Polydeoxyribonucleotide ameliorates lipopolysaccharide-induced acute lung injury via modulation of the MAPK/NF-κB signaling pathway in rats.

Acute lung injury (ALI) is characterized by disruption of the alveolar-capillary membrane resulting in pulmonary edema and accumulation of associated proteinaceous alveolar exudate. Initiation of ALI upregulates tumor necrosis factor-α (TNF-α), which activates nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) that induce various pro-inflammatory mediators. Polydexyribonucleotide (PDRN) is an adenosine A2A receptor agonist that exerts anti-inflammatory effects by suppressing the production of pro-inflammatory cytokines and apoptosis. We investigated the therapeutic efficiency of PDRN on ALI induced by lipopolysaccharide (LPS) in rats. ALI was induced by intratracheal instillation of LPS (5 mg/kg) in 200 µL saline. The PDRN treatment group received a single intraperitoneal injection of 500 µL saline including PDRN (8 mg/kg) 1 h after ALI induction. To confirm the involvement of the adenosine A2A receptor in PDRN, 8 mg/kg 7-dimethyl-1-propargylxanthine (DMPX) was applied with PDRN treatment. Rats were then sacrificed 12 h after PDRN and DMPX treatments. Intratracheal administration of LPS caused lung tissue damage and significantly increased the lung injury scores and levels of pro-inflammatory cytokines, and apoptotic factors. In addition, MAPK/NF-κB signaling factors were increased by ALI initiation. PDRN treatment potently suppressed expressions of MAPK/NF-κB signaling factors compared to the PDRN + DMPX co-treated group. These alterations led to a reduction of pro-inflammatory cytokines, apoptotic factors, and NF-κB and MAPK signaling, which promoted the recovery of damaged lung tissue. PDRN therapy demonstrated therapeutic effects for LPS-induced ALI compared to the non-treated and DMPX-treated groups. Therefore, PDRN may be used as a therapy for initial treatment of ALI.

Sequence-selective binding of phenazinium dyes phenosafranin and safranin O to guanine-cytosine deoxyribopolynucleotides: spectroscopic and thermodynamic studies.

The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O.

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA-dT)](2), [poly(dG-dC)] (2), and calf thymus DNA.

The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for [CuL'](+), which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH(2) group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.

Modifications in small interfering RNA that separate immunostimulation from RNA interference.

Synthetic small interfering RNA (siRNA) can suppress the expression of endogenous mRNA through RNA interference. It has been reported that siRNA can induce type I IFN production from plasmacytoid dendritic cells, leading to off-target effects. To separate immunostimulation from the desired gene-specific inhibitory activity, we designed RNA strands with chemical modifications at strategic positions of the ribose or nucleobase residues. Substitution of uridine residues by 2'-deoxyuridine or thymidine residues was found to decrease type I IFN production upon in vitro stimulation of human PBMC. Thymidine residues in both strands of a siRNA duplex further decreased immunostimulation. Fortunately, the thymidine residues did not affect gene-silencing activity. In contrast, 2'-O-methyl groups at adenosine and uridine residues reduced both IFN-alpha secretion and gene-silencing activity. Oligoribonucleotides with 2'-O-methyladenosine residues actively inhibited IFN-alpha secretion induced by other immunostimulatory RNAs, an effect not observed for strands with 2'-deoxynucleosides. Furthermore, neither 5-methylcytidine nor 7-deazaguanosine residues in the stimulatory strands affected IFN-alpha secretion, suggesting that recognition does not involve sites in the major groove of duplex regions. The activity data, together with structure prediction and exploratory UV-melting analyses, suggest that immunostimulatory sequences adopt folded structures. The results show that immunostimulation can be suppressed by suitable chemical modifications without losing siRNA potency by introducing seemingly minor structural changes.

Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts).

BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences.

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.

Unusual features of poly[dT-dG].[dC-dA] stretches in CDS-flanking regions of Trypanosoma cruzi genome.

In trypanosomatids, the mechanisms of gene expression regulation are not yet well understood. The genes are organized into long polycistronic transcription units separated by intergenic regions that may contain the signaling information for nucleic acid processing. Poly-dinucleotides are frequent in these regions and have been proposed to be involved in gene expression regulation. We analyzed their frequency in CDS-flanking sequences of sense strands in Trypanosoma cruzi and established that all but poly[dC-dC], poly[dC-dG], and poly[dG-dG] are significantly more frequent than expected by chance. Poly[dT-dG].[dC-dA] is among the longest and most frequent poly-dinucleotides and shows a remarkable strand asymmetry. Furthermore, electrophoretic mobility shift assays using T. cruzi epimastigotes nuclear extracts demonstrated the existence of at least, one sequence specific single-strand binding activity for each strand. These results strongly suggest that poly[dT-dG].[dC-dA] sequence is involved in regulatory mechanisms of relevance for the parasite biology.

