Different protein tyrosine phosphatase superfamilies resulting from different gene reading frames

Protein tyrosine phosphatases (PTPs) are comprised of two superfamilies, the phosphatase I superfamily containing a single low-molecular-weight PTP (lmwPTP) family and the phosphatase II superfamily including both the higher-molecular-weight PTP (hmwPTP) and the dual-specificity phosphatase (DSP) families. The phosphatase I and H superfamilies are often considered to be the result of convergent evolution. The PTP sequence and structure analyses indicate that lmwPTPs, hmwPTPs, and DSPs share similar structures, functions, and a common signature motif, although they have low sequence identities and a different order of active sites in sequence or a circular permutation. The results of this work suggest that lmwPTPs and hmwPTPs/DSPs are remotely related in evolution. The earliest ancestral gene of PTPs could be from a short fragment containing about 90similar to120 nucleotides or 30similar to40 residues; however, a probable full PTP ancestral gene contained one transcript unit with two lmwPTP genes. All three PTP families may have resulted from a common ancestral gene by a series of duplications, fusions, and circular permutations. The circular permutation in PTPs is caused by a reading frame difference, which is similar to that in DNA methyltransferases. Nevertheless, the evolutionary mechanism of circular permutation in PTP genes seems to be more complicated than that in DNA methyltransferase genes. Both mechanisms in PTPs and DNA methyltransferases can be used to explain how some protein families and superfamilies came to be formed by circular permutations during molecular evolution.

毛蕊异黄酮类似物的合成及其对JAK/STAT信号途径调控的构效关系研究

干扰素（IFNs）是最早发现的具有广泛用途的一类细胞因子，IFN-α通过JAK/STAT信号途径调控机体一系列生理和病理反应。至今尚未发现类干扰素的小分子。我们前期研究发现天然产物毛蕊异黄酮可激活干扰素诱导的JAK/STAT信号途径。为发现类干扰素小分子、获得小分子探针，本课题拟建立成熟的JAK/STAT信号途径的筛选模型，合成毛蕊异黄酮及其类似物，研究这些化合物的构效关系，进而尝试通过共价键标记生物素或香豆素来直接研究它们与相关受体的作用。 从异香草醛出发经7步合成反应得到了毛蕊异黄酮。采用平行合成策略得到异黄酮类化合物；采用分支式合成策略，以取代苯乙酸作为合成砌块，获得具有与异黄酮类似结构的香豆素、3-芳基喹诺酮。与分离得到的黄酮类化合物，构建了一个包括异黄酮、黄酮、香豆素、3-芳基喹诺酮在内的化合物库。 建立了包含IFN-α刺激反应元件 (ISRE)的荧光素酶报告基因体系，通过筛选化合物库中的化合物，发现异黄酮骨架为激活JAK/STAT信号途径必须结构、毛蕊异黄酮7-位酚羟基被取代后活性丧失。根据以上结果，对毛蕊异黄酮3′-位标记物的合成进行了初步尝试。 发现山茱萸科植物青荚叶(Helwingia japonica (Thunb.) Dietr.)有抑制蛋白酪氨酸磷酸酯酶1B（PTP1B）的活性。从其地上部分95%乙醇提取物的乙酸乙酯部分分离得到5个化合物,应用波谱方法及与已知品对照的手段鉴定它们为p-menth-2-en-1β, 4β, 8-triol (Z-1)、blumenol A (Z-2)、2′,3′,4′,5′,6′-五羟基查尔酮(Z-3)、洋芹素7-O-β-D-吡喃葡萄糖苷(Z-4)、木犀草素7-O-β-D-吡喃葡萄糖苷(Z-5). Interferons (IFNs) are one kind of cytokines with broad functions. IFN-α mediates series physiological and pathological changes of human body via JAK/STAT pathway. Untill now, no IFNs-like small molecules are discovered. In our preliminary experiment, the natural product calycosin has been observed to activate JAK/STAT pathway. Therefore, we establish a luciferase reporter gene system and synthesize calycosin and its analogues to reveal their structure-activity relationship (SAR). Besides, in order to prove that calycosin activates JAK/STAT pathway through IFN receptor, we attempted to tag it with biotin or coumarin by covalent bonding. Calycosin was synthesized from isovanillin via seven steps. Other isoflavones were obtained by parallel synthesis; coumarins and quinolones were prepared through divergent synthesis, using substituted phenylacetic acids as building blocks. Combing with natural flavones, a small molecule library was established. A luciferase reporter gene system, consisting of 5 copies of the ISRE (interferon-stimulated response element), was used for screening of small molecules from that library. We found that the core-structure of isoflavone was necessary, and if the 7-OH is substituted, the activity slumps. According to our observation, we tried to tag biotin or coumarin at 3′-OH of calycosin. The 95% ethanol extract of the aerial parts of Helwingia japonica (Thunb.) Dietr. showed protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Five compounds were isolated. On the basis of spectral data or by comparison with authentic samples, they were identified as p-menth-2-en-1β,4β,8-triol (1), blumenol A (2), 2′,3′,4′,5′,6′-pentahydroxychalcone (3), apigenin 7-O-β-D-glucopyranoside (4), and luteolin 7-O-β-D-glucopyranoside (5).

