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PubMed1993

The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences.

The inverted terminal repeat (ITR) of adenovirus type 5 (Ad5) is 103 bp in length and contains the origin of DNA replication. Cellular transcription factors NFI/CTF and NFIII/OCT-1 bind to sites within the ITR and participate in the initiation of viral DNA replication in vitro. The ITR also contains multiple copies of two conserved sequence motifs that bind the cellular transcription factors SP1 and ATF. We have analyzed a series of viruses that carry deletions at the left terminus of Ad5. A virus carrying a deletion of the NFIII/OCT-1, SP1, and ATF sites within the ITR (mutant dl309-44/107) was wild type for virus growth. However, the deletion of these elements in addition to sequences immediately flanking the ITR (mutant dl309-44/195) resulted in a virus that grew poorly. The analysis of growth parameters of these and other mutants demonstrate that the NFIII/OCT-1 and adjacent SP1 sites augment the accumulation of viral DNA following infection. The function of these elements was most evident in coinfections with a wild-type virus, suggesting that these sites enhance the ability of a limiting trans-acting factor(s), that stimulates viral DNA replication, to interact with the ITR. The results of these analyses indicate functional redundancy between different transcription elements at the left terminus of the Ad5 genome and demonstrate that the NFIII/OCT-1 site and adjacent SP1 site, previously thought to be nonessential for adenovirus growth, play a role in viral DNA replication in vivo.

Adenoviruses, Human↗
PubMed2002

A 72-bp internal deletion in the left inverted terminal repeat of the bovine adenovirus type 3 genome does not affect virus replication.

The genome of bovine adenovirus type 3 (BAV3) is flanked by 195-base pair (bp) inverted terminal repeats (ITR). We isolated a BAV3 mutant (BAV3c29) having an internal deletion within the left ITR. The deletion eliminated 72 bp between nucleotides (nt) 89 and 162, including most of the GC-rich sequences located close to the end of the ITR sequences. This deletion did not seem to have any affect on the virus plaque size or morphology and the kinetics of viral replication compared to wild-type (wt) BAV3. The nt sequence of the right ITR of BAV3c29 remained identical to the right or left ITR of wt BAV3. These results indicate that the cis-acting sequences present within the 72 bp between nt 89 and 162 of the left ITR are not essential for BAV3 DNA replication in cultured cells.

Animals↗
PubMed1993

Intertrial responses in defensive instrumental learning.

Data by the author, as well as from other laboratories, show residual fear as the main source of intertrial responses (ITR) during defensive instrumental training in rats, cats and dogs. Between-subject differences in ITR rate arise in the course of training. The differences depend on training conditions and on subject's emotionality. Under the same conditions higher ITR rates correspond to more rapid avoidance learning. The increase of ITR rate is regularly observed in the following circumstances: at early stages of training, after transfer to a less salient discriminative stimulus, and after a change of the task requirements. The decrease of ITR rate corresponds to the plateau level of avoidance or escape performance. Both a lower ITR rate and reduced flexibility of behavior are found after lesions of the amygdala nuclei.

Aggression↗
PubMed2026

Modified Plasmids and Inverted Terminal Repeats Enhance Adeno-Associated Virus Production and Performance.

Recombinant adeno-associated virus (rAAV) is a preferred vector in gene therapy, although high production costs inhibit widespread adoption. The most common approach for rAAV production involves transfection of HEK293 cells with three plasmids: pTransgene, pRep/Cap and pHelper. Producing sufficient amounts of these plasmids accounts for up to 40% of total batch costs. Initially, this work aimed to increase plasmid yields by replacing the backbones. While this approach increased pHelper yields, pRep/Cap and pTransgene yields were unaffected. A possible reason was identified: pTransgene contains inverted terminal repeat (ITR) sequences that are essential for rAAV production. ITRs have strong secondary structures (including hairpin loops termed B and C arms) that likely interfere with plasmid production. Therefore, targeted deletions were performed within the ITRs. Partial deletions in both the B and C arms of the ITR were most beneficial, as both plasmid yield and transgene expression increased. Importantly, partial deletions did not reduce rAAV yield, as had been previously observed when the B and C arms were fully deleted. In summary, we report a 140% increase in pHelper plasmid production, while the most successful ITR variant increased pTransgene plasmid yields by 57% and transgene expression by 28%, without reducing rAAV yields or transduction efficiency.

Humans↗
PubMed1992

Cortical connections of subdivisions of inferior temporal cortex in squirrel monkeys.

