The role of mineral surfaces in the origin of life

Adsorption of nucleoside phosphates on the surfaces of volcanic rocks has been studied. Differences in the adsorption of some nucleoside phosphates on the surface of basalt cinder have been found. Differences in the adsorption of similarmolecules on different mineral surfaces have also been shown. Different adsorptive capacities may have served as a mechanism for the selection of organic molecules during prebiotic evolution.     

Pentose phosphates in nucleoside interconversion and catabolism

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.     

Imaging erythrocytes under physiological conditions by atomic force microscopy

Since its invention in the mid 1980s atomic force microscopy has revolutionised the way in which surfaces can be imaged. Close to atomic resolution has been achieved for some materials and numerous images of molecules on surfaces have been recorded. Atomic force microscopy has also been of benefit to biology where protein molecules on surfaces have been studied and even whole cells have been investigated. Here we report a study of red blood cells which have been imaged in a physiological medium. At high resolution, the underlying cytoskeleton of the blood cell has been resolved and flaws in the cytoskeleton structure may be observed. Comparison of the normal 'doughnut' shaped cells with swollen cells has been undertaken. Differences in both the global properties of the cells and in the local features in cytoskeleton structure have been observed.     

Recent progress in artificial receptors for phosphate anions in aqueous solution

Phosphate esters exist ubiquitously in nature in the form of nucleoside phosphates (nucleotides) as components of RNA (or DNA), sugar nucleotides for glycosylation of oligosaccharides or proteins, activated form of proteins responding to extracellular signals, and chemical mediators playing central roles in intracellular signaling signals. Phosphorylation of anti-viral nucleoside analogues by intracellular kinases yields nucleoside phosphates (nucleotide) as biologically active forms as anti-viral agents. Development of artificial phosphate receptors would afford new methodologies for detection, separation, or transport of biologically important phosphates. Herein, a recent progress of artificial phosphate receptors is reviewed with special focus on macrocyclic polyamines and their metal complexes as a new prototype. In comparison to most of the previous artificial receptors (most of them are organic molecules), our system characteristically works in aqueous solution at neutral pH with extremely strong affinities with phosphate anions. Moreover, zinc(II)-macrocyclic tetraamine (cyclen) complexes were discovered to selectively bind thymine and uracil, so that nucleotides of these bases are specifically recognized by the bis(Zn2+-cyclen) complexes.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Methods for the determination of intracellular levels of ribose phosphates

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.     

Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.     

PNA microarrays for hybridisation of unlabelled DNA samples

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.     

昆虫几丁质合成酶及其抑制剂

几丁质合成酶(CS)是几丁质合成的关键酶,它具有3个结构域:结构域A、结构域B和结构域C,其中结构域B是催化域。根据氨基酸序列的差异,几丁质合成酶分为两类:CS-A及CS-B,分别在表皮及围食膜基质中催化合成几丁质。关于几丁质合成有2种假想模型。有多种抑制剂可以抑制几丁质的合成,其中核苷肽抗生素类及核苷磷酸类作用于CS的催化部位,是竞争性抑制剂,其它抑制剂的作用机理仍不明确。     

Analysis of human ITPase nucleobase specificity by site-directed mutagenesis

Inosine triphosphate (ITP) pyrophosphohydrolase, or ITPase, is an intracellular enzyme that is responsible for the hydrolysis of the acidic anhydride bond between the alpha and beta phosphates in ITP, and other noncanonical nucleoside triphosphates, producing the corresponding nucleoside monophosphate and pyrophosphate. This activity protects the cell by preventing noncanonical nucleoside triphosphates from accumulating in (deoxy) nucleoside triphosphate ((d)NTP) pools and/or being integrated into nucleic acids. This enzyme is encoded by the ITPA gene in mammals. It has been reported that Itpa homozygous-null knock-out mice die before weaning and have gross cardiac abnormalities. Additionally, certain variations in the human ITPA gene have been linked to adverse reactions to the immunosuppressive prodrugs azathioprine and 6-mercaptopurine and protection against ribavirin-induced hemolytic anemia. These drugs are bioactivated to form noncanonical nucleoside triphosphates. Human ITPase enzymes engineered to modulate nucleobase specificity may be valuable tools for studying the role of ITPase in heart development and drug metabolism or developing gain-of-function mutants or inhibitory molecules. Based on x-ray crystallography and amino acid sequence data, a panel of putative human ITPase nucleobase specificity mutants has been generated. We targeted eight highly conserved amino acid positions within the ITPase sequence that correspond to amino acids predicted to directly interact with the nucleobase or help organize the nucleobase binding pocket. The ability of the mutants to protect against exogenous and endogenous noncanonical purines was tested with two Escherichia coli complementation assays. Nucleobase specificity of the mutants was investigated with an in vitro biochemical assay using ITP, GTP and ATP as substrates. This methodology allowed us to identify gain-of-function mutants and categorize the eight amino acid positions according to their ability to protect against noncanonical purines as follows: Glu-22, Trp-151 and Arg-178, essential for protection; Phe-149, Asp-152, Lys-172 and Ser-176, intermediate protection; His-177, dispensable for protection against noncanonical purines.     