尼泊尔水东哥、水麻、木姜冬青和青荚叶的化学成分研究

活性筛选中发现尼泊尔水东哥 (Saurauia napaulensis DC.) 树皮95%乙醇提取物具有α-淀粉酶抑制活性、水麻(Debregeasia orientalis) 枝叶95%乙醇提取物显示血管紧张素转化酶(ACE)抑制活性、青荚叶(Helwingia japonica (Thunb.) Dieter.) 95%乙醇提取物的中小极性部分显示蛋白酪氨酸磷酸酯酶(PTP)1B抑制活性。为全面了解它们的成分及相关活性成份，主要运用硅胶柱层析方法从这三个植物分离得到39个化合物，通过波谱分析或与已知品对照的方法对其进行了鉴定。对木姜冬青(Ilex litseaefolia Hu et Tang)的成分做了进一步的研究，取得了如下结果。 1. 从尼泊尔水东哥树皮的95%乙醇提取物分离并鉴定12个化合物： auranamide、aurantiamide benzoate、齐墩果酸、β-谷甾醇、β-胡萝卜甙、乌苏酸、2α,3α-二羟基-12-烯-28-乌苏酸、2α,3β,24-三羟基-12-烯-28-乌苏酸、(2S,3S,4R,10E)-2-[(2'R)-2' -hydroxytetracosanoylamino] -10-octadecene -1,3,4-triol、 2α,3α,24-三羟基-12-烯-28-齐墩果酸、2α,3β-二羟基-12-烯-28-乌苏酸和2α,3α,24-三羟基-12-烯-28-乌苏酸。 2. 从水麻枝叶的95%乙醇提取物分离并鉴定了18个化合物：棕榈酸、二十烷酸、二十烷酸甲酯、β-谷甾醇、Monogynol A、桦木酸、Hederagenin、β-胡萝卜甙、18αH-19(29)-烯-3-酮-乌苏烷、3，4-开环-20(30)-烯-乌苏烷-3-酸、Pomolic acid，表儿茶素、儿茶素、槲皮素、槲皮素-3-O-β-D-吡喃葡萄糖苷、紫丁香苷、紫丁香酚苷和山萘酚-3-O-芸香糖。儿茶素、槲皮素和槲皮素-3-O-β-D-吡喃葡萄糖苷为具有ACE抑制活性的成分。 3. 从木姜冬青95%乙醇提取物的乙酸乙酯部分分离并鉴定了5个化合物： 2-O-β-D-吡喃葡萄糖-6,2´-二羟基-4,4´-二香草酰氧甲基-1,1´-二苯醚（冬青苷）和四个已知化合物：七叶内酯、香草酸、3,4-二甲氧基苯乙酸和vanilloylcalleryanin。冬青苷为新化合物。 4. 从青荚叶95%乙醇提取物的中小极性部分分离并鉴定了9个化合物：β-谷甾醇、β-胡萝卜苷、羽扇豆醇、桦木醇、桦木酸、棕榈酸甘油酯、桂皮酸、6αH-4-烯-3-酮-豆甾醇和6βH-4-烯-3-酮-豆甾醇。 5. 对1985-2006年间天然二苯醚类化合物及活性研究进展进行综述. The in vitro test indicated that the 95% ethanolic extract of the barks of Saurauia napaulensis DC showed α-amylase inhibitory activity, the 95% ethanolic extract of the whole plants of Debregeasia. orientalis showed angiotensin converting enzyme (ACE) inhibitory activity and some fractions of the 95% ethanolic extract of the aerial parts of Helwingia japonica showed protein tyrosine phosphatase (PTP)1B inhibitory activity. In order to investigate components and active compounds of the three plants, they were chemically studied mainly using. Thirty-nine compounds were isolated predominantly by column chromatography identified by spectral methods or comparing them with authentic samples. Further investigation of Ilex