Patterns of cortical connections and architectonics were used to determine subdivisions of inferior temporal (IT) cortex of squirrel monkeys. Single or multiple injections of the tracers wheat germ agglutinin-horseradish peroxidase, Fast Blue, Diamidino Yellow, Fluoro-Gold, and 3H-amino acids were placed into IT cortex. Most injections were placed in caudal IT cortex in the region previously shown to receive input from the caudal subdivision of the Dorsolateral Area, DLC; additional injections were placed in more rostral IT cortex. The results indicate the presence of two major regions: a caudal region, ITC, and a rostral region, ITR. An intermediate region of cortex along the ITC-ITR border that displays some connections of ITC and some connections of ITR may be another area. ITC contains a more myelinated dorsal area, ITCd, and a larger ventral area, ITCv. Both ITCd and ITCv receive a major projection from DLC; additional input from DLR, MT, and VII; and send strong projections to ITR, the lateral bank of the superior temporal sulcus, and dorsolateral prefrontal cortex. Only ITCd has strong connections with DLR and cortex in the depths of the superior temporal sulcus, and only ITCv has connections with lateral orbital cortex. The overall pattern of connections between ITC and DLC suggests that ITC has a crude topographic organization, with dorsal cortex representing the lower field and ventral cortex representing the upper field. ITR differs from ITC by receiving little if any input from DLC; projecting to inferior temporal polar cortex, the rostral Sylvian fissure, and medial orbital cortex; and having a less distinct layer IV. Comparison of subdivisions of inferior temporal cortex defined in the present study in squirrel monkeys and those reported in other primates suggests that ITC of squirrel monkeys may correspond to area TEO of macaque monkeys.

Animals↗
PubMed1986

Teratogen update: vitamin A congeners.

Despite animal vitamin A congener teratogenicity in animal studies since 1954, striking human findings only arose in 1983 following isotretinoin (ITR) marketing for oral treatment of severe acne. By November 1985, 44 outcomes with central nervous system (CNS), cardioaortic (CV), microtia, facial palsy, micrognathia, cleft palate, and/or thymic aplasia defects, and 33 spontaneous abortions have been reported. The critical period for exposure appears to be two to five weeks postconception, although this is clinically inexact. ITR half life is less than a day, although a teratogenic metabolite, 4-oxo-isoretinoin, has a half life of several days. Seven defect outcomes and one stillbirth have been reported with another congener, etretinate (ETR), used for psoriasis. Three of these had meningomyeloceles. Half life of several months makes levels cumulative. Only one additional defect, which may have occurred by chance, is reported with use stopped before conception (4 months). Other discontinuations 1 to 6 months before conception had 11 normal outcomes and two spontaneous abortions. ITR and ETR dose ranged from 0.5 to 1.5 mg/kg. Normal outcomes are reported both with ITR and ETR, but some of these appear not to have been exposed during the critical period. Less striking defects, abortions, and normal outcomes are less well reported. Because vitamin A analogs are therapeutically important and unplanned outcome not always avoidable, further animal research is needed for better risk/benefits. Megadose vitamin A (retinol) use is widespread, but experience poorly observed. Eighteen suspicious birth defect outcomes have been reported from pregnancies with high dose exposure. Twelve had findings similar to those seen in animals and in human retinoid syndromes, e.g., CNS, CV, microtia, and clefts. Epidemiological controls are lacking to establish human teratogenicity, but based on animal studies and experience with ITR and ETR, avoiding long term megadose Vitamin A use in fertile women is warranted.

Abnormalities, Drug-Induced↗
PubMed1996

Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) disposition following pulmonary, oral, dermal, and parenteral exposures to rats.

