models of HSA domains that correlate highly with the structure of HSA solved by x-ray crystallography. With this method, we show that our pipeline can be used to analyze the structure of HSA domains from HSA not only in it''s purified form, but additionally unpurified and in its native environment, human blood serum.     

Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum

Molecular Biology - Serum amyloid A is an inflammatory biomarker whose concentration changes during infectious and inflammatory diseases. SAA’s tendency for aggregation and complex formation...     

Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA,Guthrie Blood Spot,and Whole Blood

Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer’s disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.     

2,3-Diphosphoglycerate Content of Human Arterial and Venous Blood

THE glycolytic intermediate, 2,3-diphosphoglycerate, is an intracellular regulator of the oxygen affinity of haemoglobin^1,2. At high altitudes there is a direct relationship between the decreased oxygen affinity of haemoglobin and the increased concentration of diphosphoglycerate in the blood³. This was explained by Benesch et al.⁴ and Chanutin et al.⁵, who found that the binding of diphosphoglycerate to haemoglobin reduces the oxygen affinity and by our finding that the concentration of diphosphoglycerate increases when the red cells are incubated under low oxygen tension^6,7, thereby releasing oxygen from haemoglobin. For the same reason, the oxygen tension is reduced during the circulation of blood from the pulmonary alveoli to the tissues; the decreased level of the diphosphoglycerate facilitates the binding of oxygen to haemoglobin in the pulmonary alveoli and the increased level of the diphosphoglycerate in the blood of the capillaries decreases the affinity of haemoglobin for oxygen. We have measured the amount of 2,3-diphosphoglycerate and other glycolytic intermediates in arterial and venous blood to test this supposition.     

THz Exposure of Whole Blood for the Study of Biological Effects on Human Lymphocytes

The aim of the present study is toinvestigate the genotoxic effect of THzradiation in human peripheral bloodlymphocytes following 20 minutes exposureto 1 mW average power Free Electron Laserradiation in the frequency range 120–140GHz. For this purpose 9 healthy donors wereemployed and cytokinesis block techniquewas applied to study micronucleusfrequency and cell proliferation. Theresults obtained indicate that all theelectromagnetic conditions adopted so far do not alter the investigated parameters,suggesting absence of direct chromosomaldamage and alteration of cell cyclekinetics (two tailed paired Student's test:p> 0.05 in all cases).     

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16⁺ cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.     

Micro-Buffy Coats of Whole Blood: A Method for the Electron Microscopic Study of Mononuclear Cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.     

Simultaneous Determination of Cd, Pb, Cu, Zn, and Se in Human Blood of Jordanian Smokers by ICP-OES

A total of 73 blood samples (56 from smokers and 17 from nonsmokers) were collected to determine the concentrations of selected heavy metal in the whole blood of smokers and nonsmokers living in and around the city of Amman, Jordan. Analysis of heavy metals in the whole blood samples of various groups took in consideration the number of cigarettes smoked per day. The analysis of blood samples was carried out using inductively coupled plasma optical emission spectrometry. This study aimed to evaluate the blood metal levels in smokers and nonsmokers and to assess the influence of smoking cigarettes on blood metal levels. The results were compared with those from a control group. The results indicated that the average concentrations of cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), and selenium (Se) were 0.0313, 0.344, 2.328, 3.214, and 0.332 mg/L, respectively. Statistical analysis of results indicated that these average concentrations were significantly higher compared with the average concentrations in nonsmokers (P < 0.05). Moreover, the correlations between blood metal and other blood metal levels in smokers, the correlations between blood metal and other blood metal levels in nonsmokers, and the correlations between blood metal concentration in smokers and its concentration in nonsmokers were calculated. The standard reference material (blood serum National Institute of Standards and Technology 1598) and the quality control were used to validate the reliability of the method used for the estimation of heavy metals in blood samples. Results revealed that there was an agreement between the certified values and the measured values.