Double-stranded Configuration Research Articles

Brain Metabolic DNA: A Long Story and Some Conclusions.

We have previously outlined the main properties of brain metabolic DNA (BMD) and its involvement in circadian oscillations, learning, and post-trial sleep. The presence of BMD in certain subcellular fractions and their behavior in cesium gradients have suggested that BMD originates from cytoplasmic reverse transcription and subsequently acquires a double-stranded configuration. More recently, it has been reported that some DNA sequences of cytoplasmic BMD in learning mice are different from that of the control animals. Furthermore, BMD is located in vicinity of the genes involved in different modifications of synaptic activity, suggesting that BMD may contribute to the brain's response to the changing environment. The present review outlines recent data with a special emphasis on reverse transcription of BMD that may recapitulate the molecular events at the time of the "RNA world" by activating mitochondrial telomerase and generating RNA templates from mitochondrial transcripts. The latter unexpected role of mitochondria is likely to promote a better understanding of mitochondrial contribution to cellular interactions and eukaryotic evolution. An initial step regards the role of human mitochondria in embryonic BMD synthesis, which is exclusively of maternal origin. In addition, mitochondrial transcripts involved in reverse transcription of BMD might possibly reveal unexpected features elucidating mitochondrial involvement in cancer events and neurodegenerative disorders.

Brain metabolic DNA: recent evidence for a mitochondrial connection.

This review highlights recent data concerning the synthesis of brain metabolic DNA (BMD) by cytoplasmic reverse transcription and the prompt acquisition of the double-stranded configuration that allows its partial transfer to nuclei. BMD prevails in the mitochondrial fraction and is present in presynaptic regions and astroglial processes where it undergoes a turnover lasting a few weeks. Additional data demonstrate that BMD sequences are modified by learning, thus indicating that the modified synaptic activity allowing proper brain responses is encoded in learning BMD. In addition, several converging observations regarding the origin of BMD strongly suggest that BMD is reverse transcribed by mitochondrial telomerase.

Structure-based derivation and intramolecular cyclization of peptide inhibitors from PD-1/PD-L1 complex interface as immune checkpoint blockade for breast cancer immunotherapy

The interaction event between programmed death receptor-1 (PD-1) and its ligand (PD-L1) functions as an essential immune checkpoint against cytotoxic T effector cell activation. Previously, a number of small-molecule inhibitors and antibody drugs have been successfully developed to block the PD1/PDL1 signaling axis for breast cancer immunotherapy. Here, we attempt to directly disrupt the formation of PD-1/PD-L1 complex by using a self-inhibitory peptide (SIP) strategy. In the procedure, the complex crystal structure is examined systematically with energetic analysis and alanine scanning. Two double-stranded segments I and II in PD-L1 active finger are identified as hotspot regions; they directly interact with the amphipathic pocket of PD-1 to form the complex system. The segments are derived from PD-L1 to define two SIP peptides, namely, DS-I and DS-II, which are thought to have capability of rebinding at the complex interface, thus disrupting PD-1/PD-L1 interaction as a new immune checkpoint blockade. A further analysis reveals that the free linear DS-I and DS-II peptides are highly flexible without protein context support, which would incur a large entropy penalty (unfavorable indirect readout effect) when rebinding to PD-1. Next, intramolecular cyclization is applied to constraining the intrinsically disordered conformation of free DS-II peptide into native ordered double-stranded configuration, which can be substantiated by molecular dynamics simulation and circular dichroism spectroscopy. Several cyclized counterparts of linear DS-II peptide are designed and their affinities to PD-1 are determined using fluorescence polarization assays. As might be expected, three designed cyclic peptides DS-II[c111–127], ΔDS-II[c111–127] and ΔDS-II[c110–128] exhibit considerably increased potency (Kd = 28.0 ± 4.2, 17.5 ± 3.1 and 11.6 ± 2.3 μM, respectively) relative to linear DS-II peptide (Kd = 109 ± 15 μM).

