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Biomedical subjects

L Stewart

Publications and source records attributed to L Stewart.

At least 91 records · Page 5Linked to original sources
PubMed1996

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.

Adenosine Triphosphate↗
PubMed1996

The domain organization of human topoisomerase I.

Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies.

Base Sequence↗
PubMed1996

Stories from mental health.

Mental health nursing has always been a particularly challenging area in which to work. Not only in providing quality nursing care for clients who can be unpredictable, demanding and at times aggressive, but also in adjusting to changes in philosophy, focus and even contexts of work. It would be fair to say that mental health services in NSW are in a state of crisis. Mental health nurses are feeling the effects, but despite the difficulties remain strongly committed to their speciality. The Association and the membership benefit greatly from the energy, enthusiasm and ideas that mental health nurses generate. This issue of The Lamp acknowledges the important contribution of mental health nurses to the profession of nursing.

Humans↗
PubMed1995

Preferential binding of human topoisomerase I to superhelical DNA.

Eukaryotic type I DNA topoisomerase provides swivels for removing torsional strain from the DNA helix during transcription and DNA replication. Previously it has been shown that the enzyme is associated with actively transcribed genes and replicating DNA. Using an inactive mutant form of the protein containing phenylalanine instead of tyrosine at position 723, we have investigated the binding properties of the protein as a function of substrate topology. A series of filter binding assays indicated that the protein strongly prefers to bind superhelical over completely relaxed SV40 DNA. The ability of a supercoiled DNA to compete against a relaxed DNA for binding increases as the number of superhelical turns in the DNA increases. Since positively supercoiled DNA is bound with the same preference as negatively supercoiled DNA, we hypothesize that topoisomerase I binds preferentially at the nodes created by the crossing of two duplex helices. The preference for binding superhelical DNA is also exhibited by the conserved core domain (amino acids 175-659) which is missing the active site region located near the C-terminus. These results suggest that this core domain may target the enzyme in vivo to regions of torsionally strained superhelical DNA.

Binding Sites↗
PubMed1995

Human immunodeficiency virus seroprevalence in community-based primary care practices, 1990-1992. A report from the Ambulatory Sentinel Practice Network.

OBJECTIVE: To estimate the seroprevalence of human immunodeficiency virus type 1 (HIV-1) infection in primary care practices. METHODS: Fifty-four practices in the United States participated in an anonymous, unlinked HIV seroprevalence study between January 1990 and December 1992. Residual blood samples drawn for routine clinical tests from patients 15 to 49 years of age were centrally tested for the HIV-1 antibody for 1 month of each quarter. Information about patient demographics, clinician-recognized risk factors, the known HIV status of the patient, and whether the blood was drawn for HIV testing was recorded with each specimen. RESULTS: Of 21,998 specimens collected, 99 (0.45%) were seropositive. Of these 99 seropositive persons, 31.3% (a seroprevalence of 0.15%) were not suspected by their clinicians of being infected with HIV. Seroprevalences in men (0.96%) exceeded those in women (0.22%), and rates in rural practices (0.18%) were lower than in urban practices (0.71%). Among patients with unsuspected HIV infection, however, the gender differences, especially in rural areas, were less pronounced. Risk factors for HIV infection were infrequently noted. There was an increase in the overall seroprevalence during the 1990 to 1992 study period (0.36% to 0.53%); however, this trend was not statistically significant. CONCLUSIONS: Within a 3-year period, clinicians in at least two of five primary care practices can expect to encounter patients infected with HIV, regardless of practice location. Also, nearly one third of the patients with HIV infection will not be suspected of having this condition by their clinician.

Adolescent↗
PubMed1995

Bile duct injuries during laparoscopic cholecystectomy. Factors that influence the results of treatment.

