Analysis of the biased distribution of topoisomerase I break sites on replicating simian virus 40 DNA.

We previously mapped the locations of breaks introduced by eukaryotic topoisomerase I (topo I) in replicating simian virus 40 (SV40) DNA and observed an approximate 3:1 bias in the distribution of the break sites for the template strand for discontinuous DNA synthesis. In the present study, this bias has been confirmed by the mapping of additional sites utilizing a standard primer extension assay and a sensitive repetitive primer extension (RPE) method. No new sites could be detected on either strand of SV40 by the RPE method, despite the 10 to 20 fold greater sensitivity of the technique. To investigate the nature of the bias, a detailed analysis of the SV40 DNA sequence was undertaken. A set of 17 pentanucleotide sequences derived from those sites observed to be broken in the viral DNA extracted from SV40-infected cells define an in vivo consensus sequence. We show that the observed strand bias is likely due to the intrinsic asymmetric distribution of these consensus sequences on the two strands of SV40 DNA. To confirm these observations, double-stranded oligonucleotides containing previously identified in vivo topo I break sites were introduced in both orientations into SV40 to generate insertion mutants. Mapping experiments utilizing these mutants revealed that the inserted topo I break sites were broken in vivo regardless of their orientation, confirming that the SV40 sequence is the major, if not the sole determinant, of the observed strand bias. The possible origins of the strand bias are discussed in relation to the evolution of the virus.

Struggling behavior in shackled male and female broiler chickens.

Anecdotal evidence suggests that the struggling behavior of shackled broiler chickens may be positively related to compression of the shank and the probable associated discomfort: birds with large shanks tend to struggle more violently than do those with smaller shanks. Males are generally heavier and have thicker shanks than females. Therefore, we tested the hypothesis that, because the leg gaps of shackles are fixed in size, male broilers would struggle more than females. At 42 d of age, 264 floor-reared broilers were cooped in groups of 12 (six males and six females) and were transported from the university farm to the abattoir. Eighty of these served as test birds (n = 40/sex) and were shackled on a moving processing line with a bird of randomly selected sex on either side. Upon shackling, the latencies to struggle, numbers of struggling bouts, and total time spent struggling were recorded during a 1-min test period. Subsequently, the BW and circumference of the right shank (CRS) of each test bird were measured. Male birds were heavier and had thicker shanks than females (both P < 0.0001); they also struggled sooner (P < 0.01) and longer (P < 0.008). When data from males and females were pooled, CRS was negatively correlated with latency to struggle (r = -0.30; P < 0.006) and positively associated with SB (r = 0.23; P < 0.04) and total time spent struggling (r = 0.23; P < 0.04). However, there were no detectable correlations within sex. Body weight was not significantly correlated with any of the struggling behavior measures. Although other gender-related factors may be influential, an interpretation of our findings based on sex differences in CRS seems the most parsimonious. We conclude that use of shackles of fixed leg-gap size may contribute to increased struggling behavior in male broilers.

Behavioral assessment for pediatric intensive care units.

Two studies were conducted to analyze behaviors of staff and patients on a Pediatric Intensive Care Unit (PICU). In the first study, behavioral observation procedures were employed to assess patient state, physical position, affect, verbal behaviors, visual attention and activity engagement, and staff verbal behavior. On the average, one-third of the patients were judged to be conscious and alert but markedly nonengaged with their environment. In the second study, a member of the hospital staff provided alert patients with individual activities to determine whether a simple environmental manipulation could positively affect behavior of children in intensive care. Employing a reversal design, the activity intervention was found to increase attention and engagement and positive affect, and to decrease inappropriate behavior. Both studies demonstrate that behavioral assessment procedures can provide an empirical basis for designing PICU routines affecting children's psychosocial status, and, thus, complement current procedures designed to provide quality medical care.

Operant treatment of orofacial dysfunction in neuromuscular disorders.

The popularity and reported success of biofeedback treatment for neuromuscular disorders has occurred despite a lack of research identifying the critical variables responsible for therapeutic gain. In this study, we assessed the degree to which severe neurological dysfunction could be improved by using one of the components present in all biofeedback treatment, contingency management. Three cases of orofacial dysfunction were treated by reinforcing specific improvements reliably detectable without the use of biofeedback equipment. The results showed that contingency management procedures alone were sufficient to improve overt motor responses but, unlike biofeedback treatment, did not produce decreases in the hypertonic muscle groups associated with the trained motor behavior. The findings suggest that sophisticated, expensive biofeedback equipment may not be necessary in treating some neuromuscular disorders and that important clinical gains may be achieved by redesigning the patient's daily environment to be contingently therapeutic, rather than only accommodating the disabilities of the physically handicapped.

Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin.

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.

Biochemical and biophysical analyses of recombinant forms of human topoisomerase I.

Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells. Each protein is inhibited by camptothecin, topotecan, and 9-aminocamptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine. The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration. Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory. The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different. Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended. A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1-174 (approximately 21 kDa) of Topo I are largely if not completely unfolded. This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity.

Biofeedback treatment of fecal incontinence in patients with myelomeningocele.

Approximately six hours of biofeedback training was given to eight fecally incontinent children with myelomeningocele in order to establish bowel control. Their ages ranged from five to 15 years. The patients were shown a polygraph tracing of the external anal sphincter while they were being encouraged voluntarily to contract the sphincter when the rectum was distended with progressively larger volumes of air in a balloon. Seven of the eight patients showed normal sensation for rectal distension. Following this training period, five of the children had no incontinent periods, and two of these had discontinued enemas or suppositories. A sixth patient had an 80 per cent reduction in the frequency of incontinence. The remaining two did not benefit. At follow-up between 13 and 24 months later, four children were incontinent once a month or less often; two others were incontinent once per day, a considerable decrease from the pretraining period. The two children who failed to learn were still incontinent at follow-up. These results show that fecally incontinent patients with myelomeningocele can learn to evacuate normally or to reduce soiling after a relatively short period of biofeedback training.

Comparison of non-radioactive DNA hybridization probes to detect human immunodeficiency virus nucleic acid.

Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.

Detection of anti-topoisomerase I antibodies using a full length human topoisomerase I recombinant protein purified from a baculovirus expression system.

Topoisomerase I (topo I) is a major systemic sclerosis (SSc)-associated autoantigen. A cDNA construct encoding full length human topo I in a recombinant baculovirus transfer vector was used to infect insect cells in culture from which recombinant protein was purified. An ELISA using recombinant protein was evaluated in 340 sera including sera from 134 patients with SSc, of whom 33 had anti-topo I antibodies detected by immunodiffusion. A high yield of pure topo I of expected molecular mass and catalytic activity was obtained. The recombinant topo I ELISA was 92% sensitive and 98% specific in detecting anti-topo I antibodies which were present almost exclusively in patients with SSc. Therefore, the potential advantages of expressing human autoantigens in eukaryotic systems for diagnostic purposes were confirmed.