Brain Metabolic DNA Is Reverse Transcribed in Cytoplasm: Evidence by Immunofluorescence Analysis.

In a previous study (Mol Neurobiol 55:7476-7486, 2017), newly synthesized brain metabolic DNA (BMD) from rat subcellular fractions has been shown to behave as a DNA-RNA hybrid when analyzed in cesium gradients at early [3H] thymidine incorporation times but to assume the double-stranded configuration at later times. Conversely, BMD from purified nuclei displayed the dsDNA configuration even at early incorporation times. The results were interpreted to support the BMD origin by reverse transcription in the cytoplasm and its later acquisition of the double-stranded configuration before the partial transfer to the nuclei. This interpretation has now been confirmed by immunofluorescence analyses of newly synthesized BrdU-labeled BMD from the mouse brain that demonstrates its cytoplasmic localization and colocalization with DNA-RNA hybrids. In addition, BrdU-labeled BMD has been shown to colocalize with astroglial anti-GFAP antibodies and with presynaptic anti-synaptophysin antibodies.

Effect of configuration on the biomechanical performance of three suture materials used in combination with a metallic bone anchor

To determine whether different suture configurations could improve the biomechanical performance of 3 suture materials used with bone anchors. 3 suture materials (60-lb test nylon leader line, size 2 polyblend polyethylene composite suture, and 150-lb test ultrahigh-molecular weight spun polyethylene). PROCEDURES-Each suture material was looped through the eyelet of a metallic bone anchor and constructs were evaluated by use of an acute uniaxial load. Three configurations were tested for each suture material: single stranded (SS), double stranded (DS), and single stranded plus plastic insert (SSP). Force at failure, extension at failure, force at 3 mm of extension, stiffness, and site of failure of the suture were recorded for each test. For all sutures, the DS configuration was the stiffest and yielded significantly higher forces at failure and forces at 3 mm of extension. The SS configuration had the lowest forces at failure. The SSP configuration yielded greater forces at failure for all suture materials, compared with the SS configuration, with a comparable stiffness. All sutures failed at the eyelet in the SS and DS configurations. In the SSP configuration, 60-lb test nylon leader line and 150-lb test ultrahigh-molecular weight spun polyethylene failed at the eyelet less frequently than did the polyblend composite suture. Among the tested constructs, a DS suture configuration used in combination with the metallic bone anchor gave the best biomechanical results for all suture materials. Considering that the SSP configuration yielded greater forces at failure, compared with the SS configuration, covering metallic edges in bone anchors with softer materials might protect sutures and result in increased forces at failure.

247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrioTM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm was then cryopreserved using BotuCrioTM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9a v. 72.6 ± 8.4a, 31.6 ± 9.2a v. 35.3 ± 10.32a, and 49.3 ± 14.33a v. 59.7 ± 13.59a. At 8 h, the results were 26.0 ± 21.6b v. 54.7 ± 12.2a, 6.1 ± 6.4b v. 17.4 ± 8.54a, and 13.7 ± 14.85b v. 37.6 ± 14.15a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41a v. 5.7 ± 1.60a at 0 h and 8.3 ± 1.78b v. 7.2 ± 1.19b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation. FAPESP for financial support and Botupharma for donation of BotuSemenTM and BotuCrioTM extender.

Site‐Specific Recognition of Guanosine by Manganese(III) Corroles in DNA Non‐Duplex Regions through Active Oxygen Transfer

DNA damage plays an important role in cellular processes. Besides natural protein nucleases, different types of efficient agents for DNA damage have been developed over recent decades in the search for new anticancer and antiviral drugs. In addition to the double-stranded configuration, DNA structures also include some non-duplex regions, which are considered to be from spontaneous errors in DNA replication, thus playing an important role for cells. Herein, we focused on these non-duplex regions of DNA and generated manganese(III) corroles, which exhibit a highly selective cleavage ability for guanosine units located at non-duplex portions, such as loops and bulges. The cleavage mechanism was demonstrated to be a manganese-induced oxidation process. The results given herein show a molecular approach that could specifically probe the guanosine units in DNA non-duplex structures, thus representing a promising step in the construction of tools to target non-duplex structures in chromosomes.

Anomalous zipping dynamics and forced polymer translocation

We investigate by means of Monte Carlo simulations the zipping and unzipping dynamicsof two polymers connected at one end and subject to an attractive interaction betweencomplementary monomers. In zipping, the polymers are quenched from a hightemperature equilibrium configuration to a low temperature state, such that the twostrands zip up by closing up a ‘Y’-fork. In unzipping, the polymers are broughtfrom a low temperature double-stranded configuration to high temperatures,such that the two strands separate. Simulations show that the unzipping time,τu, scales as a function of the polymer length asτu ∼ L, while the zipping is characterized by the anomalous dynamicsτz ∼ Lα withα = 1.37(2). Thisexponent is in good agreement with simulation results and theoretical predictions for the scaling ofthe translocation time of a forced polymer passing through a narrow pore. We find that the exponentα is robust against variations of parameters and temperature, whereas the scaling ofτz as a function of the driving force shows the existence of two different regimes: the weak forcing (τz ∼ 1/F) and strongforcing (τz independent of F) regimes. The crossover region is possibly characterized by a non-trivial scaling inF, matching the prediction of recent theories of polymer translocation. Although thegeometrical setups are different, zipping and translocation share thus the same type ofanomalous dynamics. Systems where this dynamics could be experimentally investigatedinclude DNA (or RNA) hairpins: our results imply an anomalous dynamics for the hairpins’closing times, but not for the opening times.

Novel plant-GARP-like transcription factors in Giardia lamblia

GARP homologues constitute a large family of DNA-binding proteins in plants that may be needed for a variety of key cellular functions including regulation of transcription, phosphotransfer signaling, and differentiation. However, no member of this gene family has been reported to date in yeast, animals, or protozoan parasites. We have identified four genes with putative GARP domains in the Giardia lamblia genome (GARP-like protein or GLP). The glp1 mRNA levels increased slightly during encystation. Epitope-tagged GLP1 localized to both nuclei and the proportion of stained Giardia cells increased by 10-fold during encystation. Recombinant GLP1 specifically bound to both the regulated cwp1 and constitutive ran gene promoters in their double-stranded configurations. The C-terminal region of GLP1 containing the GARP-like domain encoded the DNA binding activity. Mutation analysis revealed that an (A/G)ATCN sequence was required for binding of GLP1 to these promoters. We also found that GLP2 recognized similar binding sites. Using mutated plasmids and transfection assays, we demonstrated that the GLP1/2 binding sites are positive cis-acting elements of the cwp1 and ran gene promoters in both trophozoites and encysting cells. GLP1 is the first GARP family gene found in protozoan parasites. Our results suggest that GLP1 may be an important transcriptional activator and that its binding sites are positive promoter elements for certain Giardia genes.

Mechanical comparison of six loop fixation methods with monofilament nylon leader line

SummaryMonofilament nylon leader line (MNL) is a popular prosthetic material used by veterinary surgeons for extra-capsular cranial cruciate ligament repair. Careful examination of MNL fixation methods has been reported, but a comprehensive comparison has not been done. This study evaluated three unpublished novel methods of MNL loop fixation [Harris wire tightener (knotter), Securos® crimp clamp system, self-locking knot] to traditional methods of MNL loop fixation (clamped square knot, sliding half hitch knot). Loops (27.3 kgt and 36.4 kgt) were distracted at 500 mm/min until failure by breaking or slipping. Ultimate force, elongation and stiffness were used for comparison of loop fixation methods. All of the loops failed by breaking within 3.0 mm of knot or clamp. The Harris knotter had significantly greater elongation than all other loops, except for the self-locking knot. The self-locking knot required the most force to failure; however, it was not superior if the doublestranded configuration was considered. If the mechanical properties of the knot method is considered in light of the subjective handling characteristics, the Securos® crimp clamp system had equivalent strength measurement for the 27.3 kgt MNL loops and was stronger than traditional methods using the 36.4 kgt MNL. The Securos® system recommends use of the larger diameter MNL and it allows a surgeon to overcome potential difficult handling characteristics previously encountered with knot formation and security.

