小鼠骨骼肌干细胞蛋白标志JAK的发现

目的通过骨骼肌卫星细胞的体外培养,探讨骨骼肌干细胞蛋白表达标志Pax7,发现新的与骨骼肌干细胞的生长、分化密切相关的表达蛋白。方法采用两种小鼠骨骼肌干细胞分离、纯化及培养方法,一种是采取成年小鼠的趾短屈肌(FDB)分离切割后,体外培养出单个骨骼肌干细胞;另一种是取成年小鼠FDB,分离出单个肌纤维丝(fiber)并体外培养。应用免疫荧光染色法对骨骼肌干细胞进行鉴定。运用RNAi技术将JAK1蛋白敲除。结果发现Pax7表达阳性的细胞质内有JAK1蛋白表达,并运用RNAi技术将JAK1蛋白敲除,结果显示骨骼肌干细胞迅速分化。结论 FDB肌纤维的体外培养是一个良好的体内肌肉状态的模型,同时发现了一种新的与骨骼肌增殖分化有关的骨骼肌干细胞蛋白标志JAK1。     

Cumulative 3-nitrotyrosine in specific muscle proteins is associated with muscle loss during aging

Post-translational oxidative protein modifications which are more marked during aging and/or high-calorie (HC) diets affect protein function and metabolism. Protein function and metabolism are different according to the type of muscle proteins. Oxidative muscle protein modifications may thus be associated with age-related sarcopenia, and HC may be implicated in the development of sarcopenia by emphasizing protein modifications. Understanding the role of protein modifications in the process of sarcopenia and metabolism associated with a high fat diet may be elucidated by investigations with skeletal muscle protein subfractionations. To study this hypothesis, carbonylated protein (CP) and 3-nitrotyrosine (3-NT) levels were measured in mixed, sarcoplasmic, myofibrillar and mitochondrial protein fractions of quadriceps in rats aged 6months (A) and 25months (O) fed a normal calorie (NC) or HC diet for 3months (AN, AH, ON, OH n=7-8). Muscle weight was lower in the older rats (AN: 0.79±0.03g, ON: 0.43±0.12g, P<0.05), but no HC effect was observed. CP did not differ between groups while 3-NT accumulated significantly in ON compared with AN, especially in mitochondria (2.4±0.5, 1.3±0.1, 1.9±0.4, 2.9±1.2 -fold in mixed, sarcoplasmic, myofibrillar and mitochondrial fractions respectively, P<0.05). 3-NT in mixed protein was negatively correlated with muscle mass (r(2)=-0.812). 3-NT accumulation during HC was observed only in specific proteins of mitochondria (100kDa) (1.0±0.6, 1.7±0.9, 3.3±1.4 and 7.0±2.5 -fold in AN, AH, ON and OH, respectively, P<0.05). Hence cumulative 3-NT in skeletal muscle protein appears associated with the development of age-related muscle loss. Mitochondrial proteins are more prone to nitration during aging and nutritional stress.     

Pentosidine: a molecular marker for the cumulative damage to proteins in diabetes, aging, and uremia.

Collagen undergoes progressive browning with age and diabetes characterized by yellowing, fluorescence, and cross-linking. The present research was undertaken in order to investigate the nature of the collagen-linked fluorescence. Human collagen was exhaustively cleaved into peptides by enzymatic digestion. Upon purification, a highly fluorescent chromophore was identified and purified from old human collagen. Structure elucidation revealed the presence of an imidazo [4,5-b] pyridinium-type structure acting as a cross-link between arginine, lysine, and a pentose. This advanced glycosylation end-product and protein cross-link results from the reaction of pentoses with proteins and was named pentosidine. Further work indicated that long-term glycosylation of proteins with hexoses also leads to pentosidine formation through sugar fragmentation. The proposed mechanism of pentosidine formation involves the dehydration of the pentose-derived Amadori compound to form an intermediate which is attacked under base catalysis by the guanido group of arginine. The strict requirement for the Amadori rearrangement is uncertain. However, oxidation is definitely involved since pentosidine is not formed in the absence of oxygen. Five-carbon sugars contributing to pentosidine formation could be formed from larger sugars by oxidative fragmentation or from trioses, tetroses, and ketoses by condensation and/or reverse aldol reactions. Pentosidine increases exponentially in human skin at autopsy. Mean age-adjusted skin levels were significantly increased in subjects with uremia and especially in type 1 diabetics with uremia vs. controls. In skin biopsy, levels were significantly elevated in all diabetic (type 1) vs. control subjects. The highest degree of association was with the cumulative grade of diabetic complication (retinopathy, nephropathy, arterial stiffness, and joint stiffness). Pentosidine also forms in various proteins other than collagen, although to a much lesser extent. In blood, pentosidine is mainly associated with plasma proteins and is highly elevated during uremia. In the lens, it is associated with both water-soluble and -insoluble protein fractions and is especially elevated during brunescent cataract formation. The origin of pentosidine in vivo is uncertain. Evidence suggests that the pentoses are the most reactive sugars in pentosidine formation in vitro; however, the origin and importance of free pentoses in vivo, especially during the diabetic state, are not certain. Possible origins include hemolysis and/or a defect in the primary pentose metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survivin: a novel neuroendocrine marker for pheochromocytoma

