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At least 163 records · Page 9Linked to original sources
PubMed1990

DNA conformational analysis in solution by uranyl mediated photocleavage.

Uranyl mediated photocleavage of double stranded DNA is proposed as a general probing for DNA helix conformation in terms of minor groove width/electronegative potential. Specifically, it is found that A/T-tracts known to constitute strong distamycin binding sites are preferentially photocleaved by uranyl in a way indicating strongest uranyl binding at the center of the minor groove of the AT-region. The A-tracts of kinetoplast DNA show the highest reactivity at the 3'-end of the tract--as opposed to cleavage by EDTA/Fell--in accordance with the minor groove being more narrow at this end. Finally, uranyl photocleavage of the internal control region (ICR) of the 5S-RNA gene yields a cleavage modulation pattern fully compatible with that obtained by DNase I which also--in a more complex way--senses DNA minor groove width.

Animals↗
PubMed2006

Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2.

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.

Animals↗
PubMed1989

Antisense peptide recognition of sense peptides: sequence simplification and evaluation of forces underlying the interaction.

Structural principles were studied which underlie the recognition of sense peptides (sense DNA encoded) by synthetic peptides encoded in the corresponding antisense strand of DNA. The direct-readout antisense peptides corresponding to ribonuclease S-peptide bind to an affinity matrix containing immobilized S-peptide with significant selectivity and with dissociation constants in the range of 10(-6) M as judged by analytical affinity chromatography. Synthetic, sequence-modified forms of antisense peptides also exhibit substantial binding affinity, including a "scrambled" peptide in which the order of residue positions is changed while the overall residue composition is retained. The antisense mutants, as the original antisense peptides, bind at saturation with greater than 1:1 stoichiometry to immobilized S-peptide. The data suggest significant sequence degeneracy in the interaction of antisense with sense peptide. In contrast, selectivity was confirmed by the inability of several control peptides to bind to immobilized S-peptide. The idea was tested that the hydropathic pattern of the amino acid sequence serves to induce antisense peptide recognition. A hydropathically sequence-simplified mutant of antisense peptide was made in which all strongly hydrophilic (charged) residues were replaced by Lys, all strongly hydrophobic residues by Leu, and all weakly hydrophilic and hydrophobic residues by Ala, except Gly which was unchanged. This "KLAG" mutant also binds to immobilized S-peptide, with an affinity only an order of magnitude less than that with the original antisense peptide and with multiple stoichiometry. Mutants of the KLAG model, in which the hydropathic pattern was changed substantially, exhibited a lower binding affinity for S-peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3.

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Delta diploids confirmed allelism. The pso9-1 mutant allele contains a -1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.

Alleles↗
PubMed2003

Nanocrystals modified with peptide nucleic acids (PNAs) for selective self-assembly and DNA detection.

Gold nanocrystals modified with peptide nucleic acids (PNAs) have been prepared and applied to self-assembly and DNA sensing. Experiments with different PNA structural motifs show that (1). the versatility in PNA synthetic design can be used to modulate the electrostatic surface properties of nanocrystals, presenting an opportunity to control assembly rate and aggregate size, (2). short (6 base) PNAs can hybridize effectively while attached to nanoparticles, providing a route to generating materials with small interparticle spacings, and (3). the superior base pair mismatch selectivity of PNAs is further enhanced on nanosurfaces, enabling PNA-modified nanoparticles to act as highly selective nanoscale sensors, as well as synthons for defect-free self-assembly. This last feature was coupled with a substantial change in colloidal stability upon DNA hybridization to develop a novel colorimetric DNA assay that detects the presence of single base imperfections within minutes. Various modes of PNA hybridization, including the first practical application of PNA-PNA interactions, were used to direct the assembly of nanoparticles into macroscopic arrangements. Shorter duplex interconnects and greater specificity in assembly were obtained compared to similar experiments with DNA-modified nanocrystals.

Base Pair Mismatch↗
PubMed2004

A corticotropin-releasing hormone antisense oligodeoxynucleotide reduces spontaneous waking in the rat.