OBJECTIVE: To analyze the treatment of bile duct injuries during laparoscopic cholecystectomy to discern the factors affecting outcome. DESIGN: An analysis of the treatment of 88 patients with laparoscopic bile duct injuries. SETTING: A university hospital. PATIENTS: Eighty-eight patients with major bile duct injuries following laparoscopic cholecystectomy. MAIN OUTCOME MEASURES: Success of treatment, morbidity rate, mortality rate, and length of illness. RESULTS: Operations to repair bile duct injuries were unsuccessful in 27 (96%) of 28 procedures when cholangiograms were not obtained preoperatively, and they were unsuccessful in 69% when cholangiographic data were incomplete. In some cases, lack of complete cholangiographic information led to an inappropriate and harmful operation. When cholangiographic data were complete, the first repair was successful in 16 (84%) of 19 patients. A primary end-to-end repair over a T tube (13 patients) was unsuccessful in every case in which the duct had been divided. Direct closure of a partial defect in the duct was successful in four of seven patients. Fifty-four (63%) of 84 Roux-en-Y hepaticojejunostomies were successful. Factors responsible for the unsuccessful outcomes were the following: incomplete excision of the scarred duct, use of nonabsorbable suture material, use of two-layer anastomosis, and failure to eradicate subhepatic infection before the attempted repair. Dilatation and stenting was uniformly unsuccessful as primary treatment (three patients) and was unsuccessful in only seven of 26 patients following a previous operative repair. Patients first treated by the primary surgeon had an average length of illness of 222 days (P < .01). Only 17% of primary repair attempts and no secondary repair attempts performed by the laparoscopic surgeon were successful. Patients whose first repair was performed by tertiary care biliary surgeons had a length of illness of 78 days (P < .01), and 45 (94%) of 48 repairs by tertiary care biliary surgeons were successful. CONCLUSIONS: Surgeons who specialize in the repair of bile duct injuries achieve much better results than those with less experience. The worse results of other surgeons could be attributed in many instances to specific correctable errors. Nonsurgical treatment was usually unsuccessful and substantially increased the duration of disability.

Adult↗
PubMed1995

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.

Animals↗
PubMed1994

Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo.

In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.

Amino Acid Sequence↗
PubMed1994

Propofol sedation in severe head injury fails to control high ICP, but reduces brain metabolism.

We have compared the effects of an intravenous infusion of propofol with those of morphine and midazolam on global brain metabolism (AVDO2) and brain perfusion following severe head injury. Fifteen patients were sedated with either a continuous infusion of propofol (mean rate 232 mg/h, range 150-400 mg/h) or infusions of morphine (mean rate 2.3 mg/h, range 0-4 mg/h) and midazolam (mean rate 2.8 mg/h, range 0-5 mg/h). Both groups were well matched for sex, age and level of coma (Glasgow coma scale) prior to sedation. Continuous data collection of AVDO2, mean arterial blood pressure (MABP), intracranial pressure (ICP), and cerebral perfusion pressure (CPP) began at 12 hours post injury and continued for a mean period of 40 hours. Morphine and midazolam did not have a significant effect on any of the measured parameters. Propofol led to a fall in AVDO2 from 6.0 +/- 2.6 ml/dl to 3.0 +/- 0.6 ml/dl at 4 hours. However, there was no effect on MABP, ICP or CPP. Outcome was similar in the 2 groups.

Adolescent↗
PubMed1994

Magnesium sulfate versus phenytoin for seizure prevention in amygdala-kindled rats.

OBJECTIVE: Magnesium sulfate is widely used for seizure prophylaxis in preeclampsia-eclampsia. However, its anticonvulsant effects in other types of seizures have not been proved. Diphenylhydantoin has been widely characterized as the "gold standard" of anticonvulsants. In this study we compared the anticonvulsant effects of therapeutic blood levels of magnesium sulfate and phenytoin in seizures generated in amygdala-kindled rats. STUDY DESIGN: Eighteen male rats had a bipolar electrode stereotaxically implanted into the central nucleus of the amygdala. After recovery an electrical seizure threshold was determined for each rat. Rats were stimulated twice daily at their seizure thresholds (i.e., kindling) until three consecutive generalized tonic-clonic seizures occurred. Kindled rats randomly received one of the following intravenous injections in a volume of 1.5 ml/kg: saline solution, magnesium sulfate (30, 60, or 90 mg/kg), or phenytoin (12.5, 25, or 50 mg/kg). Fifteen minutes after injection rats were stimulated at their seizure thresholds, and electrical and behavioral seizure activity was assessed. Statistical comparisons were made by analysis of variance and post hoc comparisons when appropriate. RESULTS: Magnesium sulfate had no effect on any of the seizure parameters assessed. Phenytoin significantly reduced seizure duration (p < 0.01), duration of postictal depression (p < 0.01), and behavioral seizure stage (p < 0.01). CONCLUSION: Amygdala-kindled seizures are more potently inhibited by phenytoin than by magnesium sulfate.

Amygdala↗
PubMed1993

Continuous monitoring of jugular bulb oxygen saturation and the effect of drugs acting on cerebral metabolism.

