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PubMed1991

Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family.

Nucleotide sequencing data from a region extending 35 kb inward from the right inverted terminal repeat (ITR) of the vaccinia virus (VV) genome established the presence of VV homologues of the Shope fibroma virus (SFV) ITR proteins. The nucleotide sequences, comprising a total of 8.6 kb, and the amino acid translations for nine predicted open reading frames (ORFs) (designated SalF4L, SalF 19R, SalF21R, B4R, B8R, B9R, B10R, and B14R) are presented. Eight of the nine VV genes and all the SFV ORFs are transcribed towards their genomic termini. However, the relative positions of the VV genes (genus Orthopoxvirus) are different than those of the corresponding ORFs in SFV (genus Leporipoxvirus), indicating complex rearrangements of DNA in the genome of one or both of these viruses subsequent to their divergence from a common ancestor. Several other features of the VV ORFs were noted. SalF4L, B7R, B8R, and B9R have hydrophobic amino-terminal signal sequences but lack discernible membrane anchor domains suggesting that the proteins may be secreted. VV ORF SalF19R has a single cysteine-rich region homologous to the multiple domains of nerve growth factor receptor (NGFR), CD40, OX40 (a glycoprotein from the surface of activated murine T lymphocytes), and the recently described tumor necrosis factor receptors. Just downstream of the ORF SalF19R and in a different reading frame, there are another two related cysteine-rich domains, indicating that SalF19R was once a larger gene. B4R has homology to the host range gene of cowpox virus and to related genes near the opposite end of the vaccinia virus genome, and contains regions homologous to the repeat domains of erythrocyte ankyrin. In addition, several of the VV ORFs have homology to ORFs from near the opposite end of the VV genome, thus increasing the number of known VV gene families.

Amino Acid Sequence↗
PubMed1985

Dose-related effects of pentobarbital on the genetic differences seen between paired, Roman high- or low-avoidance rats in a shuttle box.

The effects of low doses of pentobarbital (PB) were measured on the activity levels, shock-induced fighting and avoidance or escape behavior of paired rats of two psychogenetically-selected lines, in multiple shuttle box sessions, following shock-induced fighting or two-way avoidance training. Each pair served as its own control, by receiving drug injections only every second week. Independent of training conditions, the RLA/Verh pairs showed about 90% freezing behavior and no fighting, whereas all RHA/Verh pairs preferred avoidance or escape behavior to fighting. Although their intertrial (shuttling) responses (ITRs) were reduced at the higher doses of PB used, RHA/Verh rats were still capable of most behavioral responses even at 24 mg/kg, whereas all RLA/Verh rats slept at that dose. On the other hand, the ITRs and avoidance responses of the (less active) RLA/Verh rats were increased by injections of 8 and 16 mg/kg PB. The results, especially those pertaining to freezing behavior and changes in activity levels, were discussed in comparison to other selected rat strains which have shown certain similarities to the Roman lines in regard to "emotionality" and associated neurochemical status.

Aggression↗
PubMed1988

Adenovirus DNA replication in vitro: duplication of single-stranded DNA containing a panhandle structure.

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.

Adenoviridae↗
PubMed1982

A common sequence in the inverted terminal repetitions of human and avian adenoviruses.

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.

Adenoviridae↗
PubMed1983

Mutational mapping of a cloned adenovirus origin.

We have developed a standardized, quantitative assay to study the function of a cloned adenovirus origin. We have shown that the adenovirus origin is located within the first 20 bp of the adenovirus inverted terminal repetition (ITR), a region containing a sequence conserved among human, simian, murine, and avian adenoviruses. Deletions removing or penetrating from either direction into the conserved sequence inactivated the cloned adenovirus origin. A point mutation within the conserved sequence impaired the adenovirus origin, but point mutations outside the conserved sequence had no effect. These results strongly suggest that the conserved sequence within the first 20 bp of the ITR alone constitutes the adenovirus origin (ori) signal.

Adenoviridae↗
PubMed2005

Characterization of multiple lineages of Tc1-like elements within the genome of the amphibian Xenopus tropicalis.

We have used genomic sequencing data extracted from the first assembly of the Xenopus tropicalis genome combined with a degenerated PCR approach to identify multiple lineages of Tc1 related transposable elements. Full-length elements were isolated in each lineage and were characterized. Most of them exhibit the typical characteristics of Tc1-like elements (TLEs). An open reading frame (ORF) encoding a 340-350 aa transposase containing a [D, D(34)E] signature was found as well as conserved inverted terminal repeats (ITRs) at each extremities. These ITRs could vary in length, depending on the TLE lineage. These new TLEs were named Eagle, Froggy, Jumpy, Maya, Xeminos, XtTXr and XtTXz. Phylogenetic analyses indicate that their closest relatives are present in the genomes of actinopterygian and amphibian. Interestingly, Maya and Xeminos share remarkable characteristics. Maya contains a [D,D(36)E] motif but is not related to any described TLE so far. Xeminos is the first vertebrate TLE strongly related to an invertebrate lineage. Finally, we have identified for most of these TLEs, copies containing an intact transposase ORF suggesting that these elements may still be active.

