Human Prostatic Acid Phosphatase: Structure,Function and Regulation

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.     

Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle,Epicauta chinensis

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.     

有机结合态磷肥对土壤微生物及磷酸酶的影响

土壤培养和盆栽试验结果表明：与无机磷肥相比,有机结合态磷肥能够大幅提高土壤微生物数量和土壤磷酸酶含量;有机结合态磷原产物与磷矿粉配合的有机结合态磷肥对提高土壤微生物量的效果最好,比无机磷肥提高23倍,对提高土壤磷酸酶含量的效果也最好,比无机磷肥提高约6倍;有机结合态磷原产物与缓释氮配合的效果次之,但与前者差异幅度不大.有机结合态磷肥具有降低土壤pH的效果,对pH较高的石灰性土壤效果良好,在pH低的红壤上应选用磷矿粉与有机结合态磷原产物复合的肥料效果较好.     

Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.     

Raw Milk-Induced Protection against Food Allergic Symptoms in Mice Is Accompanied by Shifts in Microbial Community Structure

The mechanism underlying the allergy-protective effects of raw cow’s milk is still unknown, but the modulation of the gut microbiome may play a role. The effects of consuming raw cow’s milk or processed milk on fecal microbial communities were therefore characterized in an experimental murine model. C3H/HeOuJ mice were treated with raw milk, pasteurized milk, skimmed raw milk, pasteurized milk supplemented with alkaline phosphatase (ALP), or phosphate-buffered saline (PBS) for eight days prior to sensitization and challenge with ovalbumin (OVA). Fecal samples were collected after milk exposure and after OVA sensitization, and microbiomes were characterized using 16S ribosomal RNA gene amplicon sequencing. Treatment with raw milk prior to OVA sensitization increased the relative abundance of putative butyrate-producing bacteria from the taxa Lachnospiraceae UCG-001, Lachnospiraceae UCG-008, and Ruminiclostridium 5 (Clostridial clusters XIVa and IV), while it decreased the relative abundance of Proteobacterial genera such as Parasutterella, a putative pro-inflammatory bacterial genus. This effect was observed after eight days of raw milk exposure and became more pronounced five weeks later, after allergic sensitization in the absence of milk. Similar trends were observed after treatment with skimmed raw milk. Conversely, the feeding of pasteurized milk led to a loss of allergy protection and a putative dysbiotic microbiome. The addition of ALP to pasteurized milk restored the protective effect observed with raw milk and mitigated some of the microbial community alterations associated with milk pasteurization. Raw milk-induced protection against food allergic symptoms in mice is accompanied by an increased relative abundance of putative butyrate-producing Clostridiales and a decreased relative abundance of putative pro-inflammatory Proteobacteria. Given the safety concerns regarding raw milk consumption, this knowledge is key for the development of new, microbiologically safe, preventive strategies to reduce the incidence of allergic diseases.     

Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases,UBE2d3, and the C-Terminus of c-Jun

Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.     

引江济太对太湖水体碱性磷酸酶活性的影响

本文通过对2003年7—9月引江济太期间太湖水体中碱性磷酸酶活性的测定．以及总磷、总氮、正磷酸盐和叶绿素等其它水化学因子的同步监测，初步探讨了太湖水体在引入长江水之后，其碱性磷酸酶活性、营养盐和叶绿素含量的变化趋势，以及碱性磷酸酶活性与营养盐、叶绿素含量之间的关系。研究表明：(1)调水期间，碱性磷酸酶活性的最大反应速率(Vmax)值降低，而米氏常数(Km)值普遍增高，表明引入长江水后，太湖水体碱性磷酸酶的总催化效率降低；(2)太湖水体碱性磷酸酶的Vmax/Km与叶绿素含量之间具有良好的相关性．叶绿素含量随着酶活性的降低而降低。     

一株高产碱性磷酸酶菌株的分离鉴定及其培养基配方优化

为获得高产碱性磷酸酶微生物,采集土样进行筛选,并优化获得最佳产酶发酵条件。经过碱性磷酸酶筛选平板初筛和发酵复筛,分离得到一株优势产酶菌株。通过菌株形态观察、生理生化特征和分子生物学鉴定,确定该菌为醋杆菌奥尔兰亚种(Acetobacter orleanensis),编号S377。以碱性磷酸酶活力为响应值,在单因素实验的基础上,以果糖、尿素、pH、碳酸钙为实验因素,采用中心组合实验方法进行响应面优化设计。结果表明,最优发酵条件为果糖8.9 g/L,尿素0.4 g/L,碳酸钙4.5 g/L,初始pH9.2。利用该最优组合培养,碱性磷酸酶活性达到(28600±65)U,比优化前酶活力提高6.4倍。Acetobacter orleanensis S377产酶活力明显高于其它报道的产碱性磷酸酶微生物,为碱性磷酸酶生产提供了新的微生物资源,对该酶的工业化生产具有重要意义。     

鲨鱼单域抗体-碱性磷酸酶融合蛋白的表达及热稳定性分析

酶标抗体的稳定性对酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)至关重要,但常规化学偶联方法制备的酶标抗体具有热稳定性不足的缺点,这限制了酶标抗体在ELISA中的应用。本研究利用基因工程技术克隆表达出碱性磷酸酶与特异性鲨鱼单域抗体的融合蛋白,并对融合蛋白的亲和力和热稳定性进行了研究。结果表明:在16 ℃下,0.05 mmol/L的异丙基β-D-1-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导可实现融合蛋白的可溶性表达,表达出的融合蛋白分子量约为63 kDa,与目标抗原的亲和常数可达7.80×10^-8 mol/L,具有良好的抗原结合能力;融合蛋白在58 ℃处理180 min后热稳定性良好,证明该融合蛋白可以作为ELISA检测中一种潜在的免疫诊断剂。

     

Activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen scaffold

We investigated the effect of gamma-irradiation on the activities of MC3T3-E1 cells cultured on gamma-irradiated salmon atelocollagen (SAC) scaffolds. SAC-cultured samples were irradiated at doses of 10, 15, and 25 kGy. Gamma-irradiation had a significant impact on the activities of MC3T3-E1 cells. The proliferation rates and alkaline phosphatase activities of MC3T3-E1 cells increased with gamma-irradiation dose.