Pivotal role of inosine triphosphate pyrophosphatase in maintaining genome stability and the prevention of apoptosis in human cells

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.     

Structure insights into mechanisms of ATP hydrolysis and the activation of human heat-shock protein 90

The activation of molecular chaperone heat-shock protein 90 (Hsp90) is dependent on ATP binding and hydrolysis, which occurs in the N-terminal domains of protein. Here, we have determined three crystal structures of the N-terminal domain of human Hsp90 in native and in complex with ATP and ATP analog, providing a clear view of the catalytic mechanism of ATP hydrolysis by Hsp90. Additionally, the binding of ATP leads the N-terminal domains to be an intermediate state that could be used to partially explain why the isolated N-terminal domain of Hsp90 has very weak ATP hydrolytic activity.     

Type III polyketide synthases in natural product biosynthesis

Polyketides represent an important class of biologically active and structurally diverse compounds in nature. They are synthesized from acyl-coenzyme A substrates by polyketide synthases (PKSs). PKSs are classified into three groups: types I, II, and III. This article introduces recent studies on type III PKSs identified from plants, bacteria, and fungi, and describes the catalytic functions of these enzymes in detail. Plant type III PKSs have been widely studied, as exemplified by chalcone synthase, which plays an important role in the synthesis of plant metabolites. Bacterial type III PKSs fall into five groups, many of which were identified from Streptomyces, a genus that has been well known for its production of bioactive molecules and genetic alterability. Although it was believed that type III PKSs exist exclusively in plants and bacteria, recent fungal genome sequencing projects and biochemical studies revealed the presence of type III PKSs in filamentous fungi, which provides a new chance to study fungal secondary metabolism and synthesize "unnatural" natural products. Type III PKSs have been used for the biosynthesis of novel molecules through precursor-directed and structure-based mutagenesis approaches.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.     

云雾山草坡和泾川刺槐林坡面土壤电阻率和含水率的空间差异

采用盆栽控水的方法,研究干旱胁迫（80%FC、60%FC、40%FC和20%FC）及施氮（N0 0 g·pot-1、Nl 1.2 g·pot-1、Nm 3.6 g·pot-1和Nh 6.0 g·pot-1）对麻疯树幼苗叶、茎和根部主要渗透调节物质积累的影响.结果表明： 干旱胁迫条件下,麻疯树幼苗茎和根部的游离脯氨酸、可溶性蛋白和茎部可溶性糖大量积累,叶片中脯氨酸含量也随干旱胁迫程度的增加大幅度上升;Na+、Ca2+和Mg2+在麻疯树幼苗叶、茎和根中大量积累,而K+仅在茎中大量积累,叶片和根部K+含量略微上升.施氮对植株渗透调节物质的影响与干旱胁迫强度和施氮水平有关.在80%FC和60%FC水分条件下,增加N肥施用量能明显促进麻疯树幼苗各组分渗透调节物质的积累;在40%FC水分条件下,Nh处理对渗透调节物质积累的促进作用减弱;而在20%FC条件下,Nl处理植株的渗透调节能力较高,Nm和Nh处理对植株渗透调节的促进作用不明显甚至转为抑制.     

硫酸乙酰肝素3-O硫酸基转移酶5的表达、纯化及酶学性质研究

经过PCR克隆得到硫酸乙酰肝素3-O硫酸基转移酶5(3-OST-5)的基因,将其与大肠杆菌表达载体pET-15b连接后,在大肠杆菌BL21(DE3)中诱导表达,使用镍亲和层析柱纯化得到具有活性的3-OST-5。经测定纯化后的3-OST-5比活达到0.58 U/mg,是纯化前的5.27倍,回收率达80.4%。在此基础上,研究了该酶的酶学性质,酶反应的最适温度为35℃,稳定范围为20-40℃;最适pH为7.0,在pH7.0-9.0范围内稳定。在反应液中加入终浓度为1 mmol/L的K+、Ca2+、Ba2+对酶促反应有一定的促进作用。     

Study on the Effect of Kidney Transplantation on the Health of the Patients’ Offspring: A Report on 252 Chinese Children

Even though the incidence of pregnancies in the female recipients is lower and also chronic renal disease in male patients is associated with impaired spermatogenesis, the health of the children born to these patients was not studied. In this report, we discuss information on the growth and development of offspring of 248 male and female kidney recipient patients. Physical and routine clinical measurements of the 252 offspring (129 male and 123 female) born to these transplantation patients were made along with the intelligence tests. In some of these children chest X-ray and immune indices were assessed. Among the recipients, 219 males fathered 223 children with an average birth weight of 3,255 ± 374 g and 29 female recipients gave birth to 29 children with an average birth weight of 2,923 ± 551. While most of these children were normal, we noticed a case of soft double toe, a case of short tongue tie, five cases of marginal mental retardation, three cases of proteinuria, six cases of microscopic hematuria, 15 cases of low hemoglobin, and 21 cases with recurrent respiratory tract infections. We conclude that kidney transplantation has no significant impact on the growth, development, health, and intelligence of the offspring born to recipients.     

Bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats

Whether environmental exposure to bisphenol A (BPA) may induce reproductive disorders is still controversial but certain studies have reported that BPA may cause meiotic abnormalities in C. elegans and female mice. However, little is known about the effect of BPA on meiosis in adult males. To determine whether BPA exposure at an environmentally relevant dose could induce meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 20 μg/kg body weight (bw)/day for 60 consecutive days. We found that BPA significantly increased the proportion of stage VII seminiferous epithelium and decreased the proportion of stage VIII. Consequently, spermiation was inhibited and spermatogenesis was disrupted. Further investigation revealed that BPA exposure delayed meiosis initiation in the early meiotic stage and induced the accumulation of chromosomal abnormalities and meiotic DNA double-strand breaks (DSBs) in the late meiotic stage. The latter event subsequently activated the phosphatidylinositol 3-kinase-related protein kinase (ATM). Our results suggest that long-term exposure to BPA may lead to continuous meiotic abnormalities and ultimately put mammalian reproductive health at risk.