In evaluating human health risks posed by dioxins, it is necessary to accurately predict systematic dosimetry or the fate of these chemicals. Pharmacokinetic parameters pertaining to inhalation, ingestion, and dermal absorption may be estimated using animal models. The present study was designed to assess absorption, tissue distribution, and elimination of TCDD following intratracheal instillation (itr.), oral gavage (p.o.), or intravenous administration (i.v.) of 1 nmol [3H]TCDD/kg to male rats; experimental conditions were chosen to permit comparison to a previous dermal disposition study (Banks and Birnbaum, Toxicol. Appl. Pharmacol. 107, 302-310, 1991). After treatment, rats were housed in individual metabolism cages for 3 days with daily excreta collection. Following termination, radioactivity was quantified in tissues and excreta. By 3 days postexposure, fecal excretion accounted for 22 (i.v.), 26 (itr.), and 32% (p.o.) of dose, while urinary excretion was only 2.2 (i.v.), 1.3 (itr), and 1.4% (p.o.). Pulmonary absorption was calculated as 95% of administered dose, while oral absorption was 88%. Dermal absorption of an equivalent administered dose was 40% (Banks and Birnbaum, 1991). For all exposure routes by 3 days, major tissue depots for absorbed dose were liver and fat. Distribution of absorbed dose was 37% (i.v.) and 35% (itr.) to liver and 21% (i.v.) and 16% (itr.) to fat. Oral gavage-treated rats had similar dosimetry (28-30% absorbed dose) in both liver and fat. In contrast following dermal exposure, distribution to liver and fat was 52 and 22%, respectively (Banks and Birnbaum, 1991). Results suggest that inhalation can be an important route for systemic absorption of dioxins. Moreover, all environmentally relevant routes of exposure (oral, dermal, and respiration) must be uniquely considered as important routes of systemic exposure for TCDD and related compounds.

Adipose Tissue↗
PubMed1995

Topography of variola smallpox virus inverted terminal repeats.

We examined the nucleotide sequences of the inverted terminal repeat (ITR) regions adjacent to the covalently closed hairpin end sequences of three variola major and four minor strains from smallpox outbreaks in Europe, Asia, Africa, and South America. The ITR regions ranged in size from 581 to 1051 base pairs (bp) and contained no apparent open reading frames. Two nonrepetitive sequence elements, NR1 and NR2, were conserved and resembled nonrepetitive elements in the ITRs of other orthopoxviruses. Depending on strain, the terminally positioned NR1 and the more internal NR2 flanked a direct repeat region containing from none to four copies of a 69-bp sequence and one copy of a 54-bp related sequence partial repeat. A distinctive pattern of ITR topography of NR1 and NR2 flanking a single copy of the 69-bp unit characterized each of three examined alastrim variola minor strains. A nonalastrim African minor strain from the last natural case of smallpox in Somalia in 1977 showed the largest ITR region of the examined viruses because of a second direct repeat cluster following NR2.

Africa↗
PubMed2001

Retention of a small replicase gene segment in tomato bushy stunt virus defective RNAs inhibits their helper-mediated trans-accumulation.

Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.

DNA, Complementary↗
PubMed1997

Esophageal replacement with colon in children using either the intrathoracic or retrosternal route: an analysis of both surgical and long-term results.

A total of 28 colon esophageal replacements performed in children for long gap esophageal atresia (22 patients), and intractable caustic stricture (6 patients) were reviewed. Emphasis was placed on identifying the pros and cons of the different reconstruction techniques: intrathoracic route (ITR) (19 patients) and retrosternal route (RSR) (9 patients). No hospital mortality occurred, whereas a higher morbidity rate occurred among patients operated on using the ITR as opposed to the RSR (68% vs 55%; P not significant). Six patients developed an anastomotic fistula (21% with the ITR vs 22% with the RSR; P not significant), whereas an anastomotic stenosis occurred in 13 patients (67% with the RSR, and 37% with the ITR; P < 0.07). Overall, dysphagia was the most prevalent symptom at 3 months follow-up, but had significantly decreased at the final follow-up (54% vs 16%; P < 0.0027). Functional results improved significantly during the follow-up (score 1-2 vs score 3-4; Fisher test: P = 0.001). However, despite the higher morbidity rate, better functional results were achieved using the ITR as opposed to the RSR.

Anastomosis, Surgical↗
PubMed1990

Sequence analysis of the inverted terminal repetition in the genome of the parapoxvirus, orf virus.

Two BamHI fragments from the right-hand terminal region of the orf virus genome have been sequenced. The bulk of the inverted terminal repetition (ITR) sequence is contained within these fragments and makes up 3388 bp of the 4425-bp sequence reported. The overall base composition of the larger sequence is 59.4% G + C and of the ITR, 60.2% G + C. An extremely G/C-rich (83.2%) block of sequence was found spanning the ITR/unique sequence junction. The bulk of the ITR could be divided into three blocks of directly repeated sequences. One block begins about 250 nucleotides from the terminus and is a direct repeat 15 bp long, repeated 14 times. The other blocks contain seven sequence sets ranging from 16 to 36 nucleotides which are repeated 2 to 4 times, interspersed with one another, interrelated in sequence, and sometimes separated by unique sequence. Eight open reading frames (ORFs), each with the potential to code for polypeptides of 50 residues or more, were identified. Three were found within the ITR, four spanned the ITR/unique sequence junction and one was found outside the ITR. A search for putative poxvirus transcriptional control signals indicated that three of the eight ORFs are likely to be transcribed early, all in the same direction toward the right end of the genome. Sequences of the type T(A)3-5T were found only twice in the sequence and only one preceded an ORF.

