Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.     

Update on current surgical management of idiopathic macular holes

Idiopathic macular holes were generally considered an untreatable condition until 1991 when the first papers reported successful closure of macular holes and visual improvement using pars plana vitrectomy, peeling of the cortical vitreous and face-down positioning. Since that time, the original pathogenesis theory has been refined as well as surgical methods and techniques. Currently, macular hole surgery is considered the most successful vitreoretinal surgery, with a greatly improved prognosis for patients. This article will review the general characteristics of macular holes and the clinical trials and outcomes that have led to the latest techniques in idiopathic macular hole surgery.     

Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

Diabetes can lead to serious microvascular complications like proliferative diabetic retinopathy (PDR), which is the leading cause of blindness in adults. The proteomic changes that occur during PDR cannot be measured in the human retina for ethical reasons, but could be reflected by proteomic changes in vitreous humor. Thus, we considered that comparisons between the proteome profiles of the vitreous humors of PDR and nondiabetic controls could lead to the discovery of novel pathogenic proteins and clinical biomarkers. In this study, the authors used several proteomic methods to comprehensively examine vitreous humor proteomes of PDR patients and nondiabetic controls. These methods included immunoaffinity subtraction (IS)/2-DE/MALDI-MS, nano-LC-MALDI-MS/MS, and nano-LC-ESI-MS/MS. The identified proteins were subjected to the Trans-Proteomic Pipeline validation process. Resultantly, 531 proteins were identified, i.e., 415 and 346 proteins were identified in PDR and nondiabetic control vitreous humor samples, respectively, and of these 531 proteins, 240 were identified for the first time in this study. The PDR vitreous proteome was also found to contain many proteins possibly involved in the pathogenesis of PDR. The proteins described provide the most comprehensive proteome listing in the vitreous humor samples of PDR and nondiabetic control patients.     

Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole

Two-dimensional gel electrophoresis and mass spectrometry were used to make a catalogue of soluble proteins in the human vitreous humor (VH). Fifty-one different proteins were identified on silver-stained two-dimensional (2D) gel patterns with VH proteins obtained from diabetic retinopathy and macular hole. Thirty of these have not been listed in the reported 2D profiles of plasma. Immunoglobulin (Ig), alpha1-antitrypsin, alpha2-HS glycoprotein,and complement C(4) fragment showed stronger spots in VH with diabetic retinopathy patient samples than those with macular hole. Pigment epithelium-derived factor, a potent inhibitor of angiogenesis in the cornea and vitreous, was clearly detected in VH with diabetes. It is impressive that the inhibitor increases in the vitreous with proliferative angiogenesis.     

Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.     

Proteomics of Vitreous Humor of Patients with Exudative Age-Related Macular Degeneration

Background

There is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD).

Methods and Findings

Eighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity.

Conclusions

Proteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.     

The role of SOCS2 in recombinant human growth hormone (rhGH) regulating lipid metabolism in high-fat-diet-induced obesity mice

In addition to regulate body growth and development process, growth hormone (GH) also involved in lipid metabolism, decreasing fat mass and improving lipolysis. To normal mice, GH could reduce their fat content, but events turned uncertain coming to the pattern of feeding high-fat-diet. In order to investigate the role of GH in adipogenesis of mice with high-fat-diet, the high-fat-diet feeding mice were randomly assigned into three groups and treated with recombinant human growth hormone (rhGH) and the somatostatin analogue octreotide respectively. Results demonstrated that both rhGH and octreotide could reduce the body weight but the trends diminished in the end. HDL-C level was increased in octreotide treated groups but the activity of lipase was increased significantly in both two groups. RhGH remarkable increased the expression of SOCS2, FAS (P < 0.01) and SREBP-1c (P < 0.05), decreased the expression of SOCS1, SOCS3 (P < 0.05) and HSL (P < 0.01) in subcutaneous fat mass. In visceral fat tissue, all genes were increased except SOCS2 (P < 0.01), at the same time the visceral fat mass was decreased. The protein phosphorylation of JAK2 and STAT5 which were treated with octreotide were increased in subcutaneous fat, visceral fat and liver (P < 0.01) and were increased significant in visceral fat by rhGH treated (P < 0.01). In liver, only JAK2 protein phosphorylation was raised (P < 0.01). In conclusion, rhGH and octreotide could decrease the whole body mass before 6 days; the trend was weaken in later period with high-fat-diet. RhGH could increase the subcutaneous fat mass and reduce the visceral fat mass, and SOCS2 might be involved in regulation of the mechanism through JAK2/STAT5 signaling pathway.     

Proteomic Insight into the Molecular Function of the Vitreous

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.     

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.     

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. ^1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. ^1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.     

Neuroadaptations in the Striatal Proteome of the Rat Following Prolonged Excessive Sucrose Intake

Obesity is a contemporary health problem of rapidly increasing prevalence. One possible cause of obesity is loss of control over consumption of highly palatable foodstuffs, perhaps mirroring the processes involved in drug addiction. Accordingly, the striatum may be a key neural substrate involved in both food and drug craving. We hypothesised here that prolonged exposure to 10 % sucrose solution might cause neuroadaptations in the striatum that are analogous to those previously reported following prolonged exposure to alcohol or recreational drugs. Male Wistar rats were given constant access to 10 % sucrose solution (in addition to normal lab chow and tap water) for 8 months and were compared with control rats receiving no sucrose access. Rats in the sucrose group typically drank more than 100 ml of sucrose solution per day and showed 13 % greater body weight than controls at the end of the 8 months. Striatal dopamine (DA) concentrations were decreased in the sucrose group rats relative to controls. Differential expression of 18 proteins was identified in the striatum of the sucrose group rats relative to controls. Down regulated proteins included pyridoxal phosphate phosphatase, involved in DA synthesis, and glutathione transferase, involved in free radical scavenging. Up regulated proteins included prolactin (which is under negative regulation by DA) and adipose differentiation-related protein, involved in fat synthesis. We hypothesise that DA-related neuroadaptations in the striatum caused by prolonged sucrose intake may partly drive compulsive intake and seeking of high palatability foodstuffs, in a similar way to that observed with drug and alcohol addictions.     