We have previously hypothesized that corticotropin-releasing hormone (CRH) is involved in the regulation of physiological waking. In this study, we tested the hypothesis that reduction of CRH peptide would reduce spontaneous wakefulness of rats. We administered intracerebroventricularly into rats at several circadian time points antisense or sense DNA oligodeoxynucleotides (ODNs) corresponding to the initiation codon of CRH mRNA and determined subsequent effects on wakefulness and sleep of the rat. Our results indicate that CRH antisense oligodeoxynucleotides reduce spontaneous wakefulness during the dark (active) period, but not during the light (rest) period of the light/dark cycle. The alterations in time spent awake are due to reduced wake bout numbers, rather than a change in wake bout duration. These reductions in wakefulness were mirrored by increases in slow-wave sleep, while rapid eye movement sleep was not affected. Corticosterone, used as an index of CRH in the hypothalamus, was reduced by CRH antisense oligodeoxynucleotides during the same time that spontaneous wakefulness was reduced, suggesting CRH peptide modulation as the mediator of this response. In contrast, CRH sense oligodeoxynucleotides did not alter any parameter of this study during either the dark or light period. These findings provide additional support for the hypothesis that CRH is involved in the regulation/modulation of wakefulness.

Animals↗
PubMed2004

Electrochemical impedance behavior of DNA biosensor based on colloidal Ag and bilayer two-dimensional sol-gel as matrices.

A novel method for fabrication of DNA biosensors has been developed by means of self-assembling colloidal Ag (Ag) to a thiol-containing sol-gel network. The thiol groups of 3-mercaptopropyltrimethoxysilane (MPTS) serve as binding sites for the covalent attachment to gold electrode surface. Then the one-dimensional network of silane unites (1dMPTS) was combined together into a two-dimensional sol-gel network (2dMPTS) by dipping into aqueous NaOH. The second silane layer (B2dMPTS) was formed by immersing electrodes back into the MPTS solution overnight, and then the Ag nanoparticles were chemisorbed onto the thiol groups of the second silane layer. Finally, the mercapto oligonucleotide was self-assembled onto the surface via the Ag nanoparticles. The modified process was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In addition, we utilized the impedance spectroscopy as a platform for DNA sensing assay. The factors influencing the performance of the resulting biosensor were studied in detail. The linear range of the biosensor was from 8.0 x 10(-9) to 1.0 x 10(-6) M with a detection limit of 4.0 x 10(-9) M at 3sigma. In addition, the experiment results indicate that oligonucleotide immobilized on this way exhibits a good sensitivity and selectivity, high stability and a long-term maintenance of bioactivity.

Binding Sites↗
PubMed2003

Evolutionary clues to DNA polymerase III beta clamp structural mechanisms.

The prokaryotic DNA polymerase III beta homodimeric clamp links the replication complex to DNA during polynucleotide synthesis. This clamp is loaded onto DNA and unloaded by the clamp loader complex, the delta subunit of which by itself can bind to and open the clamp. beta Clamps from diverse bacteria were examined using contrast hierarchical alignment and interaction network (CHAIN) analysis, a statistical approach that categorizes and measures the evolutionary constraints imposed on protein sequences by natural selection. Some constraints are subtle inasmuch as they are unique to certain bacteria. Examination of corresponding molecular interactions within structures of the Escherichia coli beta dimeric and delta-beta complexes reveals that N320, Y323 and R176, which are subject to very strong constraints, form a substructure that may serve as a platform for leveraging and directing delta-induced conformational changes. N320 may play a prominent role, as it is strategically situated between this substructure and regions linked to delta binding and opening of beta's dimeric interface. R176 appears to act as a relay between the delta binding site and the clamp's central hole. Other residues subject to strong constraints are likewise associated with structurally important features. For example, two pairs of interacting residues, R269/E304 and K74/E300, form salt bridges at the dimeric interface, while the C-terminal residues M362, P363, M364 and R365 appear to play key roles in delta binding. Q149 and K198 appear to sense DNA within the clamp's central hole while other residues may relay this information to the delta binding site. Mutagenesis experiments designed to explore possible mechanisms are proposed.