Amino Acid Sequence↗
PubMed1987

Phylogenetic relationships between adenoviruses as inferred from nucleotide sequences of inverted terminal repeats.

The nucleotide (nt) sequences of inverted terminal repeats (ITR) from human adenovirus (Ad) 19, bovine Ad1 (BAd1), bovine Ad3 (BAd3), canine Ad2 (CAd2) and an avian Ad, EDS-76, were determined. The length of the ITR sequence was 160 bp in Ad19, 159 bp in BAd1, 195 bp in BAd3, 196 bp in CAd2 and 52 bp in EDS-76. CAd2 had the longest ITR among the examined Ads, BAd3 the second longest, and EDS-76 had the shortest ITR. A TAAT sequence located between the 10th and 13th nt counted from the ends was conserved in all Ads examined so far. To determine phylogenetic relationships among human and animal Ads, sequences of their ITRs were compared, and a phylogenetic tree was constructed by using the maximum-likelihood method. It is the method involving statistical analysis of computing the probability of a particular set of sequences on a given tree and maximizing this probability over all evolutionary trees [Felsenstein, J. Mol. Evol. 17 (1981) 368-376]. From these analyses, it was found that members belonging to the same human Ad subgenus are related closely to each other, whereas representatives of different human subgenera are distributed rather divergently among animal Ads.

Adenoviridae↗
PubMed1989

Characterization of the simian adenovirus type 30 inverted terminal repeat.

The presence of an inverted terminal repeat (ITR), which plays an important role in the initiation of DNA replication, is one of the characteristic properties of adenoviruses (Ads). We have established the nucleotide (nt) sequences for the ITR of simian adenovirus type 30 (SV30), a subgroup-III oncogenic virus. This repeat consists of 185 nt, representing the longest ITR found in an Ad so far. It contains multiple copies of internal repeats, as well as the consensus sequences of the putative binding sites for replication and transcription factors. The conserved features of the known ITRs are also found in SV30. Interestingly, the ITR of SV30 is more closely related to that of Ad5 (human), than to that of SA7 (simian).

Adenoviridae↗
PubMed2004

Genetically-based behavioral traits influence the effects of Shuttle Box avoidance overtraining and extinction upon intertrial responding: a study with the Roman rat strains.

In the present study, we evaluated the effects of extended exposure procedures to contextual fear cues as a way of reducing intertrial responses (ITRs), a characteristic stereotyped behavior displayed in 2-way Shuttle Box avoidance overtraining. We used rat strains psychogenetically selected for efficient (Roman high-avoidance; RHA) versus poor 2-way Shuttle Box performance (Roman low-avoidance; RLA), the former being fearless/impulsive relative to the latter. Additionally, Sprague-Dawley (SD) rats were compared with RHAs to have a measure of the ITRs exhibited by another, non-selected group of animals. The main findings were that after extended exposure RHAs diminished their ITRs without affecting avoidance performance, although they emitted a pattern of ITRs somewhat excessive as compared with SDs. We concluded that three factors could influence the expression of ITRs in RHA rats: (1) the repetitive and stereotyped responding induced by 2-way Shuttle Box overtraining; (2) contextual fear conditioning; and (3) a genetic background associated with high impulsiveness.

Animals↗
PubMed2004

The inositol 1,4,5-trisphosphate receptor regulates epidermal cell migration in Caenorhabditis elegans.

Polarized migration and spreading of epithelial sheets is important during many processes in vivo, including embryogenesis and wound healing. However, the signaling pathways that regulate epithelial migrations are poorly understood. To identify molecular components that regulate the spreading of epithelial sheets, we performed a screen for mutations that perturb epidermal cell migration during embryogenesis in Caenorhabditis elegans. We identified one mutant (jc5) as a weak mutation in itr-1, which encodes the single inositol 1,4,5-trisphosphate receptor (ITR) in C. elegans. During the migration of the embryonic epidermis, jc5 embryos display defects including misdirected migration or premature cessation of migration. Cells that halt their migration have disorganized F-actin and display reduced filopodial protrusive activity at their leading edge. Furthermore, some filopodia formed by epidermal cells in itr-1(jc5) embryos exhibit abnormally long lifetimes. Pharmacological studies with the inositol 1,4,5-trisphosphate antagonist xestospongin C phenocopy these defects, confirming that ITR function is important for proper epidermal migration. Our results provide the first molecular evidence that movements of embryonic epithelial cell sheets can be controlled by ITRs and suggest that such regulation may be a widespread mechanism for coordinating epithelial cell movements during embryogenesis.

