Application of two-dimensional electrophoresis in the research of retinal proteins of diabetic rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.     

立体定向脑内移植神经干细胞改善颅脑损伤大鼠的神经运动功能

BACKGROUND: Cell replacement therapy as an effective strategy for reconstruction of the central nervous system has very broad application prospects. OBJECTIVE: To investigate the effect of stereotactic transplantation of neural stem cells into the brain on the neuromotor function of craniocerebral trauma rats. METHODS: Twenty male Sprague-Dawley rats were equivalently randomized into study and control groups. Animal models of craniocerebral trauma were made using the improved free-fall method in the rats. Then, model rats in the study and control groups were given parenchymal transplantation of embryonic neural stem cells and the same volume of culture medium with no stem cells at 1 day after injury, respectively. Neuromotor function of rats was assessed based on the neurological severity scores. At 2 weeks after transplantation, brain tissues were taken for hematoxylin-eosin staining, anti-BrdU, glial fibrillary acidic protein, β-tubulin III and tyrosine hydroxylase immunohistochemistry staining. RESULTS AND CONCLUSION: The neurological severity scores in the study group were significantly lower than those in the control group at 1 and 2 weeks after injury (P < 0.05). In the study group, there were many BrdU-positive neural stem cells in the brain tissues, some of which were positive for glial fibrillary acidic protein, β-tubulin III and tyrosine hydroxylase; while in the control group, there was no BrdU-positive cell in the brain tissues. Experimental findings show that neural stem cells stereotactically transplanted into the brain can proliferate and differentiate in the brain lesion, and thereby notably improve the neuromotor function of rats with craniocerebral trauma.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

Perineuronal germinal cells in the rat cerebral cortex

The generation and renewal of cells in the adult mammalian central nervous system maintains brain functions, including plasticity. Even in the cerebral cortex of adult mammals, glial cells are thought to be replaced with newly generated cells every 100 days. Recently, we demonstrated that this proliferation is stimulated by neural activity. However, whether any germinal areas exist in the cortical parenchyma is unknown. Here, we examined the proliferating cell dynamics in the cerebral cortex of adult rats using BrdU labeling and immunohistochemistry for NeuN and lamin B1. At 2 h after a single injection of BrdU, more than 80% of BrdU-labeled cells were observed in the perineuronal territory in which the BrdU-labeled nuclei were located within 5 μm from neuronal nuclei. The ratio of perineuronal cells to nonperineuronal cells in BrdU-labeled cells gradually decreased over the 2 weeks following BrdU injection. These observations indicate that numerous cortical cells proliferate in the perineuronal territory, the germinal soil, and that part of these newly generated cells migrate from the perineuronal territory into the surrounding areas during the 2 weeks following mitosis.     

Interaction between various 14-3-3β segments and PrP In Vitro

Objective To study the potential interaction between PrP protein.Methods The supernatant of health and scrapie-infected hamsters' brain homogenate was prepared,while various recombinant 14-3-3β or PrP proteins were purified.The possible molecular interaction between 14-3-3β proteins and PrP was tested by pull-down and immunoprecipitation assays.Results Both native PrPc and its protease-resistant isoform(PrPsc)formed complexes with 14-3-3β.The full-length recombinant 14-3-3β proteins interacted with PrP.The domain responsible for interacting 14-3-3β was located at N-terminal of 14-3-3β(residues 1 to 38).Conclusion The studies of the association of PrP with 14-3-3β may further provide insight into a potential role of 14-3-3β in the biological function of PrP and the pathogenesis of prion disease.     

Interaction between various 14-3-3β segments and PrP In Vitro

Objective To study the potential interaction between PrP protein.Methods The supernatant of health and scrapie-infected hamsters' brain homogenate was prepared,while various recombinant 14-3-3β or PrP proteins were purified.The possible molecular interaction between 14-3-3β proteins and PrP was tested by pull-down and immunoprecipitation assays.Results Both native PrPc and its protease-resistant isoform(PrPsc)formed complexes with 14-3-3β.The full-length recombinant 14-3-3β proteins interacted with PrP.The domain responsible for interacting 14-3-3β was located at N-terminal of 14-3-3β(residues 1 to 38).Conclusion The studies of the association of PrP with 14-3-3β may further provide insight into a potential role of 14-3-3β in the biological function of PrP and the pathogenesis of prion disease.     

