Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

锌指结构：最普遍的核酸识别元件

锌指是最大的DNA结合蛋白家庭,是识别DNA最有效、最成功的一种结构元件。其模块性结构特点及与核酸作用的相对简单性,使其成为研究蛋白-核酸相互作用的理想材料,以及人为设计筛选新的核酸结合蛋白的最佳元件。     

STRUCTURAL STUDIES ON LNA QUADRUPLEXES

LNAs (locked nucleic acids) are new DNA analogues with higher binding affinities toward nucleic acids than the canonical counterparts mainly due to the characteristic conformational restriction arising from the 2′-O, 4′-C methylene bridge. In light of the promising therapeutic applications and considering the advantageous characteristics of LNAs, such as their high water solubility, easy handling, and synthetic accessibility through the conventional phosphoramidite chemistry, we undertook a study concerning the capability of these nucleic acid analogues to form quadruplex structures. Particularly, we have been investigating the LNA/DNA chimeras corresponding to the well-known DNA sequences 5′-GGTTGGTGTGGTTGG-3′, capable of forming an unimolecular quadruplex. This article deals with the study of the sequence 5′-ggTTggTGTggTTgg-3′ (upper and lower case letters represent DNA and LNA residues, respectively), which, according to CD spectroscopy, is able to fold into a quadruplex structure.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Effect of Nitrosative Stress on Extracellular Nucleotide Metabolism in Endothelial Cells

Mechanisms of free radical injury involve chemical modification of proteins, lipid derivatives and nucleic acids and consequent loss of its function. However, specific targets and exact sequence of events has not been fully clarified. We determined whether extracellular enzymes that are involved in adenosine formation such as ecto-5′nucleotidase (e5N) and removal such as extracellular form of adenosine deaminase (eADA) could be affected by peroxynitrite. We used intact cell assay system that involves exposure of cultured HMEC-1 cells to substrates followed by HPLC analysis of conversion of substrates into products. We found that e5N and ADA activities decreased by 20–40% after incubation for 20 or 60 minutes with 30 μM peroxynitrite. Decrease of cellular ATP and NAD was also observed. We conclude that besides other cytotoxic effects modification of extracellular enzymes of nucleotide metabolism could be important target for free radical injury.     

基因重组与基因合成实验设计软件系统的建立

本文报导了用于基因重组与基因合成实验设计的软件系统的建立.此系统由30个功能模块组成,为研究者提供了包括在DNA分子上寻找限制性内切酶位点、核酸分子片段之间同源性比较,基因化学合成的实验设计、特定顺序分析引物及核酸杂交探针的设计、阅读框的查找等功能.此外,本系统可以对外来数据库的资料进行援引和进一步分析,为分子生物学的研究提供有价值的信息.     

Kinase-catalyzed biotinylation of DNA

Prior work documented use of γ-phosphate modified ATP analogs to label DNA using T4 polynucleotide kinases (T4PNK), although applications have been limited. To fully characterize kinase-catalyzed labeling of nucleic acids, we explored use of ATP-biotin as a cosubstrate with T4PNK. T4PNK accepted ATP-biotin to 5′-label single stranded DNA. However, T4PNK-mediated labeling of double stranded substrates was low yielding. In addition, the phosphoramidate bond connecting the biotin group to the DNA was unstable. These results suggest that kinase-catalyzed biotinylation will be useful with single stranded DNA substrates and mild reaction conditions. By revealing the scope and limitations of kinase-catalyzed biotinylation, these studies provide a foundation for future development and application of kinase-catalyzed labeling to DNA-based biological studies.     

Effect of cyclic adenosine-3', 5'-monophosphate on cells of experimental lympholeukemia L-5178]

A study was made of the effect of cyclic adenosine-3',5'-monophosphate (cAMP) dibutyril-cAMP and theophylline (phosphoesterase inhibitor - an enzyme transforming adenosine-3'-5'-monophosphate into adenosine-5'-monophosphate) on the intensity of proliferation (by the increase in the content of nucleic acids in the culture), DNA synthesis (by the H3-thymidine incorporation) and on the transplantation properties (the capacity to repopulation in the animal organism) of leukemic cells of the L-5178 strain. It was found that cAMP in a concentration of 0.8 mM considerably inhibited the H3-thymidine incorporation, retarded the proliferation and decreased the transplantation capacity of leukemic cells. Theophylline and dibutyril-cAMP had a comparatively low inhibitory capacity on the DNA synthesis, proliferative activity and the transplantation properties of the cells.     

