海藻糖酶抑制剂--一种新型农药先导结构

海藻糖是细菌、真菌以及许多无脊椎动物主要的能源供应物质之一,它可以被海藻糖酶水解成为两分子葡萄糖单体,进而参与氧化供能。因此,海藻糖酶可以作为创新农药的作用靶点。该扼要介绍了几种海藻糖酶抑制剂,讨论了它们的结构一活性关系,以期对研发新型海藻糖酶抑制剂有所帮助。     

海藻糖——昆虫的血糖

海藻糖(trehalose)是由2个葡萄糖分子通过α,α-1,1糖苷键连接的一种非还原性双糖。海藻糖作为昆虫的血糖,对于生物的能量代谢和抗逆等方面具有重要的作用。文章从昆虫海藻糖的发现、海藻糖的化学性质、昆虫中海藻糖的生理作用、代谢途径等方面进行综述,并对昆虫中海藻糖的进一步研究作了展望。     

昆虫海藻糖与海藻糖酶的特性及功能研究

海藻糖作为昆虫的血糖,对昆虫的能量代谢、滞育、抗逆等具有重要作用。海藻糖酶是海藻糖代谢过程中一个重要的酶类,特异性地将一分子海藻糖水解为两分子葡萄糖而被昆虫利用,其基因表达和酶活性与昆虫各项生理过程密切相关。本文从昆虫海藻糖与海藻糖酶的特性、代谢途径以及它们在昆虫体内的重要作用进行综述,并对海藻糖和海藻糖酶在授粉昆虫方面的研究作了展望。     

海藻糖合酶的研究进展

海藻糖是一种天然存在的非还原性二糖, 对生物膜和蛋白质等大分子有独特的保护作用, 在食品、医药、化妆品等多个领域中都有广泛的发展空间。海藻糖合酶(TreS)是一类分子内转糖苷酶, 专一性地以麦芽糖为底物, 一步转化生成海藻糖, 操作工艺简单、底物价格低廉、应用前景良好。本文综述了海藻糖合酶的酶学性质、催化机理、基因工程以及目前存在的主要问题和拟解决方案。     

面包酵母海藻糖积累条件的研究

对面包酵母的海藻糖积累条件及补料分批发酵进行了研究。结果表明 pH为 5 .0 ,温度 37℃有利于海藻糖积累。少量多次的补加葡萄糖可持续增加海藻糖的含量 ,但随着发酵时间的延长 ,葡萄糖对海藻糖转化率有逐渐减少的趋势。     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

分析植物组织中海藻糖的气质联用及毛细管气相色谱法

介绍一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中海藻糖等糖类物质进行乙酰化衍生化后的气相色谱分离、质谱鉴定的分析方法.以核糖醇为内标,通过校准曲线对植物组织中的海藻糖进行定量分析.此法测定海藻糖的最低量可达8.17×10-11g,适于植物样品中微量海藻糖的分析测定.     

酵母内源性海藻糖代谢调控研究进展

作为一种天然稳定剂的双糖,海藻糖(Trehalose)在逆境下对生物体活性的保护功能既吸引了广泛的研究兴趣,也使其具有良好的应用价值和潜力。本文聚焦重要模式微生物和工业应用微生物酵母,结合组学研究最新进展,从海藻糖代谢途径、应激条件下的海藻糖代谢和转录特征以及提高胞内海藻糖含量策略等方面,对内源性海藻糖研究新进展进行了综述。     

干旱和低温胁迫影响烟草幼苗海藻糖代谢的差异比较

为了明确海藻糖代谢在干旱和低温胁迫下的响应及其差异,以及海藻糖在烟草耐旱和耐冷性中的作用,选用烟草品种云烟203幼苗为实验材料,通过对烟草幼苗进行干旱和4℃低温胁迫处理,研究叶片中海藻糖含量及代谢相关酶(海藻糖-6-磷酸合成酶TPS、海藻糖-6-磷酸磷酸酶TPP、海藻糖酶THase)活性及基因表达变化情况。结果表明,在干旱和低温胁迫下,海藻糖含量表现出先升高后降低趋势,均在处理2 d时海藻糖积累达到最大,且干旱胁迫下海藻糖含量高于低温胁迫。TPS、TPP活性在干旱和低温胁迫下先升高后降低,且干旱高于低温;THase活性在干旱和低温胁迫下均不断升高,低温下的THase活性高于干旱。TPS、TPP、THase基因在干旱下的表达量均高于0 d对照,低温下低于0 d对照。以上结果表明,干旱和低温胁迫均能促使烟草体内海藻糖的积累,干旱更能诱导相关基因的表达、相关酶活性及海藻糖含量的升高,表明海藻糖对干旱响应更敏感。     