Animals↗
PubMed2006

Effects of intertrigeminal region NMDA and non-NMDA receptors on respiratory responses in rats.

Respiratory disturbance, including apnea, can be induced by microinjection of glutamate into the intertrigeminal region (ITR) of the lateral pons, a region that is anatomically coupled to both the dorsal and ventral respiratory groups of the medulla. We showed that the ITR plays a functional role in regulating both vagal reflex apnea and spontaneous sleep-related apnea in rats, but the mechanisms have not been determined. This study shows that functional NMDA receptors are expressed in the ITR since the blockade of these receptors by AP5, a specific NMDA receptor antagonist, was fully effective in blocking apnea induced by glutamate injection within this region. Selective blockade of ITR NMDA receptors had no effect on the immediate apnea evoked by an intravenous 5-HT bolus, whereas the nonspecific glutamate receptor antagonist kynurenic acid significantly increased the duration of this vagal reflex apnea. These findings are of interest because pontine NMDA receptors participate in inspiratory off-switch mechanisms and have been implicated in various short- and long-term potentiation and depression phenomena. These data support the involvement of ITR non-NMDA receptors in modulation of reflex apnea per se, whereas NMDA receptors play a role in damping respiratory responses to transient disturbances.

2-Amino-5-phosphonovalerate↗
PubMed2004

Construction and sequencing of an infectious clone of the human parvovirus B19.

Human parvovirus B19 has a nonenveloped, icosahedral capsid packaging a linear single-stranded DNA genome of 5.6 kb with long inverted terminal repeats (ITR) at both the 5' and 3' end. Previous attempts to construct a full-length B19 clone were unsuccessful due to deletions in the ITR sequences. We cloned the complete parvovirus B19 genome with intact ITRs from an aplastic crisis patient. Sequence analysis of the complete viral genome indicated that both 5' and 3' ITRs have two sequence configurations and several base changes within the ITRs compared to previous published sequences. After transfection of the plasmid into permissive cells, spliced and non-spliced viral transcripts and viral capsid proteins could be detected. Southern blot analysis of the DNA purified from the plasmid-transfected cells confirmed parvovirus B19 DNA replication. Production of infectious virus by the B19 plasmid was shown by inoculation of cell lysate derived from transfected cells into fresh cells. Together, these results indicate the first successful production of an infectious clone for parvovirus B19 virus.

3' Untranslated Regions↗
PubMed2003

Intertrigeminal region attenuates reflex apnea and stabilizes respiratory pattern in rats.

The goal of the present study was to determine whether the newly described anatomical pathway involving the pontine intertrigeminal region (ITR), (Chamberlin and Saper, 1998), has a physiological role in mediating or modulating vagally-induced reflex apnea. We explored the ITR impact on vagal reflex apnea elicited by intravenuously injected 5-HT in ten anesthetized rats. The animals had a catheter inserted into the femoral vein for administration of 5-HT (0.00375 mg) and respiration was recorded by piezo-electric crystal. Multibarrel pipettes were used to pressure inject glutamate (5-10 nl, 10 mM), kynurenic acid (10 nl, 50 mM, a glutamate receptor antagonist), and red dye into the ITR, unilaterally and bilaterally. Intravenous administration of 5-HT produced an immediate 3-s apnea. Microinjections of glutamate into the ITR produced apneas, while microinjections of kynurenic acid blocked the glutamate effect. Following glutamate antagonism, subsequent administration of 5-HT produced apneas of much longer duration (8 s). Acute ponto-medullary transection in two animals yielded even greater prolongation of 5-HT-induced apnea. We conclude that a physiological role for the ITR in respiration is to attenuate vagally-induced reflex apneas. This finding is in agreement with the general modulatory role of pontine structures in autonomic activities including respiration, heart rate and regulation of blood pressure. In addition, our data indicate that medullary circuits, independent of pontine structures, are sufficient to produce 5-HT induced reflex apnea.

Animals↗