OBJECTIVE: To study survivin expression in human adrenal medulla and in benign and malignant pheochromocytoma tissue as a tool to predict tumor metastatic potential and prognosis. DESIGN: Blinded study to assess the role of the anti-survivin antibody in chromaffin cells. METHODS: We performed immunohistochemistry with a purified rabbit-polyclonal anti-survivin antibody on 39 formalin-fixed and paraffin-embedded pheochromocytoma/paraganglioma specimens, and on 10 normal adrenal medulla samples from patients unaffected by a chromaffin cell tumor. Fourteen samples were from 14 patients with benign pheochromocytoma (<8 year follow-up, mean 5.2 years), 18 specimens were from 12 patients with malignant pheochromocytoma (<13 year follow-up, mean 6.3 years), 5 samples were from 2 patients with malignant paraganglioma (<6 year follow-up, mean 4 years), and 2 specimens from 2 patients with benign paraganglioma (<7 year follow-up, mean 5.5 years). Malignancy was defined by metastases in non-chromaffin tissues. Staining intensity with the anti-survivin antibody was scored from 0 (none) to 3+ (heavy). Tissues from human kidney, breast, and melanoma served as controls. RESULTS: All pheochromocytoma/paraganglioma specimens stained either 2+ or 3+. By analysis of variance (ANOVA), there was no statistically significant difference between the staining intensity of benign and malignant samples. All normal adrenal medulla specimens stained positively with anti-survivin but to a lesser degree than the chromaffin cell tumors (P<0.01). CONCLUSIONS: Based on these findings, we conclude that (i) survivin may represent a novel neuroendocrine marker for chromaffin cell tumors, and (ii) survivin does not appear to reliably distinguish benign from malignant pheochromocytomas/paragangliomas and thus does not identify patients at risk of recurrent disease.     

Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients

Background

The major complications of “treated” Human Immunodeficiency Virus (HIV) infection are cardiovascular disease, malignancy, renal disease, liver disease, bone disease, and perhaps neurological complications, which are phenomena of the normal aging process occurring at an earlier age in the HIV-infected population. The present study is aimed to explore protein carbonyl content as a biomarker for detecting oxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDS patients.

和肽素:一项新的脑血管病预后指标

和肽素是精氨酸加压素原羧基末端的一部分,与多种疾病预后均具有相关性.近期研究发现,和肽素是一项新的卒中独立预后指标,并可能改善现有的卒中风险分层方案.     

Detection of differentially expressed genes in lymphomas using cDNA arrays: identification of clusterin as a new diagnostic marker for anaplastic large-cell lymphomas

This study reports the first use of gene array technology for the identification of a tumor-specific marker in lymphoid neoplasms. The differential gene expression of 31 hematopoietic cell lines, representing most major lymphoma subgroups of B- and T-cell origin, was assessed by hybridizing labeled complementary DNA to Atlas human expression arrays containing 588 genes. Genes known to be specific for B, T, or myelomonocytic lineages were appropriately identified in the arrays, validating the general utility of this approach. One gene, clusterin, not previously known to be expressed in lymphoid neoplasms, was specifically found in all 4 anaplastic large-cell lymphoma (ALCL) cell lines, but not in any of the 27 remaining tumor lines. Using a monoclonal antibody against clusterin, its differential expression was confirmed by Western blotting and immunohistochemistry. A total of 198 primary lymphomas (representing most major lymphoma subtypes), including 36 cases of systemic ALCL, were surveyed for clusterin expression by immunohistochemistry and Western blotting. All of the 36 ALCL cases marked for clusterin, with most cases showing moderate to strong staining in the majority of neoplastic cells. Clusterin expression was not related to expression of anaplastic lymphoma kinase-1. With 2 exceptions, none of the remaining 162 non-ALCL cases marked with the clusterin antibody, including Hodgkin disease and primary cutaneous ALCL. In reactive lymphoid tissues, only follicular dendritic cells and fibroblastic reticular cells exhibited staining. Clusterin is a highly conserved glycoprotein implicated in intercellular and cell matrix interactions, regulation of the complement system, lipid transport, stress responses, and apoptosis. Although its function in ALCL is unknown, the unique expression of clusterin within this category of lymphoma provides an additional marker for the diagnosis of ALCL. This study illustrates the enormous potential of gene array technologies for diagnostic marker discovery. (Blood. 2000;96:398-404)     