产海藻糖酿酒酵母培养基优化及生理学研究

采用单因子和均匀试验对酿酒酵母产海藻糖的培养基组成进行初步研究,并对发酵过程作了分析。结果表明,培养基组成、碳源浓度以及氮源的浓度比等都对细胞生物量和海藻糖积累有影响。在初步优化培养基条件下,酒酵母产海藻糖的细胞干重8g／L,海藻糖的含量为10％。通过观测发酵过程生理现象的变化,认为海藻糖的产生是对数生长期后期的稳定期,呈现不规律变化。     

Twist 在胸腺上皮性肿瘤中的表达及预后意义

目的:检测Twist在胸腺上皮性肿瘤的表达及其与预后的关系。方法:应用免疫组织化学pv6000法检测Twist在87例胸腺上皮性肿瘤(胸腺瘤71例,胸腺癌16例)中的表达情况,分析其与患者各项临床病理指标及预后的关系。结果:Twist蛋白在胸腺癌的阳性表达(81.3%)显著高于胸腺瘤组织(11.3%),其阳性表达的差异存在统计学意义(P<0.001),其在胸腺瘤各亚型之间表达的差异无统计学意义(P>0.05)。Twist阳性表达与胸腺上皮性肿瘤术后发生远处转移呈正相关关系(r=0.40,P=0.001)。Kaplan-Meier生存分析显示Twist阳性表达患者术后生存期低于Twist阴性表达患者(P<0.001)。结论:检测Twist蛋白表达情况对于鉴别胸腺瘤与胸腺癌可能有重要的参考价值,Twist阳性表达可能与术后发生远处转移有关并影响患者术后生存期。     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

结直肠癌mP53 和PCNA 表达的相关性及临床意义

目的:探讨结直肠癌中突变型P53基因(mP53)和增殖细胞核抗原(PCNA)表达的相关性及临床意义。方法:应用免疫组化二步法,检测60例结直肠癌组织及20例正常肠粘膜中mP53、PCNA的表达,结合临床病理资料进行统计分析。结果:60例结直肠癌中mP53阳性表达率65.0%,PCNA阳性表达率78.3%,20例正常肠粘膜中mP53、PCNA表达均为阴性(P<0.05)。mP53和PCNA阳性表达率在低分化组、浆膜层浸润组、淋巴结转移组均较高(P均<0.05)。mP53和PCNA表达呈正相关(r=0.58,P<0.05)。结论:mP53和PCNA在结直肠癌中表达均增高,二者与结直肠癌病理学分级、浸润深度和淋巴结转移有关,可作为判断结直肠癌预后的参考指标。     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

PCNA and P53 expression in relation to clinicopathological features of oral papilloma

Although papilloma is the most frequent benign epithelial tumour of oral cavity, its biological potential for malignant transformation is still to be evaluated. The aim of the study was to correlate PCNA and P53 expression in 55 oral papillomas with some clinicopathological variables. The tissue samples were stained with H+E and by immunohistochemistry for PCNA and P53 protein. Staining patterns were assessed semiquantitatively and correlated with each other and grade of tumour epithelial dysplasia, tumour size, localization well patient age and sex. PCNA immunostaining was positive 43 (78%) oral papillomas. P53 immunohistochemical reaction was positive in 38 (69%) out of 55 epithelial tumours. Positive relationship between PCNA and P53 expression was observed as well as between PCNA immunostaining and grade of epithelial dysplasia. There was no statistically significant relationships between PCNA, P53 immunohistochemical positivity and papilloma size, site, patient age and sex. The results of this study suggest that immunohistochemical P53 overexpression is valuable marker of early neoplastic transformation and together with PCNA are presumed predictors for malignant transformation of oral papillomas.     

Immunohistochemical expression of p53, p21/waf1, rb, p16, cyclin D1, p27, Ki67, cyclin A, cyclin B1, bcl2, bax and bak proteins and apoptotic index in normal thymus