Cytotoxicity, apoptosis, and in vitro DNA damage induced by potassium chromate.

Cr(6+) is a known human cytotoxic and carcinogenic agent that requires intracellular reduction for activation. We have analyzed the cytotoxic and DNA binding properties of K(2)CrO(4) (Cr(6+)) in comparison with those of Cl(3)Cr (Cr(3+)). The results indicate that K(2)CrO(4) exhibits higher cytotoxicity than Cl(3)Cr in several human and murine cell lines. The cytotoxic activity of K(2)CrO(4) is also indicated by the fact that is able to produce cell killing through apoptosis in cisplatin-resistant cells transformed by H-ras oncogene. Moreover, in vitro DNA binding experiments show that, in the presence of ascorbate (the major intracellular reductant of Cr(6+)), K(2)CrO(4) induces both interstrand cross-links and strand breaks. Because the chromate anion is by itself unreactive toward DNA, these data suggest that the cytotoxicity of K(2)CrO(4) may be associated with the DNA binding of reactive intermediate chromium species resulting from reduction of Cr(6+).

Efficient synthesis of DNA dumbbells using template-induced chemical ligation in double-stranded polynucleotides closed by minihairpin fragments.

The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.

Application of molecular typing methods to dermatophyte species that cause skin and nail infections.

Typing methods utilising DNA technology were applied to a collection of Trichophyton mentagrophytes and T. rubrum isolates from skin and nail infections. The methods included restriction enzyme analysis (REA), hybridisation with the DNA probe poly (dG-dT), randomly amplified polymorphic DNA (RAPD) by PCR and restriction analysis of a segment of PCR-amplified rDNA. All these tests successfully differentiated the species, but few intra-species differences were detected. REA demonstrated some isolate variation, but this was limited and difficult to interpret, making it unsuitable as a typing tool. RAPD demonstrated few variations amongst T. mentagrophytes and none in T. rubrum.

Distinct frequency-distributions of homopolymeric DNA tracts in different genomes.

The unusual base composition of the genome of the human malaria parasite Plasmodium falciparum prompted us to systematically investigate the occurrence of homopolymeric DNA tracts in the P. falciparum genome and, for comparison, in the genomes of Homo sapiens , Saccharomyces cerevisiae , Caenorhabditis elegans , Arabidopsis thaliana , Escherichia coli and Mycobacterium tuberculosis. Comparison of theobserved frequencies with the frequencies as expected for random DNA revealed that homopolymeric (dA:dT) tracts occur well above chance in the eukaryotic genome. In the majority of these genomes, (dA:dT) tract overrepresentation proved to be an exponential function of the tract length. (dG:dC) tract overrepresentation was absent or less pronounced in both prokaryotic and eukaryotic genomes. On the basis of our results, we propose that homopolymeric (dA:dT) tracts are expanded via replication slippage. This slippage-mediated expansion does not operate on tracts with lengths below a critical threshold of 7-10 bp.

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae.

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

A chloroplast transcript lacking the 3' inverted repeat is degraded by 3'-->5' exoribonuclease activity.

Chlamydomonas reinhardtii strains harboring deletions of the chloroplast atpB 3' inverted repeat (IR) are weakly phototrophic due to reduced accumulation of discrete atpB transcripts and the chloroplast ATPase beta-subunit protein. A sequence of 18 guanosine residues, which can impede a 3'-->5' exoribonuclease in vitro, is able to substitute for the atpB IR in vivo. Strains containing the poly-guanosine tract in place of the atpB 3' IR are phototrophic and accumulate near wild-type levels of discrete atpB transcripts and the ATPase beta-subunit protein. Because these atpB transcripts contain the 18 guanosine residues, and the poly-guanosine tract is not a terminator of transcription, the accumulation of discrete atpB transcripts is likely the result of impediment of 3'-->5' exoribonuclease activity. These findings support a model in which atpB transcripts lacking the 3' IR are degraded by 3'-->5' exoribonuclease activity, and demonstrate that the poly-guanosine tract can be used to study chloroplast RNA metabolism in vivo.

Sequence of the polypyrimidine tract of the 3'-terminal 3' splicing signal can affect intron-dependent pre-mRNA processing in vivo.