Amino Acid Sequence↗
PubMed2026

cGAS-STING signaling in aging and age-related diseases: therapeutic promise and precaution.

Endogenous cytoplasmic DNA (cytoDNA) is increasingly recognized as a mediator of tissue dysfunction and disease progression during aging. As a major cytosolic DNA-sensing pathway, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway can translate aging-associated cytoDNA accumulation into innate immune and inflammatory programs. This review summarizes the evolutionary and signaling features of the cGAS-STING cascade and critically discusses its crosstalk with aging-associated intracellular molecular threats, including nuclear genomic and chromatin stress, mitochondrial dysfunction, oxidative-metabolic stress, and defective clearance of nucleic acids or damaged organelles. We further synthesize evidence linking dysregulated cGAS-STING activation to inflammatory remodeling, senescence-associated changes, cell injury, fibrosis, and tissue dysfunction, while highlighting the context-dependent roles of this pathway across physiological aging and ARDs. Finally, we discuss the therapeutic potential and limitations of cGAS-STING modulation, emphasizing that successful translation will require context-defined therapeutic windows, tissue- and cell-specific targeting, subcellular compartmentalization, and long-term safety assessment.

Humans↗
PubMed1997

Genomic deletion and p53 inactivation in cervical carcinoma.

The tumor-suppressor gene p53 acts as "the guardian of the genome", sensing DNA damage and initiating protective responses. To examine the hypothesis that p53 abnormality leads to increased genomic alterations in primary tumor cells, our study utilized 51 primary tumors of cervical carcinoma and 10 microsatellite markers. These markers were mapped to the short arms of chromosomes 3 and 5, covering the regions 3p13-25 and 5p15.1-15.3. Genomic deletion on 3p and 5p was correlated with genetic or epigenetic p53 inactivation pathways, including p53 mutation, genetic deletion of p53 and cervical infection with human papillomavirus. The proportion of abnormal p53 was found to be significantly higher in the cases exhibiting loss of heterozygosity (LOH) on 5p (p < 0.001), supporting the hypothesis of the presence of a p53-dependent pathway to cervical tumorigenesis. In contrast, however, LOH on 3p was found to be independent of p53 inactivation. A common deletion region, 3p22-24, was identified in 44% of informative cases, and genomic loss at this specific region was correlated with early tumorigenic onset and poor grade of tumor differentiation. Diversity within the patterns of genomic alteration in the same form of cancer suggests different sets of risk/tumorigenic profiles, molecular pathogenesis, as well as prognosis and outcome.

Adult↗
PubMed2000

Effects of gonadotropin-releasing hormone analog on expression of genes encoding the growth hormone/prolactin/somatolactin family and a pituitary-specific transcription factor in the pituitaries of prespawning sockeye salmon.

Gonadotropin-releasing hormone (GnRH) is a possible secretagogue of growth hormone (GH) and somatolactin (SL) in teleosts. Effects of GnRH on the levels of pituitary mRNAs encoding GH, prolactin (PRL), and SL were therefore examined in prespawning sockeye salmon (Oncorhynchus nerka). A capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon for 3 weeks. The levels of hormonal mRNAs were then determined by a quantitative dot blot analysis using single-stranded sense DNA of the same sequence of mRNA as the standard. Further, we analyzed effects of GnRHa on expression of the genes encoding pituitary-specific transcription factor (Pit-1/GHF-1). Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis, which showed specific 2- and 3-kb bands of mRNAs. GnRHa significantly increased the level of SL mRNA in the males, but not in the females, compared to the control fish. It did not induce significant increases in the levels of GH and PRL mRNAs in both the males and the females. The levels of Pit-1/GHF-1 mRNAs in the control males tended to be higher than those in the initial controls, so that GnRHa might not be effective in enhancing expression of Pit-1/GHF-1 gene, except for the level of 3-kb Pit-1/GHF-1 mRNA in the females treated with 150 microg GnRHa. The pattern of changes in the levels of Pit-1/GHF-1 mRNAs were similar to those of GH and PRL mRNAs in both the males and the females and to that of SL mRNA in the females. These results indicate that, in prespawning sockeye salmon, GnRH can stimulate SL gene expression, but probably not through the Pit-1/GHF-1-dependent system.