litseaefolia Hu et Tang was carried out. Major results are as follows: 1. Twelve compounds were isolation from the 95% ethanolic extract of the barks of S. napaulensis DC. They were identified as auranamide, aurantiamide benzoate, oleanolic acid, β-sitosterol, β-daucosterol, ursolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyurs-12-en-28-oic acid, (2S,3S,4R,10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1,3,4-triol, 2α,3α,24 -trihydroxyolean-12-en-28-oic acid, 2α,3β-dihydroxyurs-12-en-28-oic acid, and 2α,3α,24-trihydroxyurs-12-ene-28-oic acid, respectively, by spectral methods or comparing them with authentic samples. 2. Eighteen compounds were isolation from the 95% ethanolic extract of the whole plants of D. orientalis. They were identified as palmitic acid, henicosanoic acid, henicosanoic acid methyl ester, β-sitosterol, monogynol, betulinic acid, hederagenin, β-daucosterol, 18αH-urs-20(30)-en-3-one, 3,4-seco-urs-20(30)-en-3-oic acid, pomolic acid, (-)-epicatechin, (+)-catechin, quercetin, quercetin 3-O-β-D-glucopyranoside, syringin, syringiaresinol digloside and kaempferol-3-O-rutinose. (+)-Catechin, quercetin and quercetin 3-O-β-D-glucopyranoside were the ACE inhibitory active components. 3. Further phytochemical investigation of the ethyl acetate parts of 95% ethanolic extract of the whole plant of I. litseaefolia afforded 2-O-β-D-glucopyranose-4,4´-di-vanilloyloxymethyl-2,6´-dihydroxy-1,1´-diphenyl ether (ilexiside), esculetin, vanillic acid, 3,4-dimethoxybenzylacetic acid and vanilloylcalleryanin. Ilexiside was new compound. 4. Nine compounds were isolation from the 95% ethanolic extract of the whole plant of H. japonica: β-sitosterol, β-daucosterol, lupeol, betulin, betulinic acid, glycerol monopalmitate, cinnamic acid, stignast-4-en-6β-3-one and stignast-4-en-6α-3-one 5.Diphenyl ether compounds from nature between 1985-2006 were summarized.