The testis-specifically expressed gene Trim69 is not essential for fertility in mice

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis.The tripartite motif(TRIM)family proteins,most of which have E3 ubiquitin ligase activity,are highly conserved in mammals.They are involved in important cellular processes such as embryonic development,immunity,and fertility.Our previous studies indicated that Trim69,a testis-specific expressed TRIM family gene,potentially participates in the spermatogenesis by mediating testicular cells apoptosis.In this study,we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology.Here,we reported that the male Trim69 knockout mice had normal fertility.The adult knockout mice have shown that the appearance of testes,testis/body weight ratios,testicular histomorphology,and the number and quality of sperm were consistent with wild-type mice.These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility,and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis.This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.     

神经生长相关蛋白在脑损伤修复中的作用

Neuronal growth associated protein (GAP-43 ) is a membrane bound phosphoprotein found in the axonal growth cones of sprouting central nervous system axons. It is neuron specific and is proposed to play a critical role in growth cone function during development of the nervous system or the regenerative recovery from axon injury. The injury leads to the transfection of fibroblasts with the GAP-43 gene, which result in increasing GAP-43 expression. GAP-43 is expressed at high levels during the course of recovery of brain injury. GAP-43 acts with calmodulin and G protein to regulate metabolic responses that initates axonal growth. It is important to reestablish neuronal structure and axonal sprouting. The level of GAP-43 returns to basal level once sprouting has finished. So more and more researchers attach importance to the study of GAP-43, which will improve the level of treatment with brain injury.     

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 (Ctomp2) is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species . To purify the protein and to prepare monoclonal antibodies (mAbs) against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni2 -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1:40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans . It had high specifity. In comparison with gold standard test-cell culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein , but also to the establishment of the method for its clinical application, for it had not been reported before.     

Expression of IL-10 and TNF-α in Rats with Cerebral Infarction after Transplantation with Mesenchymal Stem Cells

We investigated the effects of bone marrow-derived mesenchymal stem cells （MSCs） transplantation on the recovery of neurological functions in rat＇s MCAO （middle cerebral artery occlusion） model and its mechanism. MSCs were isolated from bone marrow of male Sprague Dawley （SD） rats. Female adult SD rats were randomly assigned into 4 groups： sham-operated group, MCAO group, vehicle group and MCAO ＋ MSCs-treated group. MSCs were injected into the lateral ventricle of rats in the MSCs-treated group and the same volume of PBS was given to the vehicle group. The expressions of IL-10 and TNF-α were assayed by RT-PCR and ELISA detections at day 1 and 4 after MCAO. The infarction volume was measured by TTC-staining. All rats underwent behavioral tests before, as well as 1, 4, and 14 days after MCAO. MSCs significantly improved functional recovery compared with the control at day 14 after transplantation. Compared with the MCAO group and the vehicle group, the expression of IL-10 mRNA and its protein level in the MSCs group significantly upregulated. However, the expression of TNF-α at day 4 after MCAO in the MSCs group significantly decreased compared with that of the MCAO group and the vehicle group. As a result, transplantation with MSCs significantly decreased infarct volume at day 1 and 4. This study strongly suggested transplantation with MSCs could reduce neuronal injury post focal cerebral ischemia in rats partly by regulating the expressions of IL-10 and TNF-α in the brain.     

过磷酸化tau蛋白在阿尔茨海默病患者脊髓中的表达

目的 观察过磷酸化tau蛋白在阿尔茨海默病(AD)患者脊髓中的表达及其在AD发病中的可能机制.方法 常规取神经病理证实的3例AD患者脊髓和7例老龄非痴呆性且无重要神经系统疾病对照患者脊髓,胸2、胸8、胸10、腰4和骶2平面取材.常规制片,行Gallyas-Braak染色及过磷酸化tau蛋白(AT8)免疫组织化学染色(SP法),光镜观察过磷酸化tau蛋白在AD及各对照组脊髓的分布情况.结果 3例AD患者中2例在脊髓前角出现过磷酸化tau蛋白阳性神经元,3例出现过磷酸化tau蛋白阳性轴索或轴索样结构,3例出现胶质细胞内过磷酸化tau蛋白表达,过磷酸化tau蛋白阳性程度与脑内神经原纤维缠结程度相关;对照组中仅1例脊髓胸2平面可见过磷酸化tau蛋白阳性的点状结构.结论 过磷酸化tau蛋白可以在AD患者脊髓内表达,提示轴浆运输障碍可能在AD的发病机制中具有一定作用.