Apelin: a novel marker for the patients with first ST-elevation myocardial infarction

To date, only animal studies have been concerned with apelin involvement in acute myocardial ischemia. The aim of this study was to investigate apelin measurements in low-risk patients with first ST-elevation myocardial infarction (STEMI) and to assess if apelin may feature as a marker of left ventricular (LV) injury and prognosis. In 78 consecutive patients (mean age 67 ± 11.5 years, 24 women) with first STEMI treated with primary percutaneous coronary intervention, plasma apelin-36 concentrations were measured twice: on admission and on the 5th day of hospitalization. Left ventricle ejection fraction (LVEF) was applied as marker of LV injury. Composite endpoint (CEP), which included death, stroke, and recurrent ischemic event, was assessed after 1 year follow-up. On the first day, median apelin-36 concentration was 2138.5 pg/ml and on the 5th day was significantly lower, 2008.3 pg/ml (P = 0.002). There were no significant differences found in apelin-36 concentrations between patients with normal and low LVEF. In both groups significant reductions were found in apelin-36 concentrations measured in 5-day intervals (P = 0.04 and P = 0.008, respectively). After a 1-year follow-up, only one patient died and 19 patients (24.3%) had reached CEP. No difference in baseline apelin-36 concentrations were found in the group of patients who reached CEP compared with those without CEP. However, in both groups concentrations significantly decreased after 5 days (P = 0.04 and P = 0.013, respectively). Apelin-36 concentrations are reduced in lowrisk first STEMI patients during the first days regardless of the degree of LV dysfunction and prognosis.     

自身免疫性肝炎小鼠模型差异表达基因的筛查及柴胡皂苷D对部分差异表达基因表达的影响

目的对自身免疫性肝炎(AIH)小鼠模型的差异表达基因进行基因芯片筛查,并观察柴胡皂苷D(SS-d)对部分差异表达基因表达的影响,探讨AIH的发病机制及SS-d对该病的治疗作用机制。方法健康、雌性SPF级C57BL/6小鼠40只[体质量(20±2)g],分为基因芯片组(n=8)和SS-d治疗组(n=32)。基因芯片组小鼠又分为正常对照组(n=4)和模型组(n=4),正常对照组常规饲养,模型组小鼠按15 mg/kg剂量给予尾静脉注射刀豆蛋白A(Con A),12 h后处死并提取肝组织,按Agilent芯片说明书进行差异表达基因的筛选,采用荧光定量PCR技术对部分差异表达基因进行验证。SS-d治疗组小鼠随机分为正常组(常规饲养)、模型组(按15 mg/kg剂量给予尾静脉注射Con A)、SS-d低剂量组和SS-d高剂量组(分别按2.5 mg/kg和5.0 mg/kg剂量给予腹腔注射SS-d预处理,8 h后按15 mg/kg剂量给予尾静脉注射Con A)(每组8只)。12 h后收集各组小鼠静脉血,ELISA法检测血清ALT、AST。无菌条件下提取小鼠肝组织,部分多聚甲醛固定后切片,并进行HE染色;部分肝组织用于提取RNA,采用荧光定量PCR技术检测部分差异表达基因(IFNγ、IL-4、IL-5、IL-13和IL-17)的mRNA水平变化。多组间比较采用单因素方差分析,进一步两两比较采用LSD-t法检验;两组间比较采用t检验。结果基因芯片组小鼠共筛查差异表达基因11512个,其中上调5189个,下调6323个,显著富集于138条信号通路;荧光定量PCR验证结果显示,与正常对照组比较,模型组小鼠IFNγmRNA和IL-17 mRNA的表达升高(P值均<0.01),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达下调(P值均<0.01),与芯片筛查结果一致。在SS-d治疗组中,与正常对照组比较,模型组小鼠血清ALT、AST水平均升高(P值均<0.01);肝组织内可见大量淋巴细胞浸润;IFNγmRNA和IL-17 mRNA的表达水平明显升高(P值均<0.05),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平明显降低(P值均<0.05)。与模型组比较,SS-d低剂量组和SS-d高剂量组小鼠血清ALT、AST水平均降低(P值均<0.05),肝组织内淋巴细胞浸润程度减轻;IFNγmRNA和IL-17 mRNA的表达水平均降低(P值均<0.05),IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平均升高(P值均<0.05),上述基因的含量变化差异在SS-d高剂量组与SS-d低剂量组之间具有统计学意义(P<0.05)。结论AIH的发生发展系大量基因异常表达的共同作用结果。其中IFNγ、IL-4、IL-5、IL-13、IL-17与AIH的发病密切相关,同时也是SS-d发挥免疫调节及肝损伤保护作用的生物学靶点。     