PTP1B抑制剂的制备与构效关系研究

蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase, PTP1B）是蛋白酪氨酸磷酸酶（protein tyrosine phosphatases, PTPs）家族中的一个经典的非受体型酪氨酸磷酸酶，在胰岛素信号通路中起着重要的负调控作用，是目前公认的一个新颖的糖尿病和肥胖症治疗靶点。寻找PTP1B的高活性抑制剂对糖尿病和肥胖症治疗有着重要的应用前景。 双-(2,3-二溴-4,5-二羟基苯基)-甲烷（BDDPM）是从松节藻醇提物中分离鉴定出的溴酚类化合物，体外活性筛选发现，它具有极强的蛋白酪氨酸磷酸酶1B(PTP1B)抑制活性(IC50=2.4μmol/L)。采用高脂饮食-链脲佐菌素诱导的大鼠模型（STZ-DM）对富含BDDPM的松节藻醇提物进行动物实验，发现中、高剂量组同样表现出惊人的活性，降糖效果优于阳性对照临床药物文迪雅，并呈剂量依赖性。于是拟采用STZ-DM大鼠模型对单一组分BDDPM进行药理、药效学等体内降糖活性研究，但体内动物实验需要30g以上BDDPM，所以首先要解决药源的问题。本文尝试从天然海藻提取分离和化学合成两种途径来解决BDDPM制备的问题。 首先本文尝试从松节藻中提取分离BDDPM的制备方法。通过正相硅胶色谱、凝胶Sephadex LH-20色谱和重结晶等纯化手段分离纯化目标化合物BDDPM，并借助IR，MS和NMR等技术确定了其化学结构。最终从常温风干的50kg松节藻干样品中分离得到7.8g BDDPM。由于松节藻藻体构成复杂，给分离纯化BDDPM带来极大困难，致使分离纯化过程耗费大量时间和金钱；并且原材料松节藻的采集也易受季节和原料短缺等自然因素的影响。所以，从天然海藻中分离纯化的方法不适宜用于BDDPM的制备。 本文的重点是对BDDPM（4e）的化学合成途径进行研究。本文通过5步合成法（Friedel-Craftz酰基化反应、苯环逐级溴代、羰基还原、羟基脱保护）成功地合成了BDDPM，合成总产率为23.6%。同时本文采用上述合成路线获得了四个系列共计20个溴酚系列衍生物（4e为目标产物BDDPM，其余19个为溴酚系列衍生物），其中10个为新化合物。合成的20个化合物经1H NMR、13C NMR、MSEI和IR进行了结构鉴定。合成的20个化合物在体外活性筛选中均表现出不同程度的PTP1B抑制活性，其中合成的目标产物4e具有与天然分离纯化获得的BDDPM同等效率的PTP1B抑制作用。 另外，通过比较四个系列化合物PTP1B抑制活性间的差异，对此类溴酚化合物的构效关系作了初步分析，结果表明：1．羰基官能团的存在会明显降低此类化合物的PTP1B抑制率；2．化合物中的羟基官能团被甲氧基保护后，PTP1B抑制活性会得到一定程度的加强；3．化合物苯环上溴原子取代基数目增多时，其PTP1B抑制率也会随之增强。但是，筛选结果中也有少部分化合物的PTP1B抑制作用与上述规则相违背。因此，本文总结的初步构效关系还需要进一步的实验研究加以验证。 最后本文通过化学合成的方法，经过5步反应成功地制备出了30g BDDPM，为后续的药理、药效学研究奠定了基础。

Total synthesis of bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane as potent PTP1B inhibitor

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator and has been proved to be an effective target for the treatment of type 2 diabetes mellitus. Bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane 7 was first reported as a natural bromophenol with significant inhibition against PTP1B which was isolated from red algae Rhodomela confervoides. Intrigued by its astonishing activity (IC50 = 2.4 mu mol/L), compound 7 was synthesized with the overall yield of 24% and evaluated for its PTP1B inhibitory activity compared with natural compound. (C) 2008 Li Jun Han. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of Rhodomela confervoides extract in diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator in insulin signaling pathways. PTP1B is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2',3,3'-tetrabromo-4,4',5,5'-tetra-hydroxydiphenyl methane (1), 3-bormo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl) pyrocatechol (2), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (3) and 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxy-methyldiphenylmethane (4) showed significant inhibitory activity against PTP1B (IC50 were 2.4, 1.7, 1.5 and 0.84 mu mol/L, respectively) as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from R. confervoides on streptozotocin-induced diabetes (STZ-diabetes) in male Wistar rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose (FBG) as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the inhibitory actions against PTP1B of the bromophenol derivatives and that may be of clinical importance in improving the management of type 2 diabetes mellitus.