Abstract：

Objective To study the expression of tau-related protein in spinal cord of Chinese patients with Alzheimer's disease. Methods Gallays-Braak stain and immunohistochemical study for tau protein (AT8) were carried out in the spinal cord tissue (T2, T8, T10, L2 and S2 segments) of 3 Chinese patients with Alzheimer's disease. Seven age-matched cases without evidence of dementia or neurologic disease were used as controls. Results Neurofibrillary tangles were identified in the neurons of anterior horn in 2 Alzheimer's disease cases but none was observed in the controls. Tau-positive axons and astroglia were detected in all Alzheimer's disease cases. Tau immunoreactivity in spinal cord of the patients correlated with that in brain tissue. Conclusion The expression of tau-related protein is demonstrated in the spinal cord of Alzheimer's disease patients suggesting that axonal transport defect may play a role in the pathogenesis of Alzheimer's disease.     

Lateralization of choline acetyltransferase (ChAT) activity in fetus and adult human brain

Choline acetyltransferase (ChAT) activity was measured in the first temporal gyrus (Brodmann area 22) and in the gyrus precentrale (Brodmann area 4) of both hemispheres in post-mortem human fetus and adult brains. In fetal brains the ChAT values were higher in the right first temporal gyrus in comparison to the left one (P < 0.05). Conversely in adult brains ChAT activity was higher in the left first temporal gyrus than in the controlateral one (P < 0.05). Absolute values of ChAT were similar in the right first temporal gyrus of fetus and adult brains. No asymmetry was found in the fetal and adult gyrus precentrale. These findings could be interpreted in terms of differential rates of development of cholinergic synapses in left and right human temporal lobes.     

Proteomic analysis of human omental adipose tissue in the polycystic ovary syndrome using two-dimensional difference gel electrophoresis and mass spectrometry

BACKGROUND: Our aim was to study the protein expression profiles of omental adipose tissue biopsies obtained from morbidly obese women with or without polycystic ovary syndrome (PCOS) at the time of bariatric surgery to evaluate the possible involvement of visceral adiposity in the development of PCOS. METHODS: Ten PCOS patients and nine control samples were included. We used two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel digestion, and mass spectrometry (MS) of selected protein spots. RESULTS: The 2D-DIGE technology allowed the analysis of approximately 1840 protein spots in the comparative study of control and patient proteomes, revealing 15 statistically significant spot changes (>2-fold, P < 0.05). Unambiguous protein identification was achieved for 9 of these 15 spots by MS. This preliminary study revealed differences in expression of proteins that may be involved in lipid and glucose metabolism, oxidative stress processes and adipocyte differentiation; they include proapolipoprotein Apo-A1, annexin V, glutathione S-transferase M3 (GSTM3), triosephosphate isomerase, peroxiredoxin 2 isoform a, actin and adipocyte plasma membrane-associated protein. The most relevant finding was an increase of GSTM3 in the omental fat of PCOS patients confirming previous studies conducted by our group. CONCLUSIONS: Proteomic analysis of omental fat reveals differential expression of several proteins in PCOS patients and non-hyperandrogenic women presenting with morbid obesity. The application of this novel methodology adds further evidence to support the role of visceral adiposity in the pathogenesis of PCOS.     

New allelic variant of triosephosphate isomerase found in cultured tumor cells of human prostate

A proteomic study of the proteins of the cell lines that model malignant and benign human prostate tumors (LNCaP, DU145, PC3, BPH-1) revealed a new allelic variant of the triosephosphate isomerase caused by the amino acid substitution 233G → D in the LNCaP. The study of the exon region 7 in TPI1 gene showed that the cells of the LNCaP line were heterozygous for a single-nucleotide substitution 2765 G/A, which gives rise to two gene expression products, such as allelic isoforms of triosephosphate isomerase that differ in the amino acid substitution 233G → D.     

增殖细胞核抗原和C-Fos蛋白在人胎胃壁组织中的表达

背景：增殖细胞核抗原表达增高可促进细胞DNA合成增加,反映细胞增殖状况。C-Fos与DNA结合可直接调节具有转录活性的转录因子,在胚胎细胞的发育过程中,对细胞的生长、分化及增殖起促进作用。 目的：观察增殖细胞核抗原和C-Fos蛋白在人胎胃组织中的表达。 方法：应用免疫组织化学法和图像分析软件检测第2,3,4三个月龄段,人胎胃组织细胞中PCNA和C-Fos蛋白阳性表达的积分吸光度。 结果与结论：第2~4个月胎龄段,增殖细胞核抗原和C-Fos蛋白在人胎胃壁各层组织细胞均呈阳性表达。随着胎龄的增大,胃壁组织中增殖细胞核抗原蛋白阳性表达先升高再降低,C-Fos蛋白阳性表达则逐渐增高(P < 0.01)。提示增殖细胞核抗原和C-Fos蛋白在人胚胎早期胃组织细胞的生长发育过程中起重要的调节作用。     

Identification of wheat flour allergens by means of 2-dimensional immunoblotting

BACKGROUND: Wheat flour proteins are allergens for 60% to 70% of bakers with workplace-related respiratory symptoms. OBJECTIVE: The aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized bakers and to identify the most frequently recognized allergens. METHODS: Water/salt-soluble wheat flour proteins from the cultivar Bussard were separated by using 2-dimensional gel electrophoresis with immobilized pH gradients. IgE-reactive proteins were identified by means of immunoblotting with sera of 10 subjects with baker's asthma. Mass spectrometric fingerprinting was used to identify the proteins most frequently recognized by IgE. RESULTS: The IgE immunoblots obtained with 10 different sera exhibited a remarkable heterogeneity. Each patient showed an individual IgE-binding pattern with 4 to 50 different allergen spots. Altogether, more than 100 IgE-binding protein spots were detected. Nine of the predominant IgE-binding protein spots were identified by using mass spectrometric fingerprinting. The obtained masses matched 2 different isoforms of glycerinaldehyde-3-phosphate dehydrogenase from Hordeum vulgare, triosephosphate isomerase from H vulgare, and serpin, a serine proteinase inhibitor from Triticum aestivum. CONCLUSIONS: The results show a great interindividual variation of IgE-binding patterns of wheat flour proteins in baker's asthma. The clinical relevance of the identified 4 new allergens will be further investigated in the near future.     

Changes in neuronal protein expression in LP-BM5-infected mice

Murine acquired immunodeficiency syndrome (MAIDS) induced by LP-BM5 murine leukemia virus is used as a model of human immunodeficiency virus (HIV)-related neurologic dysfunction. Mice infected with LP-BM5 have mnemonic abnormalities (i.e., spontaneous alternation behavior in the Y-maze and performance in the Morris water maze) and biochemical alternations (i.e., cytokines, platelet-activating factor, quinolinate, glutamate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) that produce neurologic symptoms similar to those observed in HIV-related neurologic dysfunction. To identify proteins associated with dysmnesia in the MAIDS model, we examined the expression of neuronal proteins in LP-BM5-infected mice using two-dimensional polyacrylamide gel electrophoresis (2-DE). Neuronal protein expression in LP-BM5-infected mice was compared with that in non-infected mice using the Image Master 2D. We detected approximately 800 protein spots, of which 35 were distinguishable between non-infected and LP-BM5-infected mice. Most of these spots were downregulated in LP-BM5-infected mice. Three of the spots were identified as 14-3-3 protein zeta/delta, synapsin 2 and protein disulfide isomerase using a capillary nanoliquid chromatography tandem mass spectrometric system. We verified the expression levels of these proteins by Western blot. Analysis of these 35 spots could provide insight into mechanisms of dysmnesia in the MAIDS model of HIV-related neuronal dysfunction.     