Animals↗
PubMed1993

Production of alpha-1-antichymotrypsin by PSA-containing cells of human prostate epithelium.

Prostate-specific antigen (PSA) is a chymotrypsin-like serine protease exclusively produced by the prostate epithelium, and abundant in seminal fluid. In serum, PSA is predominantly complexed to a liver-derived serine protease inhibitor, alpha-1-antichymotrypsin (ACT). A higher proportion of serum PSA is complexed to ACT in prostate cancer than in benign prostate hyperplasia. Since the molecular basis for this is unclear, we have investigated whether or not ACT may be produced in the prostate gland. Immunocytochemistry, using either monoclonal or polyclonal IgGs, demonstrated specific immunostaining for ACT in normal PSA-containing prostate epithelium. Production of ACT in the normal PSA-producing prostate epithelium was demonstrated by means of nonradioactive in situ hybridization using 30-mer anti-sense DNA probes for ACT and for PSA. The ACT and PSA coding transcripts, as detected by in situ hybridization, were distributed perinuclearly, in contrast to the specific immunostaining for ACT and PSA which was most intense in the apical portion of the secretory cells. The results strongly suggest local production and release of ACT by the normal prostate epithelium that may be important for interaction between PSA and ACT in extracellular compartments.

Culture Techniques↗
PubMed1988

Sequence requirements for premature termination of transcription in the human c-myc gene.

We have used the Xenopus oocyte injection system to investigate the sequence requirements of premature termination of transcription within the human c-myc gene. We show that in the oocyte, truncated RNAs are produced by RNA polymerase II with 5' ends at the P1 and P2 promoters and 3' ends at two T stretches (sites I and II) near the exon 1/intron 1 junction. The location of these 3' ends is consistent with the site of the block to c-myc transcription identified by nuclear runoff assays in human cells and confirmed in dissected nuclei of injected oocytes. Evidence is presented that transcriptional termination rather than RNA processing produces these short c-myc RNAs. Deletion analysis of site I reveals that sequences upstream of the T stretch determine the site of 3' end formation, and that the stretch of T's on the sense DNA strand is not required for termination. The sequences specifying termination reside within a 95 base region located -130 to -35 relative to the exon 1/intron 1 boundary. The termination activity of these sequences is orientation-dependent and functions downstream of the HSV-TK promoter.

Animals↗
PubMed2005

Chemical stress sensitive luminescent human cells: molecular biology approach using inducible Drosophila melanogaster hsp22 promoter.

A whole-cell bioassay has been developed for the total toxicity testing of liquid samples. The method is based on the induction of the bioluminescent activity of genetically manipulated mammalian cells. For that purpose, transfection was used to introduce, in HeLa cells, a DNA sensing element that responds to chemical stress agents (heavy metals, genotoxic agents, and endocrine-disrupting chemicals). Such element was designed to direct the expression of a reporting gene (firefly luciferase) through the activation of Drosophila melanogaster hsp22 promoter. A molecular approach was conducted to optimize hsp22 promoter element in order to decrease the background expression level of the reporting gene and to increase the sensitivity of the bioassay for testing endocrine disruptors. As a result, in the presence of 20-100 microM cadmium chloride, a 6-fold increase in luciferase expression was obtained using a specially designed truncated hsp22 promoter construction. The following chemicals known to be found in the polluted samples were tested: CdCl2, Cd(NO3)2, NaAsO2, alachlore, fentine acetate, thiram, and maneb. The stressing effect of each of them was sensitively detected by the present bioassay in the 0.05-50 microM concentration range.

Animals↗
PubMed2026

Proteomics-based analysis of the defense mechanisms of disease-resistant grass carp against Aeromonas veronii.