FLUCTUATION OF SIZE-FRACTIONATED ALKALINE PHOSPHATASE AFTER BLOOM DISAPPEARANCE IN TWO SHALLOW PONDS

The temporal and vertical fluctuations of size fractionated alkaline phosphatase activity (APA) and kinetics parameters as well as orthophosphate (o-P) and chlorophyll concentrations were investigated after bloom disappearance in two shallow ponds A and B from 27 October 2001 to 15 April 2002. Pond A (Microcystis) bloomed seriously but pond B did not. The data of o-P and chlorophyll suggested that phosphorus was the principal limiting nutrimental element and its vertical flux should be regarded as an important driving factor for algal growth. In pond A, the accumulation of algae-derived detritus after bloom disappearance in overlying water stimulated excretion of algal fraction APA, mainly produced by attached bacteria responsible for detritus decomposition, whereas bacterial fraction APA preferred to function in surface water. Interestingly, completely contrary phenomena were observed in pond B. In season, even though no obvious difference for size-fractionated APA in both ponds, the total APA in pond A peaked earlier showing higher activity and efficiency (low K-m and high V-max values) as a result of algal-derived detritus input. In summary, it is suggested that the excretion of alkaline phosphatase with strongly catalyzing efficiency and high activity should be taken as important contributor to algal-derived detritus decomposition, further fueling nutrient recycle and accelerating algal development next year. Furthermore, some inhibitors and surfactants were testified to be good tools to identify the origin of dissolved alkaline phosphatase.

Phosphorus fractions and alkaline phosphatase activity in sediments of a large eutrophic Chinese lake (Lake Taihu)

Spatial, vertical, and seasonal variations in phosphorus fractions and in alkaline phosphatase activity (APA) were investigated in sediments in a large-shallow eutrophic Chinese lake (Lake Taihu) in 2003-2004. The phosphorus content was highest in the most seriously polluted lake area. Iron-bound phosphorus (Fe(OOH)-P) dominated (47% on average) among the phosphorus fractions determined according to Golterman (Hydrobiologia 335:87-95, 1996). Notably, organically-bound P comprised a further significant additional portion (acid-soluble + hot NaOH-extractable organic P = 25%), which was highest at the most polluted sites. The Fe(OOH)-P content was the lowest in spring (April, 2004), suggesting that degradation of organic matter led to the release of iron-bound phosphates. Sediment APA showed a significant positive relationship with both organically-bound P and Fe(OOH)-P. Consequently, organically-bound P is an important portion of the sediment phosphorus in Lake Taihu. It is mainly derived from freshly-settled autochthonous particles and from external discharges. Organically-bound P induces APA and may lead to the release of bioavailable phosphates from the organic sediments, thereby accelerating lake eutrophication.

Contributions of phosphatase and microbial activity to internal phosphorus loading and their relation to lake eutrophication

Phosphatase may accelerate the process of lake eutrophication through improving phosphorus bioavailability. This mechanism was studied in three Chinese eutrophic shallow lakes (Lake Taihu, Lake Longyang,and Lake Lianhua). Phosphatase activity was related to the concentration of soluble reactive phosphorus (SRP) and chlorophyll a. Stability of dissolved phosphatase in reverse micelles may be attributed to molecular size, conformation and active residues of the enzyme. At the site with Microcystis bloomed in Lake Taihu, dissolved phosphatase activity was higher and more stable in micelles, SRP concentrations were lower in interstitial water, the contents of different forms of phosphorus and the amounts of aerobic bacteria were lower while respiration efficiency was higher in sediments. Phosphobacteria, both inorganic and organic and other microorganisms were abundant in surface water but rare in sediments. Therefore, internal phosphorus may substantially flux into water column by enzymatic hydrolysis and anaerobic release, together with mobility of bacteria, thereby initiating the bloom. In short, biological mechanism may act in concert with physical and chemical factors to drive the internal phosphorus release and accelerate lake eutrophication.