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.     

Platelet glycoprotein VI: a novel marker for acute coronary syndrome

The platelet collagen receptor glycoprotein (GP) VI is critical for the formation of arterial thrombosis. GPVI platelet surface expression was examined in patients with stable angina and in patients with acute coronary syndrome (ACS). Surface expression of platelet activation markers such as P-selectin, GPIbalpha, and platelet GPVI was determined by flow cytometry. Patients with ACS showed a significantly enhanced GPVI expression compared with patients with stable angina and healthy controls. The expression of GPVI correlated well with CD62P. Elevated platelet GPVI expression was associated with ACS independent of markers of myocardial necrosis such as troponin and creatine kinase. In ACS, platelet surface GPVI expression was already elevated several hours before troponin and creatine kinase indicated myocardial injury. We conclude that the determination of the platelet-specific thrombotic marker GPVI may help to identify patients at risk before myocardial ischemia is evident.     

Secretagogin is a novel marker for neuroendocrine differentiation

Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed in neuroendocrine cells and nerve cells in normal mucosa of the digestive tract. Tissues adjacent to benign hyperplasic polyps and adenomas showed a decreased number of secretagogin-expressing neuroendocrine cells. Secretagogin co-localized with neuroendocrine markers (chromogranin A, neuron-specific enolase, synaptophysin) in neuroendocrine cells in crypts of normal mucosa, and in tumor cells of carcinoids. Secretagogin was strongly expressed in the cytosol and the nucleus of 19 well-differentiated neuroendocrine carcinoids and carcinoid metastases, as well as in neuroendocrine tumors from the lung, pancreas and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids and undifferentiated carcinomas. We conclude that secretagogin is a novel marker for neuroendocrine differentiation.     

Plasma retinol: a novel marker for cardiovascular disease mortality in Australian adults

Background and aimsVitamin A affects inflammation and immune function and is thus a factor of interest in relation to cardiovascular disease (CVD). As vitamin A circulates in the plasma in the form of retinol, this study aims to describe the relationship between plasma retinol and the 5-year incidence of CVD mortality.Methods and resultsCommunity-dwelling adults (n = 441, 45% with type 2 diabetes) were recruited in Melbourne, assessed at baseline and followed for 5 years. At baseline, CVD risk factors were assessed by clinical evaluation, by personal lifestyle questionnaire and from biochemistry (plasma fasting glucose, lipids, total homocysteine, C-reactive protein, retinol and carotenoids plus the urinary albumin excretion rate over 24 h.). Dietary intake was assessed by a validated food frequency questionnaire. CVD mortality over 5-years was determined by consulting state or national registries. The majority of participants had adequate plasma retinol concentrations (≥30 μg/dL). The final Cox regression model indicated that those in the highest tertile of plasma retinol (mean ± SD) 76 ± 14 μg/dL) had a significantly lower risk of 5-year CVD mortality (hazard ratio 0.27 [95% confidence interval 0.11, 0.68], P = 0.005), an effect that was not readily explained in terms of traditional CVD risk factors or dietary intake.ConclusionIn well-nourished older Australian adults, plasma retinol was inversely associated with CVD mortality via mechanisms apparently unrelated to established CVD risk factors and dietary intake.