与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析

目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1与UL128-642 bp编码蛋白相互作用.结论 成功构建pGBKT7 UL128-519 bp和pGBKT7 UL128-642 bp,并应用酵母双杂交系统分别筛选出EFEMP2和THY-1与UL128-519 bp和UL128-642 bp编码蛋白相互作用,在所筛选得到的相互作用蛋白之间未发现有相同的蛋白,UL128-519 bp和UL128-642 bp所编码的蛋白在HCMV感染致病过程中可能发挥不同的作用.     

胎儿心肌细胞cyclin E和p16蛋白的表达

目的探讨细胞周期素cyc lin E和p16蛋白表达与胎儿心肌细胞增殖、分化的关系。方法应用S-P免疫组化方法分别对不同月份的胎儿心肌细胞cyc lin E和p16蛋白表达进行检测。结果3个月胎儿左心室心肌细胞核中cyc lin E的表达高于5个月和6个月胎儿,8个月表达为阴性;3、5和6个月胎儿左心室只有很少部分心肌细胞核有p16蛋白的表达,8个月p16蛋白表达的心肌细胞数量明显增加。结论随月份增加细胞周期素cyc lin E表达下调和p16表达上升提示二者可能参与调控细胞周期和诱导心肌细胞的分化。     

神经纤维丝蛋白、神经烯醇化酶和突触素在人胚胎舌组织中的表达

目的 探讨神经纤维丝蛋白（NF）、神经烯醇化酶(NSE)和突触素(SYN)在人胚胎舌组织不同发育阶段的分布特征。 方法 应用免疫组织化学法,检测第2～4个月胎龄段共16份人胚胎舌组织内NF、NSE和SYN蛋白的表达,分析其变化规律。 结果 第2～4个月龄段,NF、NSE和SYN蛋白在人胚舌组织内均有阳性表达。随着胎龄的增大,NF、NSE和SYN在舌组织内阳性表达数量增多,表达强度逐渐增强。第2个月龄时,NF、NSE和SYN蛋白呈少量弱阳性表达,阳性表达强度值分别是135.83±24.62、136.57±15.23和139.84±21.40。第3个月龄时,NF、NSE和SYN阳性表达强度值分别是96.04±23.37、94.89±22.52和90.65±21.08。第4个月胎龄时,NF、NSE和SYN阳性表达强度值分别是79.02±20.90、76.78±21.27和83.43±25.90。应用 One-Way ANOVA和 LSD-t统计学方法,分析第2～4个月龄段人胚胎舌组织内NF、NSE和SYN蛋白的各自阳性表达强度值,P＜0.01。 结论 第2～4个月龄段,人胚胎舌组织内NF、NSE和SYN的表达强度值随胎龄增大而降低,它们均参与调控人胚胎舌内神经系统和舌肌的分化发育。     

Proteomic analysis of salivary glands of female Anopheles barbirostris species A2 (Diptera: Culicidae) by two-dimensional gel electrophoresis and mass spectrometry

Salivary gland proteins of adult female Anopheles barbirostris species A2, a potential vector of Plasmodium vivax in Thailand, were analyzed using a proteomic approach (two-dimensional gel electrophoresis followed by nanoLC-MS). Two-dimensional gel electrophoresis revealed approximately 75 well-resolved spots on the reference gel. Most of the protein spots displayed relative molecular masses from 14 to 85?kDa and isoelectric points ranging from 3.9 to 10. The proteome profiles of A. barbirostris species A2 female salivary glands were affected by aging. The typical electrophoretic pattern of the female salivary glands was reached in 48?h post emergence, suggesting the maturation of salivary glands and saliva contents for blood feeding. Proteins involved in blood feeding, i.e., putative 5' nucleotidase/apyrase, anti-platelet protein, long form D7 salivary protein, D7-related 1 protein, and gSG6 salivary protein, start to accumulate from emergence and gradually increase becoming predominant within 48?h. There are different salivary components expressed within each region of the female glands. The blood-feeding proteins were detected in the distal-lateral lobes and/or medial lobes. Proteins detected and/or identified by this approach could be tested in strategies developed to control pathogen and disease transmission. Moreover, the information of a 2D map of the female salivary gland could be used for comparison with other related species in the A. barbirostris complex to distinguish species members in the complex.