Amino Acid Motifs↗
PubMed2005

Mutant Mos1 mariner transposons are hyperactive in Aedes aegypti.

The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K and R, respectively. The effects of these mutations on the transpositional activities of Mos1-derived transposon constructs were evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti. The transpositional activities of two Mos1 transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3' ITRs, were increased when the mutant transposases were supplied in trans in E. coli. The use of the perfect repeat transposon with wild type transposase did not result in an increase in transposition frequency in Ae. aegypti. However, an improvement in the integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular evolution of Mos1, and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1 mediated germ line transformation of Ae. aegypti.

Aedes↗
PubMed2005

Vapor-induced film formation from low-Tg particles for different solvent compositions.

The photon transmission method was used to study latex film formation from poly(vinyl acetate) (PVAc) particles induced by two different solvents. Films with the same latex content were prepared from PVAc particles and exposed to vapor of ethanol-water and acetone-water mixtures in various compositions. Transmitted photon intensities, Itr, from these films increased with increasing vapor exposure time. The increase in Itr is attributed to the increase in crossing density at the polymer-polymer junction. The Prager-Tirrell model was employed to obtain the back-and-forth frequency, nu, of the reptating polymer chain during film formation induced by solvent vapor. It was observed that the produced nu values increase as the solvent content is increased for both solvents. Abilities of both solvents to form films were interpreted with the solubility parameters of the solvents and the PVAc.

Journal Article↗
PubMed2005

Effect of an aqueous extract of dried immature fruit of Poncirus trifoliata (L.) Raf. on intestinal transit in rodents with experimental gastrointestinal motility dysfunctions.

The effect of an aqueous extract of the dried immature fruit of Poncirus trifoliata Raf. (Rutaceae) (PF-W) on gastrointestinal (GI) motor function was investigated by measuring the intestinal transit rate (ITR) of Evans blue in rats and mice with experimental GI motility dysfunctions (GMDs). GMD was induced by appropriate surgery or by the administration of acetic acid, atropine, L-DOPA, or morphine to the animals. The ITR in these GMD animals was significantly retarded compared to normal animals. The retardation, however, was significantly inhibited by the intragastric administration of PF-W (0.1-1g/kg) in a dose dependent manner for all GMD models except for L-DOPA-induced GMD. The above results suggest that PF-W has the potential for development as a prokinetic agent that may prevent or alleviate GMD in human patients.

Animals↗
PubMedDate not supplied

Closure and summary of Ninth International Small Bowel Transplantation Symposium.

The highlights of The Ninth International Small Bowel Transplantation Symposium included the latest results from the Intestinal Transplant Registry (ITR), which demonstrated that the number of transplants performed annually continued to rise to 180 per year, most of which were performed in the United States, where the greatest number of home parenteral nutrition (PN) patients live (a prevalence of around 40 per million or 10,000 individuals). The three largest programs in the United States all use anti-CD25 monoclonal antibodies, but three different forms of lymphocyte depletion are employed in induction protocols: (1) humanized monoclonal anti-thymocyte globulin; (2) pretreatment of the recipient with Campath; (3) pretreatment of the small bowel allograft with anti-lymphocyte globulin. The main gain in these new approaches has been in the reduction in tacrolimus and steroid exposure. Indeed, the Pittsburgh team reduced tacrolimus to alternate-day dosing and stopped steroids altogether at 6 months in some patients, recording 100% survival in the past 2 years. The ITR demonstration of a difference in survival between adult patients who are hospitalized (40% to 60%) versus those who are still at home (80% to 100% survival) when called to transplant makes it almost unethical to delay evaluating patients for small bowel transplantation once they start experiencing complications on home PN. The input of gastroenterologists is also crucial in making recommendations about isolated liver transplantation for individuals with a potential to come off PN. Several important papers with useful prognostic clinical data with respect to selecting patients for isolated liver transplant were presented. It is evident from demographic surveys that only 2% to 5% adult patients and 5% to 15% children in large, well-resourced PN programs will be unlucky enough to develop life-threatening complications, but they must have rapid access to small bowel transplantation in this event. It is therefore important that the collaboration between gastroenterologists and the intestinal transplant teams continues. These excellent results for small bowel transplantation mean that the time is right for a large cost-effectiveness study comparing small bowel transplantation with PN.

Humans↗
PubMed2004

Expression strategy of densonucleosis virus from Mythimna loreyi.

The genome of Mythimna loreyi densovirus (MlDNV) was cloned into the pEMBL(19)+ vector. This clone was infectious upon transfection, both in LD cells and larvae. The genome possessed ITRs of 543 nucleotides of which the distal 126 nucleotides could form a hairpin. The nonstructural (NS) and structural (VP) genes were located on the 5'-halves of the complementary strands and their transcripts started 27 nts downstream of the ITRs. These transcripts had an overlap of 57 nucleotides in middle of the genome. The NS cassette consisted of three genes with NS1 and the overlapping NS2 downstream of NS3. The NS3 gene was spliced out from a fraction of the NS transcripts to allow leaky scanning translation of the downstream bicistronic NS1 and NS2 genes. The four VPs were similarly generated by leaky scanning translation of unspliced mRNA. The 5'-untranslated region of the VP transcript was only seven nucleotides long.