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. However, not all introns can provide this function. For example, transcripts synthesized from a variant of the human beta-globin gene lacking its second intervening sequence (IVS2), yet retaining its first intervening sequence (IVS1), exhibit multiple defects in mRNA biogenesis. To investigate why, we transfected into monkey cells plasmids containing the human beta-globin gene and variants of it altered in (i) IVS1, (ii) the 3'-terminal exon, and (iii) the polyadenylation signal. The beta-globin RNAs accumulated in these cells were analyzed by quantitative S1 nuclease mapping for nuclear accumulation, intron excision, polyadenylation and cytoplasmic accumulation. We found that the 3' splicing signal of IVS1, with multiple purines interrupting its polypyrimidine tract, could efficiently function as an internal 3' splicing signal; however, it could not efficiently function as the 3'-terminal 3' splicing signal for any of these steps in intron-dependent mRNA biogenesis unless (i) its polypyrimidine tract was made uninterrupted in pyrimidines, or (ii) specific sequences were deleted from the 3'-terminal exon. We conclude that whether an intron can provide the function necessary for efficient processing of intron-dependent pre-mRNA is dependent upon the ability of its 3' splicing signal to define the 3'-terminal exon. On the practical side, this finding means one needs to consider both the sequence and location of the intron to be included in an intron-dependent gene to obtain efficient expression in vivo.

Renal uptake of an 18-mer phosphorothioate oligonucleotide.

Renal uptake of a 35S labeled 18-mer phosphorothioate oligodeoxynucleotide (molecular wt approximately 6,000) was evaluated following intravenous infusion into rats. The kidneys contained 21 +/- 3% of the infused dose at five hours after infusion and 3 +/- 1% of the infused dose at four days after infusion. The concentration of oligonucleotide was greater in the kidney than in the liver, spleen, or plasma at both intervals. Urine excretion of oligonucleotide label averaged 17 +/- 1%, 35 +/- 5%, and 64 +/- 3% of the infused dose at five hours, one day, and four days after infusion. Electrophoresis (PAGE) showed that oligonucleotide was retained in the kidney was the intact 18-mer at both five hours and four days after infusion, while full size oligonucleotide was not found in the urine at either interval. Light microscopic autoradiography showed that oligonucleotide uptake was most prominent in the early proximal tubule. Electron microscopic autoradiography indicated that oligonucleotide was not confined to the brush border or endocytic-lysosomal pathway. Micropuncture studies showed that the tubule fluid to plasma concentration ratios of oligonucleotide label averaged 7 +/- 3% in Bowman's space and 6 +/- 2% in the distal tubule. Despite restriction of filtration by plasma protein binding, as indicated by the low Bowman's space to plasma concentration ratio, the calculated tubular reabsorption rate for oligonucleotide was sufficient to account for the large amount of oligonucleotide found in the kidney after intravenous infusion. These results indicate that the proximal tubule plays a prominent role in the disposition of intravenously infused oligonucleotide, and raise the possibility that oligonucleotides could exert antisense effects in this nephron segment.

Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure.

Many yeast promoters contain homopolymeric dA:dT sequences that affect nucleosome formation in vitro and are required for wild-type levels of transcription in vivo. Here, we show that poly(dA:dT) is a novel promoter element whose function depends on its intrinsic structure, not its interaction with sequence-specific, DNA-binding proteins. First, poly(dA:dT) stimulates Gcn4-activated transcription in a manner that is length dependent and inversely related to intracellular Gcn4 levels. Second, Datin, the only known poly(dA:dT)-binding protein, behaves as a repressor through poly(dA:dT) sequences. Third, poly(dG:dC), a structurally dissimilar homopolymer that also affects nucleosomes, has transcriptional properties virtually identical to those of poly(dA:dT). Three probes of chromatin structure including HinfI endonuclease cleavage in vivo indicate that poly(dA:dT) increases accessibility of the Gcn4 binding site and adjacent sequences in physiological chromatin. These observations suggest that, by virtue of its intrinsic structure, poly(dA:dT) locally affects nucleosomes and increases the accessibility of transcription factors bound to nearby sequences.

Isolation and functional characterization of DNA-derived aptamers that act as thrombin inhibitors in human platelets and coagulation assays.

Aptamer sequences were isolated from defibrotide, a single-stranded, commercial DNA preparation and studied for thrombin inhibitory activity. Three different aptamers were identified, sequenced and their biological activity was determined in platelet aggregation and coagulation assays. All aptamers were potent inhibitors of thrombin-induced platelet aggregation and thromboxane formation and prolonged the thrombin time in human plasma. There was no effect of any of these compounds when a thromboxane mimetic (U 46.1619), collagen or thrombin activating peptide (TRAP-6) were used as agonists, excluding a nonspecific binding of the compounds to the thrombin receptor. The data suggest that thrombin-inhibitory aptamers are present in the mammalian genome and may constitute an endogenous antithrombin system.