Vertical variation in dissolved alkaline phosphatase activity in a shallow eutrophic lake determined in reverse micelles

We evaluated the feasibility of microencapsulating dissolved alkaline phosphatase of a water body into reverse micelle systems prepared by hexadecyltrimethylammonium bromide as a surfactant in cyclohexane and 1-butanol as co-surfactant. The dissolved alkaline phosphatase activity within the micelle was described, including its kinetic parameters and the effects of pH and temperature on catalytic activity in surface, overlying and interstitial water of Lake Donghu. We found the similarities on the behavior of dissolved alkaline phosphatase of surface and interstitial water in reverse micelles, which was distinctly different from its behavior in the overlying water. This difference likely reflected the different origins of the dissolved alkaline phosphatase in the vertical profile of the lake. This system provides a novel tool with which to study the diversity and ecological significance of extracellular enzymes in aquatic environments.

Discharges and sediments as sources of extracellular alkaline phosphatase in a shallow Chinese eutrophic lake

Total alkaline phosphatase activity (APA) and soluble reactive phosphorus (SRP) concentrations were measured in municipal wastewater, and a shallow Chinese freshwater lake receiving it. Activities of Dissolved alkaline phosphatase ( ADAP) in overlying and interstitial water were also analyzed monthly at three sites for several years. The lake was enriched with SRP and alkaline phosphatase by discharge of the wastewater, indicating that the inclusion of APA for estimating water pollution was reasonable. Annual data showed that APA in coarser fraction was significantly higher at the site receiving more wastewaters, both in surface and overlying water, suggesting that resuspension of enzyme most likely occurred in the basin heavily discharged. ADAP was an order of magnitude higher in the wastewater than those in lake waters, and was generally higher in interstitial water, a feature more striking at the site receiving more discharges. Besides, it was irrespectively inhibited by Na2WO4, L-cysteine and EDTA-Na, but stimulated by Cu2+, Zn2+, CTAB and Triton X-100 in interstitial, overlying and surface waters. This similarity of responding patterns to the stressors indicated an analogy between dissolved alkaline phosphatase in water column and that in interstitial water, supporting the hypothesis that the polluted sediments act as source of dissolved alkaline phosphatase in eutrophic lakes.

Temporal and spatial variations in kinetics of alkaline phosphatase in sediments of a shallow Chinese eutrophic lake (Lake Donghu)

Monthly sediment and interstitial water samples were collected in a shallow Chinese freshwater lake (Lake Donghu) from three areas to determine if alkaline phosphatase activity (APA) plays an important role in phosphorus cycling in sediment. The seasonal variability in the kinetics of APA and other relevant parameters were investigated from 1995-1996. The phosphatase hydrolyzable phosphorus (PHP) fluctuated seasonally in interstitial water, peaking in the spring. A synchronous pattern was observed in chlorophyll a contents in surface water in general. The orthophosphate (o-P) concentrations in the interstitial water increased during the spring. An expected negative relationship between PHP and V-max of APA is not evident in interstitial water. The most striking feature of the two variables is their co-occurring, which can be explained in terms of an induction mechanism. It is argued that phosphatase activity mainly contributes to the driving force of o-P regeneration from PHP in interstitial water, supporting the development of phytoplankton biomass in spring. The V-max values in sediment increased during the summer, in Conjunction with lower K-m values in interstitial water that suggest a higher affinity for the substrate. The accumulation of organic matter in the sediment could be traced back to the breakdown of the algal spring bloom, which may stimulate APA with higher kinetic efficiency, by a combination of the higher V-max in sediments plus lower K-m values in interstitial water, in Summer. In summary, a focus On phosphatase and its substrate in annual scale may provide a useful framework for the development of novel P cycling, possible explanations for the absence of a clear relationship between PHP and APA were PHP released from the sediment which induced APA, and the presence of kinetically higher APA both in sediment and interstitial water which permitted summer mineralization of organic matter derived from the spring bloom to occur. The study highlighted the need for distinguishing functionally distinct extracellular enzymes between the sediment and interstitial water of lakes. (C) 2002 Elsevier Science Ltd. All rights reserved.