Abiogenic nucleoside synthesis in the presence of phosphorus salts

By means of UV-spectroscopy, gel-filtration, ion-exchange and thin layer chromatography it has been shown that the action of ionizing radiation on the mixture of dry preparations of adenine and deoxyribose in the presence of the K, Na and Ca phosphates results in the formation of nucleoside-like substances. The phosphates catalyze or inhibit the nucleoside synthesis but they are not phosphorylating agents. The data obtained indicate the reality of abiogenic synthesis of nucleoside-like substances from the mixture of dry preparations of adenine and deoxyribose in the lithosphere during chemical evolution.     

Molecular dynamics simulations of dinucleoside and dinucleoside-drug crystal hydrates.

Molecular dynamics simulations have been performed on the dinucleoside monophosphates rGpC and dCpG, the latter in its intercalation complex with the acridine drug proflavine. The simulations were performed on the crystal structures, with crystallographically-located solvent molecules. It was found that satisfactory results were best obtained with restraints placed on the movements of the water molecules. Motions of individual atoms have been examined in terms of rms fluctuations and anisotropy and correlation functions. Relative motions of groups (phosphates, sugars, bases and proflavine molecules) have been analysed.     

Protoplast formation and leakage of intramembrane cell components: induction by the competence activator substance of pneumococci.

Treatment of pneumococci with activator (a protein that induces bacterial "competence" to absorb deoxyribonucleic acid molecules and undergo genetic transformation) can cause either protoplast formation or leakage of intracellular components to the medium depending on postincubation conditions. The leaked intracellular components include nucleoside phosphates, beta-galactosidase, deoxyribonuclease, autolysin, and hemolysin. Leakage and protoplast formation are induced by the electrophoretically pure activator, and these phenomena require the same conditions as induction of competence for genetic transformation, namely, genetic capacity for competence, protein synthesis, incorporation of choline, and the optimal pH for activation. It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space. The activity of these autolysin molecules from within would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.     

Recharacterization of fungal dinucleoside polyphosphate (HS3)

Three polyphosphorylated dinucleosides given the pseudonyms of HS3, HS2, and HS1 that were erroneously described as diguanosine polyphosphates (LéJohn, H. B., Cameron, L. E., McNaughton, D. R. & Klassen, G. R. (1975) Biochem, Biophys, Res, Commun. 66, 460-467) have been repurified and partially recharacterized. They have proved to be extremely complex molecules; chemical (HCl and KOH hydrolysis), physical (ultraviolet-light spectral analysis and ion-exchange chromatography), and enzymic (nucleotide pyrophosphatase and bacterial alkaline phosphatase hydrolysis) studies showed that (i) all three HS compounds are uracil rich and (ii) only HS3 contains a purine nucleoside and glutamate. The partial structure of HS3 was deciphered as a moiety of ADP--sugar X--glutamate (the mode of attachment of glutamate is obscure) that is covalently linked to another moiety composed of UDP, mannitol, and four phosphates. Sugar X had chromatographic characteristics of ribitol, but the chromatographic isolate also contained a ninhydrin-sensitive entity presumed to be an amino group. Sugar X, THEREFore, may be an amino sugar polyol. Only the general chemical compositions of HS2 and HS1 were determined. Each contained two uridines and HS2 had 10 phosphates whereas HS1 had 12.     

Regulation of nuclear histone acetyltransferase by nucleic acids,histone · DNA complex,and chromatin

Nuclear histone acetyltransferase is found to be inhibited by various nucleic acids and components. Of the adenosine phosphates, the order of inhibitory potency is ATP>ADP>AMP. Among the nucleoside triphosphates, GTP seems to be the best inhibitor, followed by ATP, CTP, and UTP. Deoxymononucleotides have the same order of inhibition potential as their ribonucleotide counterparts, with inhibition constants in the low millimolar range. Oligonucleotides and polynucleotides are much better inhibitors than mononucleotides. The inhibition constants of the DNA molecules are size dependent. Molecules larger than 40 base pairs have inhibition constants less than 18 µg/ml, whereas molecules with decreasing numbers of base pairs have increasing magnitudes of inhibition constants. However, acetyltransferase has a lower affinity for free DNA molecules than for DNA · histone complexes as revealed by its interaction with DNA-Sepharose and histone · DNA-Sepharose columns. Furthermore, native chromatin depleted of endogenous histone acetyltransferase activity shows no inhibitory effect on the enzyme. Yet heated chromatin not only loses substrate activity but also becomes an inhibitor for the enzyme. Since unmodified sea urchin sperm chromatin has been shown to be a potent acetyltransferase inhibitor, it seems possible that DNA · histone complexes may be the true inhibitory species and that the conformational states of such complexes may serve as a regulatory mechanism in the control of the enzyme activity.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Sequence specific fragments in the field desorption mass spectra of dinucleoside phosphates

In field desorption mass spectrometry of ribodinucleoside phosphates the formation of nucleoside cyclophosphates can be used to determine the base sequence.     

Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase

The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides.