Enhanced Product Stability in the Hammerhead Ribozyme†

PubMed Central

Shepotinovskaya, Irina; Uhlenbeck, Olke C.

2010-01-01

The rate of dissociation of P1, the 5′ product of hammerhead cleavage, is 100–300-fold slower in full-length hammerheads than in hammerheads that either lack or have disrupting mutations in the loop-loop tertiary interaction. The added stability requires the presence of residue 17 at the 3′ terminus of P1 but not the 2′, 3′ terminal phosphate. Since residue 17 is buried within the catalytic core of the hammerhead in the x-ray structure, we propose that the enhanced P1 stability is the result of the cooperative folding of the hammerhead around this residue. However, since the P1 is fully stabilized above 2.5 mM MgCl2 while hammerhead activity continues to increase with increasing MgCl2, it is clear that the hammerhead structure in the transition state must differ from that of the product complex. The product stabilization assay is used to test our earlier proposal that different tertiary interactions modulate the cleavage rate by differentially stabilizing the core. PMID:20423112

Studying Hammerheads in Hawaii

ERIC Educational Resources Information Center

Handler, Alex; Duncan, Kanesa

2006-01-01

In this article, the author discusses the High School Scalloped Hammerhead Shark Tagging Program in Hawaii which is an example of a successful partnership research collaboration. High school students and teachers worked with biologists from the University of Hawaii-Manoa (UHM) to conduct research on the life history of scalloped hammerhead sharks…

Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor.

PubMed Central

Murray, J B; Arnold, J R

1996-01-01

A screening of a range of common laboratory antibiotics for inhibition of the hammerhead ribozyme has shown that in addition to certain aminoglycosides (most notably neomycin B) the tetracyclines are also effective inhibitors, with chlorotetracycline being more effective than tetracycline. Inhibition by chlorotetracycline is not as strong as that by neomycin B but is more complicated, with at least two binding sites apparent. As with hammerhead inhibition by neomycin B, chlorotetracycline inhibition can be overcome by raising the concentration of the Mg2+ ion cofactor. We find that around six Mg2+ ions will displace neomycin B, compared with twelve for chlorotetracycline. Inhibition observed in the presence of mixtures of neomycin B and chlorotetracycline is consistent with separate binding sites on the hammerhead for these two classes of antibiotic. Under certain conditions of the mixing order and low concentration of chlorotetracycline, enhancement of single-turnover hammerhead cleavage by up to 20% is observed, with higher concentrations of antibiotic being inhibitory. We have also found that the presence of 2.5% (v/v) DMSO causes a 30% enhancement of the single-turnover cleavage. These results thus extend the range of known inhibitors of hammerhead cleavage, and also demonstrate how the cleavage can be accelerated. PMID:8760373

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2013 CFR

2013-10-01

... Mexico blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran Lemon... isodon Gag grouper, Mycteroperca microlepis Lane snapper, Lutjanus synagris Lemon shark, Negaprion...

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2014 CFR

2014-10-01

... Mexico blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran Lemon... isodon Gag grouper, Mycteroperca microlepis Lane snapper, Lutjanus synagris Lemon shark, Negaprion...

A minimally invasive technique to assess several life-history characteristics of the endangered great hammerhead shark Sphyrna mokarran.

PubMed

O'Connell, C P; Leurs, G

2016-03-01

A dorsal-fin photo-identification technique paired with a non-invasive parallel laser photogrammetry technique was used to non-invasively identify individual Sphyrna mokarran over time. Based on the data collected over a duration of 59 days, 16 different S. mokarran (mean ± S.D. pre-caudal length: 220·82 ± 13·66 cm; mean ± S.D. cephalofoil width: 71·38 ± 7·94 cm) were identified using dorsal-fin photo-identification, with a mean ± S.D. shark re-sighting frequency of 4·05 ± 3·06 at-sea days. The results illustrate a high S. mokarran sighting rate and therefore, the utilization of parallel laser photogrammetry and dorsal-fin photo-identification may be a plausible multi-year approach to aid in non-invasively determining the growth rate and inter-annual site fidelity of these animals. © 2016 The Fisheries Society of the British Isles.

Probing the hammerhead ribozyme structure with ribonucleases.

PubMed Central

Hodgson, R A; Shirley, N J; Symons, R H

1994-01-01

Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; GL2.1 in the stem II loop and G12 in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at GL2.1 in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G12 site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural. Images PMID:8202361

Inhibition of the hammerhead ribozyme by neomycin.

PubMed Central

Stage, T K; Hertel, K J; Uhlenbeck, O C

1995-01-01

A series of antibiotics was tested for stimulation or inhibition of the hammerhead ribozyme cleavage reaction. Neomycin was found to be a potent inhibitor of the reaction with a Kl of 13.5 microM. Two hammerheads with well-characterized kinetics were used to determine which steps in the reaction mechanism were inhibited by neomycin. The data suggest that neomycin interacts preferentially with the enzyme-substrate complex and that this interaction leads to a reduction in the cleavage rate by stabilizing the ground state of the complex and destabilizing the transition state of the cleavage step. A comparison of neomycin with other aminoglycosides and inhibitors of hammerhead cleavage implies that the ammonium ions of neomycin are important for the antibiotic-hammerhead interaction. PMID:7489494

Efficient Ligation of the Schistosoma Hammerhead Ribozyme †

PubMed Central

Canny, Marella D.; Jucker, Fiona M.; Pardi, Arthur

2011-01-01

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2–3 at physiological Mg2+ ion concentrations (0.1 –1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme. PMID:17319693

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2010 CFR

2010-10-01

..., Thunnus thynnus Dolphin fish, Coryphaena hippurus Oceanic whitetip shark, Carcharhinus longimanus... Tables Table 1 of Appendix A to Part 635—Oceanic Sharks A. Large Coastal Sharks 1. Ridgeback sharks... sharks: Blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran...

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2011 CFR

2011-10-01

..., Thunnus thynnus Dolphin fish, Coryphaena hippurus Oceanic whitetip shark, Carcharhinus longimanus... Tables Table 1 of Appendix A to Part 635—Oceanic Sharks A. Large Coastal Sharks 1. Ridgeback sharks... sharks: Blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran...

Characterization of a native hammerhead ribozyme derived from schistosomes

PubMed Central

OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

2005-01-01

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

76 FR 23935 - Atlantic Highly Migratory Species; Atlantic Shark Management Measures

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-29

.... 110120049-1144-01] RIN 0648-BA69 Atlantic Highly Migratory Species; Atlantic Shark Management Measures... retention, transshipping, landing, storing, or selling of hammerhead sharks in the family Sphyrnidae (except for Sphyrna tiburo) and oceanic whitetip sharks (Carcharhinus longimanus) caught in association with...

Specificity of hammerhead ribozyme cleavage.

PubMed Central

Hertel, K J; Herschlag, D; Uhlenbeck, O C

1996-01-01

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA

PubMed Central

Martick, Monika; Horan, Lucas H.; Noller, Harry F.; Scott, William G.

2008-01-01

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR1. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes4–7. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads8–10, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the poly-adenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals. PMID:18615019

Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection.

PubMed Central

Tang, J; Breaker, R R

1997-01-01

We have designed a self-cleaving ribozyme construct that is rendered inactive during preparative in vitro transcription by allosteric interactions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a ribozyme whose catalytic rate can be controlled by ATP. Upon purification by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of "allosteric delay" was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for the hammerhead. Using in vitro selection, this comprehensive RNA pool was screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the core of naturally occurring hammerhead dominate the population of selected RNAs, indicating that the natural consensus sequence of this ribozyme is optimal for catalytic function. PMID:9257650

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

PubMed Central

Wang, L; Ruffner, D E

1997-01-01

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly. PMID:9336468

Community structure of elasmobranchs in estuaries along the northwest Gulf of Mexico

NASA Astrophysics Data System (ADS)

Plumlee, Jeffrey D.; Dance, Kaylan M.; Matich, Philip; Mohan, John A.; Richards, Travis M.; TinHan, Thomas C.; Fisher, Mark R.; Wells, R. J. David

2018-05-01

Estuaries promote high levels of productivity and biodiversity by providing habitat for many biological communities due to their wide range of environmental conditions. Estuarine systems serve as nurseries, areas for parturition, and feeding grounds for elasmobranchs. However, estuaries face an array of anthropogenic pressures, including overfishing, altered flow regimes, pollution, and habitat destruction. Given the vulnerability of estuarine ecosystems, observing long-term changes in community structure is essential to understanding the effects of anthropogenic stressors. Elasmobranch community structure was analyzed among eight estuaries in the northwest Gulf of Mexico to evaluate spatial and temporal variability in species abundance and diversity using bi-annual fisheries independent gillnet survey data over three decades (1985-2014). Ten species comprised 99.4% of elasmobranchs caught which included 35.3% bull sharks (Carcharhinus leucas), 18.1% bonnetheads (Sphyrna tiburo), 17.0% cownose rays (Rhinoptera bonasus), 13.4% blacktip sharks (Carcharhinus limbatus), 5.9% Atlantic stingrays (Dasyatis sabina), 3.1% Atlantic sharpnose sharks (Rhizoprionodon terraenovae), 2.7% spinner sharks (Carcharhinus brevipinna), 2.1% scalloped hammerheads (Sphyrna lewini), 1.7% finetooth sharks (Carcharhinus isodon), and 0.7% lemon sharks (Negaprion brevirostris). During the study period, elasmobranch community structure changed among estuaries and among decades. Bull sharks, bonnetheads, cownose rays, blacktip sharks, and spinner sharks all increased in abundance during the study period, whereas finetooth sharks and lemon sharks decreased over time. Higher latitude estuaries were dominated by bull sharks while lower latitude estuaries were dominated by cownose rays. Salinity was the most important environmental variable in predicting individual elasmobranch species abundance (deviance explained: 14.4 ± 6.5 SD), while temperature and depth also played a role in shaping community

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

PubMed Central

Chartrand, P; Harvey, S C; Ferbeyre, G; Usman, N; Cedergren, R

1995-01-01

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer. Images PMID:7479070

Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

PubMed Central

Hendrix, C; Anné, J; Joris, B; Van Aerschot, A; Herdewijn, P

1996-01-01

Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location. PMID:8670082

Active-site monovalent cations revealed in a 1.55-Å-resolution hammerhead ribozyme structure.

PubMed

Anderson, Michael; Schultz, Eric P; Martick, Monika; Scott, William G

2013-10-23

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

Shifting elasmobranch community assemblage at Cocos Island--an isolated marine protected area.

PubMed

White, Easton R; Myers, Mark C; Flemming, Joanna Mills; Baum, Julia K

2015-08-01

Fishing pressure has increased the extinction risk of many elasmobranch (shark and ray) species. Although many countries have established no-take marine reserves, a paucity of monitoring data means it is still unclear if reserves are effectively protecting these species. We examined data collected by a small group of divers over the past 21 years at one of the world's oldest marine protected areas (MPAs), Cocos Island National Park, Costa Rica. We used mixed effects models to determine trends in relative abundance, or probability of occurrence, of 12 monitored elasmobranch species while accounting for variation among observers and from abiotic factors. Eight of 12 species declined significantly over the past 2 decades. We documented decreases in relative abundance for 6 species, including the iconic scalloped hammerhead shark (Sphyrna lewini) (-45%), whitetip reef shark (Triaenodon obesus) (-77%), mobula ray (Mobula spp.) (-78%), and manta ray (Manta birostris) (-89%), and decreases in the probability of occurrence for 2 other species. Several of these species have small home ranges and should be better protected by an MPA, which underscores the notion that declines of marine megafauna will continue unabated in MPAs unless there is adequate enforcement effort to control fishing. In addition, probability of occurrence at Cocos Island of tiger (Galeocerdo cuvier), Galapagos (Carcharhinus galapagensis), blacktip (Carcharhinus limbatus), and whale (Rhincodon typus) sharks increased significantly. The effectiveness of MPAs cannot be evaluated by examining single species because population responses can vary depending on life history traits and vulnerability to fishing pressure. © 2015 Society for Conservation Biology.

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

PubMed Central

Grasby, J A; Jonathan, P; Butler, G; Gait, M J

1993-01-01

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage. PMID:8233777

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

The Hydrodynamics and Odorant Transport Phenomena of Olfaction in the Hammerhead Shark

NASA Astrophysics Data System (ADS)

Rygg, Alex; Craven, Brent

2013-11-01

The hammerhead shark possesses a unique head morphology that is thought to facilitate enhanced olfactory performance. The olfactory organs, located at the distal ends of the cephalofoil, contain numerous lamellae that increase the surface area for olfaction. Functionally, for the shark to detect chemical stimuli, water-borne odors must reach the olfactory sensory epithelium that lines these lamellae. Thus, odorant transport from the aquatic environment to the sensory epithelium is the first critical step in olfaction. Here we investigate the hydrodynamics and odorant transport phenomena of olfaction in the hammerhead shark based on an anatomically-accurate reconstruction of the head and olfactory chamber from high-resolution micro-CT and MRI scans of a cadaver specimen. Computational fluid dynamics (CFD) simulations of water flow in the reconstructed model reveal the external and internal hydrodynamics of olfaction during swimming. Odorant transport in the olfactory organ is investigated using a multi-scale approach, whereby molecular dynamics (MD) simulations are used to calculate odorant partition coefficients that are subsequently utilized in macro-scale CFD simulations of odorant deposition. The hydrodynamic and odorant transport results are used to elucidate several important features of olfactory function in the hammerhead shark.

Origins of the temperature dependence of hammerhead ribozyme catalysis.

PubMed Central

Peracchi, A

1999-01-01

The difficulties in interpreting the temperature dependence of protein enzyme reactions are well recognized. Here, the hammerhead ribozyme cleavage was investigated under single-turnover conditions between 0 and 60 degrees C as a model for RNA-catalyzed reactions. Under the adopted conditions, the chemical step appears to be rate-limiting. However, the observed rate of cleavage is affected by pre-catalytic equilibria involving deprotonation of an essential group and binding of at least one low-affinity Mg2+ion. Thus, the apparent entropy and enthalpy of activation include contributions from the temperature dependence of these equilibria, precluding a simple physical interpretation of the observed activation parameters. Similar pre-catalytic equilibria likely contribute to the observed activation parameters for ribozyme reactions in general. The Arrhenius plot for the hammerhead reaction is substantially curved over the temperature range considered, which suggests the occurrence of a conformational change of the ribozyme ground state around physiological temperatures. PMID:10390528

Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures.

PubMed Central

Hernández, C; Flores, R

1992-01-01

Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs. Images PMID:1373888

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

PubMed Central

Bassi, G S; Murchie, A I; Lilley, D M

1996-01-01

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. PMID:8752086

Entropy-Driven Folding of an RNA Helical Junction: An Isothermal Titration Calorimetric Analysis of the Hammerhead Ribozyme†

PubMed Central

Mikulecky, Peter J.; Takach, Jennifer C.; Feig, Andrew L.

2008-01-01

Helical junctions are extremely common motifs in naturally occurring RNAs, but little is known about the thermodynamics that drive their folding. Studies of junction folding face several challenges: non-two-state folding behavior, superposition of secondary and tertiary structural energetics, and drastically opposing enthalpic and entropic contributions to folding. Here we describe a thermodynamic dissection of the folding of the hammerhead ribozyme, a three-way RNA helical junction, by using isothermal titration calorimetry of bimolecular RNA constructs. By using this method, we show that tertiary folding of the hammerhead core occurs with a highly unfavorable enthalpy change, and is therefore entropically driven. Furthermore, the enthalpies and heat capacities of core folding are the same whether supported by monovalent or divalent ions. These properties appear to be general to the core sequence of bimolecular hammerhead constructs. We present a model for the ion-induced folding of the hammerhead core that is similar to those advanced for the folding of much larger RNAs, involving ion-induced collapse to a structured, non-native state accompanied by rearrangement of core residues to produce the native fold. In agreement with previous enzymological and structural studies, our thermodynamic data suggest that the hammerhead structure is stabilized in vitro predominantly by diffusely bound ions. Our approach addresses several significant challenges that accompany the study of junction folding, and should prove useful in defining the thermodynamic determinants of stability in these important RNA motifs. PMID:15134461

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

PubMed Central

Wang, Bingbing; Salavati, Reza; Heidmann, Stefan; Stuart, Kenneth

2002-01-01

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing. PMID:11991648

Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis

PubMed Central

Chi, Young-In; Martick, Monika; Lares, Monica; Kim, Rosalind; Scott, William G; Kim, Sung-Hou

2008-01-01

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures. PMID:18834200

Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.

PubMed Central

Sigurdsson, S T; Tuschl, T; Eckstein, F

1995-01-01

Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure. PMID:7489517

Coupling of Fast and Slow Modes in the Reaction Pathway of the Minimal Hammerhead Ribozyme Cleavage

PubMed Central

Radhakrishnan, Ravi

2007-01-01

By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg2+) ions, proceeds via an in-line-attack by CYT 17 O2′ on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack. PMID:17545240

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

PubMed Central

McDowell, S. Elizabeth; Jun, Jesse M.; Walter, Nils G.

2010-01-01

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼10-fold higher than physiologic concentrations of Mg2+ ions. Extended versions containing native loop–loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg2+ concentrations, for reasons that are still ill-understood. Here, we use Mg2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop–loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop–loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop–loop interactions in extended hammerhead ribozymes—and Mg2+ ions that bind to minimal ribozymes—may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state. PMID:20921269

Drivers of abundance and spatial distribution of reef-associated sharks in an isolated atoll reef system

PubMed Central

Letessier, Tom B.; Koldewey, Heather J.; Meeuwig, Jessica J.

2017-01-01

We investigated drivers of reef shark demography across a large and isolated marine protected area, the British Indian Ocean Territory Marine Reserve, using stereo baited remote underwater video systems. We modelled shark abundance against biotic and abiotic variables at 35 sites across the reserve and found that the biomass of low trophic order fish (specifically planktivores) had the greatest effect on shark abundance, although models also included habitat variables (depth, coral cover and site type). There was significant variation in the composition of the shark assemblage at different atolls within the reserve. In particular, the deepest habitat sampled (a seamount at 70-80m visited for the first time in this study) recorded large numbers of scalloped hammerhead sharks (Sphyrna lewini) not observed elsewhere. Size structure of the most abundant and common species, grey reef sharks (Carcharhinus amblyrhynchos), varied with location. Individuals at an isolated bank were 30% smaller than those at the main atolls, with size structure significantly biased towards the size range for young of year (YOY). The 18 individuals judged to be YOY represented the offspring of between four and six females, so, whilst inconclusive, these data suggest the possible use of a common pupping site by grey reef sharks. The importance of low trophic order fish biomass (i.e. potential prey) in predicting spatial variation in shark abundance is consistent with other studies both in marine and terrestrial systems which suggest that prey availability may be a more important predictor of predator distribution than habitat suitability. This result supports the need for ecosystem level rather than species-specific conservation measures to support shark recovery. The observed spatial partitioning amongst sites for species and life-stages also implies the need to include a diversity of habitats and reef types within a protected area for adequate protection of reef-associated shark assemblages

Drivers of abundance and spatial distribution of reef-associated sharks in an isolated atoll reef system.

PubMed

Tickler, David M; Letessier, Tom B; Koldewey, Heather J; Meeuwig, Jessica J

2017-01-01

We investigated drivers of reef shark demography across a large and isolated marine protected area, the British Indian Ocean Territory Marine Reserve, using stereo baited remote underwater video systems. We modelled shark abundance against biotic and abiotic variables at 35 sites across the reserve and found that the biomass of low trophic order fish (specifically planktivores) had the greatest effect on shark abundance, although models also included habitat variables (depth, coral cover and site type). There was significant variation in the composition of the shark assemblage at different atolls within the reserve. In particular, the deepest habitat sampled (a seamount at 70-80m visited for the first time in this study) recorded large numbers of scalloped hammerhead sharks (Sphyrna lewini) not observed elsewhere. Size structure of the most abundant and common species, grey reef sharks (Carcharhinus amblyrhynchos), varied with location. Individuals at an isolated bank were 30% smaller than those at the main atolls, with size structure significantly biased towards the size range for young of year (YOY). The 18 individuals judged to be YOY represented the offspring of between four and six females, so, whilst inconclusive, these data suggest the possible use of a common pupping site by grey reef sharks. The importance of low trophic order fish biomass (i.e. potential prey) in predicting spatial variation in shark abundance is consistent with other studies both in marine and terrestrial systems which suggest that prey availability may be a more important predictor of predator distribution than habitat suitability. This result supports the need for ecosystem level rather than species-specific conservation measures to support shark recovery. The observed spatial partitioning amongst sites for species and life-stages also implies the need to include a diversity of habitats and reef types within a protected area for adequate protection of reef-associated shark assemblages.

Global versus local causes and health implications of high mercury concentrations in sharks from the east coast of South Africa.

PubMed

McKinney, Melissa A; Dean, Kylie; Hussey, Nigel E; Cliff, Geremy; Wintner, Sabine P; Dudley, Sheldon F J; Zungu, M Philip; Fisk, Aaron T

2016-01-15

Conservation concern regarding the overharvest of global shark populations for meat and fin consumption largely surrounds documented deleterious ecosystem effects, but may be further supported by improved knowledge of possibly high levels in their edible tissues (particularly meat) of the neurotoxin, methylmercury (CH3Hg). For many regions, however, little data exist on shark tissue Hg concentrations, and reasons for Hg variation within and among species or across regions are poorly understood. We quantified total Hg (THg) in 17 shark species (total n=283) from the east coast of South Africa, a top Hg emitter globally. Concentrations varied from means of around 0.1 mg kg(-1) dry weight (dw) THg in hardnose smoothhound (Mustelus mosis) and whale (Rhincodon typus) sharks to means of over 10 mg kg(-1) dw in shortfin mako (Isurus oxyrinchus), scalloped hammerhead (Sphyrna lewini), white (Carcharodon carcharias) and ragged-tooth (Carcharias taurus) sharks. These sharks had higher THg levels than conspecifics sampled from coastal waters of the North Atlantic and North, mid-, and South Pacific, and although sampling year and shark size may play a confounding role, this result suggests the potential importance of elevated local emissions. Values of THg showed strong, species-specific correlations with length, and nearly half the remaining variation was explained by trophic position (using nitrogen stable isotopes, δ(15)N), whereas measures of foraging habitat (using carbon stable isotopes, δ(13)C) were not significant. Mercury concentrations were above the regulatory guidelines for fish health effects and safe human consumption for 88% and 70% of species, respectively, suggesting on-going cause for concern for shark health, and human consumers of shark meat. Copyright © 2015 Elsevier B.V. All rights reserved.

Deep-sea observations at hydrocarbon drilling locations: Contributions from the SERPENT Project after 120 field visits

NASA Astrophysics Data System (ADS)

Gates, Andrew R.; Benfield, Mark C.; Booth, David J.; Fowler, Ashley M.; Skropeta, Danielle; Jones, Daniel O. B.

2017-03-01

The SERPENT Project has been running for over ten years. In this time scientists from universities and research institutions have made more than 120 visits to oil rigs, drill ships and survey vessels operated by 16 oil companies, in order to work with the industry's Remotely Operated Vehicles (ROV). Visits have taken place in Europe, North and South America, Africa and Australasia at water depths from 100 m to nearly 3000 m. The project has directly produced >40 peer reviewed publications and data from the project's >2600 entry online image and video archive have been used in many others. The aim of this paper is to highlight examples of how valuable data can be obtained through collaboration with hydrocarbon exploration and production companies to use existing industry infrastructure to increase scientific discovery in unexplored areas and augment environmental monitoring of industrial activity. The large number of industry ROVs operating globally increases chance encounters with large, enigmatic marine organisms. SERPENT video observations include the deepest known records of species previously considered epipelagic such as scalloped hammerhead (Sphyrna lewini) and southern sunfish (Mola ramsayi) and the first in situ observations of pelagic species such as oarfish (Regalecus glesne). Such observations enable improvements to distribution records and description of behaviour of poorly understood species. Specimen collection has been used for taxonomic descriptions, functional studies and natural products chemistry research. Anthropogenic effects been assessed at the local scale using in situ observations and sample collection at the time of drilling operations and subsequent visits have enabled study of recovery from drilling. Future challenges to be addressed using the SERPENT approach include ensuring unique faunal observations by industry ROV operators are reported, further study of recovery from deep-water drilling activity and to carry out in situ studies to

Plasticity of trophic interactions among sharks from the oceanic south-western Indian Ocean revealed by stable isotope and mercury analyses

NASA Astrophysics Data System (ADS)

Kiszka, Jeremy J.; Aubail, Aurore; Hussey, Nigel E.; Heithaus, Michael R.; Caurant, Florence; Bustamante, Paco

2015-02-01

Sharks are a major component of the top predator guild in oceanic ecosystems, but the trophic relationships of many populations remain poorly understood. We examined chemical tracers of diet and habitat (δ15N and δ13C, respectively) and total mercury (Hg) concentrations in muscle tissue of seven pelagic sharks: blue shark (Prionace glauca), short-fin mako shark (Isurus oxyrinchus), oceanic whitetip shark (Carcharhinus longimanus), scalloped hammerhead shark (Sphyrna lewini), pelagic thresher shark (Alopias pelagicus), crocodile shark (Pseudocarcharias kamoharai) and silky shark (Carcharhinus falciformis), from the data poor south-western tropical Indian Ocean. Minimal interspecific variation in mean δ15N values and a large degree of isotopic niche overlap - driven by high intraspecific variation in δ15N values - was observed among pelagic sharks. Similarly, δ13C values of sharks overlapped considerably for all species with the exception of P. glauca, which had more 13C-depleted values indicating possibly longer residence times in purely pelagic waters. Geographic variation in δ13C, δ15N and Hg were observed for P. glauca and I. oxyrinchus. Mean Hg levels were similar among species with the exception of P. kamoharai which had significantly higher Hg concentrations likely related to mesopelagic feeding. Hg concentrations increased with body size in I. oxyrinchus, P. glauca and C. longimanus. Values of δ15N and δ13C varied with size only in P. glauca, suggesting ontogenetic shifts in diets or habitats. Together, isotopic data indicate that - with few exceptions - variance within species in trophic interactions or foraging habitats is greater than differentiation among pelagic sharks in the south-western Indian Ocean. Therefore, it is possible that this group exhibits some level of trophic redundancy, but further studies of diets and fine-scale habitat use are needed to fully test this hypothesis.

Recent developments in the hammerhead ribozyme field.

PubMed Central

Vaish, N K; Kore, A R; Eckstein, F

1998-01-01

Developments in the hammerhead ribozyme field during the last two years are reviewed here. New results on the specificity of this ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of ribozyme technology examples of the application of this ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction. PMID:9826743

Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

PubMed Central

Hermann, T; Auffinger, P; Scott, W G; Westhof, E

1997-01-01

In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA. PMID:9254698

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

PubMed Central

Gao, Run-Ping; Brigstock, David R

2009-01-01

AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase. PMID:19673024

Hammerhead Shark Research Immersion Program: Experiential Learning Leads to Lasting Educational Benefits

ERIC Educational Resources Information Center

Handler, Alex; Duncan, Kanesa

2006-01-01

High school students (n = 45) participated in a 5-day research immersion study on juvenile scalloped hammerhead sharks in Kaneohe Bay, Oahu, Hawaii. Self-surveys were used to evaluate scientific concepts and skills taught during the program. There was a significant shift in students' perceived level of understanding for all categories of concepts…

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

PubMed Central

Shields, T P; Mollova, E; Ste Marie, L; Hansen, M R; Pardi, A

1999-01-01

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product. PMID:10496226

Population genetics of four heavily exploited shark species around the Arabian Peninsula

PubMed Central

Spaet, Julia L Y; Jabado, Rima W; Henderson, Aaron C; Moore, Alec B M; Berumen, Michael L

2015-01-01

The northwestern Indian Ocean harbors a number of larger marine vertebrate taxa that warrant the investigation of genetic population structure given remarkable spatial heterogeneity in biological characteristics such as distribution, behavior, and morphology. Here, we investigate the genetic population structure of four commercially exploited shark species with different biological characteristics (Carcharhinus limbatus, Carcharhinus sorrah, Rhizoprionodon acutus, and Sphyrna lewini) between the Red Sea and all other water bodies surrounding the Arabian Peninsula. To assess intraspecific patterns of connectivity, we constructed statistical parsimony networks among haplotypes and estimated (1) population structure; and (2) time of most recent population expansion, based on mitochondrial control region DNA and a total of 20 microsatellites. Our analysis indicates that, even in smaller, less vagile shark species, there are no contemporary barriers to gene flow across the study region, while historical events, for example, Pleistocene glacial cycles, may have affected connectivity in C. sorrah and R. acutus. A parsimony network analysis provided evidence that Arabian S. lewini may represent a population segment that is distinct from other known stocks in the Indian Ocean, raising a new layer of conservation concern. Our results call for urgent regional cooperation to ensure the sustainable exploitation of sharks in the Arabian region. PMID:26120422

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

PubMed Central

Zhou, Jing-Min; Zhou, De-Min; Takagi, Yasuomi; Kasai, Yasuhiro; Inoue, Atsushi; Baba, Tadashi; Taira, Kazunari

2002-01-01

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses. PMID:12034824

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

PubMed Central

Borda, Emily J.; Markley, John C.; Sigurdsson, Snorri Th.

2003-01-01

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes. PMID:12736309

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

PubMed Central

Tabler, M; Homann, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. Images PMID:7937118

Efficient trans-cleavage by the Schistosoma mansoni SMα1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus

PubMed Central

Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M.; Berenguer, José; Cedergren, Robert

2002-01-01

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMα DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme–product dissociation step. The optimal temperature for trans-cleavage was 70°C. This result was confirmed when both the SMα1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMα1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMα1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo. PMID:11917021

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

PubMed Central

Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

2014-01-01

The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

PubMed

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

In vitro activity of minimised hammerhead ribozymes.

PubMed Central

Hendry, P; McCall, M J; Santiago, F S; Jennings, P A

1995-01-01

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2. The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes. PMID:7479037

Evaluation of a Petition Requesting National Marine Fisheries Service (NMFS) to List the Smooth Hammerhead Shark (Sphryna zygaena) as a Threatened or Endangered Species Under the Endangered Species Act (ESA)

NASA Astrophysics Data System (ADS)

Sturm, A. B.

2016-12-01

The wildlife conservation organization, Defenders of Wildlife, petitioned NMFS to list the smooth hammerhead shark, Sphryna zygaena, as endangered or threatened throughout its range under the ESA. The petition was critically evaluated to determine if the petitioners presented substantial scientific or commercial information indicating that the smooth hammerhead shark may warrant listing under the ESA. The petition and the cited scientific literature (as well as scientific literature readily available in NMFS files) were evaluated to determine if the smooth hammerhead shark may be threatened or endangered because of any one or a combination of the following five ESA section 4(a)(1) factors: (1) present or threatened destruction, modification, or curtailment of its habitat or range; (2) over utilization for commercial, recreational, scientific, or educational purposes; (3) disease or predation; (4) inadequacy of existing regulatory mechanisms; (5) or other natural or manmade factors affecting its continued existence. The available scientific literature indicates that the smooth hammerhead shark populations have declined in multiple regions. Smooth hammerhead sharks may warrant listing due to ongoing threats of over utilization for commercial purposes by global fisheries that target and retain incidental catch of these species to obtain their high-value fins, possible inadequacies in global regulatory mechanisms to control this level of exploitation, and natural factors (such as inherent biological vulnerabilities) that may be exacerbating these threats. Based on these findings, the smooth hammerhead shark may warrant listing as a threatened or endangered species under the ESA and a status review of the species is currently being conducted.

Evaluation of a Petition Requesting National Marine Fisheries Service (NMFS) to List the Smooth Hammerhead Shark (Sphryna zygaena) as a Threatened or Endangered Species Under the Endangered Species Act (ESA)

NASA Astrophysics Data System (ADS)

Sturm, A. B.

2016-02-01

The wildlife conservation organization, Defenders of Wildlife, petitioned NMFS to list the smooth hammerhead shark, Sphryna zygaena, as endangered or threatened throughout its range under the ESA. The petition was critically evaluated to determine if the petitioners presented substantial scientific or commercial information indicating that the smooth hammerhead shark may warrant listing under the ESA. The petition and the cited scientific literature (as well as scientific literature readily available in NMFS files) were evaluated to determine if the smooth hammerhead shark may be threatened or endangered because of any one or a combination of the following five ESA section 4(a)(1) factors: (1) present or threatened destruction, modification, or curtailment of its habitat or range; (2) over utilization for commercial, recreational, scientific, or educational purposes; (3) disease or predation; (4) inadequacy of existing regulatory mechanisms; (5) or other natural or manmade factors affecting its continued existence. The available scientific literature indicates that the smooth hammerhead shark populations have declined in multiple regions. Smooth hammerhead sharks may warrant listing due to ongoing threats of over utilization for commercial purposes by global fisheries that target and retain incidental catch of these species to obtain their high-value fins, possible inadequacies in global regulatory mechanisms to control this level of exploitation, and natural factors (such as inherent biological vulnerabilities) that may be exacerbating these threats. Based on these findings, the smooth hammerhead shark may warrant listing as a threatened or endangered species under the ESA and a status review of the species is currently being conducted.

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

PubMed Central

Roychowdhury-Saha, Manami; Burke, Donald H.

2007-01-01

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role. PMID:17456566

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

PubMed Central

de Silva, Chamaree; Walter, Nils G.

2009-01-01

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer. PMID:19029309

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

PubMed Central

Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

1995-01-01

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

PubMed Central

Kore, A R; Vaish, N K; Kutzke, U; Eckstein, F

1998-01-01

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH. PMID:9722629

76 FR 72891 - Endangered and Threatened Wildlife; 90-Day Finding on a Petition To List the Scalloped Hammerhead...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-28

... hammerhead shark as threatened or endangered under the Endangered Species Act (ESA), and to designate... NatureServe for listing under the U.S. Endangered Species Act'' because NatureServe assessments ``have... Headquarters Office (see ADDRESSES). Authority The authority for this action is the Endangered Species Act of...

The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

PubMed Central

Przybilski, Rita; Hammann, Christian

2007-01-01

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. PMID:17666711

Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

NASA Astrophysics Data System (ADS)

Patwardhan, Anjali A.

The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.

PubMed

Bellon, L; Maloney, L; Zinnen, S P; Sandberg, J A; Johnson, K E

2000-08-01

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine. Copyright 2000 Academic Press.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

NASA Astrophysics Data System (ADS)

El-Murr, Nizar; Maurel, Marie-Christine; Rihova, Martina; Vergne, Jacques; Hervé, Guy; Kato, Mikio; Kawamura, Kunio

2012-09-01

The "RNA world" hypothesis proposes that—early in the evolution of life—RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg2+, which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg2+ on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na+ or K+), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

Serum relaxin concentrations and reproduction in male bonnethead sharks, Sphyrna tiburo.

PubMed

Gelsleichter, James; Steinetz, Bernard G; Manire, Charles A; Ange, Cristal

2003-06-01

Relaxin is a 6-kd polypeptide hormone that is responsible for regulating several reproductive processes in female vertebrates, but its role in male reproduction remains unclear. To aid in clarifying this role, the objective of the present study was to investigate changes in endogenous relaxin levels associated with reproductive events in male elasmobranchs, which represent one of only three vertebrate groups known to possess this hormone. Serum relaxin concentrations were measured in 27 immature and 66 mature male bonnethead sharks (Sphyrna tiburo), a species with a well-characterized, seasonal reproductive cycle. Temporal changes in serum relaxin concentrations of immature male S. tiburo were not observed. In contrast, a temporal cycle in serum relaxin concentrations of mature male S. tiburo was observed in individuals from two sampling locations. Significant increases (P<0.05) in serum relaxin concentrations of mature male S. tiburo from both collection sites occurred during late spermatogenesis and the mating period, two critical stages of the reproductive cycle. The results from this study suggest that relaxin may play an important role in regulating semen quality, or other aspects of reproduction in male sharks. This is the first study to demonstrate a temporal pattern in endogenous serum Rlx concentrations associated with reproductive events in feral vertebrates. As such, it strengthens earlier hypotheses that suggested a role for this hormone in regulating male vertebrate fertility and copulatory success.

Hammerhead Ribozyme against γ‐Glutamylcysteine Synthetase Attenuates Resistance to Ionizing Radiation and Cisplatin in Human T98G Glioblastoma Cells

PubMed Central

Tani, Masaharu; Goto, Shinji; Kamada, Kensaku; Mori, Katsuharu; Urata, Yoshishige; Ihara, Yoshito; Kijima, Hiroshi; Ueyama, Yoshito; Shibata, Shobu

2002-01-01

Glioblastoma cells are highly malignant and show resistance to ionizing radiation, as well as anti‐cancer drugs. This resistance to cancer therapy is often associated with a high concentration of glutathione (GSH). In this study, the effect of continuous down‐regulation of γ‐glutamylcysteine synthetase (γ‐GCS) expression, a rate‐limiting enzyme for GSH synthesis, on resistance to ionizing radiation and cisplatin (CDDP) was studied in T98G human glioblastoma cells. We constructed a hammerhead ribozyme against a γ‐GCS heavy subunit (γ‐GCSh) mRNA and transfected it into T98G cells. (1) The transfection of the ribozyme decreased the concentration of GSH and resulted in G1 cell cycle arrest of T98G cells. (2) The transfection of the ribozyme increased the cytotoxicity of ionizing radiation and CDDP in T98G cells. Thus, hammerhead ribozyme against γ‐GCS is suggested to have potential as a cancer gene therapy to reduce the resistance of malignant cells to ionizing radiation and anti‐cancer drugs. PMID:12079521

Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

PubMed Central

Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

2000-01-01

The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere

Characteristics of trapping various organophosphorus pesticides with a ferritin reactor of shark liver (Sphyrna zygaena).

PubMed

Huang, He-Qing; Xiao, Zhi-Qun; Lin, Qing-Mei; Chen, Ping

2005-03-15

A reactor is composed of liver ferritin of Sphyrna zygaena (SZLF) and an oscillating bag. A reactive procedure for trapping various organphosphorus pesticides (OPs) with the SZLF reactor in the flowing water is described in detail, showing the maximal trapping numbers of 28 +/-1.0 dichlorovos/SZLF, 42 +/- 1.0 dimethoate/SZLF, and 55 +/- 1.0 methamido-phos/SZLF determined by a improved spectrophotometric method in 12 h. In addition, it is found that the OP numbers trapped by the reactor increase along with the incubation time and its concentration increment in the flowing water (or seawater), respectively. This trapping capacity is considered to depend on the composition of amino acids on the surface of the ferritin shell interior rather than the available volume within the shell. A novel pathway for trapping various OPs with the ferritin is suggested in reference to unstable characteristics of the protein subunits. We claim that the ferritin reactor will be employed to monitor the contamination level of various OPs in the flowing water continuously.

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

PubMed Central

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

2016-01-01

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction. PMID:26398724

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

PubMed

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L

2015-10-20

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

PubMed Central

Conaty, J; Hendry, P; Lockett, T

1999-01-01

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system. PMID:10325431

Great hammerhead sharks swim on their side to reduce transport costs

PubMed Central

Payne, Nicholas L.; Iosilevskii, Gil; Barnett, Adam; Fischer, Chris; Graham, Rachel T.; Gleiss, Adrian C.; Watanabe, Yuuki Y.

2016-01-01

Animals exhibit various physiological and behavioural strategies for minimizing travel costs. Fins of aquatic animals play key roles in efficient travel and, for sharks, the functions of dorsal and pectoral fins are considered well divided: the former assists propulsion and generates lateral hydrodynamic forces during turns and the latter generates vertical forces that offset sharks' negative buoyancy. Here we show that great hammerhead sharks drastically reconfigure the function of these structures, using an exaggerated dorsal fin to generate lift by swimming rolled on their side. Tagged wild sharks spend up to 90% of time swimming at roll angles between 50° and 75°, and hydrodynamic modelling shows that doing so reduces drag—and in turn, the cost of transport—by around 10% compared with traditional upright swimming. Employment of such a strongly selected feature for such a unique purpose raises interesting questions about evolutionary pathways to hydrodynamic adaptations, and our perception of form and function. PMID:27457414

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

PubMed Central

Homann, M; Tabler, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo. Images PMID:7524030

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

PubMed Central

KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

2005-01-01

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3' end blocked for transesterification.

PubMed Central

Grosshans, C A; Cech, T R

1991-01-01

The L-21 Scal form of the Tetrahymena ribozyme acts as a sequence-specific endonuclease. This ribozyme has a homogeneous 5' end but a somewhat heterogeneous 3' end, as is typical of RNA synthesized by transcription in vitro. To produce a more homogeneous ribozyme for both structural and enzymological studies, a hammerhead ribozyme was inserted at the 3' end of the Tetrahymena ribozyme. During transcription the hammerhead moiety self-cleaves to produce the L-21 A Tetrahymena ribozyme, which ends at A410 with a 2',3'-cyclic phosphate terminus. The new ribozyme has endoribonuclease activity equivalent to that of L-21 Scal under conditions where binding of substrate is rate-limiting, as well as under conditions where chemical cleavage by guanosine is rate-limiting. However, the L-21 A has lost activity in oligo(C) disproportionation (e.g., 2 pC5----pC4 + pC6), consistent with the previous proposal that this reaction occurs predominantly through a covalent ribozyme-substrate intermediate involving the 3'-terminal hydroxyl group of the ribozyme. Formation of such an intermediate would be prevented by the 2',3'-cyclic phosphate terminus. Thus the L-21 A ribozyme has simplified enzymatic activity, being fully active as an endonuclease but blocked for disproportionation. Images PMID:1650453

Diversity and Abundance of a Tropical Fishery on the Pacific Shelf of Michoacán, México

NASA Astrophysics Data System (ADS)

Madrid, J.; Sánchez, P.; Ruiz, A.

1997-10-01

This study was undertaken on the Pacific coast of Mexico. Some 22 523 individual fish were sampled, representing two classes, 154 genera, 64 families and 257 species in a total area of 50 km 2. The species Lutjanus guttatus, Scomberomorus sierra, Cynoscion reticulatus, Sphyrna lewini, Arius platypogonand Caranx caninuswere the most numerically abundant, amounting to 53·2% of the whole sample. Two models for sample size analysis and richness prediction were used. From these, the forecast for a theoretical doubling of the level of sampling effort (90 days) was 297 and 331. The Shannon diversity indexes in summer (June-August) and winter (December-February) were significantly different (paired t-test, P<0·0005). Of four families compared, three gave significant differences in the summer and winter periods (the Carangidae, the Haemulidae and the Sciaenidae). The classification (Euclidean Distance and UPGMA) indicated two climatic periods and three species assemblages. In addition, the nodal fidelity analysis agreed with the importance of the winter season in the study area. Two El Niño/southern oscillation incidences from 1986-87 and 1991-93 occurred during the sampling period.

Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

PubMed Central

PENEDO, J. CARLOS; WILSON, TIMOTHY J.; JAYASENA, SUMEDHA D.; KHVOROVA, ANASTASIA; LILLEY, DAVID M.J.

2004-01-01

It has been shown that the activity of the hammerhead ribozyme at μM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the μM range of magnesium ion concentration. Disruption of the loop–loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions. PMID:15100442

Insight into the role of Mg2+ in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation

PubMed Central

Lee, Tai-Sung; López, Carlos Silva; Martick, Monika; Scott, William G.; York, Darrin M.

2008-01-01

Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg2+ ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg2+ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg2+ spends significant time closely associated with the 2′OH of G8, but remains fairly distant from the leaving group O5′ position. In the early TS mimic simulation, where the nucleophilic O2′ and leaving group O5′ are equidistant from the phosphorus, the Mg2+ ion remains tightly coordinated to the 2′OH of G8, but is positioned closer to the O5′ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg2+ ion undergoes a transition whereby the coordination with the 2′OH of G8 is replace by the leaving group O5′ that has developed significant charge. At the same time, the 2′OH of G8 forms a hydrogen bond with the leaving group O5′ and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg2+ ion in catalysis that is consistent with both crystallographic and biochemical data. PMID:19079784

Complete mitochondrial DNA genome of bonnethead shark, Sphyrna tiburo, and phylogenetic relationships among main superorders of modern elasmobranchs

PubMed Central

Díaz-Jaimes, Píndaro; Bayona-Vásquez, Natalia J.; Adams, Douglas H.; Uribe-Alcocer, Manuel

2015-01-01

Elasmobranchs are one of the most diverse groups in the marine realm represented by 18 orders, 55 families and about 1200 species reported, but also one of the most vulnerable to exploitation and to climate change. Phylogenetic relationships among main orders have been controversial since the emergence of the Hypnosqualean hypothesis by Shirai (1992) that considered batoids as a sister group of sharks. The use of the complete mitochondrial DNA (mtDNA) may shed light to further validate this hypothesis by increasing the number of informative characters. We report the mtDNA genome of the bonnethead shark Sphyrna tiburo, and compare it with mitogenomes of other 48 species to assess phylogenetic relationships. The mtDNA genome of S. tiburo, is quite similar in size to that of congeneric species but also similar to the reported mtDNA genome of other Carcharhinidae species. Like most vertebrate mitochondrial genomes, it contained 13 protein coding genes, two rRNA genes and 22 tRNA genes and the control region of 1086 bp (D-loop). The Bayesian analysis of the 49 mitogenomes supported the view that sharks and batoids are separate groups. PMID:27014583

Ubiquitous presence of the hammerhead ribozyme motif along the tree of life

PubMed Central

de la Peña, Marcos; García-Robles, Inmaculada

2010-01-01

Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements. PMID:20705646

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro

PubMed Central

Jiang, Shan; Xie, Qing; Zhang, Wei; Zhou, Xia-Qiu; Jin, You-Xin

2005-01-01

AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neorpBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with [α-32P]UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37°C in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg2+). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE (containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37°C, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis. PMID:15996037

Molecular identification and functional characteristics of peptide transporters in the bonnethead shark (Sphyrna tiburo).

PubMed

Hart, Hannah R; Evans, Andrew N; Gelsleichter, James; Ahearn, Gregory A

2016-10-01

Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark (Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H(+)-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies.

DNA barcode identification of commercial fish sold in Mexican markets.

PubMed

Sarmiento-Camacho, Stephanie; Valdez-Moreno, Martha

2018-04-24

The substitution of high-value fish species for those of lower value is common practice. Although numerous studies have addressed this issue, few have been conducted in Mexico. In this study, we sought to identify fresh fillets of fish, sharks, and rays using DNA barcodes. We analyzed material from "La Viga" in Mexico City, and other markets located on the Gulf and Caribbean coasts of Mexico. From 134 samples, we obtained sequences from 129, identified to 9 orders, 28 families, 38 genera, and 44 species. The most common species were Seriola dumerili, Pangasianodon hypophthalmus, Carcharhinus falciformis, Carcharhinus brevipinna, and Hypanus americanus. Pangasianodon hypophthalmus was most commonly used as a substitute for higher-value species. The substitution rate was 18% of the total. A review of the conservation status of the specimens identified against the IUNC list enabled us to establish that some species marketed in Mexico are threatened: Makaira nigricans, Lachnolaimus maximus, Hyporthodus flavolimbatus, and Isurus oxyrinchus are classified as vulnerable; Lopholatilus chamaeleonticeps and Sphyrna lewini are endangered; and the status of Hyporthodus nigritus is critical. These results will demonstrate to the Mexican authorities that DNA barcoding is a reliable tool for species identification, even when morphological identification is difficult or impossible.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

PubMed Central

Hammann, Christian; Norman, David G.; Lilley, David M. J.

2001-01-01

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range. PMID:11331743

Gill lesions associated with Erpocotyle tiburonis (Monogenea: Hexabothriidae) on wild and aquarium-held bonnethead sharks (Sphyrna tiburo).

PubMed

Bullard, S A; Frasca, S; Benz, G W

2001-10-01

Gill lesions associated with infections of Erpocotyle tiburonis (Brooks, 1934) (Monogenea: Hexabothriidae) on wild bonnethead sharks (Sphyrna tiburo (L., 1758) (Carcharhiniformes: Sphyrinidae)) were compared with those on aquarium-held ones using light and scanning electron microscopy. Uninfected gill filaments had slender, triangular, smooth-surfaced lamellae and interlamellar water channels that were approximately equal in size. Four wild sharks were each infected by 3-11 widely separated adult E. tiburonis, and 1 of these sharks hosted a juvenile specimen. Lamellae flanking or touching adult E. tiburonis were pushed aside or bent, but were otherwise identical to those of uninfected filaments. Two aquarium-held sharks were each infected by hundreds of juvenile and adult E. tiburonis. In these sharks, lamellae near juveniles were pushed apart or bent, but were otherwise normal, whereas a thick, ragged-surfaced layer of hyperplastic epithelium both filled interlamellar water channels and partially or completely covered lamellae near adults. Results of this study suggest that the intense infections of E. tiburonis were facilitated by captivity and caused severe hyperplastic lesions that ultimately led to the death of the sharks by reducing or blocking the respiratory water flow over lamellae and thus reducing the exchange of gases and ions across the lamellar epithelium. In contrast, the wild sharks were infected by fewer worms and exhibited relatively minor lesions.

Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue.

PubMed

Mir, Aamir; Golden, Barbara L

2016-02-02

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

Robust Sub-harmonic Mixer at 340 GHz Using Intrinsic Resonances of Hammer-Head Filter and Improved Diode Model

NASA Astrophysics Data System (ADS)

Wang, Cheng; He, Yue; Lu, Bin; Jiang, Jun; Miao, Li; Deng, Xian-Jin; Xiong, Yong-zhong; Zhang, Jian

2017-11-01

This paper presents a sub-harmonic mixer at 340 GHz based on anti-parallel Schottky diodes (SBDs). Intrinsic resonances in low-pass hammer-head filter have been adopted to enhance the isolation for different harmonic components, while greatly minimizing the transmission loss. The application of new DC grounding structure, impedance matching structure, and suspended micro-strip mitigates the negative influences of fabrication errors from metal cavity, quartz substrate, and micro-assembly. An improved lumped element equivalent circuit model of SBDs guarantees the accuracy of simulation, which takes current-voltage (I/V) behavior, capacitance-voltage (C/V) behavior, carrier velocity saturation, DC series resistor, plasma resonance, skin effect, and four kinds of noise generation mechanisms into consideration thoroughly. The measurement indicates that with local oscillating signal of 2 mW, the lowest double sideband conversion loss is 5.5 dB at 339 GHz; the corresponding DSB noise temperature is 757 K. The 3 dB bandwidth of conversion loss is 50 GHz from 317 to 367 GHz.

PLASMA PROTEIN ELECTROPHORESIS AND SELECT ACUTE PHASE PROTEINS IN HEALTHY BONNETHEAD SHARKS (SPHYRNA TIBURO) UNDER MANAGED CARE.

PubMed

Hyatt, Michael W; Field, Cara L; Clauss, Tonya M; Arheart, Kristopher L; Cray, Carolyn

2016-12-01

Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks ( Sphyrna tiburo ) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at -80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, β globulin, and γ globulin. These fractions were classified as fractions 1-5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.

Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir, Aamir; Golden, Barbara L.

2015-11-09

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the p K A of G12. Finally, on the basis ofmore » this crystal structure as well as a wealth of biochemical studies, in this paper we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.« less

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

PubMed Central

Inoue, Atsushi; Takagi, Yasuomi; Taira, Kazunari

2004-01-01

Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst. PMID:15302920

Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

PubMed Central

Nakano, Shu-ichi; Kitagawa, Yuichi; Miyoshi, Daisuke; Sugimoto, Naoki

2014-01-01

Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol), small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds. PMID:25161873

Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

PubMed Central

Ludwig, J; Blaschke, M; Sproat, B S

1998-01-01

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2. PMID:9580675

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena).

PubMed

Huang, He-Qing; Xiao, Zhi-Qun; Chen, Xu; Lin, Qing-Mei; Cai, Zong-Wei; Chen, Ping

2004-11-01

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

PubMed Central

Lee, Tai-Sung; Wong, Kin-Yiu; Giambasu, George M.; York, Darrin M.

2016-01-01

Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis. PMID:24156941

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

PubMed Central

Mikulecky, Peter J.; Feig, Andrew L.

2004-01-01

In proteins, empirical correlations have shown that changes in heat capacity (ΔCP) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔCP on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔCPs of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔCP of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔCP by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔCP. We weigh the strengths and limitations of this method for determining ΔCP values. Finally, we discuss the data in light of the physical origins of the ΔCPs for RNA folding and consider their impact on biological function. PMID:15282329

Serum steroid hormones including 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone in juvenile and adult bonnethead sharks, Sphyrna tiburo.

PubMed

Manire, C A; Rasmussen, L E; Gross, T S

1999-10-01

Previous studies in the placental viviparous bonnethead shark, Sphyrna tiburo, have correlated 17 beta-estradiol, progesterone, testosterone, and dihydrotestosterone with reproductive events in both males and females. However, several key reproductive events, including implantation, maintenance of pregnancy, and parturition, did not correlate with these four steroid hormones. Therefore, the present study investigated three steroid hormones, 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone, which have demonstrably important roles in the reproductive cycles of teleosts. It was hypothesized that one or more of these three hormones would correlate with specific reproductive events in S. tiburo. Concurrently, developmental (growth and/or maturation) analyses of these three steroids plus 17 beta-estradiol, progesterone, testosterone, and dihydrotestosterone were investigated in juvenile bonnethead sharks. Serum dihydroprogesterone concentrations were highest in mature females and 11-ketotestosterone concentrations were highest in mature males. In mature females, 11-ketoandrostenedione levels were elevated from the time of mating, through six months of sperm storage and another four months of gestation. At parturition concentrations became significantly lower and remained lower until mating occurred again in another two to three months. Serum 11-ketotestosterone concentrations were the highest at implantation though not significant. In mature males, significantly elevated serum levels of dihydroprogesterone occurred in April and May, near the start of annual testicular development. During growth in males, testosterone and dihydrotestosterone increased progressively and in females, testosterone increased progressively. At maturity in males, significant increases occurred in testosterone and 11-ketotestosterone concentrations while, in females, dihydroprogesterone, 11-ketotestosterone, 17 beta-estradiol, progesterone, testosterone, and

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

Disseminated fungal infection in two species of captive sharks.

PubMed

Marancik, David P; Berliner, Aimee L; Cavin, Julie M; Clauss, Tonya M; Dove, Alistair D M; Sutton, Deanna A; Wickes, Brian L; Camus, Alvin C

2011-12-01

In this report, two cases of systemic mycosis in captive sharks are characterized. These cases were progressive and ultimately culminated in terminal disease. Paecilomyces lilacinus, an uncommon pathogen in human and veterinary medicine, was associated with areas of necrosis in the liver, heart, and gill in a great hammerhead shark (Sphyrna mokarran). Fungal growth was observed from samples of kidney, spleen, spinal fluid, and coelomic cavity swabs. Dual fungal infection by Exophiala pisciphila and Mucor circinelloides was diagnosed in a juvenile zebra shark (Stegostoma fasciatum). Both fungi were present in the liver, with more severe tissue destruction associated with E. pisciphila. E. pisciphila also produced significant necrosis in the spleen and gill, while M. circinelloides was associated with only minimal tissue changes in the heart. Fungal cultures from liver, kidney, and spleen were positive for both E. pisciphila and M. circinelloides. Identification of P. lilacinus and M. circinelloides was based on colonial and hyphal morphology. E. pisciphila was identified by sequence analysis of the 28S rRNA D1/D2 region and the internal transcribed spacer (ITS) region between the 18S and 28S rRNA subunit. These cases, and a lack of information in the literature, highlight the need for further research and diagnostic sampling to further characterize the host-pathogen interaction between elasmobranchs and fungi.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water

NASA Technical Reports Server (NTRS)

Sarma, R. H.; Sarma, M. H.; Rein, R.; Shibata, M.; Setlik, R. S.; Ornstein, R. L.; Kazim, A. L.; Cairo, A.; Tomasi, T. B.

1995-01-01

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Inducible Transgenic Models of BRCA1 Function

DTIC Science & Technology

1999-10-01

inducible expression vectors were created to conditionally express four different hammerhead ribozymes designed to specifically cleave the Brca...transcript. Hammerhead ribozymes are catalytic RNAs that efficiently cleave RNA and thereby down- regulate gene expression. Hammerhead ribozymes can cleave...any RNA containing its 5’-UH-3’ consensus sequence where U can be replaced by a C, and H=C, U or A. Hammerhead ribozymes effectively and selectively

Inducible Transgenic Models of BRCA1 Function

DTIC Science & Technology

2000-10-01

four different hammerhead ribozymes designed to specifically cleave the Brcal transcript. Hammerhead ribozymes are catalytic RNAs that efficiently...cleave RNA and thereby down- regulate gene expression. Hammerhead ribozymes can cleave any RNA containing a 5’-UH-3’ consensus sequence where U can be...replaced by C, and H=C, U or A. Hammerhead ribozymes have been shown to effectively and selectively inhibit gene expression in bacteria, plants, cell

Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

PubMed Central

Jankowsky, E; Strunk, G; Schwenzer, B

1997-01-01

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme

NASA Technical Reports Server (NTRS)

Setlik, R. F.; Shibata, M.; Sarma, R. H.; Sarma, M. H.; Kazim, A. L.; Ornstein, R. L.; Tomasi, T. B.; Rein, R.

1995-01-01

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.

PubMed Central

Williams, D M; Pieken, W A; Eckstein, F

1992-01-01

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed. Images PMID:1736306

78 FR 42021 - Atlantic Highly Migratory Species; Commercial Gulf of Mexico Aggregated Large Coastal Shark and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-15

... Large Coastal Shark and Gulf of Mexico Hammerhead Shark Management Groups AGENCY: National Marine... coastal sharks (LCS) and hammerhead sharks in the Gulf of Mexico region. This action is necessary because... aggregated LCS and Gulf of Mexico hammerhead shark management groups are closed effective 11:30 p.m. local...

Baseline health parameters and species comparisons among free-ranging Atlantic sharpnose (Rhizoprionodon terraenovae), bonnethead (Sphyrna tiburo), and spiny dogfish (Squalus acanthias) sharks in Georgia, Florida, and Washington, USA.

PubMed

Haman, Katherine H; Norton, Terry M; Thomas, Austen C; Dove, Alistair D M; Tseng, Florina

2012-04-01

Sharks are of commercial, research, conservation, and exhibition importance but we know little regarding health parameters and population status for many species. Here we present health indicators and species comparisons for adults of three common wild-caught species: 30 Atlantic sharpnose sharks (Rhizoprionodon terraenovae) and 31 bonnethead sharks (Sphyrna tiburo) from the western Atlantic, and 30 spiny dogfish sharks (Squalus acanthias) from the eastern Pacific. All animals were captured during June-July 2009 and 2010. Median values and preliminary reference intervals were calculated for hematology, plasma biochemistry, trace nutrients, and vitamin A, E, and D concentrations. Significant differences, attributable to physiologic differences among the species, were found in the basic hematologic and plasma biochemistry variables. Significant species differences in arsenic and selenium plasma concentrations were found and appear to coincide with diet and habitat variability among these three species. Vitamin E was significantly higher in the bonnethead shark, again related to the foraging ecology and ingestion of plant material by this species. The Atlantic sharpnose had significantly higher vitamin A concentrations, supported by the higher proportion of teleosts in the diet. Vitamin D was below the limit of quantification in all three species. These preliminary reference intervals for health variables can be used to assess and monitor the population health and serve as indicators of nutritional status in these populations of wild elasmobranchs.

Mercury accumulation in sharks from the coastal waters of southwest Florida.

PubMed

Rumbold, Darren; Wasno, Robert; Hammerschlag, Neil; Volety, Aswani

2014-10-01

As large long-lived predators, sharks are particularly vulnerable to exposure to methylmercury biomagnified through the marine food web. Accordingly, nonlethal means were used to collect tissues for determining mercury (Hg) concentrations and stable isotopes of carbon (δ(13)C) and nitrogen (δ(15)N) from a total of 69 sharks, comprising 7 species, caught off Southwest Florida from May 2010 through June 2013. Species included blacknose (Carcharhinus acronotus), blacktip (C. limbatus), bull (C. leucas), great hammerhead (Sphyrna mokarran), lemon (Negaprion brevirostris), sharpnose (Rhizoprionodon terraenovae), and tiger sharks (Galeocerdo cuvier). The sharks contained Hg concentrations in their muscle tissues ranging from 0.19 mg/kg (wet-weight basis) in a tiger shark to 4.52 mg/kg in a blacktip shark. Individual differences in total length and δ(13)C explained much of the intraspecific variation in Hg concentrations in blacknose, blacktip, and sharpnose sharks, but similar patterns were not evident for Hg and δ(15)N. Interspecific differences in Hg concentration were evident with greater concentrations in slower-growing, mature blacktip sharks and lower concentrations in faster-growing, young tiger sharks than other species. These results are consistent with previous studies reporting age-dependent growth rate can be an important determinant of intraspecific and interspecific patterns in Hg accumulation. The Hg concentrations observed in these sharks, in particular the blacktip shark, also suggested that Hg may pose a threat to shark health and fitness.

A revision of Phoreiobothrium (Tetraphyllidea: Onchobothriidae) with descriptions of five new species.

PubMed

Caira, J N; Richmond, C; Swanson, J

2005-10-01

Five new species of Phoreiobothrium (Cestoda: Tetraphyllidea) are described from carcharhiniform sharks collected from a diversity of localities throughout the world. These are Phoreiobothrium perilocrocodilus n. sp. from Negaprion acutidens, Phoreiobothrium anticaporum n. sp. from Negaprion brevirostris, Phoreiobothrium lewinense n. sp. from Sphyrna lewini, Phoreiobothrium robertsoni n. sp. from Carcharhinus brachyurus, and Phoreiobothrium blissorum n. sp. from C. plumbeus, This brings the number of described species of Phoreiobothrium to 17. The new species from the lemon sharks, P. perilocrocodilus n. sp. and P. anticaporum n. sp., both possess more bothridial subloculi than do all species from hammerhead sharks, and fewer subloculi than do all species from sharks of Carcharhinus spp. Phoreiobothrium anticaporum n. sp. is unique among Phoreiobothrium species in that its genital pore is located in the extreme anterior portion of the proglottid. Phoreiobothrium lewinense n. sp. is unique in its possession of a combination of 8-11 subloculi and basal hook prongs that lack internal channels. Phoreiobothrium robertsoni n. sp. possesses 25-29 subloculi, triangular bothridia that flare posteriorly, and hooks that extend well beyond the middle of the bothridia. Phoreiobothrium blissorum n. sp. is most similar to Phoreiobothrium lasium in that it possesses rectangular bothridia with greater than 22 subloculi and long filitriches on its scolex. However, it differs from the latter species in that rather than articulating with one another, the bases of the medial and lateral hooks are widely separated; it also possesses 5-6 (vs.) 7-8 columns of testes. Phoreiobothrium lasium and Phoreiobothrium tiburonis are redescribed. The host associations of P. lasium are reevaluated and a neotype is designated for this species. The mature proglottid of P. tiburonis is described for the first time from voucher material collected from the type host and locality. These data suggest that

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

PubMed Central

Daròs, J A; Marcos, J F; Hernández, C; Flores, R

1994-01-01

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7809126

Nervus terminalis ganglion of the bonnethead shark (Sphyrna tiburo): evidence for cholinergic and catecholaminergic influence on two cell types distinguished by peptide immunocytochemistry.

PubMed

White, J; Meredith, M

1995-01-16

The nervus terminalis is a ganglionated vertebrate cranial nerve of unknown function that connects the brain and the peripheral nasal structures. To investigate its function, we have studied nervus terminalis ganglion morphology and physiology in the bonnethead shark (Sphyrna tiburo), where the nerve is particularly prominent. Immunocytochemistry for gonadotropin-releasing hormone (GnRH) and Leu-Pro-Leu-Arg-Phe-NH2 (LPLRFamide) revealed two distinct populations of cells. Both were acetylcholinesterase positive, but LPLR-Famide-immunoreactive cells consistently stained more darkly for acetylcholinesterase activity. Tyrosine hydroxylase immunocytochemistry revealed fibers and terminal-like puncta in the ganglion, primarily in areas containing GnRH-immunoreactive cells. Consistent with the anatomy, in vitro electrophysiological recordings provided evidence for cholinergic and catecholaminergic actions. In extracellular recordings, acetylcholine had a variable effect on baseline ganglion cell activity, whereas norepinephrine consistently reduced activity. Electrical stimulation of the nerve trunks suppressed ganglion activity, as did impulses from the brain in vivo. During electrical suppression, acetylcholine consistently increased activity, and norepinephrine decreased activity. Muscarinic and, to a lesser extent, alpha-adrenergic antagonists both increased activity during the electrical suppression, suggesting involvement of both systems. Intracellular recordings revealed two types of ganglion cells that were distinguishable pharmacologically and physiologically. Some cells were hyperpolarized by cholinergic agonists and unaffected by norepinephrine; these cells did not depolarize with peripheral nerve trunk stimulation. Another group of cells did depolarize with peripheral trunk stimulation; a representative of this group was depolarized by carbachol and hyperpolarized by norepinephrine. These and other data suggest that the bonnethead nervus terminalis ganglion

Ribozyme Targeting the Novel Fusion Junction of EGFRvIII in Breast Cancer

DTIC Science & Technology

2003-07-01

targeting the novel junction of EGFRvyII. * Demonstrate the therapeutic efficacy of an anti-EGFRvIll hammerhead ribozyme targeting the endogenous...first demonstration of the therapeutic efficacy of an anti-EGFRvlII hammerhead ribozyme targeting the endogenous EGFRvAII expression against human...202-687-7505.designed and generated a tumor specific hammerhead ribozyme E-mail: Tangc@georgetown.edu targeted to the novel fusion junction of

Digestive enzyme activities in the guts of bonnethead sharks (Sphyrna tiburo) provide insight into their digestive strategy and evidence for microbial digestion in their hindguts.

PubMed

Jhaveri, Parth; Papastamatiou, Yannis P; German, Donovan P

2015-11-01

Few investigations have studied digestive enzyme activities in the alimentary tracts of sharks to gain insight into how these organisms digest their meals. In this study, we examined the activity levels of proteases, carbohydrases, and lipase in the pancreas, and along the anterior intestine, spiral intestine, and colon of the bonnethead shark, Sphyrna tiburo. We then interpreted our data in the context of a rate-yield continuum to discern this shark's digestive strategy. Our data show anticipated decreasing patterns in the activities of pancreatic enzymes moving posteriorly along the gut, but also show mid spiral intestine peaks in aminopeptidase and lipase activities, which support the spiral intestine as the main site of absorption in bonnetheads. Interestingly, we observed spikes in the activity levels of N-acetyl-β-D-glucosaminidase and β-glucosidase in the bonnethead colon, and these chitin- and cellulose-degrading enzymes, respectively, are likely of microbial origin in this distal gut region. Taken in the context of intake and relatively long transit times of food through the gut, the colonic spikes in N-acetyl-β-D-glucosaminidase and β-glucosidase activities suggest that bonnetheads take a yield-maximizing strategy to the digestive process, with some reliance on microbial digestion in their hindguts. This is one of the first studies to examine digestive enzyme activities along the gut of any shark, and importantly, the data match with previous observations that sharks take an extended time to digest their meals (consistent with a yield-maximizing digestive strategy) and that the spiral intestine is the primary site of absorption in sharks. Copyright © 2015 Elsevier Inc. All rights reserved.

Knocking on FXR's door: the "hammerhead"-structure series of FXR agonists - amphiphilic isoxazoles with potent in vitro and in vivo activities.

PubMed

Gege, Christian; Kinzel, Olaf; Steeneck, Christoph; Schulz, Andreas; Kremoser, Claus

2014-01-01

The Farnesoid X Receptor (FXR) was recently validated in clinical studies using the bile acid analogue Obeticholic Acid (OCA) as an attractive drug target for liver diseases such as Primary Biliary Cirrhosis (PBC) or Non-alcoholic Steatohepatitis (NASH). OCA, however, turned out to induce cholesterol- related side effects upon prolonged treatment and it shows bile acid like pharmacokinetics. The quest for synthetic non-steroidal FXR agonists with general drug likeliness and improved pharmacokinetic and - dynamic properties has started more than a decade ago: The first non-steroidal and selective FXR agonist with decent submicromolar potency, GW4064, was patented in 1998 and published in 2000. Since then, many pharmaceutical companies have taken GW4064 as a structural template for their efforts in identifying novel patentable FXR agonists with the GW-derived trisubstituted isoxazole general structure. However, so far only one compound out of these different series has made it into the early stages of clinical development: The Px-102/Px-104 from Phenex is currently tested in a phase IIa study in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). In this review we try to summarize from the patent and scientific literature the attempts to improve the GW4064 structure into different directions. Furthermore, we suggest directions for further improvements of this special class of synthetic FXR agonists which all display the typical "hammerhead"-conformation in the FXR ligand binding pocket that provides the basis for their impressive in vitro and in vivo potencies.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

PubMed Central

Lott, William B.; Pontius, Brian W.; von Hippel, Peter H.

1998-01-01

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 μM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and >50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg2+–La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction. PMID:9435228

Study the Pathogenic Role of ErbB-3, ErbB-4 and their Ligand Heregulin in Human Breast Cancer Cell

DTIC Science & Technology

1999-07-01

implicated in human breast cancers. To delineate the biological function of ErbB-4 receptors in breast cancer, we employed a hammerhead ribozyme strategy to...receptors for neuregulin (23, 24). Activation cancer, we generated three specific hammerhead ribozymes targeted to of ErbB-2 by NRGI-a is thought to...generated three specific hammerhead ribozymes targeted to specific sites within ErbB-4 mRNA. These ErbB-4 ribozymes (Rz6, INTRODUCTION Rz2I, and Rz29

Rolling Circle Transcription of Ribozymes Targeted to ras and mdr-1

DTIC Science & Technology

2001-09-01

ssDNA) to direct transcription of an tion-PCR, and recyclization were carried out to optimize active hammerhead ribozyme in E. coli cells. transcription...transcription I hammerhead ribozyme I in vitro selection and 12.5 units/ml RNase inhibitor (Promega), in a total reaction volume of 15 tk1. After a...sequence encoding a ssDNA, and splint ssDNA were ethanol-precipitated and used as hammerhead ribozyme . templates to begin the next round of in vitro

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

PubMed Central

Holm, P. S.; Scanlon, K. J.; Dietel, M.

1994-01-01

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line. Images Figure 1 Figure 3 Figure 2 PMID:7914421

Study the Pathogenic Role of ErbB-3, ErbB-4 and their Ligand Heregulin in Human Breast Cancer Cells.

DTIC Science & Technology

1997-07-01

characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes , Rz6...proposal we plan to utilize hammerhead ribozymes which target HRG and its receptors (ErbB-3, ErbB-4) to interrupt their signaling. We will then study...cancer. Results: To assist in characterizing the role of ErbB-4 in breast cancer, we have generated three specific hammerhead ribozymes (Rz6, Rz2 1

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

PubMed Central

Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

1993-01-01

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

Publications - GMC 116 | Alaska Division of Geological & Geophysical

Science.gov Websites

hydrocarbon data) of cuttings from OCS Y-0849-1 (Hammerhead #1) well Authors: Unocal Corporation Publication data) of cuttings from OCS Y-0849-1 (Hammerhead #1) well: Alaska Division of Geological &

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1998-06-01

and tested a hammerhead ribozyme [20, 37] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the GUC triplet at nucleotides 18-20...UACACGG- 3’ "A UACICGG31 AAGGCC AUGUGCC-5’ IIII C AGCCGG U A G GA U FIG. 1. CR-l-specific hammerhead ribozyme . (A) Diagram of the processed CR-i mRNA...the CR-1 message and ribozyme is shown RT-PCR reactions were electrophoresed through 1.5% agarose-TAE in Figure 1A, and the folded hammerhead , bound

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1999-06-01

4). T.O. 2 We have designed and tested a hammerhead ribozyme [22, 23] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the GUC...III C AGCCGG U A G GA U FIG. 1. CR-i-specific hammerhead ribozyme . (A) Diagram of the processed CR-1 mRNA, showing the ribozyme recognition sequence...diagram of the CR-1 message and ribozyme is shown RT-PCR reactions were electrophoresed through 1.5% agarose-TAE in Figure 1A, and the folded hammerhead

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3′ end can be exported efficiently and specifically to the cytoplasm in mammalian cells

PubMed Central

Kuwabara, Tomoko; Warashina, Masaki; Koseki, Shiori; Sano, Masayuki; Ohkawa, Jun; Nakayama, Kazuhisa; Taira, Kazunari

2001-01-01

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNAVal-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3′ end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5′ and 3′ ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells. PMID:11433023

Shark fisheries in the Southeast Pacific: A 61-year analysis from Peru

PubMed Central

Gonzalez-Pestana, Adriana; Kouri J., Carlos; Velez-Zuazo, Ximena

2016-01-01

Peruvian waters exhibit high conservation value for sharks. This contrasts with a lag in initiatives for their management and a lack of studies about their biology, ecology and fishery. We investigated the dynamics of Peruvian shark fishery and its legal framework identifying information gaps for recommending actions to improve management. Further, we investigated the importance of the Peruvian shark fishery from a regional perspective. From 1950 to 2010, 372,015 tons of sharks were landed in Peru. From 1950 to 1969, we detected a significant increase in landings; but from 2000 to 2011 there was a significant decrease in landings, estimated at 3.5% per year. Six species represented 94% of landings: blue shark ( Prionace glauca), shortfin mako ( Isurus oxyrinchus), smooth hammerhead ( Sphyrna zygaena), common thresher ( Alopias vulpinus), smooth-hound ( Mustelus whitneyi) and angel shark ( Squatina californica). Of these, the angel shark exhibits a strong and significant decrease in landings: 18.9% per year from 2000 to 2010. Peru reports the highest accumulated historical landings in the Pacific Ocean; but its contribution to annual landings has decreased since 1968. Still, Peru is among the top 12 countries exporting shark fins to the Hong Kong market. Although the government collects total weight by species, the number of specimens landed as well as population parameters (e.g. sex, size and weight) are not reported. Further, for some genera, species-level identification is deficient and so overestimates the biomass landed by species and underestimates the species diversity. Recently, regional efforts to regulate shark fishery have been implemented to support the conservation of sharks but in Peru work remains to be done. PMID:27635216

Big catch, little sharks: Insight into Peruvian small-scale longline fisheries.

PubMed

Doherty, Philip D; Alfaro-Shigueto, Joanna; Hodgson, David J; Mangel, Jeffrey C; Witt, Matthew J; Godley, Brendan J

2014-06-01

Shark take, driven by vast demand for meat and fins, is increasing. We set out to gain insights into the impact of small-scale longline fisheries in Peru. Onboard observers were used to document catch from 145 longline fishing trips (1668 fishing days) originating from Ilo, southern Peru. Fishing effort is divided into two seasons: targeting dolphinfish (Coryphaena hippurus; December to February) and sharks (March to November). A total of 16,610 sharks were observed caught, with 11,166 identified to species level. Of these, 70.6% were blue sharks (Prionace glauca), 28.4% short-fin mako sharks (Isurus oxyrinchus), and 1% were other species (including thresher (Alopias vulpinus), hammerhead (Sphyrna zygaena), porbeagle (Lamnus nasus), and other Carcharhinidae species (Carcharhinus brachyurus, Carcharhinus falciformis, Galeorhinus galeus). Mean ± SD catch per unit effort of 33.6 ± 10.9 sharks per 1000 hooks was calculated for the shark season and 1.9 ± 3.1 sharks per 1000 hooks were caught in the dolphinfish season. An average of 83.7% of sharks caught (74.7% blue sharks; 93.3% mako sharks) were deemed sexually immature and under the legal minimum landing size, which for species exhibiting k-selected life history traits can result in susceptibility to over exploitation. As these growing fisheries operate along the entire Peruvian coast and may catch millions of sharks per annum, we conclude that their continued expansion, along with ineffective legislative approaches resulting in removal of immature individuals, has the potential to threaten the sustainability of the fishery, its target species, and ecosystem. There is a need for additional monitoring and research to inform novel management strategies for sharks while maintaining fisher livelihoods.

Big catch, little sharks: Insight into Peruvian small-scale longline fisheries

PubMed Central

Doherty, Philip D; Alfaro-Shigueto, Joanna; Hodgson, David J; Mangel, Jeffrey C; Witt, Matthew J; Godley, Brendan J

2014-01-01

Shark take, driven by vast demand for meat and fins, is increasing. We set out to gain insights into the impact of small-scale longline fisheries in Peru. Onboard observers were used to document catch from 145 longline fishing trips (1668 fishing days) originating from Ilo, southern Peru. Fishing effort is divided into two seasons: targeting dolphinfish (Coryphaena hippurus; December to February) and sharks (March to November). A total of 16,610 sharks were observed caught, with 11,166 identified to species level. Of these, 70.6% were blue sharks (Prionace glauca), 28.4% short-fin mako sharks (Isurus oxyrinchus), and 1% were other species (including thresher (Alopias vulpinus), hammerhead (Sphyrna zygaena), porbeagle (Lamnus nasus), and other Carcharhinidae species (Carcharhinus brachyurus, Carcharhinus falciformis, Galeorhinus galeus). Mean ± SD catch per unit effort of 33.6 ± 10.9 sharks per 1000 hooks was calculated for the shark season and 1.9 ± 3.1 sharks per 1000 hooks were caught in the dolphinfish season. An average of 83.7% of sharks caught (74.7% blue sharks; 93.3% mako sharks) were deemed sexually immature and under the legal minimum landing size, which for species exhibiting k-selected life history traits can result in susceptibility to over exploitation. As these growing fisheries operate along the entire Peruvian coast and may catch millions of sharks per annum, we conclude that their continued expansion, along with ineffective legislative approaches resulting in removal of immature individuals, has the potential to threaten the sustainability of the fishery, its target species, and ecosystem. There is a need for additional monitoring and research to inform novel management strategies for sharks while maintaining fisher livelihoods. PMID:25360274

Shark fisheries in the Southeast Pacific: A 61-year analysis from Peru.

PubMed

Gonzalez-Pestana, Adriana; Kouri J, Carlos; Velez-Zuazo, Ximena

2014-01-01

Peruvian waters exhibit high conservation value for sharks. This contrasts with a lag in initiatives for their management and a lack of studies about their biology, ecology and fishery. We investigated the dynamics of Peruvian shark fishery and its legal framework identifying information gaps for recommending actions to improve management. Further, we investigated the importance of the Peruvian shark fishery from a regional perspective. From 1950 to 2010, 372,015 tons of sharks were landed in Peru. From 1950 to 1969, we detected a significant increase in landings; but from 2000 to 2011 there was a significant decrease in landings, estimated at 3.5% per year. Six species represented 94% of landings: blue shark ( Prionace glauca), shortfin mako ( Isurus oxyrinchus), smooth hammerhead ( Sphyrna zygaena), common thresher ( Alopias vulpinus), smooth-hound ( Mustelus whitneyi) and angel shark ( Squatina californica). Of these, the angel shark exhibits a strong and significant decrease in landings: 18.9% per year from 2000 to 2010. Peru reports the highest accumulated historical landings in the Pacific Ocean; but its contribution to annual landings has decreased since 1968. Still, Peru is among the top 12 countries exporting shark fins to the Hong Kong market. Although the government collects total weight by species, the number of specimens landed as well as population parameters (e.g. sex, size and weight) are not reported. Further, for some genera, species-level identification is deficient and so overestimates the biomass landed by species and underestimates the species diversity. Recently, regional efforts to regulate shark fishery have been implemented to support the conservation of sharks but in Peru work remains to be done.

Toxic Neuronal Death by Glyeraldehyde-3-Phosphate Dehydrogenase and Mitochondria

DTIC Science & Technology

2003-08-01

Neuroreport, 10(5), 1149-1153. Sioud, M., & Jespersen, L. (1996). Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase...1996) Enhancemen t of hammerhead r ibozyme cata lysis by glycera ldehyde-3- phospha te dehydrogenase. J Mol Biol 257:775–789. Sirover MA (1997) Role of

Publications - GMC 90 | Alaska Division of Geological & Geophysical Surveys

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a -1 (Hammerhead) well Authors: Unknown Publication Date: 1988 Publisher: Alaska Division of Geological for the Union Oil Company OCS-Y-0849-1 (Hammerhead) well: Alaska Division of Geological &

Publications - GMC 118 | Alaska Division of Geological & Geophysical

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a Company OCS Y-0849-2 (Hammerhead #2) well Authors: Unknown Publication Date: 1989 Publisher: Alaska reflectance data of cuttings from the Union Oil Company OCS Y-0849-2 (Hammerhead #2) well: Alaska Division of

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

Molecular Identification of Shark Meat From Local Markets in Southern Brazil Based on DNA Barcoding: Evidence for Mislabeling and Trade of Endangered Species.

PubMed

Almerón-Souza, Fernanda; Sperb, Christian; Castilho, Carolina L; Figueiredo, Pedro I C C; Gonçalves, Leonardo T; Machado, Rodrigo; Oliveira, Larissa R; Valiati, Victor H; Fagundes, Nelson J R

2018-01-01

Elasmobranchs, the group of cartilaginous fishes that include sharks and rays, are especially vulnerable to overfishing due to low fecundity and late sexual maturation. A significant number of elasmobranch species are currently overexploited or threatened by fisheries activities. Additionally, several recent reports have indicated that there has been a reduction in regional elasmobranch population sizes. Brazil is an important player in elasmobranch fisheries and one of the largest importers of shark meat. However, carcasses entering the shark meat market have usually had their fins and head removed, which poses a challenge to reliable species identification based on the morphology of captured individuals. This is further complicated by the fact that the internal Brazilian market trades several different elasmobranch species under a common popular name: "cação." The use of such imprecise nomenclature, even among governmental agencies, is problematic for both controlling the negative effects of shark consumption and informing the consumer about the origins of the product. In this study, we used DNA barcoding (mtDNA, COI gene) to identify, at the species level, "cação" samples available in local markets from Southern Brazil. We collected 63 samples traded as "cação," which we found to correspond to 20 different species. These included two teleost species: Xiphias gladius ( n = 1) and Genidens barbus ( n = 6), and 18 species from seven elasmobranch orders (Carcharhiniformes, n = 42; Squaliformes, n = 3; Squatiniformes, n = 2; Rhinopristiformes, n = 4; Myliobatiformes, n = 3; Rajiformes, n = 1; and Torpediniformes, n = 1). The most common species in our sample were Prionace glauca ( n = 15) and Sphyrna lewini ( n = 14), while all other species were represented by four samples or less. Considering IUCN criteria, 47% of the elasmobranch species found are threatened at the global level, while 53% are threatened and 47% are critically endangered in Brazil. These

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

Selective Androgen Receptor Downregulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2006-10-01

of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol, 12: 1558...cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol...used to down-regulate the AR include antisense oligonucleotides (9, 10), ribozyme treatments (11, 12), AR dominant negatives (13) and small

Methylmercury in dried shark fins and shark fin soup from American restaurants.

PubMed

Nalluri, Deepthi; Baumann, Zofia; Abercrombie, Debra L; Chapman, Demian D; Hammerschmidt, Chad R; Fisher, Nicholas S

2014-10-15

Consumption of meat from large predatory sharks exposes human consumers to high levels of toxic monomethylmercury (MMHg). There also have been claims that shark fins, and hence the Asian delicacy shark fin soup, contain harmful levels of neurotoxic chemicals in combination with MMHg, although concentrations of MMHg in shark fins are unknown. We measured MMHg in dried, unprocessed fins (n=50) of 13 shark species that occur in the international trade of dried shark fins as well as 50 samples of shark fin soup prepared by restaurants from around the United States. Concentrations of MMHg in fins ranged from 9 to 1720 ng/g dry wt. MMHg in shark fin soup ranged from <0.01 to 34 ng/mL, with MMHg averaging 62 ± 7% of total Hg. The highest concentrations of MMHg and total Hg were observed in both fins and soup from large, high trophic level sharks such as hammerheads (Sphyrna spp.). Consumption of a 240 mL bowl of shark fin soup containing the average concentration of MMHg (4.6 ng/mL) would result in a dose of 1.1 μg MMHg, which is 16% of the U.S. EPA's reference dose (0.1 μg MMHg per 1 kg per day in adults) of 7.4 μg per day for a 74 kg person. If consumed, the soup containing the highest measured MMHg concentration would exceed the reference dose by 17%. While shark fin soup represents a potentially important source of MMHg to human consumers, other seafood products, particularly the flesh of apex marine predators, contain much higher MMHg concentrations and can result in substantially greater exposures of this contaminant for people. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Role of mp 170 Seprase in Breast Cancer.

DTIC Science & Technology

1999-07-01

endogenous Seprase/FAPa in MDA-436 cells. Previously, three hammerhead ribozyme constructs targeting Seprase/FAPa at nucleotide residues 703, 892...breast cancer cells using the ribozyme approach. In contrast, I also constructed two hammerhead ribozymes targeting FGF-BP2 at nucleotide residues 223 and...molecule for further characterization, however, with little success. Ribozyme constructs targeting Seprase/FAPa were made, and despite their in vitro

Publications - GMC 197 | Alaska Division of Geological & Geophysical

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a (9,134-9,147', and 11,129') of Northstar #1 well, from unwashed cuttings (5,280-5,400) of OCS Y-0849-1 (Hammerhead #1) well, and from unwashed cuttings (5,100-5,520') of OCS Y-0849-2 (Hammerhead #2) well Authors

Inhibitory Ah Receptor-Androgen Receptor Crosstalk in Prostate Cancer

DTIC Science & Technology

2006-02-01

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate...therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [14] T. Dorai, C.A. Olsson, A.E. Katz, R. Buttyan, Development of a hammerhead ... ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer, Prostate 32 (1997) 246

Development of RNAi Libraries for Target Validation and Therapeutics

DTIC Science & Technology

2006-03-01

met using a hammerhead ribozyme transgene reduces in vitro invasion and migration in prostate cancer cells. Prostate, 60: 317-324, 2004. 49...Watkins, G., Mason, M.D., Jiang, W.G. (2004) Targeting the HGF/SF receptor c-met using a hammerhead ribozyme transgene reduces in vitro invasion...reduction of tumor growth (27). In addition, when c-met was knocked-down using ribozymes , cell invasion and metastasis were inhibited both in vitro

Development of Lead Hammerhead Ribozyme Candidates against Human Rod Opsin mRNA for Retinal Degeneration Therapy

PubMed Central

Abdelmaksoud, Heba E.; Yau, Edwin H.; Zuker, Michael; Sullivan, Jack M.

2011-01-01

To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p < 0.05). Successful lead candidates (hhRz CUC↓ 266, hhRz CUC↓ 1411, hhRz AUA↓ 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48 hr paradigm of testing. These results validate a rigorous computational

Molecular Identification of Shark Meat From Local Markets in Southern Brazil Based on DNA Barcoding: Evidence for Mislabeling and Trade of Endangered Species

PubMed Central

Almerón-Souza, Fernanda; Sperb, Christian; Castilho, Carolina L.; Figueiredo, Pedro I. C. C.; Gonçalves, Leonardo T.; Machado, Rodrigo; Oliveira, Larissa R.; Valiati, Victor H.; Fagundes, Nelson J. R.

2018-01-01

Elasmobranchs, the group of cartilaginous fishes that include sharks and rays, are especially vulnerable to overfishing due to low fecundity and late sexual maturation. A significant number of elasmobranch species are currently overexploited or threatened by fisheries activities. Additionally, several recent reports have indicated that there has been a reduction in regional elasmobranch population sizes. Brazil is an important player in elasmobranch fisheries and one of the largest importers of shark meat. However, carcasses entering the shark meat market have usually had their fins and head removed, which poses a challenge to reliable species identification based on the morphology of captured individuals. This is further complicated by the fact that the internal Brazilian market trades several different elasmobranch species under a common popular name: “cação.” The use of such imprecise nomenclature, even among governmental agencies, is problematic for both controlling the negative effects of shark consumption and informing the consumer about the origins of the product. In this study, we used DNA barcoding (mtDNA, COI gene) to identify, at the species level, “cação” samples available in local markets from Southern Brazil. We collected 63 samples traded as “cação,” which we found to correspond to 20 different species. These included two teleost species: Xiphias gladius (n = 1) and Genidens barbus (n = 6), and 18 species from seven elasmobranch orders (Carcharhiniformes, n = 42; Squaliformes, n = 3; Squatiniformes, n = 2; Rhinopristiformes, n = 4; Myliobatiformes, n = 3; Rajiformes, n = 1; and Torpediniformes, n = 1). The most common species in our sample were Prionace glauca (n = 15) and Sphyrna lewini (n = 14), while all other species were represented by four samples or less. Considering IUCN criteria, 47% of the elasmobranch species found are threatened at the global level, while 53% are threatened and 47% are critically endangered in Brazil

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

PubMed Central

Cho, Suhyung; Kim, Ji-Eun; Lee, Bo-Rahm; Kim, June-Hyung; Kim, Byung-Gee

2005-01-01

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal. PMID:16314308

78 FR 36525 - Fisheries of the Atlantic and the Gulf of Mexico; Southeast Data, Assessment, and Review (SEDAR...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-18

...) Atlantic Sharpnose (Rhizoprionodon terraenovae) and Bonnethead (Sphyrna tiburo) sharks. SUMMARY: The SEDAR 34 assessment of HMS Atlantic Sharpnose and Bonnethead sharks will consist of an in-person workshop... for determining the status of fish stocks in the Southeast Region. SEDAR is a multi-step process...

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Miocene Shark and Batoid Fauna from Nosy Makamby (Mahajanga Basin, Northwestern Madagascar)

PubMed Central

Andrianavalona, Tsiory H.; Ramihangihajason, Tolotra N.; Rasoamiaramanana, Armand; Ward, David J.; Ali, Jason R.; Samonds, Karen E.

2015-01-01

Madagascar is well known for producing exceptional fossils. However, the record for selachians remains relatively poorly known. Paleontological reconnaissance on the island of Nosy Makamby, off northwest Madagascar, has produced a previously undescribed assemblage of Miocene fossils. Based on isolated teeth, ten taxonomic groups are identified: Otodus, Carcharhinus, Galeocerdo, Rhizoprionodon, Sphyrna, Hemipristis, Squatina, Rostroraja, Himantura and Myliobatidae. Six are newly described from Madagascar for the Cenozoic (Galeocerdo, Rhizoprionodon, Sphyrna, Squatina, Rostroraja and Himantura). In association with these specimens, remains of both invertebrates (e.g., corals, gastropods, bivalves) and vertebrates (e.g., bony fish, turtles, crocodylians, and sirenian mammals) were also recovered. The sedimentary facies are highly suggestive of a near-shore/coastal plain depositional environment. This faunal association shares similarities to contemporaneous sites reported from North America and Europe and gives a glimpse into the paleoenvironment of Madagascar’s Miocene, suggesting that this region was warm, tropical shallow-water marine. PMID:26075723

The shallow-water fish assemblage of Isla del Coco National Park, Costa Rica: Structure and patterns in an isolated, predator-dominated ecosystem

USGS Publications Warehouse

Friedlander, Alan M.; Zgliczynski, Brian J.; Ballesteros, Enric; Aburto-Oropeza, Octavio; Bolaños, Allan; Sala, Enric

2012-01-01

Fishes at Isla del Coco National Park, Costa Rica, were surveyed as part of a larger scientific expedition to the area in September 2009. The average total biomass of nearshore fishes was 7.8 tonnes per ha, among the largest observed in the tropics, with apex predators such as sharks, jacks, and groupers accounting for nearly 40% of the total biomass. The abundance of reef and pelagic sharks, particularly large aggregations of threatened species such as the scalloped hammerhead shark (up to 42 hammerheads ha-1) and large schools of jacks and snappers show the capacity for high biomass in unfished ecosystems in the Eastern Tropical Pacific. However, the abundance of hammerhead and reef whitetip sharks appears to have been declining since the late 1990s, and likely causes may include increasing fishing pressure on sharks in the region and illegal fishing inside the Park. One Galapagos shark tagged on September 20, 2009 in the Isla del Coco National Park moved 255km southeast towards Malpelo Island in Colombia, when it stopped transmitting. These results contribute to the evidence that sharks conduct large-scale movements between marine protected areas (Isla del Coco, Malpelo, Galápagos) in the Eastern tropical Pacific and emphasize the need for regional-scale management. More than half of the species and 90% of the individuals observed were endemic to the tropical eastern Pacific. These high biomass and endemicity values highlight the uniqueness of the fish assemblage at Isla del Coco and its importance as a global biodiversity hotspot.

Selenium is a Chemotherapeutic Agent for the Treatment of Prostate Cancer

DTIC Science & Technology

2006-02-01

receptor activity by a hammerhead ribozyme . Mol Endrocrinol 1998;12:1558-1566. 10. Ko YJ, Devi GR, London CA, et al. Androgen receptor down-regulation...Strategic targeting of the AR with ribozymes , antisense oligomers, and small interfering RNAs has been shown to significantly inhibit prostate cancer

The sexually dimorphic cephalofoil of bonnethead sharks, Sphyrna tiburo.

PubMed

Kajiura, Stephen M; Tyminski, John P; Forni, Jesica B; Summers, Adam P

2005-08-01

Sexually dimorphic head shape is common in vertebrates from teleosts to mammals. Herein we document that cephalic sexual dimorphism is also found in the cartilaginous fishes (Chondrichthyes). Male bonnethead sharks develop a prominent bulge along the anterior margin of the cephalofoil at the onset of sexual maturity. This contrasts with the uniformly rounded anterior margin of adult females and juveniles and embryos of both sexes. The anterior cephalic bulge is produced by elongation of the rod-like rostral cartilages, and its appearance corresponds temporally with the elongation of the rod-like cartilages of the male intromittent organs (claspers). We propose that the rostral cartilage elongation is a byproduct of endocrinological changes at the onset of sexual maturity that stimulate growth of the clasper cartilages. The basal location of the chondrichthyan fishes within the vertebrate clade extends the earliest appearance of cephalic sexual dimorphism among the vertebrates.

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

78 FR 59878 - Atlantic Highly Migratory Species; Commercial Atlantic Aggregated Large Coastal Shark (LCS...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-30

... submitted electronically on a weekly basis through a NMFS-approved electronic reporting system by the dealer... metric tons (mt) dressed weight (dw) (149,914 lb dw) of non-blacknose SCS quota from the Atlantic region... lb dw), the Atlantic hammerhead shark management group quota is 27.1 metric tons (mt) dressed weight...

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

PubMed Central

Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S

1999-01-01

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2009-10-01

CT-cells have endog- enous CT stably knocked down to undetectable levels using anti-CT hammerhead ribozymes [25]. Salmon CT (BAChem, Torrance, CA) was...and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knock- down of CTR[18]. CTR-cells have very low levels of CD44v

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2008-04-01

hammerhead ribozymes .25 Salmon CT (BAChem, Torrance, CA) was used at physiologic 50 nM dose14,16, which effectively alters CD44,6 or at 250 nM. To detect...receptor14), and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knockdown of CTR.18 CTR- cells have very low levels of

3. AERIAL VIEW OF MOBILE LAUNCHER. ON TOP OF LUT ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. AERIAL VIEW OF MOBILE LAUNCHER. ON TOP OF LUT SITS A 25 TON HAMMERHEAD CRANE. STRUCTURE ON LEFT SIDE OF LAUNCH PLATFORM IS KNOWN AS A 'MILK STOOL' AND ALLOWS A SATURN 1B ROCKET TO BE USED IN PLACE OF THE SATURN V ROCKET. - Mobile Launcher One, Kennedy Space Center, Titusville, Brevard County, FL

2002 Airborne Geophysical Survey at Pueblo of Isleta Bombing Targets, New Mexico, April 10 May 6, 2002 (Rev 1)

DTIC Science & Technology

2005-12-01

helicopter geophysical survey performed by US Army Engineering Support Center, Huntsville (USAESCH) and Oak Ridge National Laboratory ( ORNL ) over areas...Array Detection System NAD North American Datum ORAGS Oak Ridge Airborne Geophysical System ORNL Oak Ridge National Laboratory RMS Root...used by ORNL in 1999 for.....................5 Figure 2.4 ORAGS-Hammerhead airborne magnetometer system used at Badlands Bombing Range in FY2000

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Signal Propagation and Detection via Catalytically Immolative Biopolymer-Programmed Nanomaterials

DTIC Science & Technology

2015-02-09

hammerhead   ribozymes   as   therapeutic   tools   to   control  disease  genes.  Curr.  Gene  Ther.  5,  11-­‐24  (2005...Incorporation  of  Coenzymes  by   Ribozymes .  Journal   of  Molecular  Evolution  40,  551-­‐558  (1995).   36   Liou,  H.-­‐C

Lateral Mixing DRI Analysis: Submesoscale Water-Mass Spectra

DTIC Science & Technology

2013-09-30

program to determine submesoscale variability in the Sargasso Sea under weak-to-moderate mesoscale conditions. Two sites were examined, a quiet site...anomalies and dye streaks. Hammerhead carries finescale Sea -Bird sensors for temperature, conductivity and pressure as well as Chelsea and WetLab...m of dye-injection target densities. They were embedded in 35-km towyo grid surveys by Craig Lee’s Triaxus and 15-km butterfly surveys by Jody

HER-2 as a Progression Factor and Therapeutic Target in Breast Cancer.

DTIC Science & Technology

1999-06-01

used gene specific targeting of HER-2 with hammerhead - ribozyme expression constructs, a technology which we have applied successfully in the...2 in MCF-7 cells by ribozyme -targeting estradiol lost its ability to induce anchorage- independent colony formation in soft agar of the tumor cells...between estrogen and HER-2 signal transduction is ongoing. 14. SUBJECT TERMS Breast Cancer HER-2, estradiol, ribozymes , apoptosis, cell cycle, cDNA

Georgetown Institute for Cognitive and Computational Sciences

DTIC Science & Technology

2000-03-01

in neuronal apoptosis. Cerebellar granules cells (CGCs) were co-transfected with a green fluorescent protein reporter and one of several hammerhead ... ribozymes constructed to cleave caspase-3 RNA. Use of such ribozymes is highly selective. In separate experiments we co-transfected with a gene that...expressing a ribozyme against rat caspase-3. Apoptosis was assessed after 24, 36, or 48 h of serum/K+ deprivation. In negative control cells expressing ß

Pleiotrophin as a Growth Factor and Therapeutic Target in Breast Cancer.

DTIC Science & Technology

1998-10-01

Hs578T cells by stable transfection with hammerhead ribozymes as done for a number of different genes by my laboratory (for a review see reference [28...angiogenesis and metastasis. 14. SUBJECT TERMS Breast Cancer growth factor, pleiotrophin, hormones, retrovirus, ribozymes 15. NUMBER OF PAGES...of their endogenous PTN with specific ribozymes [10-12]. We were the first laboratory to purify PTN from human cancer cells (MDA-MB 231 breast

Transdominant Rev and Protease Mutant Proteins of HIV-SIV as Potential Antiviral Agents in Vitro and in Vivo (AIDS)

DTIC Science & Technology

1993-10-30

hammerhead ribozymes (7-9) and a hairpin ribozyme (10) directed against HIV-l RNA has been shown to confer significant resistance to HIV-I infection...antisense oligodeoxynucleotides (ODN) directed to the Rev Response Element (RRE) and ribozymes that target viral mRNAs. The ribozyme approach, in...particular, has yielded extremely encouraging positive data. We showed that a hairpin ribozyme designed to cleave HIV-1 RNA in the 5’ leader sequence

Selective Androgen Receptor Down-Regulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2007-10-01

PCa (9). Thus far, the techniques that have been used to down-regulate the AR include antisense oligonucleotides (10, 11), ribozyme treatments (12...Our findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or...Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol

View southwest of 350ton crane, showing one of four castings ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

View southwest of 350-ton crane, showing one of four castings which support a stationary, tapered steel girder structure called a "tower". This tower is located within an outer rotating framework designated as the "pintle" of the 350-ton crane. The whole crane pivots around bearing at the top of this tapered support tower. - Naval Base Philadelphia-Philadelphia Naval Shipyard, 350-Ton Hammerhead Crane, League Island, Philadelphia, Philadelphia County, PA

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

Advances in Integrating Autonomy with Acoustic Communications for Intelligent Networks of Marine Robots

DTIC Science & Technology

2013-02-01

Sonar AUV #Environmental Sampling Environmental AUV +name : string = OEX Ocean Explorer +name : string = Hammerhead Iver2 +name : string = Unicorn ...executable» Google Earth Bluefin 21 AUV ( Unicorn ) MOOS Computer GPS «serial» Bluefin 21 AUV (Macrura) MOOS Computer «acoustic» Micro-Modem «wired...Computer Bluefin 21 AUV ( Unicorn ) MOOS Computer NURC AUV (OEX) MOOS Computer Topside MOOS Computer «wifi» 5.0GHz WiLan «acoustic» Edgetech GPS

Repairing RNA Transcripts that Mediate Breast Cancer Susceptibility

DTIC Science & Technology

2005-08-01

is actually the yield of TES product plus the yield of cryptic is in contrast to hammerhead and hairpin ribozymes , which products. This increases the...therapeutics. To this end, we have developed a novel biomolecule (a ribozyme ) that can specifically excise regions from RNA transcripts. In this work, we...designed a ribozyme that excises an insertion mutation that is linked to breast cancer predisposition from a short mimic of the p53 transcript in a

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

2000-12-01

Task 4). T.O. 2 We have designed and tested a hammerhead ribozyme [21, 22] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the...Growth Factors, Ribozymes 23 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF...Underexpression of CR-1 will decrease tumorigenicity of highly or moderately tumorigenic cell lines. (T.O. 2) 4. Efficient delivery of a ribozyme or

Puberty, Differentiation and Breast Cancer Risk

DTIC Science & Technology

1999-09-01

tetracycline or doxocycline to culture medium. We designed hammerhead ribozymes which recognize four different regions of mBrca2, 6 Proprietary Data DAMD17...containing a ribozyme directed against the 5’ end of mBrca2 appear to show dox-dependent regulation of Brca2 levels (as measured by Western blot...for possible modest regulation), we have designed ribozymes which recognize human and mouse BRCA2 sequence, and we intend to create tet-inducible

A Novel Molecular Targeting of a Tumor-Specific Oncogenic Mutant Receptor in Human Prostate Cancer

DTIC Science & Technology

2005-02-01

in cells and can generate dominant negative mutant (15). Hammerhead ribozymes are self-cleaving RNAs whose catalytic activity has been mapped to a...specific ribozyme targeted at the fusion junction of EGFRvIII. This specific EGFRvIII ribozyme is able to effectively cleave EGFRvIII mRNA under...physiological conditions in a cell-free system. While expressing this EGFRvIII- ribozyme in 32D/EGFRvIII cell, EGFRvIII- ribozyme is capable of down-regulating

Robot-friendly connector. [space truss structures

NASA Technical Reports Server (NTRS)

Parma, George F. (Inventor); Vandeberghe, Mark H. (Inventor); Ruiz, Steve C. (Inventor)

1993-01-01

Robot friendly connectors, which, in one aspect, are truss joints with two parts, a receptacle and a joint, are presented. The joints have a head which is loosely inserted into the receptacle and is then tightened and aligned. In one aspect, the head is a rounded hammerhead which initially is enclosed in the receptacle with sloppy fit provided by the shape, size, and configuration of surfaces on the head and on the receptacle.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

PubMed Central

Zhou, De-Min; Zhang, Li-He; Taira, Kazunari

1997-01-01

In a previous examination using natural all-RNA substrates that contained either a 5′-oxy or 5′-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5′-thio group (R11S) and (ii) the departure of the 5′ leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5′-oxygen, because a switch in metal ion specificity was not observed with the 5′-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis. PMID:9405614

Distribution of pancreatic elastase and metalloproteinase in vertebrates.

PubMed

Yoshinaka, R; Sato, M; Tsuchiya, N; Ikeda, S

1986-01-01

Elastase-like enzymes were detected as zymogens in all of the pancreatic extracts from the gummy shark, bullhead shark, angel shark, smooth hammerhead, bestel, rainbow trout, carp, eel, Japanese mackerel, yellowtail, sea bass, parrotfish, bullfrog, chicken, bluewhite dolphin, hog, rat, cat, and dog. The distribution of pancreatic elastase and metalloproteinase was examined on the basis of the effect of specific inhibitors on elastase like-activity in each extract. The results indicate that pancreatic elastases are present in all the species examined and pancreatic metalloproteinases are present only in the teleost fishes.

SERUM RELAXIN CONCENTRATIONS AND REPRODUCTION IN MALE BONNETHEAD SHARKS, SPHYRNA TIBURO. (R826128)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

PubMed

Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

2015-05-15

Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches.

An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures

PubMed Central

Zandi, Kasra; Butler, Gregory; Kharma, Nawwaf

2016-01-01

Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer. PMID:27499762

Monitoring Retroviral RNA Dimerization In Vivo via Hammerhead Ribozyme Cleavage

PubMed Central

Pal, Bijay K.; Scherer, Lisa; Zelby, Laurie; Bertrand, Edouard; Rossi, John J.

1998-01-01

We have used a strategy for colocalization of Psi (Ψ)-tethered ribozymes and targets to demonstrate that Ψ sequences are capable of specific interaction in the cytoplasm of both packaging and nonpackaging cells. These results indicate that current in vitro dimerization models may have in vivo counterparts. The methodology used may be applied to further genetic analyses on Ψ domain interactions in vivo. PMID:9733882

ICTV Virus Taxonomy Profile: Avsunviroidae.

PubMed

Di Serio, Francesco; Li, Shi-Fang; Matoušek, Jaroslav; Owens, Robert A; Pallás, Vicente; Randles, John W; Sano, Teruo; Verhoeven, Jacobus Th J; Vidalakis, Georgios; Flores, Ricardo; Ictv Report Consortium

2018-05-01

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

PubMed

Zhang, Zhixiang; Qi, Shuishui; Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

2014-12-01

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

PubMed Central

Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

2014-01-01

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs. PMID:25503469

Design and Performance of a 2.7 THz Waveguide Tripler

NASA Technical Reports Server (NTRS)

Maiwald, Frank; Martin, S.; Bruston, J.; Maestrini, A.; Crawford, T.; Siegel, P. H.

2001-01-01

The design and performance of a 0.9 THz to 2.7 THz waveguide tripler are presented. An unusual split block configuration with parallel input and output waveguides accommodates a monolithic membrane diode (MoMeD) circuit. Submicron planar GaAs Schottky diodes in single and antiparallel pairs are implemented with matching filters on a 3-micrometer thick suspended substrate as part of the MoMeD structure. The filters are a combination of short hammerheads and high-low impedance elements. Only a few circuit variations have been measured to date. The best current performance shows an output power of 0.1 microW and an efficiency of 0.002% at the band center frequency of 2.55 THz.

Ventricle morphology in pelagic elasmobranch fishes.

PubMed

Emery, S H; Mangano, C; Randazzo, V

1985-01-01

Ventricle weights of the warm-bodied great white shark, Atlantic shortfin mako, and the common thresher shark (the latter presumed to be warm-bodied) are similar to those of ectothermic blue sharks, sandbar sharks, dusky sharks, tiger sharks and scalloped hammerhead sharks. Ventricle muscularity, as estimated by the ratio of cortical to spongy layer thickness, is almost twice as great in the former three species than in the latter elasmobranchs. Measurements of ventricular volumes suggest that the ventricles of the great white, Atlantic shortfin mako and common thresher sharks are better adapted to respond to demands for increases in cardiac output via increased heartbeat frequency in comparison with ectothermic species of shark.

The earliest herbivorous marine reptile and its remarkable jaw apparatus.

PubMed

Chun, Li; Rieppel, Olivier; Long, Cheng; Fraser, Nicholas C

2016-05-01

Newly discovered fossils of the Middle Triassic reptile Atopodentatus unicus call for a radical reassessment of its feeding behavior. The skull displays a pronounced hammerhead shape that was hitherto unknown. The long, straight anterior edges of both upper and lower jaws were lined with batteries of chisel-shaped teeth, whereas the remaining parts of the jaw rami supported densely packed needle-shaped teeth forming a mesh. The evidence indicates a novel feeding mechanism wherein the chisel-shaped teeth were used to scrape algae off the substrate, and the plant matter that was loosened was filtered from the water column through the more posteriorly positioned tooth mesh. This is the oldest record of herbivory within marine reptiles.

The earliest herbivorous marine reptile and its remarkable jaw apparatus

PubMed Central

Chun, Li; Rieppel, Olivier; Long, Cheng; Fraser, Nicholas C.

2016-01-01

Newly discovered fossils of the Middle Triassic reptile Atopodentatus unicus call for a radical reassessment of its feeding behavior. The skull displays a pronounced hammerhead shape that was hitherto unknown. The long, straight anterior edges of both upper and lower jaws were lined with batteries of chisel-shaped teeth, whereas the remaining parts of the jaw rami supported densely packed needle-shaped teeth forming a mesh. The evidence indicates a novel feeding mechanism wherein the chisel-shaped teeth were used to scrape algae off the substrate, and the plant matter that was loosened was filtered from the water column through the more posteriorly positioned tooth mesh. This is the oldest record of herbivory within marine reptiles. PMID:27386529

CALCITONIN-LIKE IMMUNOREACTIVITY IN SERUM AND TISSUES OF THE BONNETHEAD SHARK, SPHYRNA TIBURO. (R826128)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Central Andes mountains, Chile/Argentina as seen from STS-67

NASA Technical Reports Server (NTRS)

1995-01-01

The Chilean coastline and the arid Atacama Desert stretch the length of the view with the high Andes on the eastern margin where hundreds of volcanoes dot the landscape. The wider (250-350 kilometers) Altiplano ('plains') sector of the Andes appears in the top half of the view, and the narrow (120 kilometers) 'mountain-chain-dominated' sector to the bottom. The northern half of Chile can be seen, with the 'hammer-head' peninsula at the city of Antofagasta, top left. Up welling of cold water as the Humboldt Current immediately offshore gives rise to low stratus cloud. The extensive cloud mass on the right lies beyond the Andes in the low country of Argentina's 'pampas' grasslands and Chaco semi-desert.

Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.

PubMed

Millington-Ward, Sophia; Allers, Carolina; Tuohy, Gearóid; Conget, Paulette; Allen, Danny; McMahon, Helena P; Kenna, Paul F; Humphries, Peter; Farrar, G Jane

2002-09-15

Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.

Selection and sex-biased dispersal in a coastal shark: the influence of philopatry on adaptive variation.

PubMed

Portnoy, D S; Puritz, J B; Hollenbeck, C M; Gelsleichter, J; Chapman, D; Gold, J R

2015-12-01

Sex-biased dispersal is expected to homogenize nuclear genetic variation relative to variation in genetic material inherited through the philopatric sex. When site fidelity occurs across a heterogeneous environment, local selective regimes may alter this pattern. We assessed spatial patterns of variation in nuclear-encoded, single nucleotide polymorphisms (SNPs) and sequences of the mitochondrial control region in bonnethead sharks (Sphyrna tiburo), a species thought to exhibit female philopatry, collected from summer habitats used for gestation. Geographic patterns of mtDNA haplotypes and putatively neutral SNPs confirmed female philopatry and male-mediated gene flow along the northeastern coast of the Gulf of Mexico. A total of 30 outlier SNP loci were identified; alleles at over half of these loci exhibited signatures of latitude-associated selection. Our results indicate that in species with sex-biased dispersal, philopatry can facilitate sorting of locally adaptive variation, with the dispersing sex facilitating movement of potentially adaptive variation among locations and environments. © 2015 John Wiley & Sons Ltd.

New tropical carcharhinids (chondrichthyes, carcharhiniformes) from the late Eocene early Oligocene of Balochistan, Pakistan: Paleoenvironmental and paleogeographic implications

NASA Astrophysics Data System (ADS)

Adnet, S.; Antoine, P.-O.; Hassan Baqri, S. R.; Crochet, J.-Y.; Marivaux, L.; Welcomme, J.-L.; Métais, G.

2007-04-01

New selachians (sharks and rays) have been collected from several late Eocene and early Oligocene marine localities in the Bugti Hills (Balochistan, Pakistan). Two new species of Requiem sharks (close to the Recent "Bull shark") are described : Carcharhinus balochensis and Carcharhinus perseus. The rest of the fauna is notable for the strong representation of Carcharhiniformes. These selachian faunas represent a unique tropical association for the Oligocene period and one of the first modern tropical selachian faunas, with modern taxa such as the two new species of "Bull sharks", Negaprion sp. and one of the first occurrences of Sphyrna sp. Moreover, these faunas permit paleoenvironmental interpretation of adjacent land masses. The relatively modern aspect of these faunas, compared with other contemporaneous and younger selachian associations from Atlantic and Mediterranean seas, suggests biogeographic isolation of selachian communities living in eastern and western parts of the Tethys before its final closure during the early-middle Miocene.

Ribozyme-mediated signal augmentation on a mass-sensitive biosensor.

PubMed

Knudsen, Scott M; Lee, Joonhyung; Ellington, Andrew D; Savran, Cagri A

2006-12-20

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

PubMed

Tanaka, T; Inui, O; Dohi, N; Okada, N; Okada, H; Kikuchi, Y

2001-07-01

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

76 FR 23794 - Stock Status Determination for Atlantic Highly Migratory Scalloped Hammerhead Shark

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-28

...) published in the North American Journal of Fisheries Management a stock assessment of the Atlantic... conservation and management measures to rebuild overfished stocks within 2 years of making this determination... to rebuilding the fishery within the shortest time possible in accordance with 16 U.S.C. 1854(e)(4...

Model systems: how chemical biologists study RNA

PubMed Central

Rios, Andro C.; Tor, Yitzhak

2009-01-01

Ribonucleic acids are structurally and functionally sophisticated biomolecules and the use of models, frequently truncated or modified sequences representing functional domains of the natural systems, is essential to their exploration. Functional non-coding RNAs such as miRNAs, riboswitches, and, in particular, ribozymes, have changed the view of RNA’s role in biology and its catalytic potential. The well-known truncated hammerhead model has recently been refined and new data provide a clearer molecular picture of the elements responsible for its catalytic power. A model for the spliceosome, a massive and highly intricate ribonucleoprotein, is also emerging, although its true utility is yet to be cemented. Such catalytic model systems could also serve as “chemo-paleontological” tools, further refining the RNA world hypothesis and its relevance to the origin and evolution of life. PMID:19879179

Facile analysis of contents and compositions of the chondroitin sulfate/dermatan sulfate hybrid chain in shark and ray tissues.

PubMed

Takeda, Naoko; Horai, Sawako; Tamura, Jun-ichi

2016-04-07

The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from specific tissues of several kinds of sharks and rays. The contents and sulfation patterns of the CS/DS hybrid chain were precisely analyzed by digestion with chondroitinases ABC and AC. All samples predominantly contained the A- and C-units. Furthermore, all samples characteristically contained the D-unit. Species-specific differences were observed in the contents of the CS/DS hybrid chain, which were the highest in Mako and Blue sharks and Sharpspine skates, but were lower in Hammerhead sharks. Marked differences were observed in the ratio of the C-unit/A-unit between sharks and rays. The contents of the CS/DS hybrid chain and the ratio of the C-unit/A-unit may be related to an oxidative stress-decreasing ability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation.

PubMed

Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S; Naash, Muna I

2016-01-01

The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.

Development of a truss joint for robotic assembly of space structures

NASA Technical Reports Server (NTRS)

Parma, George F.

1992-01-01

This report presents the results of a detailed study of mechanical fasteners which were designed to facilitate robotic assembly of structures. Design requirements for robotic structural assembly were developed, taking into account structural properties and overall system design, and four candidate fasteners were designed to meet them. These fasteners were built and evaluated in the laboratory, and the Hammer-Head joint was chosen as superior overall. It had a high reliability of fastening under misalignments of 2.54 mm (0.1 in) and 3 deg, the highest end fixity (2.18), the simplest end effector, an integral capture guide, good visual verification, and the lightest weight (782 g, 1.72 lb). The study found that a good design should incorporate chamfers sliding on chamfers, cylinders sliding on chamfers, and hard surface finishes on sliding surfaces. The study also comments on robot flexibility, sag, hysteresis, thermal expansion, and friction which were observed during the testing.

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study

NASA Astrophysics Data System (ADS)

Leclerc, Fabrice; Zaccai, Giuseppe; Vergne, Jacques; Řìhovà, Martina; Martel, Anne; Maurel, Marie-Christine

2016-07-01

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

Broad and Temperature Independent Replication Potential of Filoviruses on Cells Derived From Old and New World Bat Species.

PubMed

Miller, Megan R; McMinn, Rebekah J; Misra, Vikram; Schountz, Tony; Müller, Marcel A; Kurth, Andreas; Munster, Vincent J

2016-10-15

Filoviruses are strongly associated with several species of bats as their natural reservoirs. In this study, we determined the replication potential of all filovirus species: Marburg marburgvirus, Taï Forest ebolavirus, Reston ebolavirus, Sudan ebolavirus, Zaire ebolavirus, and Bundibugyo ebolavirus. Filovirus replication was supported by all cell lines derived from 6 Old and New World bat species: the hammer-headed fruit bat, Buettikofer's epauletted fruit bat, the Egyptian fruit bat, the Jamaican fruit bat, the Mexican free-tailed bat and the big brown bat. In addition, we showed that Marburg virus Angola and Ebola virus Makona-WPGC07 efficiently replicated at 37°C, 37°-41°C, or 41°C, contrary to the hypothesis that temporal elevation in temperature due to flight affects filovirus replication in bats. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Recovery of infectious type Asia1 foot-and-mouth disease virus from suckling mice directly inoculated with an RNA polymerase I/II-driven unidirectional transcription plasmid.

PubMed

Lian, Kaiqi; Yang, Fan; Zhu, Zixiang; Cao, Weijun; Jin, Ye; Li, Dan; Zhang, Keshan; Guo, Jianhong; Zheng, Haixue; Liu, Xiangtao

2015-10-02

We developed an RNA polymerase (pol) I- and II-driven plasmid-based reverse genetics system to rescue infectious foot-and-mouth disease virus (FMDV) from cloned cDNA. In this plasmid-based transfection, the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences, which were arranged downstream of the two promoters (cytomegalovirus (CMV) and pol I promoter) and upstream of the terminators and polyadenylation signal, respectively. The utility of this method was demonstrated by the recovery of FMDV Asia1 HN/CHA/06 in BHK-21 cells transfected with cDNA plasmids. Furthermore, infectious FMDV Asia1 HN/CHA/06 could be rescued from suckling mice directly inoculated with cDNA plasmids. Thus, this reverse genetics system can be applied to fundamental research and vaccine studies, most notably to rescue those viruses for which there is currently an absence of a suitable cell culture system. Copyright © 2015 Elsevier B.V. All rights reserved.

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

PubMed Central

Liu, Y H; Symons, R H

1998-01-01

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids. PMID:9630248

Photocopy of photograph (from NBPPNSY, CSF35871264) Replogle, photographer, September 23, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Photocopy of photograph (from NBP-PNSY, CSF-3587-12-64) Replogle, photographer, September 23, 1921 view northwest from 350 - ton crane (Haer no. PA-387-F) of Philadelphia Navy Yard. Drydock no. 2 (Haer no. Pa-387-B) at right, below center of the photograph. This timber structure is 465 feet long, completed by yard labor in early 1915. The slip had dive McMyler Interstate Company rotating hammerhead cranes. It was the site where the U.S.S. Henderson, first ship built by the League Island yard, came down the ways on June 17, 1916. Between drydock no. 2 and slip no. 1 are small shipways, 210 feet long, used for construction of sea-going mine sweepers. The slip ways at the upper left of the photograph are nos. 2 and 3. They are of timber construction and were built in 1918 no. 2 was 850 and no. 3 700 feet long. - Naval Base Philadelphia-Philadelphia Naval Shipyard, League Island, Philadelphia, Philadelphia County, PA

A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing

PubMed Central

Baeyens, Katrien J.; De Bondt, Hendrik L.; Pardi, Arthur; Holbrook, Stephen R.

1996-01-01

The crystal structure of the RNA dodecamer 5′-GGCC(GAAA)GGCC-3′ has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson–Crick base pairs make up an internal loop in the middle of the duplex, including sheared G·A pairs and novel asymmetric A·A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34° from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson–Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA. PMID:8917508

Effect of Hammerhead Ribozyme against Human Thymidylate Synthase on the Cytotoxicity of Thymidylate Synthase Inhibitors

PubMed Central

Takemura, Yuzuru; Miyachi, Hayato; Skelton, Lorraine; Jackman, Ann L.

1995-01-01

One of the resistance mechanisms to folate‐based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non‐polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20‐fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus‐based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro. PMID:8567390

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

PubMed Central

Fu, D J; McLaughlin, L W

1992-01-01

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction. PMID:1570323

Electrothermal piezoresistive cantilever resonators for personal measurements of nanoparticles in workplace exposure

NASA Astrophysics Data System (ADS)

Wasisto, Hutomo Suryo; Wu, Wenze; Uhde, Erik; Waag, Andreas; Peiner, Erwin

2015-05-01

Low-cost and low-power piezoresistive cantilever resonators with integrated electrothermal heaters are developed to support the sensing module enhancement of the second generation of handheld cantilever-based airborne nanoparticle (NP) detector (CANTOR-2). These sensors are used for direct-reading of exposure to carbon engineered nanoparticles (ENPs) at indoor workplaces. The cantilever structures having various shapes of free ends are created using silicon bulk micromachining technologies (i.e, rectangular, hammer-head, triangular, and U-shaped cantilevers). For a complete wearable CANTOR-2, all components of the proposed detector can be grouped into two main units depending on their packaging placements (i.e., the NP sampler head and the electronics mounted in a handy-format housing). In the NP sampler head, a miniaturized electrophoretic aerosol sampler and a resonant silicon cantilever mass sensor are employed to collect the ENPs from the air stream to the cantilever surfaces and measuring their mass concentration, respectively. After calibration, the detected ENP mass concentrations of CANTOR-2 show a standard deviation from fast mobility particle sizer (FMPS, TSI 3091) of 8-14%.

Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae.

PubMed

Daròs, José-Antonio

2016-10-01

Eggplant latent viroid (ELVd) is the only species of the genus Elaviroid (family Avsunviroidae). All the viroids in the family Avsunviroidae contain hammerhead ribozymes in the strands of both polarities, and are considered to replicate in the chloroplasts of infected cells. This family includes two other genera: Avsunviroid and Pelamoviroid. ELVd consists of a single-stranded, circular, non-coding RNA of 332-335 nucleotides that folds in a branched quasi-rod-like minimum free-energy conformation. RNAs of complementary polarity exist in infected cells and are considered to be replication intermediates. Plus (+) polarity is assigned arbitrarily to the strand that accumulates at a higher concentration in infected tissues. HOST: To date, ELVd has only been shown to infect eggplant (Solanum melongena L.), the species in which it was discovered. A very narrow host range seems to be a common property in members of the family Avsunviroidae. ELVd infections of eggplants are apparently symptomless. ELVd is transmitted mechanically and by seed. http://subviral.med.uottawa.ca. © 2015 BSPP and John Wiley & Sons Ltd.

A conserved phosphorylation switch controls the interaction between cadherin and β-catenin in vitro and in vivo

DOE PAGES

Choi, Hee -Jung; Loveless, Timothy; Lynch, Allison M.; ...

2015-04-06

In metazoan adherens junctions, β-catenin links the cytoplasmic tail of classical cadherins to the F-actin-binding protein α-catenin. Phosphorylation of a Ser/Thr-rich region in the cadherin tail dramatically enhances affinity for β-catenin and promotes cell-cell adhesion in cell culture systems, but its importance has not been demonstrated in vivo. In this paper, we identify a critical phosphorylated serine in the C. elegans cadherin HMR-1 required for strong binding to the β-catenin homolog HMP-2. Ablation of this phosphoserine interaction produces developmental defects that resemble full loss-of-function (Hammerhead and Humpback) phenotypes. Most metazoans possess a single gene for β-catenin, which is also amore » transcriptional coactivator in Wnt signaling. Nematodes and planaria, however, have a set of paralogous β-catenins; for example, C. elegans HMP-2 functions only in cell-cell adhesion, whereas SYS-1 mediates transcriptional activation through interactions with POP-1/Tcf. Finally, our structural data define critical sequence differences responsible for the unique ligand specificities of these two proteins.« less

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

PubMed

Lafontaine, D; Beaudry, D; Marquis, P; Perreault, J P

1995-10-01

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

78 FR 24701 - Endangered and Threatened Wildlife; 90-Day Finding on Petitions To List the Great Hammerhead...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-26

... ``may be'' warranted. As a general matter, these decisions hold that a petition need not establish a... dorsal side colored dark brown to light grey or olive that shades to white on the ventral side (Compagno... this region lack the resources to provide effective or, for that matter, any enforcement, with some...

Differential co-localization with choline acetyltransferase in nervus terminalis suggests functional differences for GnRH isoforms in bonnethead sharks (Sphyrna tiburo)

PubMed Central

Moeller, John F.; Meredith, Michael

2010-01-01

The nervus terminalis (NT) is a vertebrate cranial nerve whose function in adults is unknown. In bonnethead sharks the nerve is anatomically independent of the olfactory system, with two major cell populations within one or more ganglia along its exposed length. Most cells are immunoreactive for either gonadotropin-releasing hormone (GnRH) or RFamide-like peptides. To define further the cell populations and connectivity, we used double-label immuno-cytochemistry with antisera to different isoforms of GnRH and to choline acetyltransferase (ChAT). The labeling patterns of two GnRH antisera revealed different populations of GnRH immunoreactive (ir) cell-profiles in the NT ganglion. One antiserum labeled a large group of cells and fibers, which likely contain mammalian GnRH (GnRH-I) as described in previous studies, and which were ChAT immunoreactive. The other antiserum labeled large club-like structures, which were anuclear, and a sparse number of fibers, but with no clear labeling of cell bodies in the ganglion. These club structures were choline acetyltrasferase (ChAT) negative, and preabsorption control tests suggest they may contain chicken-GnRH-II (GnRH-II) or dogfish GnRH. The second major NT ganglion cell-type was immunoreactive for RF-amides, which regulate GnRH release in other vertebrates, and may provide an intraganglionic influence on GnRH release. The immunocytochemical and anatomical differences between the two GnRH immunoreactive profile types indicate possible functional differences for these isoforms in the NT. The club-like structures may be sites of GnRH release into the general circulation since these structures were observed near blood vessels and resembled structures seen in the median eminence of rats. PMID:20950589

Differential co-localization with choline acetyltransferase in nervus terminalis suggests functional differences for GnRH isoforms in bonnethead sharks (Sphyrna tiburo).

PubMed

Moeller, John F; Meredith, Michael

2010-12-17

The nervus terminalis (NT) is a vertebrate cranial nerve whose function in adults is unknown. In bonnethead sharks, the nerve is anatomically independent of the olfactory system, with two major cell populations within one or more ganglia along its exposed length. Most cells are immunoreactive for either gonadotropin-releasing hormone (GnRH) or RF-amide-like peptides. To define further the cell populations and connectivity, we used double-label immunocytochemistry with antisera to different isoforms of GnRH and to choline acetyltransferase (ChAT). The labeling patterns of two GnRH antisera revealed different populations of GnRH-immunoreactive (ir) cell profiles in the NT ganglion. One antiserum labeled a large group of cells and fibers, which likely contain mammalian GnRH (GnRH-I) as described in previous studies and which were ChAT immunoreactive. The other antiserum labeled large club-like structures, which were anuclear, and a sparse number of fibers, but with no clear labeling of cell bodies in the ganglion. These club structures were choline acetyltrasferase (ChAT)-negative, and preabsorption control tests suggest they may contain chicken-GnRH-II (GnRH-II) or dogfish GnRH. The second major NT ganglion cell-type was immunoreactive for RF-amides, which regulate GnRH release in other vertebrates, and may provide an intraganglionic influence on GnRH release. The immunocytochemical and anatomical differences between the two GnRH-immunoreactive profile types indicate possible functional differences for these isoforms in the NT. The club-like structures may be sites of GnRH release into the general circulation since these structures were observed near blood vessels and resembled structures seen in the median eminence of rats. Copyright © 2010 Elsevier B.V. All rights reserved.

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

Multifrequency Pulsed EPR Studies of Biologically Relevant Manganese(II) Complexes

PubMed Central

Stich, T. A.; Lahiri, S.; Yeagle, G.; Dicus, M.; Brynda, M.; Gunn, A.; Aznar, C.; DeRose, V. J.; Britt, R. D.

2011-01-01

Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H2O)6]2+, Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echo-detected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented. PMID:22190766

Utility of mesohabitat features for determining habitat associations of subadult sharks in Georgia’s estuaries

USGS Publications Warehouse

Belcher, C.N.; Jennings, Cecil A.

2010-01-01

We examined the affects of selected water quality variables on the presence of subadult sharks in six of nine Georgia estuaries. During 231 longline sets, we captured 415 individuals representing nine species. Atlantic sharpnose shark (Rhizoprionodon terranovae), bonnethead (Sphyrna tiburo), blacktip shark (Carcharhinus limbatus) and sandbar shark (C. plumbeus) comprised 96.1% of the catch. Canonical correlation analysis (CCA) was used to assess environmental influences on the assemblage of the four common species. Results of the CCA indicated Bonnethead Shark and Sandbar Shark were correlated with each other and with a subset of environmental variables. When the species occurred singly, depth was the defining environmental variable; whereas, when the two co-occurred, dissolved oxygen and salinity were the defining variables. Discriminant analyses (DA) were used to assess environmental influences on individual species. Results of the discriminant analyses supported the general CCA findings that the presence of bonnethead and sandbar shark were the only two species that correlated with environmental variables. In addition to depth and dissolved oxygen, turbidity influenced the presence of sandbar shark. The presence of bonnethead shark was influenced primarily by salinity and turbidity. Significant relationships existed for both the CCA and DA analyses; however, environmental variables accounted for <16% of the total variation in each. Compared to the environmental variables we measured, macrohabitat features (e.g., substrate type), prey availability, and susceptibility to predation may have stronger influences on the presence and distribution of subadult shark species among sites.

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

Giant worms chez moi! Hammerhead flatworms (Platyhelminthes, Geoplanidae, Bipalium spp., Diversibipalium spp.) in metropolitan France and overseas French territories

PubMed Central

Winsor, Leigh; Gey, Delphine; Gros, Pierre; Thévenot, Jessica

2018-01-01

Background Species of the genera Bipalium and Diversibipalium, or bipaliines, are giants among land planarians (family Geoplanidae), reaching length of 1 m; they are also easily distinguished from other land flatworms by the characteristic hammer shape of their head. Bipaliines, which have their origin in warm parts of Asia, are invasive species, now widespread worldwide. However, the scientific literature is very scarce about the widespread repartition of these species, and their invasion in European countries has not been studied. Methods In this paper, on the basis of a four year survey based on citizen science, which yielded observations from 1999 to 2017 and a total of 111 records, we provide information about the five species present in Metropolitan France and French overseas territories. We also investigated the molecular variability of cytochrome-oxidase 1 (COI) sequences of specimens. Results Three species are reported from Metropolitan France: Bipalium kewense, Diversibipalium multilineatum, and an unnamed Diversibipalium ‘black’ species. We also report the presence of B. kewense from overseas territories, such as French Polynesia (Oceania), French Guiana (South America), the Caribbean French islands of Martinique, Guadeloupe, Saint Martin and Saint Barthélemy, and Montserrat (Central America), and La Réunion island (off South-East Africa). For B. vagum, observations include French Guiana, Guadeloupe, Martinique, Saint Barthélemy, Saint Martin, Montserrat, La Réunion, and Florida (USA). A probable new species, Diversibipalium sp. ‘blue,’ is reported from Mayotte Island (off South–East Africa). B. kewense, B. vagum and D. multilineatum each showed 0% variability in their COI sequences, whatever their origin, suggesting that the specimens are clonal, and that sexual reproduction is probably absent. COI barcoding was efficient in identifying species, with differences over 10% between species; this suggests that barcoding can be used in the future for identifying these invasive species. In Metropolitan south–west France, a small area located in the Department of Pyrénées-Atlantiques was found to be a hot-spot of bipaliine biodiversity and abundance for more than 20 years, probably because of the local mild weather. Discussion The present findings strongly suggest that the species present in Metropolitan France and overseas territories should be considered invasive alien species. Our numerous records in the open in Metropolitan France raise questions: as scientists, we were amazed that these long and brightly coloured worms could escape the attention of scientists and authorities in a European developed country for such a long time; improved awareness about land planarians is certainly necessary. PMID:29844951

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Flow over a Biomimetic Surface Roughness Microgeometry

NASA Astrophysics Data System (ADS)

Warncke Lang, Amy; Hidalgo, Pablo; Westcott, Matthew

2006-11-01

Certain species of sharks (e.g. shortfin mako and common hammerhead) have a skin structure that could result in a bristling of their denticles (scales) during increased swimming speeds (Bechert, D. W., Bruse, M., Hage, W. and Meyer, R. 2000, Fluid mechanics of biological surfaces and their technological application. Naturwissenschaften 80:157-171). This unique surface geometry results in a three-dimensional array of cavities* (d-type roughness geometry) forming within the surface and has been given the acronym MAKO (Micro-roughness Array for Kinematic Optimization). Possible mechanisms leading to drag reduction over the shark's body by this unique roughness geometry include separation control thereby reducing pressure drag, skin friction reduction (via the `micro-air bearing' effect first proposed by Bushnell (AIAA 83-0227)), as well as possible transition delay in the boundary layer. Initial work is confined to scaling up the geometry from 0.2 mm on the shark skin to 2 cm, with a scaling down in characteristic velocity from 10 - 20 m/s to 10 - 20 cm/s for laminar flow boundary layer water tunnel studies. Support for this research by NSF SGER grant CTS-0630489 and a University of Alabama RAC grant is gratefully acknowledged. * Patent pending.

Use of a fishery-independent trawl survey to evaluate distribution patterns of subadult sharks in Georgia

USGS Publications Warehouse

Belcher, C.N.; Jennings, Cecil A.

2009-01-01

We investigated the utility of a fishery-independent trawl survey for assessing a potential multispecies shark nursery in Georgia's nearshore and inshore waters. A total of 234 subadult sharks from six species were captured during 85 of 216 trawls. Catch rates and size distributions for subadult sharks and the ratio of neonates to juveniles were consistent among areas. The highest concentrations of subadult sharks occurred in creeks and sounds. Species composition varied among areas. The Atlantic sharpnose shark Rhizoprionodon terraenovae was the most abundant species in sound and nearshore stations, whereas the bonnethead Sphyrna tiburo was the most abundant species in creeks. The aggregate of other species occurred with higher frequency in the sounds and nearshore. Sampling characteristics of the trawl survey were compared with those from a fishery-independent longline survey of subadult sharks to assess the similarity of the two gears. A total of 193 subadult sharks from seven species were captured during 57 of 96 longline sets, whereas 52 subadults from four species were captured during 20 of 48 trawls. Selectivity and efficiency differed between the two gears. The trawl had lower catch rates, caught smaller sharks, and encountered a different suite of species than the longline. General seasonal trends in relative abundance also differed between the two gears; the longline showed an increasing trend in abundance, whereas the trawl showed a stable trend. Although trawls were not found to be efficient for sampling subadult sharks from most species, they can be a useful source of supplemental data.

Sexual dimorphism in sister species of Leucoraja skate and its relationship to reproductive strategy and life history.

PubMed

Martinez, Christopher M; Rohlf, F James; Frisk, Michael G

2016-01-01

Instances of sexual dimorphism occur in a great variety of forms and manifestations. Most skates (Batoidea: Rajoidei) display some level of body shape dimorphism in which the pectoral fins of mature males develop to create a distinct bell-shaped body not found in females. This particular form of dimorphism is present in each of the sister species Leucoraja erinacea and Leucoraja ocellata, but differences between sexes are much greater in the former. In order to understand the nature and potential causes of pectoral dimorphism, we used geometric morphometrics to investigate allometry of fin shape in L. erinacea and L. ocellata and its relationship to the development of reproductive organs, based on previous work on the bonnethead shark, Sphyrna tiburo. We found that allometric trajectories of overall pectoral shape were different in both species of skate, but only L. erinacea varied significantly with respect to endoskeleton development. Male maturation was characterized by a number of sex-specific morphological changes, which appeared concurrently in developmental timing with elongation of cartilage-supported claspers. We suggest that external sexual dimorphism of pectoral fins in skates is a byproduct of skeletal growth needed for clasper development. Further, the magnitude of male shape change appears to be linked to the differential life histories of species. This work reports for the first time that pectoral dimorphism is a persistent feature in rajoid fishes, occurring in varying degrees across several genera. Lastly, our results suggest that pectoral morphology may be useful as a relative indicator of reproductive strategy in some species. © 2016 Wiley Periodicals, Inc.

Stiff and tough: a comparative study on the tensile properties of shark skin.

PubMed

Creager, Shelby B; Porter, Marianne E

2018-02-01

In sharks, the skin is a biological composite with mineralized denticles embedded within a collagenous matrix. Swimming performance is enhanced by the dermal denticles on the skin, which have drag reducing properties produced by regional morphological variations and changes in density along the body. We used mechanical testing to quantify the effect of embedded mineralized denticles on the quasi-static tensile properties of shark skin to failure in four coastal species. We investigated regional differences in denticle density and skin properties by dissecting skin from the underlying fascia and muscle at 10 anatomical landmarks. Hourglass-shaped skin samples were extracted in the cranial to caudal orientation. Denticle density was quantified and varied significantly among both regions and species. We observed the greatest denticle densities in the cranial region of the body for the bonnethead, scalloped hammerhead, and bull sharks. Skin samples were then tested in tension until failure, stress strain curves were generated, and mechanical properties calculated. We found significant species and region effects for all three tensile mechanical properties. We report the greatest ultimate tensile strength, stiffness, and toughness near the cranial and lateral regions of the body for all 4 of the coastal species. We also report that denticle density increases with skin stiffness but decreases with toughness. Copyright © 2017 Elsevier GmbH. All rights reserved.

DNA barcoding of shark meats identify species composition and CITES-listed species from the markets in Taiwan.

PubMed

Liu, Shang-Yin Vanson; Chan, Chia-Ling Carynn; Lin, Oceana; Hu, Chieh-Shen; Chen, Chaolun Allen

2013-01-01

An increasing awareness of the vulnerability of sharks to exploitation by shark finning has contributed to a growing concern about an unsustainable shark fishery. Taiwan's fleet has the 4th largest shark catch in the world, accounting for almost 6% of the global figures. Revealing the diversity of sharks consumed by Taiwanese is important in designing conservation plans. However, fins make up less than 5% of the total body weight of a shark, and their bodies are sold as filets in the market, making it difficult or impossible to identify species using morphological traits. In the present study, we adopted a DNA barcoding technique using a 391-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene to examine the diversity of shark filets and fins collected from markets and restaurants island-wide in Taiwan. Amongst the 548 tissue samples collected and sequenced, 20 major clusters were apparent by phylogenetic analyses, each of them containing individuals belonging to the same species (most with more than 95% bootstrap values), corresponding to 20 species of sharks. Additionally, Alopias pelagicus, Carcharhinus falciformis, Isurus oxyrinchus, and Prionace glauca consisted of 80% of the samples we collected, indicating that these species might be heavily consumed in Taiwan. Approximately 5% of the tissue samples used in this study were identified as species listed in CITES Appendix II, including two species of Sphyrna, C. longimanus and Carcharodon carcharias. DNA barcoding provides an alternative method for understanding shark species composition when species-specific data is unavailable. Considering the global population decline, stock assessments of Appendix II species and highly consumed species are needed to accomplish the ultimate goal of shark conservation.

A retrospective study of disease in elasmobranchs.

PubMed

Garner, M M

2013-05-01

This report reviews diseases of 1546 elasmobranchs representing at least 60 species submitted to Northwest ZooPath from 1994 to 2010. Cownose rays (Rhinoptera bonasus) (78), southern rays (Dasyatis americana) (75), dusky smooth-hounds (Mustelus canis) (74), bonnethead sharks (Sphyrna tiburo) (66), and bamboo sharks (Hemiscylliidae) (56) were the most commonly submitted species. Infectious/inflammatory disease was most common (33.5%) followed by nutritional (11.9%, mostly emaciation), traumatic (11.3%), cardiovascular (5.5%, mostly shock), and toxin-associated disease (3.7%). Bacterial infections (518/1546, 15%) included sepsis (136/518, 26%), dermatitis (7%), branchitis (6%), and enteritis (4%). Fungal infections (10/1546, 0.6%) included dermatitis (30%), hepatitis (30%), and branchitis (20%). Viral or suspected viral infections or disease processes (15/1546, 1%) included papillomatosis (47%), herpesvirus (20%), and adenovirus (7%). Parasitic infections (137/1546, 9%) included nematodiasis (36/137, 26%), ciliate infections (23%), trematodiasis (20%), coccidiosis (6%), myxozoanosis (5%), amoebiasis (4%), cestodiasis (1%), and flagellate infections (1%). Inflammation of unknown cause (401/1546, 26%) included enteritis (55/401, 14%), branchitis (9%), encephalitis (9%), and dermatitis (7%). Traumatic diseases (174/1546, 11.3%) included skin trauma (103/174, 60%), stress/maladaptation (9%), and gut trauma (7%). Toxicoses (57/1546, 4%) included toxic gill disease (16/57, 26%), gas bubble disease (19%), fenbendazole (7%), ammonia (7%), chlorine (5%), and chloramine (3%). Species trends included visceral nematodiasis in black-nosed sharks (Carcharhinus acronotus) (55%); sepsis in dusky smooth-hounds (41%), blue-spotted stingrays (36%), southern rays (36%), and wobeggong sharks (Orectolobus spp) (69%); emaciation in bamboo (33%) and bonnethead (32%) sharks and freshwater stingrays (Potamotrygon motoro) (32%); and trauma in bonnethead sharks (30%).

Relationships of mercury concentrations across tissue types, muscle regions and fins for two shark species.

PubMed

O'Bryhim, Jason R; Adams, Douglas H; Spaet, Julia L Y; Mills, Gary; Lance, Stacey L

2017-04-01

Mercury (Hg) exposure poses a threat to both fish and human health. Sharks are known to bioaccumulate Hg, however, little is known regarding how Hg is distributed between different tissue groups (e.g. muscle regions, organs). Here we evaluated total mercury (THg) concentrations from eight muscle regions, four fins (first dorsal, left and right pectorals, caudal-from both the inner core and trailing margin of each fin), and five internal organs (liver, kidney, spleen, heart, epigonal organ) from two different shark species, bonnethead (Sphyrna tiburo) and silky shark (Carcharhinus falciformis) to determine the relationships of THg concentrations between and within tissue groups. Total Hg concentrations were highest in the eight muscle regions with no significant differences in THg concentrations between the different muscle regions and muscle types (red and white). Results from tissue collected from any muscle region would be representative of all muscle sample locations. Total Hg concentrations were lowest in samples taken from the fin inner core of the first dorsal, pectoral, and caudal (lower lobe) fins. Mercury concentrations for samples taken from the trailing margin of the dorsal, pectoral, and caudal fins (upper and lower lobe) were also not significantly different from each other for both species. Significant relationships were found between THg concentrations in dorsal axial muscle tissue and the fin inner core, liver, kidney, spleen and heart for both species as well as the THg concentrations between the dorsal fin trailing margin and the heart for the silky shark and all other sampled tissue types for the bonnethead shark. Our results suggest that biopsy sampling of dorsal muscle can provide data that can effectively estimate THg concentrations in specific organs without using more invasive, or lethal methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA Barcoding of Shark Meats Identify Species Composition and CITES-Listed Species from the Markets in Taiwan

PubMed Central

Liu, Shang-Yin Vanson; Chan, Chia-Ling Carynn; Lin, Oceana; Hu, Chieh-Shen; Chen, Chaolun Allen

2013-01-01

Background An increasing awareness of the vulnerability of sharks to exploitation by shark finning has contributed to a growing concern about an unsustainable shark fishery. Taiwan’s fleet has the 4th largest shark catch in the world, accounting for almost 6% of the global figures. Revealing the diversity of sharks consumed by Taiwanese is important in designing conservation plans. However, fins make up less than 5% of the total body weight of a shark, and their bodies are sold as filets in the market, making it difficult or impossible to identify species using morphological traits. Methods In the present study, we adopted a DNA barcoding technique using a 391-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene to examine the diversity of shark filets and fins collected from markets and restaurants island-wide in Taiwan. Results Amongst the 548 tissue samples collected and sequenced, 20 major clusters were apparent by phylogenetic analyses, each of them containing individuals belonging to the same species (most with more than 95% bootstrap values), corresponding to 20 species of sharks. Additionally, Alopias pelagicus, Carcharhinus falciformis, Isurus oxyrinchus, and Prionace glauca consisted of 80% of the samples we collected, indicating that these species might be heavily consumed in Taiwan. Approximately 5% of the tissue samples used in this study were identified as species listed in CITES Appendix II, including two species of Sphyrna, C. longimanus and Carcharodon carcharias. Conclusion DNA barcoding provides an alternative method for understanding shark species composition when species-specific data is unavailable. Considering the global population decline, stock assessments of Appendix II species and highly consumed species are needed to accomplish the ultimate goal of shark conservation. PMID:24260209

Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae.

PubMed

Klauser, Benedikt; Rehm, Charlotte; Summerer, Daniel; Hartig, Jörg S

2015-01-01

Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches. © 2015 Elsevier Inc. All rights reserved.

Subsea ice-bearing permafrost on the U.S. Beaufort Margin: 2. Borehole constraints

USGS Publications Warehouse

Ruppel, Carolyn D.; Herman, Bruce M.; Brothers, Laura L.; Hart, Patrick E.

2016-01-01

Borehole logging data from legacy wells directly constrain the contemporary distribution of subsea permafrost in the sedimentary section at discrete locations on the U.S. Beaufort Margin and complement recent regional analyses of exploration seismic data to delineate the permafrost's offshore extent. Most usable borehole data were acquired on a ∼500 km stretch of the margin and within 30 km of the contemporary coastline from north of Lake Teshekpuk to nearly the U.S.-Canada border. Relying primarily on deep resistivity logs that should be largely unaffected by drilling fluids and hole conditions, the analysis reveals the persistence of several hundred vertical meters of ice-bonded permafrost in nearshore wells near Prudhoe Bay and Foggy Island Bay, with less permafrost detected to the east and west. Permafrost is inferred beneath many barrier islands and in some nearshore and lagoonal (back-barrier) wells. The analysis of borehole logs confirms the offshore pattern of ice-bearing subsea permafrost distribution determined based on regional seismic analyses and reveals that ice content generally diminishes with distance from the coastline. Lacking better well distribution, it is not possible to determine the absolute seaward extent of ice-bearing permafrost, nor the distribution of permafrost beneath the present-day continental shelf at the end of the Pleistocene. However, the recovery of gas hydrate from an outer shelf well (Belcher) and previous delineation of a log signature possibly indicating gas hydrate in an inner shelf well (Hammerhead 2) imply that permafrost may once have extended across much of the shelf offshore Camden Bay.

Quantifying the Spatial Ecology of Wide-Ranging Marine Species in the Gulf of California: Implications for Marine Conservation Planning

PubMed Central

Anadón, José Daniel; D'Agrosa, Caterina; Gondor, Anne; Gerber, Leah R.

2011-01-01

There is growing interest in systematic establishment of marine protected area (MPA) networks and representative conservation sites. This movement toward networks of no-take zones requires that reserves are deliberately and adequately spaced for connectivity. Here, we test the network functionality of an ecoregional assessment configuration of marine conservation areas by evaluating the habitat protection and connectivity offered to wide-ranging fauna in the Gulf of California (GOC, Mexico). We first use expert opinion to identify representative species of wide-ranging fauna of the GOC. These include leopard grouper, hammerhead sharks, California brown pelicans and green sea turtles. Analyzing habitat models with both structural and functional connectivity indexes, our results indicate that the configuration includes large proportions of biologically important habitat for the four species considered (25–40%), particularly, the best quality habitats (46–57%). Our results also show that connectivity levels offered by the conservation area design for these four species may be similar to connectivity levels offered by the entire Gulf of California, thus indicating that connectivity offered by the areas may resemble natural connectivity. The selected focal species comprise different life histories among marine or marine-related vertebrates and are associated with those habitats holding the most biodiversity values (i.e. coastal habitats); our results thus suggest that the proposed configuration may function as a network for connectivity and may adequately represent the marine megafauna in the GOC, including the potential connectivity among habitat patches. This work highlights the range of approaches that can be used to quantify habitat protection and connectivity for wide-ranging marine species in marine reserve networks. PMID:22163013

Quantifying the spatial ecology of wide-ranging marine species in the Gulf of California: implications for marine conservation planning.

PubMed

Anadón, José Daniel; D'Agrosa, Caterina; Gondor, Anne; Gerber, Leah R

2011-01-01

There is growing interest in systematic establishment of marine protected area (MPA) networks and representative conservation sites. This movement toward networks of no-take zones requires that reserves are deliberately and adequately spaced for connectivity. Here, we test the network functionality of an ecoregional assessment configuration of marine conservation areas by evaluating the habitat protection and connectivity offered to wide-ranging fauna in the Gulf of California (GOC, Mexico). We first use expert opinion to identify representative species of wide-ranging fauna of the GOC. These include leopard grouper, hammerhead sharks, California brown pelicans and green sea turtles. Analyzing habitat models with both structural and functional connectivity indexes, our results indicate that the configuration includes large proportions of biologically important habitat for the four species considered (25-40%), particularly, the best quality habitats (46-57%). Our results also show that connectivity levels offered by the conservation area design for these four species may be similar to connectivity levels offered by the entire Gulf of California, thus indicating that connectivity offered by the areas may resemble natural connectivity. The selected focal species comprise different life histories among marine or marine-related vertebrates and are associated with those habitats holding the most biodiversity values (i.e. coastal habitats); our results thus suggest that the proposed configuration may function as a network for connectivity and may adequately represent the marine megafauna in the GOC, including the potential connectivity among habitat patches. This work highlights the range of approaches that can be used to quantify habitat protection and connectivity for wide-ranging marine species in marine reserve networks.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

PubMed Central

Sripathi, Kamali N.; Tay, Wendy W.; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G.

2014-01-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2′-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. PMID:24854621

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape.

PubMed

Sripathi, Kamali N; Tay, Wendy W; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G

2014-07-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. © 2014 Sripathi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Building oceanographic and atmospheric observation networks by composition: unmanned vehicles, communication networks, and planning and execution control frameworks

NASA Astrophysics Data System (ADS)

Sousa, J. T.; Pinto, J.; Martins, R.; Costa, M.; Ferreira, F.; Gomes, R.

2014-12-01

The problem of developing mobile oceanographic and atmospheric observation networks (MOAO) with coordinated air and ocean vehicles is discussed in the framework of the communications and control software tool chain developed at Underwater Systems and Technologies Laboratory (LSTS) from Porto University. This is done with reference to field experiments to illustrate key capabilities and to assess future MOAO operations. First, the motivation for building MOAO by "composition" of air and ocean vehicles, communication networks, and planning and execution control frameworks is discussed - in networked vehicle systems information and commands are exchanged among multiple vehicles and operators, and the roles, relative positions, and dependencies of these vehicles and operators change during operations. Second, the planning and execution control framework developed at LSTS for multi-vehicle systems is discussed with reference to key concepts such as autonomy, mixed-initiative interactions, and layered organization. Third, the LSTS tool software tool chain is presented to show how to develop MOAO by composition. The tool chain comprises the Neptus command and control framework for mixed initiative interactions, the underlying IMC messaging protocol, and the DUNE on-board software. Fourth, selected LSTS operational deployments illustrate MOAO capability building. In 2012 we demonstrated the use of UAS to "ferry" data from UUVs located beyond line of sight (BLOS). In 2013 we demonstrated coordinated observations of coastal fronts with small UAS and UUVs, "bent" BLOS through the use of UAS as communication relays, and UAS tracking of juvenile hammer-head sharks. In 2014 we demonstrated UUV adaptive sampling with the closed loop controller of the UUV residing on a UAS; this was done with the help of a Wave Glider ASV with a communications gateway. The results from these experiments provide a background for assessing potential future UAS operations in a compositional MOAO.

Marfan syndrome, magnesium status and medical prevention of cardiovascular complications by hemodynamic treatments and antisense gene therapy.

PubMed

Igondjo-Tchen, S; Pagès, N; Bac, P; Godeau, G; Durlach, J

2003-03-01

The medical management of Marfan Syndrome (MFS) mainly relies on early prevention of the aortic complications. Hemodynamic treatments try to diminish the forcefulness of cardiac contractions and to reduce blood pressure: for example long term administration of propranolol may significantly reduce the rate of increase in aortic ratio (aortic diameter/expected aortic diameter). Retardation of aortic dilatation may be most often observed by early treatment started when the baseline end-diastolic aortic root diameter is < 40 mm. It seems better to use beta-blockers without intrinsic sympathomimetic activity. Successful acceptance of beta-blockers may be limited by side-effects, but the efficiency of alternative hypotensive agents (calcium channel inhibitors, ACE inhibitors) is not yet validated. Gene therapy might constitute an etiologic specific treatment of MFS. FBN1-RZ1 hammerhead antisense ribozyme is able to suppress expression of the mutant FBN1 allele. The use of ribozymes as systemic therapeutic agents will depend on efficient delivery to its target, but the various proposed vectors raise yet unsolved problems. A hydrogel angioplasty balloon might be a possible vector for delivering an antisense ribozyme in the aortic wall specifically. Ribozymes--as deoxyribonucleotides--may be taken up by tissue upon local application. Further research should study ex vivo local application of antisense ribozyme on human aortic wall, before assessing in vivo efficiency and tolerance of this aortic local vectorisation. It is always necessary to maintain a balanced magnesium intake in patients with MFS. Firstly to prevent the multiple noxious effects of magnesium deficiency on cardiovascular targets. Secondly to ensure the best efficiency and the least toxicity of the hemodynamic drugs used as long term prophylactic treatment for cardiovascular complications and of the etiologic antisense magnesium-dependent gene therapy, in the future.

RNA Thermodynamic Structural Entropy

PubMed Central

Garcia-Martin, Juan Antonio; Clote, Peter

2015-01-01

Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner’99 and Turner’04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

PubMed Central

Tan, Chee Wah; Tee, Han Kang; Lee, Michelle Hui Pheng; Sam, I-Ching; Chan, Yoke Fun

2016-01-01

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3’ ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71. PMID:27617744

Measurement correction method for force sensor used in dynamic pressure calibration based on artificial neural network optimized by genetic algorithm

NASA Astrophysics Data System (ADS)

Gu, Tingwei; Kong, Deren; Shang, Fei; Chen, Jing

2017-12-01

We present an optimization algorithm to obtain low-uncertainty dynamic pressure measurements from a force-transducer-based device. In this paper, the advantages and disadvantages of the methods that are commonly used to measure the propellant powder gas pressure, the applicable scope of dynamic pressure calibration devices, and the shortcomings of the traditional comparison calibration method based on the drop-weight device are firstly analysed in detail. Then, a dynamic calibration method for measuring pressure using a force sensor based on a drop-weight device is introduced. This method can effectively save time when many pressure sensors are calibrated simultaneously and extend the life of expensive reference sensors. However, the force sensor is installed between the drop-weight and the hammerhead by transition pieces through the connection mode of bolt fastening, which causes adverse effects such as additional pretightening and inertia forces. To solve these effects, the influence mechanisms of the pretightening force, the inertia force and other influence factors on the force measurement are theoretically analysed. Then a measurement correction method for the force measurement is proposed based on an artificial neural network optimized by a genetic algorithm. The training and testing data sets are obtained from calibration tests, and the selection criteria for the key parameters of the correction model is discussed. The evaluation results for the test data show that the correction model can effectively improve the force measurement accuracy of the force sensor. Compared with the traditional high-accuracy comparison calibration method, the percentage difference of the impact-force-based measurement is less than 0.6% and the relative uncertainty of the corrected force value is 1.95%, which can meet the requirements of engineering applications.

Experimental Visualizations of a Generic Launch Vehicle Flow Field: Time-Resolved Shadowgraph and Infrared Imaging

NASA Technical Reports Server (NTRS)

Garbeff, Theodore J., II; Panda, Jayanta; Ross, James C.

2017-01-01

Time-Resolved shadowgraph and infrared (IR) imaging were performed to investigate off-body and on-body flow features of a generic, 'hammer-head' launch vehicle geometry previously tested by Coe and Nute (1962). The measurements discussed here were one part of a large range of wind tunnel test techniques that included steady-state pressure sensitive paint (PSP), dynamic PSP, unsteady surface pressures, and unsteady force measurements. Image data was captured over a Mach number range of 0.6 less than or equal to M less than or equal to 1.2 at a Reynolds number of 3 million per foot. Both shadowgraph and IR imagery were captured in conjunction with unsteady pressures and forces and correlated with IRIG-B timing. High-speed shadowgraph imagery was used to identify wake structure and reattachment behind the payload fairing of the vehicle. Various data processing strategies were employed and ultimately these results correlated well with the location and magnitude of unsteady surface pressure measurements. Two research grade IR cameras were positioned to image boundary layer transition at the vehicle nose and flow reattachment behind the payload fairing. The poor emissivity of the model surface treatment (fast PSP) proved to be challenging for the infrared measurement. Reference image subtraction and contrast limited adaptive histogram equalization (CLAHE) were used to analyze this dataset. Ultimately turbulent boundary layer transition was observed and located forward of the trip dot line at the model sphere-cone junction. Flow reattachment location was identified behind the payload fairing in both steady and unsteady thermal data. As demonstrated in this effort, recent advances in high-speed and thermal imaging technology have modernized classical techniques providing a new viewpoint for the modern researcher

RNA Thermodynamic Structural Entropy.

PubMed

Garcia-Martin, Juan Antonio; Clote, Peter

2015-01-01

Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

EM calibration based on Post OPC layout analysis

NASA Astrophysics Data System (ADS)

Sreedhar, Aswin; Kundu, Sandip

2010-03-01

Design for Manufacturability (DFM) involves changes to the design and CAD tools to help increase pattern printability and improve process control. Design for Reliability (DFR) performs the same to improve reliability of devices from failures such as Electromigration (EM), gate-oxide break down, hot carrier injection (HCI), Negative Bias Temperature Insatiability (NBTI) and mechanical stress effects. Electromigration (EM) occurs due to migration or displacement of atoms as a result of the movement of electrons through a conducting medium. The rate of migration determines the Mean Time to Failure (MTTF) which is modeled as a function of temperature and current density. The model itself is calibrated through failure analysis (FA) of parts that are deemed to have failed due to EM against design parameters such as linewidth. Reliability Verification (RV) of a design involves verifying that every conducting line in a design meets certain MTTF threshold. In order to perform RV, current density for each wire must be computed. Current itself is a function of the parasitics that are determined through RC extraction. The standard practice is to perform the RC extraction and current density calculation on drawn, pre-OPC layouts. If a wire fails to meet threshold for MTTF, it may be resized. Subsequently, mask preparation steps such as OPC and PSM introduce extra features such as SRAFs, jogs,hammerheads and serifs that change their resistance, capacitance and current density values. Hence, calibrating EM model based on pre-OPC layouts will lead to different results compared to post-OPC layouts. In this work, we compare EM model calibration and reliability check based on drawn layout versus predicted layout, where the drawn layout is pre-OPC layout and predicted layout is based on litho simulation of post-OPC layout. Results show significant divergence between these two approaches, making a case for methodology based on predicted layout.

Quantitative comparison of two independent lateral force calibration techniques for the atomic force microscope.

PubMed

Barkley, Sarice S; Deng, Zhao; Gates, Richard S; Reitsma, Mark G; Cannara, Rachel J

2012-02-01

Two independent lateral-force calibration methods for the atomic force microscope (AFM)--the hammerhead (HH) technique and the diamagnetic lateral force calibrator (D-LFC)--are systematically compared and found to agree to within 5 % or less, but with precision limited to about 15 %, using four different tee-shaped HH reference probes. The limitations of each method, both of which offer independent yet feasible paths toward traceable accuracy, are discussed and investigated. We find that stiff cantilevers may produce inconsistent D-LFC values through the application of excessively high normal loads. In addition, D-LFC results vary when the method is implemented using different modes of AFM feedback control, constant height and constant force modes, where the latter is more consistent with the HH method and closer to typical experimental conditions. Specifically, for the D-LFC apparatus used here, calibration in constant height mode introduced errors up to 14 %. In constant force mode using a relatively stiff cantilever, we observed an ≈ 4 % systematic error per μN of applied load for loads ≤ 1 μN. The issue of excessive load typically emerges for cantilevers whose flexural spring constant is large compared with the normal spring constant of the D-LFC setup (such that relatively small cantilever flexural displacements produce relatively large loads). Overall, the HH method carries a larger uncertainty, which is dominated by uncertainty in measurement of the flexural spring constant of the HH cantilever as well as in the effective length dimension of the cantilever probe. The D-LFC method relies on fewer parameters and thus has fewer uncertainties associated with it. We thus show that it is the preferred method of the two, as long as care is taken to perform the calibration in constant force mode with low applied loads.

Feeding biomechanics and theoretical calculations of bite force in bull sharks (Carcharhinus leucas) during ontogeny.

PubMed

Habegger, Maria L; Motta, Philip J; Huber, Daniel R; Dean, Mason N

2012-12-01

Evaluations of bite force, either measured directly or calculated theoretically, have been used to investigate the maximum feeding performance of a wide variety of vertebrates. However, bite force studies of fishes have focused primarily on small species due to the intractable nature of large apex predators. More massive muscles can generate higher forces and many of these fishes attain immense sizes; it is unclear how much of their biting performance is driven purely by dramatic ontogenetic increases in body size versus size-specific selection for enhanced feeding performance. In this study, we investigated biting performance and feeding biomechanics of immature and mature individuals from an ontogenetic series of an apex predator, the bull shark, Carcharhinus leucas (73-285cm total length). Theoretical bite force ranged from 36 to 2128N at the most anterior bite point, and 170 to 5914N at the most posterior bite point over the ontogenetic series. Scaling patterns differed among the two age groups investigated; immature bull shark bite force scaled with positive allometry, whereas adult bite force scaled isometrically. When the bite force of C. leucas was compared to those of 12 other cartilaginous fishes, bull sharks presented the highest mass-specific bite force, greater than that of the white shark or the great hammerhead shark. A phylogenetic independent contrast analysis of anatomical and dietary variables as determinants of bite force in these 13 species indicated that the evolution of large adult bite forces in cartilaginous fishes is linked predominantly to the evolution of large body size. Multiple regressions based on mass-specific standardized contrasts suggest that the evolution of high bite forces in Chondrichthyes is further correlated with hypertrophication of the jaw adductors, increased leverage for anterior biting, and widening of the head. Lastly, we discuss the ecological significance of positive allometry in bite force as a possible

Recovery of metals from waste printed circuit boards by a mechanical method using a water medium.

PubMed

Duan, Chenlong; Wen, Xuefeng; Shi, Changsheng; Zhao, Yuemin; Wen, Baofeng; He, Yaqun

2009-07-15

Research on the recycling of waste printed circuit boards (PCB) is at the forefront of environmental pollution prevention and resource recycling. To effectively crush waste PCB and to solve the problem of secondary pollution from fugitive odors and dust created during the crushing process, a wet impacting crusher was employed to achieve comminution liberation of the PCB in a water medium. The function of water in the crushing process was analyzed. When using slippery hammerheads, a rotation speed of 1470 rpm, a water flow of 6m(3)/h and a sieve plate aperture of 2.2mm, 95.87% of the crushed product was sized less than 1mm. 94.30% of the metal was in this grade of product. Using smashed material graded -1mm for further research, a Falcon concentrator was used to recover the metal from the waste PCB. Engineering considerations were the liberation degree, the distribution ratio of the metal and a way to simplify the technology. The separation mechanism for fine particles of different densities in a Falcon concentrator was analyzed in detail and the separation process in the segregation and separation zones was deduced. Also, the magnitude of centrifugal acceleration, the back flow water pressure and the feed slurry concentration, any of which might affect separation results, were studied. A recovery model was established using Design-Expert software. Separating waste PCB, crushed to -1mm, with the Falcon separator gave a concentrated product graded 92.36% metal with a recovery of 97.05%. To do this the reverse water pressure was 0.05 MPa, the speed transducer frequency was set at 30 Hz and the feed density was 20 g/l. A flow diagram illustrating the new technique of wet impact crushing followed by separation with a Falcon concentrator is provided. The technique will prevent environmental pollution from waste PCB and allow the effective recovery of resources. Water was used as the medium throughout the whole process.

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

PubMed

Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

2016-04-01

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of the Active Site Guanine in the glmS Ribozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

PubMed Central

2015-01-01

The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40(N1) or possibly A-1(O2′), which would be followed by proton transfer from G40(N1) to A-1(O2′). After this initial deprotonation, A-1(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg2+ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated pKa rather than a pKa shifted toward neutrality, although a balance among the pKa’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40. PMID:25526516

Click nucleic acid ligation: applications in biology and nanotechnology.

PubMed

El-Sagheer, Afaf H; Brown, Tom

2012-08-21

Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have

In vivo Knock-down of the HSV-1 Latency-Associated Transcript Reduces Reactivation from Latency.

PubMed

Watson, Zachary L; Washington, Shannan D; Phelan, Dane M; Lewin, Alfred S; Tuli, Sonal S; Schultz, Gregory S; Neumann, Donna M; Bloom, David C

2018-06-06

During Herpes Simplex Virus (HSV) latency, most viral genes are silenced with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long non-coding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilize an Adeno-associated Virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness. Importance Herpes Simplex Virus (HSV) establishes a life long infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This

Efficient and Robust Paramyxoviridae Reverse Genetics Systems

PubMed Central

Beaty, Shannon M.; Won, Sohui T.; Hong, Patrick; Lyons, Michael; Vigant, Frederic; Freiberg, Alexander N.; tenOever, Benjamin R.; Duprex, W. Paul

2017-01-01

ABSTRACT The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses

Expression of death decoy receptor-3 (DcR3) in human breast cancer and its functional effects on breast cancer cells in vitro.

PubMed

Ge, Zhicheng; Sanders, Andrew J; Ye, Lin; Wang, Yu; Jiang, Wen G

2011-01-01

Death Decoy Receptor-3 (DcR3), otherwise known as tumour necrosis factor receptor superfamily member 6b, is suggested to be involved in the progression and immune evasion of malignant tumours. Its ligands include FASL and LIGHT (Tumour necrosis factor ligand superfamily member 14). DcR3 has been found to be amplified in certain solid tumours. However, its role in breast tumours remains unclear. In the present study, we examined the role played by DcR3 in MCF7 and MDA-MB-231 cell lines. The expression of DcR3 was examined in MCF7 and MDA-MB-231 cell lines using immunocytochemical staining and RT-PCR. Anti-DcR3 hammerhead ribozyme transgenes were constructed and transfected into cells to create DcR3 knock-down cell sublines. The biological impact of modifying DcR3 expression in breast cancer cells was evaluated using a variety of in vitro assays, including growth, adhesion, migration and invasion models. MCF7 and MDA-MB-231 cells, usually expressing DcR3, were transfected with the anti-DcR3 ribozyme transgene. Stable transfectants containing the DcR3 ribozyme transgene (MCF7DcR3KO, MDA-MB-231DcR3KO) displayed a reduction of DcR3 expression at mRNA and protein levels. DcR3 knockdown in MCF7 cells was found to significantly reduce invasive capacity compared to pEF6 control cell lines (30.78 +/- 6.40 vs.151.67 +/- 17.67 P < 0.001). The rate of migration in MCF7DcR3KO was significantly lower than MCF7pEF6 (P < 0.001). In contrast, no such significant differences was seen between MDA-MB-231DcR3KO and MDA-MB-231pEF6. Suppressing DcR3 expression was found to have an inhibitory effect on cellular invasion and migration in MCF7 breast cancer cells. This suggests that the invasion and migration capacity of this breast cancer cell line may, at least partly, depend on DcR3. DcR3 may be regarded as a negative regulator for aggressiveness during the development and progression of certain types of breast cancer.

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

NASA Technical Reports Server (NTRS)

Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

PubMed

Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

2004-03-01

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

PubMed Central

Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

2016-01-01

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a

Mercury in the atmospheric and coastal environments of Mexico.

PubMed

Ruelas-Inzunza, Jorge; Delgado-Alvarez, Carolina; Frías-Espericueta, Martín; Páez-Osuna, Federico

2013-01-01

sediments have increased from2- to 15-fold in recent years. Since the 1940s, historical increases of Hg fluxes have resulted from higher agricultural waste releases and exhaust from the thermo electric plants. The levels of Hg in water reveal a moderate to elevated contamination of some Mexican coastal sites. In Urias lagoon (NW Mexico), moderate to high levels were found in the dissolved and suspended fraction, and these are related to shipping, the fishing industry, domestic effluents, and the presence of a thermoelectric plant. In Coatzacoalcos (SE Mexico), extremely elevated Hg levels were found during the decade of the 1970s. Low to moderate levels of Hg were measured in waters from the Alvarado lagoon (SE Mexico); those concentrations appear to be associated with river waters that became enriched with organic matter and suspended solids inthe brackish mixing zone.Regarding the Hg content in invertebrates, the use of bivalves (oysters and mussels)as biomonitors must be established along the coastal zones of Mexico, because some coastal lagoons have not been previously monitored. In addition, more research is needed to investigate shrimp farms that are associated with agricultural basins and receive effluents from several anthropogenic sources (e.g., mining activity and urban discharges). Hg residues in several vertebrate groups collected in Mexico have been studied.These include mammals, birds, reptiles, and fish. In elasmobranch species, the highest Hg concentration (27.2 flg g-1 dry wt) was found in the muscle of the smooth hammer head shark (Sphyrna zygaena). Teleost fish are the vertebrate group that has been most studied, with regard to Hg residue content; the highest value (5.67 11g g-1dry wt) was detected in the striped marlin (T. audax). Among reptiles, only marine turtles were studied; Hg levels found ranged from 0.795 in the liver to 0.0006flg g-1dry wt in the blood of L. olivacea. In birds, the highest Hg concentration (5.08 flg g-1dry wt) detected was in