Amino Acid Sequence↗
PubMed2004

Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera.

The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the inverted terminal repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the "naked" DNA vaccines encoding the LAMP/gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4(+) T cell responses. In contrast, significantly higher levels of CD8(+) and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization.

Animals↗
PubMed2006

Molecular characterization of caprine adeno-associated virus (AAV-Go.1) reveals striking similarity to human AAV5.

The complete genome of the caprine adeno-associated virus (AAV-Go.1) was sequenced, and the expression profile of AAV-Go.1 was determined following virus infection in primary lamb kidney cells. A remarkable similarity between the Rep coding region, the ITR sequence and the central intron of AAV-Go.1, and the human derived AAV5 was observed. The transcription profile of AAV-Go.1 was also quite similar to that of AAV5. AAV-Go.1 was able to efficiently infect human cell lines, following co-infection of human adenovirus, and in reciprocal experiments, AAV5 also was able to efficiently infect primary lamb cells and bovine cell lines. Recombinant AAV5 and AAV-Go.1 expressing the beta-gal gene flanked by AAV5 ITRs, showed similar transduction efficiency in various human and animal cells. Studies of AAV-Go.1 may expand our understanding of the evolutionary relationship between the AAV-5-like group of AAVs, which, in addition to human derived AAV5, includes AAV viruses from bovine, caprine, and avian species.

Animals↗
PubMed1998

CS modality transfer of two-way avoidance in rats with central and basolateral amygdala lesions.

Post-lesion acquisition of two-way avoidance and subsequent transfer to two warning signals (conditioned stimulus, CS) of different modality were investigated in 60 rats. In Experiment I the animals were originally trained with less salient (darkness) CS, then transferred to more salient compound (darkness and white noise), and finally to white noise CS. The opposite arrangement of the conditioned stimuli (CSi) during the subsequent stages was employed in Experiment II. In control animals, avoidance acquisition was faster and the intertrial responding (ITR) rate lower with the auditory than with the visual CS. Lesioned rats learned avoidance responses more slowly, independently of CS modality. The transfer to other CSi revealed dramatic between-group difference in the level and consistency of avoidance response, shuttle-box latencies and ITR rate. In control animals, transfer to more salient CSi enhanced avoidance performance, whereas change to less salient CS decreased it. Rather small changes in shuttle-box performance and consistency of avoidance response due to CS modality were seen in rats with the basolateral lesions. In contrast, central nucleus injury caused a strong deterioration in the avoidance transfer, especially when the visual CS followed the acoustic one. The results indicate differential involvement of the basolateral and central amygdala nuclei in stimulus-processing mechanisms of instrumental defensive behavior.

Acoustic Stimulation↗
PubMed1998

Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701.

The orf virus (OV) strain D1701 belongs to the genetically heterogenous parapoxvirus (PPV) genus of the family Poxviridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal possible genetic mechanisms of PPV attenuation. The present study demonstrates a genomic map of the approximately 158 kbp DNA of OV D1701 established by hybridization studies of cloned restriction fragments covering the complete viral genome. The results show an enlargement of the inverted terminal repeats (ITR) to up to 18 kbp due to recombination between nonhomologous sequences during cell culture adaptation. DNA sequencing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposition-deletion variant of the New Zealand OV strain NZ2 (Fleming et al., 1995) the two genes E3L (a homologue of dUTPase) and G1L neighbouring E2L are retained in OV D1701. DNA and RNA analyses proved the presence of E2L gene in wild-type OV isolated directly from scab material. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important viral gene in determining virulence and pathogenesis of OV.

Amino Acid Sequence↗
PubMed2002

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans.

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.

Amino Acid Sequence↗
PubMed2001

Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation.

A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by two general promoters (cytomegalovirus and medaka beta-actin) and one muscle-specific promoter (zebrafish alpha-actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, 34.6% (81/234), 10% (10/60), and 18% (38/212) showed homogenous GFP-expression for the derivative constructs of the cytomegalovirus, alpha-actin, and beta-actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was (a) extended for an entire lifetime without degradation, and (b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye.

Actins↗
PubMed1998

Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus.

Expression of transgenes within a single generation by direct DNA injection into vertebrate embryos has been plagued by inefficient and nonuniform gene expression. We report a novel strategy for efficient and stable expression of transgenes driven by both ubiquitous and tissue-specific promoters by direct DNA injection into developing Xenopus laevis embryos. This strategy involves flanking expression cassettes of interest with inverted terminal repeat sequences (ITRs) from adeno-associated virus. Our results suggest that the ITR strategy may be generally applicable to other systems, such as zebra fish and embryonic stem cells, and may enable tissue-specific expression of transgenes in problematic contexts.

Actins↗