Sustainable aquaculture of grass carp (Ctenopharyngodon idella, GC) is consistently threatened by bacterial diseases, particularly those caused by Aeromonas veronii. A disease-resistant grass carp (DR-GC) has been developed by backcrossing female gynogenetic GC with normal male GC, exhibiting improved resistance. However, the systemic molecular mechanisms of DR-GC defending against Aeromonas veronii infection remain largely unexplored. Here, a label-free quantitative proteomics approach was employed to systematically compare proteomic profiles across five tissues (intestine, liver, muscle, skin, and kidney) in DR-GC and GC under healthy and infected conditions. The intestine was identified as the central defense tissue, exhibiting the highest number of differentially abundant proteins (DAPs). In DR-GC, A0A3N0YEK7 (small ribosomal subunit protein eS28), A0A3N0YGT8 (ATP synthase-coupling factor 6) and A0A3N0YNS7 (apolipoprotein A-I) were significantly upregulated in intestine, while D5KZW6 (GCHV-induced protein), A0A3N0Z0A1 and Q8JH84 (hemoglobin subunit alpha) were significantly dysregulated across multiple tissues, which playing the critical roles in defense mechanisms at the protein level. Furthermore, cytochrome P450-associated pathways, cytosolic DNA-sensing and RIG-I-like receptor signaling pathways were identified as crucial coordinators mediating immune and metabolic responses. This study provides the first comprehensive proteomic view of multi-tissue defense mechanisms in DR-GC, and identifies key DAPs and pathways for subsequent functional validation.

Animals↗
PubMed2005

Escape from immune- and nonimmune-mediated tumor surveillance.

Surveillance against neoplastic transformation is dependent on intrinsic "nonimmune" control mechanisms that sense DNA damage and illegitimate cell growth. In addition, evidence from gene-targeted mice with a variety of immune defects demonstrates that the immune system is involved in tumor elimination. Induction of apoptosis is a common feature of most nonimmune surveillance mechanisms but also the downstream consequence of cytotoxic lymphocyte activity. Thus, mechanisms of tumor cell escape from nonimmune- and immune-surveillance share several features characterized by increased thresholds of apoptosis induction. In this review, tumor cell resistance to the effector mechanisms of cytotoxic lymphocytes are discussed in the context of the complementary actions of nonimmune- and immune-surveillance.

Humans↗
PubMed2000

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I).

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

Annexin A1↗
PubMed2001

Additional organizational features of the murine gamma-glutamyl hydrolase gene. Two remotely situated exons within the complement C3 gene locus encode an alternate 5' end and proximal ORF under the control of a bidirectional promoter.

Analysis of independently isolated clones from a mouse liver cDNA library identified a splice variant of gamma-GH mRNA with novel nucleotide sequence at the 5' end. Genomic sequencing now shows that this variant (variant II) incorporates two new alternates (exons Bla and Blb) of exon 1 in the murine gamma-GH gene remotely situated with respect to the rest of the gene. Further analysis of this variant also showed that it incorporates a small segment at the 3' end of exon A1, revealing that the previously described exon 1 consists of two individual exons (Ala and Alb) joined at a cryptic splice site. The 5' UTR and a segment of the ORF of variant II results from splicing of exon Bla to exon Blb which in turn is spliced to exon Alb and through this splicing to the rest of the exons within this gene. Remarkably, this splicing occurs even though exons Bla and Blb are located >45 Kb upstream of exons Ala and Alb. Our results also show that transcription starting at exons Bla and Blb is under the control of a separate and bidirectional promoter (promoter B). Exons Bla and Blb are located on the sense DNA strand within the complement C3 gene locus which is encoded on the antisense strand. This promoter is less efficient than the downstream promoter (promoter A) in regulating transcription at least in the context of reporter gene and primer extension assays. However, in these same contexts, this region of DNA sequence in the reverse orientation is markedly more efficient in driving transcription of an unidentified gene. Deletion of specific regions of sequence within this promoter have different effects depending upon the orientation (forward or reverse) within the reporter gene construct.

Alternative Splicing↗