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Induction of c-Ha-ras Gene Expression by Double-Stranded RNA and Interferon Requirement

The addition of double-stranded RNA (dsRNA) to NIH 3T3 cells led to an increase in the RNA levels of c-Ha-ras. The double-stranded configuration was required for the increase in c-Ha-ras mRNA levels, as heat-denatured dsRNA and single-stranded RNA did not have any effect. Nuclear run-on transcription experiments indicated that the increase in c-Ha-ras mRNA levels stimulated by dsRNA was due to transcriptional activation of the gene. The induction of c-Ha-ras gene expression by dsRNA was inhibited by anti-beta interferon antibodies, suggesting that interferon might mediate the induction.

Induction of c-Ha-ras gene expression by double-stranded RNA and interferon requirement.

The addition of double-stranded RNA (dsRNA) to NIH 3T3 cells led to an increase in the RNA levels of c-Ha-ras. The double-stranded configuration was required for the increase in c-Ha-ras mRNA levels, as heat-denatured dsRNA and single-stranded RNA did not have any effect. Nuclear run-on transcription experiments indicated that the increase in c-Ha-ras mRNA levels stimulated by dsRNA was due to transcriptional activation of the gene. The induction of c-Ha-ras gene expression by dsRNA was inhibited by anti-beta interferon antibodies, suggesting that interferon might mediate the induction.

Structural features in the 3'-terminal region of polyribosome-bound rabbit globin messenger RNAs.

A nuclease S1 mapping procedure was used to identify sites accessible to nucleases in the 3'-noncoding region of the rabbit globin mRNAs. A complex structure was evident in the alpha-globin species, with one highly accessible single-stranded site, large portions in an accessible double-stranded configuration, and a portion not accessible to any of the nucleases. In the beta-globin mRNA, the region was more uniformly accessible to RNase T1 and to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease. The patterns of cleavage were nearly identical in the deproteinized mRNAs and in the mRNAs associated with polyribosomes in reticulocyte extracts. In both species, a zone of secondary structure occurred around the poly(A) junction. In each species, virtually all the molecules had a poly(A) sequence of at least 20-25 AMP residues. A periodicity in poly(A) size distribution was observed. These results indicate that the beginning of this sequence is well protected against degradation inside the cell and that zones of partial protection occur at measured intervals. In crude extracts, where the poly(A) is covered with proteins, this sequence was protected against nuclease digestion.

Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid

Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.

S1 nuclease hydrolysis of single-stranded nucleic acids with partial double-stranded configuration.

The single-strand specific nuclease S1 from Aspergillus oryzae (EC 3.1.4.21) was purified 600-fold in 16% yield from dried mycelia. Determination of the isoelectric point of S1 nuclease as 4.3-4.4 allowed adjustment of chromatographic conditions such that the enzyme was isolated free of contaminating ribonucleases T1 and T2. S1 nuclease so purified was used for removal of single-stranded portions from the RNA of the Escherichia coli phage MS2, which has a helical content of about 65% in vitro. At 23 degrees, increasing amounts of enzyme converted the RNA to mononucleotides in about equimolar base ratios. No small intermediates of chain length 2-8 were found. At 0 degrees, MS2 RNA hydrolysis was slower and reached, in exhaustive digests, a plateau where 70% of the substrate RNA remained insoluble in 66% EtOH. With [32P]MS2 RNA, strip chart counting of 6% acrylamide-6 M urea electrophoresis patterns of such digests gave recoveries of 80-91% in the form of defined oligomer bands. On 2.5% acrylamide-0.5% agarose gels, the molecular weights of the major oligomers were found to range from 25,000 to 41,000. Similar to purified tRNAArg used as a control, these oligomers were not resistant to pancreatic RNase-RNase T1 hydrolysis at 37 degrees, and were not bound on hydroxylapatite at 50 degrees in 0.14 M sodium phosphate (pH 6.8). Melting of the oligomers gave complex profiles without a clear Tm and showed an increase in A260 of 35% at 93 degrees over that at 28 degrees. Upon formaldehyde denaturation of MS2 RNA prior to S1 nuclease hydrolysis, no resistant oligomers were found.

