Influence of carbonate on the binding of carboplatin to DNA

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO(3) for 45 h, 37 degrees, pH 8.6) with pBR322 DNA at 0 < r < 2.8, where r = [drug]/[DNA-bp], in 24 mM HEPES buffer, pH 7.4, for 24 h, followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two-buffer system, 24 mM HEPES plus 24 mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2 intrastrand cross-link on DNA. Studies with aged carboplatin and with cisplatin carried out with ca. 4.0 < r < 10.0 in the two-buffer system show that some DNA binding and unwinding occurs for both drugs. Since carbonate inhibits the binding of aged carboplatin and cisplatin to DNA, carbonate present in the body likely modulates the reactivity of these drugs with a variety of biological targets including DNA.     

Alginate modified nanostructured calcium carbonate with enhanced delivery efficiency for gene and drug delivery

To improve the performance of nanostructured calcium carbonate in gene delivery, a hydrophilic polysaccharide, alginate, was added to calcium carbonate co-precipitation systems to form alginate/CaCO(3)/DNA nanoparticles. The size and ζ-potential of the nanoparticles were measured by a zetasizer. Due to the existence of alginate chains which retarded the growth of calcium carbonate based co-precipitates, the alginate/CaCO(3)/DNA nanoparticles exhibited a decreased size and enhanced stability in the aqueous solution. To evaluate the gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles to form alginate/CaCO(3)/DNA/DOX nanoparticles. The in vitro gene transfections mediated by different nanoparticles in 293 T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. With an appropriate amount of alginate, the gene transfection efficiency of alginate modified nanoparticles could be significantly enhanced as compared with the nanoparticles without alginate modification for the gene delivery systems, as well as the gene and drug co-delivery systems. The study on in vitro cell inhibition effects showed that the cell viability decreased with increasing DOX amount loaded in alginate/CaCO(3)/DNA/DOX nanoparticles. The alginate modification is a useful strategy to improve the calcium carbonate co-precipitation technique for the preparation of gene and drug delivery systems, and the nanoparticles prepared in this study have promising applications in gene and drug delivery.     

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.     

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo‐) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro‐) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high‐throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome‐scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.     

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.     

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.     

An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts

We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants ≤100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.     

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium-rich groundwater and carbonate-rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa-inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO₂ outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

One-pot solvent free synthesis and DNA binding studies of thieno[2,3-b]-1,8-naphthyridines

With the aim of evaluating interaction between double-stranded calf thymus (ds)DNA and sulphur containing fused planar rings, the derivatives of 1,8-naphthyridine containing thiono groups were synthesized by the condensation of 2-mercapto-3-formyl[1,8]naphthyridines using 1-chloroacetone, 2-chloroacetamide, chloroaceticacid, and 2-chloro-1-phenylethanone in the presence of anhydrous potassium carbonate as s catalyst under solvent free microwave irradiation. The structures of the compounds were elucidated on the basis of elemental analysis, IR, (1)H NMR, and mass spectra. The interaction of thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (TNC) (3a) with ct-DNA was studied by UV-Vis spectrophotometry, viscosity, thermal denaturation, as well as cyclic voltammetry experiments. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. Binding parameters, determined from spectrophotometric measurements indicated a binding constant of Kb=2.1 x 10(6) M(-1). The thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (3a) increases the viscosity of sonicated rod-like DNA fragments. The binding of TNC to DNA increased the melting temperature by about 4 degrees C. The decrease in peak current heights and shifts of peak potential values are observed by the addition of calf thymus DNA in cyclic voltammetry studies.     

Pt(IV) complexes as prodrugs for cisplatin

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl₂(OH)₂(NH₃)₂], 3, and a carboxylate-modified analog, c,t,c-[PtCl₂(OH)(O₂CCH₂CH₂CO₂H)(NH₃)₂], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ¹⁹⁵Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ¹⁹⁵Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ¹³C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed.     

低Mg/Ca条件下丛毛单胞菌HJ-1菌株诱导文石的形成

【目的】为了探讨细菌对碳酸盐矿物种类和形态的影响。【方法】本文利用丛毛单胞菌HJ-1菌株进行了持续50 d的培养实验。在实验过程中,对细菌数量、沉淀物重量、培养液中Ca2+和Mg2+浓度等进行了动态监测。利用扫描电子显微镜对矿物形态进行了观察,并利用X-射线衍射仪对矿物成分进行测定。【结果】丛毛单胞菌HJ-1菌株具有显著的诱导碳酸盐矿物沉淀的能力,碳酸盐矿物的重量随着培养时间的延长而逐渐增加。X-射线衍射结果表明,形成的碳酸盐沉淀主要由文石和高镁方解石组成,其中文石的最高含量达86%。上述矿物在形态上复杂多样,主要有杆状、柱状、哑铃形、球状和板状以及不规则状和鳞片状集合体。【结论】通常,在Mg/Ca≤2并且有微生物参与的条件下极少形成文石。本文在Mg/Ca为2,不含碳酸根离子的培养基中培养HJ-1菌株的过程中发现了文石。作者认为,低Mg/Ca条件下文石的形成主要与HJ-1菌株分泌较多的胞外多糖有关。     

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.     

Volume changes accompanying thermal denaturation of deoxyribonucleic acid. II. Denaturation at alkaline pH

The change in apparent molal volume ? of DNA on thermal denaturation in carbonate buffer at pH 11.0 has been determined by the dilatometric method. It was found that ? increases sigmoidally during the helix–coil transition. Several methods, including a colorimetric technique that closely simulates the conditions used in the dilatometric experiments, were employed to estimate the protons lost by the DNA during the transition. These measurements indicated that the extent of the proton loss depends on the counterion present, increasing in the order Li⁺ < Na⁺ < K⁺ < Cs⁺. The major part of the volume changes observed during the denaturation is due to the volume changes expected to accompany the transfer of protons from the bases guanine and thym ne to carbonate ions. As has been previously reported for the denaturation of DNA at neutral pH, the volume change directly due to the change in shape of the polymer molecules is so small as to be experimentally undetectable.     

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.     

Opposite staining effect of two silver-staining techniques on sister chromatids

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.     

Phylogenetic Analysis of Bacterial Isolates from Man-Made High-pH, High-Salt Environments and Identification of Gene-Cassette–Associated Open Reading Frames

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette–specific primers. Different gene-cassette–linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.     

Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl₂ and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.     

Design and Synthesis of New Acid Cleavable Linkers for DNA Sequencing by Synthesis

A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-succinimidyl) carbonate (DSC) and modified 2′-deoxyuridine triphosphate (dUTP); the final product, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained and confirmed by ¹H-NMR, ³¹P-NMR, and HRMS with purity of up to 99%. The synthesized dye-labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved completely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.