Proteomic analysis of human reproductive fluids

Fertilization, fetal development, and delivery depend upon a coordinated series of events in the oocyte, the embryo, and the supporting tissues and fluids. Proteomic techniques which are capable of identifying and characterizing multiple proteins simultaneously have added new dimensions to the field of human reproduction. Application of these high throughput methodologies in pregnancy-related research has begun to provide a novel perspective on the biochemical pathways involved in pregnancy and its related disorders. Most of the existing research on human reproduction and gestation has focused on follicular fluid (FF) and amniotic fluid (AF). Proteome analysis of FF has yielded significant information relevant to oocyte maturation and quality. Studies performed on the protein content of AF cells and supernatant contributed to the comprehension of the underlying pathophysiology, clinical diagnosis of pregnancy-related disorders and identification of relevant disease biomarkers. Although proteome technologies in reproduction research are not as yet widely applied, characterization of the proteome of reproductive fluids can be expected to significantly improve maternal healthcare in the future.     

Proteomic analysis for human urinary proteins associated with arsenic intoxication

Arsenic is widely distributed in nature and is mainly transported in the environment by water. Consumption of artesian well water with high levels of arsenic has been associated with genitourinary cancer, especially bladder transitional cell carcinoma (TCC). To search for biomarkers that are specific for arsenic associated with the diagnosis of bladder TCC, proteins in the urine of non-cancer urological patients and patients with either bladder TCC or arsenic-associated bladder TCC were systematically examined by HPLC ESI-MS/MS. Urine specimens were collected by catheterization from patients and age- (within 5?years) and sex-matched non-cancer urological patients. A nano-HPLC-ESI-MS/MS was used to generate proteome patterns from urine specimens obtained from patients with arsenic-associated (n?=?8) and non-arsenic-associated (n?=?8) primary TCC and from sex- and age-matched non-cancer urological patients (n?=?8). Three urinary proteins were found to have significantly altered levels in patients following chronic arsenic exposure. These proteins were a disintegrin and metalloprotease (ADAM) protein, a calpain9, and ring finger protein 20. The large-scale identification of urinary proteomes using HPLC ESI-MS/MS may serve as an ideal and efficient method to establish a panel of potential arsenic-associated TCC biomarkers and may help elucidate the mechanisms involved in bladder cancer induced by chronic arsenic exposure.     

Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin

We used 2‐D DIGE to analyze the early response of NB‐4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca²⁺ binding proteins such as translationally controlled tumor protein rose and hippocalcin‐like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB‐4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.     

Proteomic analysis of a podocyte vesicle-enriched fraction from human normal and pathological urine samples

Podocytes (glomerular visceral epithelial cells) release vesicles into urine. Podocyte vesicle-enriched fractions from normal and pathological human urine samples were prepared for proteomic analysis. An immunoadsorption method was applied and enrichment of podocyte vesicles was assessed. We identified 76 unique proteins. One protein, serum paraoxonase/arylesterase 1 (PON-1), was newly identified in normal human urine sample. We confirmed this result and showed PON-1 expression in normal human kidney. These results demonstrated the potential for using the urine samples enriched in podocyte vesicles as a starting material in studies aimed at discovery of biomarkers for diseases.     

Proteomic expression analysis and comparison of protein and mRNA expression profiles in human malignant gliomas

Gliomas are highly heterogeneous and therapy resistant tumors with a poor prognosis. Novel experimental therapeutic approaches have shown some promising results, but often target specific molecular mechanisms or antigens, and careful characterization of the molecular subgroup of the tumors will therefore likely be important. Thorough investigations of gene and protein alterations are also important to better understand the tumorigenic mechanisms. We have undertaken a proteomic approach, using 2-D DIGE and LC-MS/MS protein identification, to investigate 38 human gliomas and normal brains. We show that the proteome profile can discriminate between normal brain and tumors, and between tumors of varying grade by a supervised classifier. Furthermore, an analysis of the identified proteins shows an enrichment of proteins associated to pathways known to be central in gliomas, such as MEK/Erk signaling and actin cytoskeleton. It also shows a shift between different glial fibrillary acidic protein (GFAP) representatives in different grades. In a previous study the gene expression profile was characterized in an almost identical set of tumors, which enabled a paired analysis of the gene and protein expression profiles. We show that there is often a weak correlation between the mRNA and protein level. This, together with the ability of proteomics to identify PTMs, emphasizes the benefit of characterization on a protein level.     

Proteomic approaches to the analysis of atopic dermatitis and new insights from interactomics

In this review, we summarized the recent findings regarding atopic dermatitis (AD) skin disease based on proteomic studies. AD is a chronic relapsing inflammatory skin disease typically characterized by a distribution of eczematous skin lesions with lichenification, pruritic excoriations and dry skin with wide varieties of pathophysiological aspects. We summarized the alterations of the protein expressions in the primary cultured AD cells from the patients'-biopsy samples that were mostly analyzed by 2-D PAGE and MALDI-TOF. Further, we also conducted protein-protein interaction mapping according to the obtained candidate proteins. As a result, we found that several hub proteins, i.e. heat shock 70-kDa protein 2, heat shock 70-kDa protein 9, tumor rejection antigen-1 (gp96), spermatogenesis-associated factor, protein kinase C inhibitor 1, vimentin, tenascin, semaphorin 4f (SEMA4F), complement component C1r deficiency (C1R) and apolipoprotein A (LPA), respectively, could receive important consideration in future studies. Since the mechanism of AD disease has been shown to be complex, our results may provide new clues to aid understanding of AD.     

Cyclic inelastic analysis of tubular column sections

An analytical study of the inelastic cyclic moment-thrust-curvature behavior and moment-thrust-axial strain behavior for fabricated tubular cross sections as commonly used in offshore structures is presented. The material characteristics are assumed to be elastic-perfectly plastic and typical residual stresses for such columns are included in the analysis. Based upon the tangent stiffness formulation, a computer program was developed to provide numerical results. In particular, the following two loading cases are studied numerically in detail: (1) the axial force P is held constant while the moment M is cycled, (2) P and M are both cycled proportionally. Shakedown study of the tubular sections is also given.     

Elastoplastic analysis of tubular joints of offshore platforms

The subject of this paper is the analysis and design of complex tubular joints, eventually including internal and external gussets and stiffener rings, corresponding to fixed and mobile offshore structures.Two different joints are analysed. In the first case an X joint is studied for elastic and elastoplastic behaviour, loading up to collapse in order to determine ultimate strength and safety factor. Finite elements which can reproduce the elastic and plastic singularities of the stress and the strain fields in the crack tip, are then used for the analysis of a T joint. Both direction and rate are considered in the crack propagation, and an elastoplastic analysis is carried out, to determine the crack opening displacement (COD).Finally, the consideration of fatigue effects in tubular joints is discussed, and techniques for evaluating fatigue life are outlined.     

投影寻踪在火灾后混凝土性能评价中的应用

在研究高强混凝土高温后性能的基础上,通过对影响火灾后高强混凝土结构性能的主要因素进行分析,选择抗压强度损失系数、耐久性损失系数、爆裂损失系数和裂纹损失系数作为评价指标,以投影寻踪回归理论为基础,提出综合评定高温后损伤混凝土性能的方法,建立了投影寻踪综合评价模型,编制了基于MATLAB的相应程序,采用人口迁移算法寻求最优投影方向,根据投影特征指标值对高强混凝土高温后性能进行综合评价。研究表明,该方法避免了评判专家人为确定权重,评价结果客观准确、方法简单,为混凝土结构火灾损伤的诊断评估与修复加固提供科学依据。     

Trajectory analysis of nonisothermal tubular reactor nonlinear models

The existence and uniqueness of the state trajectories (temperature and reactant concentration) are analyzed for nonisothermal plug flow and axial dispersion tubular reactor models. It is mainly shown that these trajectories exist on the whole (nonnegative real) time axis and the set of all physically feasible state values is invariant under the dynamics equations. The main nonlinearity in the model originates from the Arrhenius-type kinetics term in the model equations. The analysis essentially uses Lipschitz and dissipativity properties of the nonlinear operator involved in the dynamics and the concept of state trajectory positivity.     

Proteomic analysis of pericardial effusion: Characteristics of tuberculosis-related proteins

The aim of this study has been designed to identify the tuberculosis (TB)-related proteins in pericardial effusion by proteomic approaches. TB is one of the major infectious diseases causing pericardial effusion. This study details protein profiles in pericardial effusion from three TB patients and three heart failure patients. Pericardial effusions were analyzed using 2-DE combined with the nano-HPLC-ESI-MS/MS. Eleven protein spots with differential expression in pericardial effusion were identified between the two groups of TB and heart failure patients (the control group). Seven protein spots were upregulated and four were downregulated. The composition of the pericardial effusion proteome may reflect the pathophysiological conditions affecting the progression of tuberculous pericarditis. The proteins in the tuberculous pericardial effusion with differential expression may serve as new and direct indicators of drug treatment. A possible conclusion is indicated that fibrinogen may play an important role for fibrin assembly in tuberculous pericardial effusion.     

Proteomic analysis of alpha-1-antitrypsin in immunoglobulin A nephropathy

Immunoglobulin A nephropathy (IgAN) is recognized as the most common form of primary glomerulonephritis worldwide. It is characterized by mesangial cell proliferation with mesangial IgA deposition in the glomeruli, and is usually associated with secondary tubulointerstitial injury. Although significant progress has been made in the clarification of the pathogenesis of IgAN, the exact pathogenetic mechanism remains unclear. To find out the candidate proteins that play an important role in IgAN, renal cortex tissues and urine from IgAN patients were studied. The 2‐DE was performed on renal tissues of IgAN and normal controls. A series of spots identified as alpha‐1‐antitrypsin (AAT) by mass spectrometry, were found to be significantly increased in patients with IgAN. Up‐regulation of AAT variants was validated in renal cortex tissues of IgAN using Western blot and 2‐DE immunoblot. Lower isoforms (?48 kDa) and fragments (?33 kDa), suspected as cleavage forms by proteinase attack, were especially increased in IgAN compared to normal controls. In addition, AAT proteins modified by tyrosine nitration (approximately 57 and 48 kDa), which reflects excessive oxidative stress, were increased in IgAN tissue. Additionally in the urine of IgAN, increase of AAT variants and fragments was detected by 2‐DE immunoblot as well as Western blot. Immunohistochemical staining of IgAN kidney tissue revealed that the increase of AAT appeared to be derived from hypertrophic proximal tubules. The AAT staining in the glomerulus was not clear in IgAN. In addition, immunodepletion‐zymography showed a positive correlation between AAT and 80–110‐kDa proteinases in IgAN tissue. Further studies regarding the functional roles of AAT and the proteinases will allow better understanding of the pathogenesis of IgAN.     

Proteomic analysis of secretory proteins and vesicles in vascular research

The release of proteins and membrane vesicles in the bloodstream regulates diverse vascular processes, both physiological, such as angiogenesis and haemostasis, and pathological, such as atherosclerosis and atherothrombosis. Proteomics, beside its canonical application for the expression profiling in cells and organs, can be applied to the study of secreted proteins and microvesicles, which play a significant role in the homeostasis of the vasculature, and the development of the atherosclerotic disease.     

Proteomic analysis to improve adequacy of hemo- and peritoneal dialysis: Removal of small and high molecular weight proteins with high- and low-flux filters or a peritoneal membrane

The reduction of morbidity and mortality in patients undergoing hemo- or peritoneal dialysis is strongly related to an efficient and selective clearance of uremic toxins. We used proteomics methods to analyze and further characterize the dialytic removal of still undefined middle and high molecular weigh proteins as a basis for further improvement of dialysis assessment. Dialysates from 26 hemodialysis patients treated with different types of low- (F6HPS?) and high-flux (FX80?, APS650?, FX60?) filters as well as peritoneal fluids from 10 continuous ambulatory peritoneal dialysis (CAPD) patients were analyzed by SELDI-TOF and 2-DE. The protein patterns showed selective differences in the proteins cleared depending on the dialysis method used and the filter membrane. While SELDI analyses of dialysates from the F6HPS revealed almost no protein clearance, high-flux filters and CAPD dialysates showed protein release of different molecular weight ranges. Furthermore, 2-DE and MS analysis identified 48 different proteins from the dialysate of high-flux filters and 21 from peritoneal dialysis fluids. In F6HPS dialysates, however, only few proteins could be identified.