Extracellular Nucleotide Catabolism in Aortoiliac Bifurcation of Atherosclerotic ApoE/LDLr Double Knock Out Mice

Atherosclerosis is a consequence of diverse pathologies that could be affected by signaling mediated by nucleotides and their metabolites. Concentration of specific nucleotide derivatives in the proximity of purinergic receptors is controlled by extracellular enzymes such as ecto-nucleoside triphopsphate diphosphohydrolase (eNTPD), ecto-5′-nucleotidase (e5NT), and ecto-adenosine deaminase (eADA). To estimate changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall, aortoiliac bifurcation of ApoE/LDLr (–/–) mice was perfused with solution containing adenosine-5′-triphosphate (ATP), adenosine-5′-monophosphate (AMP) or adenosine. Formation of the product of eNTPD, e5NT or eADA was measured by high performance liquid chromatography (HPLC). The most significant difference between ApoE/LDLr (–/–) and wild-type mice was several times higher rate of conversion of adenosine to inosine catalyzed by eADA activity. This highlights potential decrease in intravascular adenosine concentration in atherosclerosis.     

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea.     

Action of tetracycline and sodium deoxycholate combinations on protein and nucleic acid synthesis in the cells of the NAG vibrio, Staphylococcus aureus and Escherichia coli]

The effect of tetracycline combination with sodium desoxycholate, a surface-active substance, on the synthesis of proteins and nucleic acids in the cells of NAG-vibrio, Staph. aureus and E. coli was studied by incorporation of 1-14C-glycine and 8-14C-adenine into proteins and nucleic acids. It was found that sodium desoxycholate suppressed the synthesis of proteins and nucleic acids in the cells of NAG-vibrio and Staph. aureus. Its combination with tetracycline resulted in summation or increase of the suppressive effects on proteins and nucleic acids as compared to the effect of the substances used alone. Sodium desoxycholate even in very high concentration, up to 12800 gamma/ml, had no effect on the synthesis of proteins and nucleic acids in the cells of E. coli and respectively it did not change the activity of tetracycline on combined use.     

2′-BIS-PYRENE MODIFIED OLIGONUCLEOTIDES: SENSITIVE FLUORESCENT PROBES OF NUCLEIC ACIDS STRUCTURE

A new type of fluorescent nucleic acid probes, 2′-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2′-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2′-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.     

Comparison of Various Procedures for Removing Proteins and Nucleic Acids from Cell Walls of Bacillus Subtilis

Several procedures were used i n an attempt to prepare clean cell walls from Bacillus subtilis. The results indicate that protein and nucleic acids are tightly bound tothe walls. cleanest wall preparations were found following trichloroacetic acid extraction at 60° or by extraction with 0.lN NaOH under a nitrogen atmosphere for 10 hrs. Protein denaturants, such as sodium dodecyl sulfate and concentrated guanidine hydrochloride were relatively ineffective in removing proteins and nucleic acids from the cell walls. Cell wall-bound DNA was biologically The active i n transformation assays.     

A bioluminescence method for determining adenosine 3'-phosphate 5'-phosphate (PAP) and adenosine 3'-phosphate 5'-sulfatophosphate (PAPS) in biological materials.

A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis, which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence.Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.     

Aminoacyl-tRNA Analogues; Synthesis,Purification and Properties of 3′-Anthraniloyl Oligoribonucleotides

Abstract

Reaction of isatoic anhydride with adenosine, adenosine 5′-phosphate, oligoribonucleotides or with the E. coli tRNA^Val led to attachment of an anthraniloyl residue at 2′-or 3′-OH groups of 3′-terminal ribose residue. No protection of the S'-hydroxyl group or internal 2′-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.