Sedimentation properties of yeast nuclear and mitochondrial DNA

The sedimentation behavior of high molecular weight nuclear and mitochondrial DNA from the yeast Saccharomyces cerevisiae has been examined and the molecular weights estimated based on a linear, double-stranded configuration. Nuclear DNA molecules exhibit a narrow range of molecular weights of the order 400 × 10 6 to 600 × 10 6 daltons, and chromosome VIII of a disomic strain has been tentatively assigned a molecular weight of 400 × 10 6 daltons. Several sedimenting DNA components can be distinguished from isolated yeast mitochondria: a major species corresponding to a molecular weight of 46 × 10 6 daltons, as well as others with molecular weights of 120 × 10 6, 55 × 10 6 and 25 × 10 6 daltons.

Interférons humains induits in vitro par des polynucléotides synthétiques

Human interferons induced in vitro by synthetic polynucleotides Optimal conditions of in vitro production of human interferon were studied, employing polyinosinic acid and polycytidylic acid complexes as inducers. Two cellular sources were utilized: leukocytes and the amniotic membrane. Greater amounts of interferon were obtained from leukocytes. In this system as little as 0.01 μg/ml of poly I + poly C was sufficient for the induction of detectable amounts of interferon in the extracellular medium. The production of interferon increased more than 100-fold when cells were pretreated with DEAE-dextran at a concentration of 10 μg/ml. In the presence of DEAE-dextran (10 μg/ml of the complex in leukocyte suspensions 10 7 cells/ml) titres of interferon between 1024 and 2048 were obtained. Under the same conditions, interferon titering 64 was recovered from the amniotic membrane. Physicochemical and biological properties of the polynucleotide-induced interferons were determined and found to be the same as those for viral-induced interferons. The molecular weight of polynucleotide-induced leukocyte interferon was 25000 (± 10 %). The same values were found using para influenza 1 (Sendaï) virus as inducer. Pre-treatment of leukocytes with actinomycin D (1 μg/ml, 1 h) reduced the production of interferon by 75 %. Biological activity of interferon in amniotic cells was inhibited 90 % by pretreatment with actinomycin D (0.1 μg/ml). The mixing curve of poly I + poly C was studied correlating the hypochromic effect and interferon production. The largest amounts of interferon and maximal hypocromic effect were found when poly I and poly C were mixed in equimolecular proportions. These results agree with the hypothesis that the double-stranded configuration of the RNA molecule is essential for the induction of interferon.

Mutagenesis in phages ϕX174 and T4 and properties of the genetic material

In vitro treatment of phages ϕX174 and T4 with nitrous acid produces mutants. Mutants of ϕX174 arise in pure clones, whereas most T4 mutants arise in mixed clones, suggesting that there is only one copy of genetic information in ϕX174 and at least two copies in T4. The alteration of one of the T4 copies yields a type of heterozygote. The results is consistent with the picture that the deoxyribonucleic acid (DNA) of T4 is in the Watson-Crick double-stranded configuration, both strands carrying the same information, whereas the DNA of ϕX174 is single-stranded. The production of mutants in ϕX174 follows a single-hit kinetics, showing that the alteration of a single nucleotide is sufficient to produce a mutant. The process of nitrous acid mutagenesis in DNA is discussed.