Design,synthesis and biological evaluation of N2,N4-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives as HIV-1 NNRTIs

A series of N₂,N₄-disubstituted-1,1,3-trioxo-2H,4H-pyrrolo[1,2-b][1,2,4,6]thiatriazine derivatives (PTTDs) was designed and synthesized by a facile route. The biological assay results showed that five most potent compounds displayed inhibitory activity against HIV-1 at low micromolar concentrations (EC₅₀ = 5.1–8.9 μM). Structure–activity relationship analysis indicated that N₂-(3-halogenated-benzyl) analogues were more potent than N₂-(unsubstituted-benzyl) analogues. The N₄-substitutions contributed to the antiviral activity in the following order: 2-/3-cyano substituted benzyl > 2-/3-halogenated benzyl > non-substituted benzyl > 4-halogenated benzyl. Docking studies of the representative compound revealed the binding conformation of these compounds and provided critical insights for the further development of PTTD analogues.     

Design,synthesis and biological evaluation of negatively charged 111In-DTPA-octreotide derivatives

Our previous studies indicated that ¹¹¹In-diethylenetriaminepentaacetic acid (¹¹¹In-DTPA)-octreotide derivatives with an additional negative charge by replacing N-terminal d-phenylalanine (d-Phe) with an acidic amino acid such as l-aspartic acid (Asp) or its derivative exhibited low renal radioactivity levels when compared with ¹¹¹In-DTPA-d-Phe¹-octreotide. On the basis of the findings, we designed, synthesized and evaluated two Asp-modified ¹¹¹In-DTPA-conjugated octreotide derivatives, ¹¹¹In-DTPA-Asp¹-octreotide and ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide. While ¹¹¹In-DTPA-Asp¹-octreotide showed negligible AR42J cell uptake, ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide exhibited AR42J cell uptake similar to that of ¹¹¹In-DTPA-d-Phe¹-octreotide. When administered to AR42J tumor-bearing mice, ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide exhibited renal radioactivity levels significantly lower than did ¹¹¹In-DTPA-d-Phe¹-octreotide at 1 and 3 h post-injection. No significant differences were observed in tumor accumulation between ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide and ¹¹¹In-DTPA-d-Phe¹-octreotide after 1 and 3 h injection. The findings in this study suggested that an interposition of an Asp at an appropriate position in ¹¹¹In-DTPA-d-Phe¹-octreotide would constitute a useful strategy to develop ¹¹¹In-DTPA-d-Phe¹-octreotide derivatives of low renal radioactivity levels while preserving tumor accumulation.     

Synthesis,and in vitro Enzymatic and Antiviral Evaluation of d4T Polyphosphate Derivatives as Chain Terminators

A series of d4T di‐ or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV‐1 RT, and also tested for their in vitro anti‐HIV activity. The steady‐state kinetic study of compounds 1 – 4 in an enzymatic incorporation assay by HIV‐1 RT follows Michaelis? Menten profile. In addition, compounds 2 – 4 are able to inhibit HIV‐1 replication to the same extent as d4T and d4TMP in MT‐4 cells, as well as in CEM/0 cells and CEM/TK^? cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.     

Design,synthesis and biological evaluation of 3-benzyloxy-linked pyrimidinylphenylamine derivatives as potent HIV-1 NNRTIs

A novel series of 3-benzyloxy-linked pyrimidinylphenylamine derivatives (8a–8s) was designed, synthesized and evaluated for their in vitro anti-HIV activity in MT-4 cell cultures. Most of the compounds inhibited wild-type (wt) HIV-1 replication in the lower micromolar concentration range (EC₅₀ = 0.05–35 μM) with high selectivity index (SI) values (ranged from 10 to >4870). In particular, 8h and 8g displayed excellent antiretroviral activity against wt HIV-1 with low cytotoxicity (EC₅₀ = 0.07 μM, CC₅₀ >347 μM, SI >4870; EC₅₀ = 0.05 μM, CC₅₀ = 42 μM, SI = 777, respectively), comparable to that of the marked drug nevirapine (EC₅₀ = 0.113 μM, CC₅₀ >15 μM, SI >133). In order to confirm the binding target, 8h was selected to perform the anti-HIV-1 RT assay. Additionally, preliminary structure activity relationship (SAR) analysis and molecular docking studies of newly synthesized compounds were also discussed, as well as the predicted physicochemical properties.     

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea

Abstract: Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

中国主要鹅品种的线粒体DNA多态性与起源分化研究

运用19种限制性内切酶对中国11个家鹅品种138个样本进行了mtDNA的限制性片段长度多态性（RFLP）分析。在使用的19种内切酶中,有7个酶检测出多态。综合27种限制性态型（restrictionmorph）,可得到6种mtDNA单倍型（hopotype）。伊犁鹅与另外10个鹅品种没有共享的单倍型,遗传距离和UPGMA聚类分析也表明,伊犁鹅与这些品种具有不同的起源。EcoRV、HaeⅡ、HincⅡ和KpnⅠ4种酶的限制性态型可作为鉴别两种起源家鹅的母系遗传标记。起源于鸿雁的10个鹅品种群体内出现一定的遗传差异,其群体多态度（π）、单倍型间平均遗传距离（P）、品种间平均净遗传距离（δnet）分别为0．025％、0．266％和0．029％。白羽鹅品种在形成过程中经历过创立者效应（foUndereffect）。这10个鹅品种可能起源于两个不同地理区的鸿雁类群。     

Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3'OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3'OH end of a DNA strand, without the need of a template. Pol lambda and pol micro are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol micro and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol micro and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Design,synthesis, and characterization of nucleosomes containing site-specific DNA damage

How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.     

High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'-deoxycytidine, and other deoxynucleosides and nucleosides in DNA digests

This work has undertaken liquid chromatographic separation of nucleosides and deoxynucleosides. Two different columns with three mobile phases (A, deionized water; B, 50 mM phosphate buffer (pH 4.0); C, methanol) and slightly different gradient programs were used. The elution order was as follows: cytidine (C), 2′-deoxycytidine (dC), uridine (U), 5-methyl-2′-cytidine (5mC), 5-methyl-2′-deoxycytidine (5mdC), guanosine (G), deoxyguanosine (dG), 2′-deoxythymidine (dT), adenosine (A), and 2′-deoxyadenine (dA). Using a Luna C18 Phenomenex column (150 × 4.6 mm, 5 μm), the separation was performed at 40 °C with a total flow rate of 1 ml/min and a run time of 10 min. The second column was an Agilent C18 (50 × 3 mm, 1.8 μm), for which the run time was 4.5 min with a flow rate of 0.6 ml/min (25 °C). In application to the DNA digests from human THP-1 cells, the quantification of C, dC, U, 5mC, 5mdC, G, dG, and A was performed. The percentages of global methylation were evaluated based on the 5mdC and dC concentrations (c_5mdC / [c_5mdC + c_dC], where c is concentration in μg/ml) and compared with those calculated from the respective peak areas (A_5mdC / [A_5mdC + A_dC], where A is peak area at 254 nm). For peak area measurements, excellent agreement was obtained with the results reported previously in the same cell line. In the quantitative approach, the results of DNA methylation were higher but consistent with the previous data obtained using mass spectrometric detection. Comparing the analytical features of the two procedures, the use of a smaller column could be recommended because it provides efficient separation (capacity factors in the range of 1.29-10.66), a short run time, and feasibility of nucleoside and deoxynucleoside quantification in real-world samples and because it also minimizes the use of reagents.