囊胚形成的基因表达与调控(英文)

囊胚形成是胚胎早期发育过程中一个重要阶段 ,涉及几个重要的生理事件 ,即细胞融合 (compaction ,亦称致密化作用 )、囊胚腔出现、囊胚腔扩张及滋养层和内细胞团的分化。在细胞间连接蛋白的作用下 ,各种细胞间连接方式逐步建立起来 ,在合子型基因组表达调控下 ,促进了最终囊胚的形成。细胞间连接蛋白和细胞粘附相关蛋白参与组建各种细胞间连接 ,参与细胞融合、囊胚腔形成、滋养层分化和囊胚扩张等过程。通过顶部的紧密连接、侧部的缝隙连接和桥粒 ,建立起细胞的连接复合体。在人胚胎 8 细胞之前 ,卵裂球细胞界限明显 ,可能以中间连接方式相互作用 ;8 细胞期发生致密化作用 ,通过紧密连接将细胞分成顶部和基部 ,使得胚胎处于半封闭状态 ,促进胚胎内部积液 ,形成囊胚腔。细胞融合的同时也产生缝隙连接。桥粒最初出现在人胚胎达到 3 2 细胞阶段 ,桥粒连接参与囊胚腔形成以及在囊胚扩张时维持滋养层的稳定性。桥粒由一些跨膜粘蛋白组成 ,包括参与细胞内粘附的桥粒子和桥粒球以及一些细胞质内蛋白 (如desmoplakins,plakoglobin ,plakophilin) ,由细胞内蛋白质形成空斑结构并介导细胞角蛋白丝固定。对植入前牛胚胎的研究表明 ,只有DcII,DcIII和plako三种桥粒蛋白参与桥粒组建。在鼠囊胚中DcII的表达部位位于     

Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat

The recent introduction of fluorescent two-dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30-day-old kittens, in order to identify proteins expressed in an age-related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two-dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.     

Influence of exogenous abscisic acid on alterations in protein expression in the proteome of Triticosecale seedlings

The aim of this study was to investigate molecular basis of germination inhibition of Triticosecale under the influence of exogenous ABA. Seedlings were isolated from seeds after 48 h of germination in water (control sample) and 100 μM ABA solution. It was observed that the applied concentration of phytohormone caused a significant inhibition of germination and a suppressed contribution of polysomes in the total ribosomal fraction. To identify proteins whose expression was affected by ABA, a proteomic approach employing 2D electrophoresis was used. Proteome maps for both control and ABA-treated samples displayed over 2,200 Coomassie Brilliant Blue-stained spots each. Protein spots showing either increase or decrease in spot intensity under the ABA influence were identified by mass spectrometry. Many of the identified proteins whose expression was stimulated by ABA proved to be stress-related, involved in nucleotide metabolism or playing a regulatory and signal transduction role, whereas proteins whose expression decreased are known to be involved in numerous metabolic pathways (including energy, carbohydrate or nucleotide metabolism) as well as related to genetic information processing and protein synthesis. Our results show that the germination inhibition caused by ABA is a result of multigene, diversified actions leading together to triticale growth suppression.     

Systems pathology analysis identifies neurodegenerative nature of age‐related vitreoretinal interface diseases

Aging is a phenomenon that is associated with profound medical implications. Idiopathic epiretinal membrane (iEMR) and macular hole (MH) are the major vision‐threatening vitreoretinal diseases affecting millions of aging people globally, making these conditions an important public health issue. iERM is characterized by fibrous tissue developing on the surface of the macula, which leads to biomechanical and biochemical macular damage. MH is a small breakage in the macula and is associated with many ocular conditions. Although several individual factors and pathways are suggested, a systems pathology level understanding of the molecular mechanisms underlying these disorders is lacking. Therefore, we performed mass spectrometry‐based label‐free quantitative proteomics analysis of the vitreous proteomes from patients with iERM and MH to identify the key proteins, as well as the multiple interconnected biochemical pathways, contributing to the development of these diseases. We identified a total of 1,014 unique proteins, many of which are linked to inflammation and the complement cascade, revealing the inflammation processes in retinal diseases. Additionally, we detected a profound difference in the proteomes of iEMR and MH compared to those of diabetic retinopathy with macular edema and rhegmatogenous retinal detachment. A large number of neuronal proteins were present at higher levels in the iERM and MH vitreous, including neuronal adhesion molecules, nervous system development proteins, and signaling molecules, pointing toward the important role of neurodegenerative component in the pathogenesis of age‐related vitreoretinal diseases. Despite them having marked similarities, several unique vitreous proteins were identified in both iERM and MH, from which candidate targets for new diagnostic and therapeutic approaches can be provided.     

Influence of Ki-ras-driven oncogenic transformation on the protein network of murine fibroblasts

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.