The immunohistochemical expression of p53, p21, Rb, p16, cyclin D1, Ki67, cyclin A, cyclin B1, p27, bcl2, bax, and bak proteins and the apoptotic index (Al) were investigated in 20 normal thymuses (8 adults, 3 adolescents, 5 infants and 4 newborns). The expressions of Rb, Ki67, cyclin A and cyclin B1 were overlapping, being high in the cortex with a tendency for decreased expression toward the medulla. Apoptotic cells were mainly detected in the cortex and the corticomedullary junction, rarely being present in Hassall's corpuscles. The mean values of Ki67, cyclin A, and cyclin B1 expression in thymuses were 77.2%, 32.2% and 21.4% (newborns), 62.4%, 33.7% and 18.5% (infants), 56.9%, 23.4% and 18.9% (adolescents) and 38.7%, 21.7% and 14.6% (adults), respectively. The mean values of AI in thymuses from newborns, infants, adolescents and adults were 1.4%, 2.9%, 2.7% and 3.8%, respectively. This decrease in proliferation and increase in apoptosis may account for the process of thymic involution. P16 expression was widespread with most of Hassall's corpuscles being p16-positive. P16-positive cells and Hassall's corpuscles increased with the increase in age, in keeping with the suggested role of p16 in cellular senescence. P27 expression was undetectable in subcapsular thymocytes with a tendency for increased expression toward the medulla. The expressions of Ki67, cyclin A and cyclin B1 were inversly related with that of p27, consistent with previous evidence that p27 concentration is reduced when the cell-cycle progresses. P21 and much less frequently p53 proteins were mainly detected in a part of the subcapsular cortical epithelial cells. These findings suggest that a) in thymocytes, the apoptotic pathway is mostly p53-independent and the function of p21 as a negative regulator of the cell cycle must be redundant to other negative regulators, such as p16 and p27 which were abundantly detected in thymocytes and b) in some thymic epithelial cells, the p21 expression may be induced by p53, but in most of them seems to be p53-independent. Most of Hassall's corpuscles were p21-positive, consistent with previous evidence that these structures represent end stages of maturation of thymic medullary epithelium and that p21 protein is involved in the process of terminal differentiation. Cyclin D1 positivity was found in some macrophages. Bcl2 expression was mainly seen in medullary thymocytes, reflecting the surviving thymocytes in this region. The expressions of Bax and bak were more widespread in both the medulla and cortex, suggesting that these proteins play a broader role than bcl2 in the regulation of thymic apoptosis.     

Immunohistochemical detection of early-stage carcinogenesis of oral leukoplakia by increased DNA-instability and various malignancy markers

The degree of DNA instability as determined by immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (the DNA instability test) was used as a marker of malignancy. The test was applied to tissues of oral leukoplakia assessed histopathologically as hyperplasia (38 cases), mild (12 cases), moderate (11 cases) and severe (8 cases) dysplasia, and invasive squamous cell carcinoma (SCC, 20 cases). Tissues were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), p53, DNA-fragmentation factor 45 (DFF45), analysis of various AgNORs parameters, and triple immunostaining for vascular endothelial growth factor (VEGF), CD34, and PCNA. The DNA instability test was positive in 20 (100%) SCC cases, 8 (100%) severe dysplasia cases, 8 (72.7%) moderate dysplasia cases, 6 (50.0%) mild dysplasia cases, and 9 (23.7%) hyperplasia cases, indicating malignancy. The proportion of lesions positive for PCNA, p53, DFF45, and values of AgNORs parameters steadily increased from hyperplasia to mild, moderate and severe dysplasia, and SCC, especially in those showing positive DNA instability test, indicative of malignancy. Based on these results, 44.9% of leukoplakia were malignant tissues, namely carcinoma in situ. The proportion of PCNA-positive vascular endothelial cells in the vicinity of VEGF-positive epithelial lesion was significantly higher than that of negative DNA instability lesions, as revealed by immunohistochemical triple staining for VEGF, CD34, and PCNA. Our results suggest that increased DNA instability, enhanced proliferative activity, p53 mutation, and induction of DFF45 and VEGF may allow cancer cell proliferation, enhance their survival by escaping apoptosis, and provide abundant nutrients during early-stage carcinogenesis of oral leukoplakia.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

广东食管癌中Epstein-Barr病毒感染与细胞增殖p53的关系

目的 :研究食管癌中 EB病毒感染情况 ,探讨其与肿瘤细胞增殖及抑癌基因 p5 3的关系。方法 :应用聚合酶链反应技术 (PCR)检测食管癌中 EB病毒 DNA的感染。用免疫组化方法检测其中 p5 3的表达及肿瘤细胞增殖。结果 :2 4例食管癌中 ,EB病毒 DNA阳性率为 95 .8% (2 3/2 4)。 2 3例阳性病例中存在 p5 3蛋白积聚者为 16例 ,积聚率为 6 9.6 % (16 /2 3) ;1例 EB病毒 DNA阴性者 ,p5 3蛋白染色阴性。 p5 3蛋白积聚与增殖细胞核抗原的表达密切相关 (P<0 .0 5 )。结论 :广东食管癌中普遍存在 EB病毒的感染。有 EB病毒感染的病例 ,亦存在 p5 3蛋白积聚。 EB病毒可能与 p5 3的改变有关 ,从而导致细胞增殖。     

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.