The laser absorption spectrophotometric technique was used to continuously monitor jugular bulb oxygen saturation (SjO2), and thus to calculate arteriovenous oxygen differences (AVDO2), in three subgroups of intensively monitored, severely head injured patients. We have used this data to address two questions: 1. How do cerebral and systemic haemodynamic changes affect SjO2 and AVDO2; and 2. Can ADVO2 measurements be used to detect therapeutic changes in brain metabolism, in response to drugs. The major haemodynamic factor affecting SjO2 and AVDO2 was intracranial pressure (ICP). Increases in ICP were associated with concomitant increases in SjO2, and decreases in AVDO2, suggesting cerebral hyperaemia in response to ICP waves. Systemic changes were less frequent, but potent influences on SjO2. The short acting anaesthetic agent propofol produced a marked increase in SjO2 (decrease in AVDO2 to below the normal range) which became less marked with time. A new high affinity glutamate antagonist produced no change in SjO2. With rigorous attention to technical factors, and exclusion of extra and intracranial haemodynamic effects, SjO2 monitoring may be a useful "surrogate end point" for the effect of drugs acting on brain metabolism.

Adolescent↗
PubMed1993

Spotted fever group rickettsial infection in south-eastern Australia: isolation of rickettsiae.

Flinders Island spotted fever (FISF), a spotted fever group (SFG) rickettsial disease first described in 1991, occurs in south-eastern Australia. The isolation of the aetiological agent is described for the first time having been obtained from the blood of two patients. An additional 22 cases are also reported. Of these patients four had positive initial serology, and 20 showed seroconversion (using Rickettsia australis as antigen). Acute phase blood specimens taken from seven patients caused neonatal mice to seroconvert to R. australis and a blood specimen from one of these patients (and one other) yielded rickettsiae. A field survey for possible reservoir and vector animals on Flinders Island, Tasmania and in Gippsland, Victoria (both in south-eastern Australia) yielded 217 vertebrates and 1445 invertebrate ectoparasites, mostly ticks. Ixodes cornuatus from humans and dogs in Gippsland produced seroconversion to SFG rickettsia when inoculated into mice but no invertebrate pools from Flinders Island produced seroconversion in mice. Haemolymph from an individual I. cornuatus removed from a human in Gippsland, yielded a SFG rickettsia on tissue culture. Sera from several species of native vertebrates, especially the bush rat, Rattus fuscipes, were positive for antibodies to SFG rickettsia.

Adolescent↗
PubMed1993

The negotiation of New Zealand alcohol policy in a decade of stabilized consumption and political change: the role of research.

Significant changes in the key alcohol policy areas of availability, advertising and taxation have taken place in New Zealand from 1985 to 1991. These changes are described in the context of the social climate which was one of extreme fiscal crisis and an unprecedented swing to the right. The very limited role played by public health research in the process of policy development is viewed in part as a consequence of this social climate. The political changes affected the strength and extent of the public health voices in the debate and the reception they received. In each of the policy areas in which change occurred, the policy-keeper was other than a public health agency and the utilization of research by public health advocates was, therefore, in response to already articulated positions. The dissemination of research relied too heavily on lengthy written submissions, and too little on media advocacy and interpersonal contact. There was also a lack of directly relevant local research to inform the policy debate. The case study illustrates the constraints on researchers' in the policy development process, but suggests that such involvement is important for the public's health.

Advertising↗
PubMed1993

Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells.

Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.

Animals↗
PubMed1993

Vascular smooth muscle response to ouabain. Relation of tissue Na+ to the contractile response.

Smooth muscle responses to Na+ pump inhibition are thought to reflect two elements: a neurogenic contribution, involving catecholamine release from nerve terminals, and a myogenic response, attributed to relations between pump activity, [Na+]i, and [Ca2+]i. In the present study, we describe the time course and magnitude of cell Na+ changes, assessed by two methods, atomic absorption and nuclear magnetic resonance spectroscopy during the myogenic contractile response of rabbit aorta strips to ouabain. A threshold concentration of 3 x 10(-7) mol/L induced a gradual rise in [Na+]i. Both methods showed an essentially identical monotonic rise over 4 to 8 hours from a baseline level of 8 to 10 mmol/L water to a peak, which was approximately fivefold higher. The neurogenic (rapid) and myogenic (delayed and gradual) contractile responses were temporally distinct. Ouabain at 10(-7) mol/L, a concentration 10- to 100-fold lower than the threshold for catecholamine-dependent rapid-onset responses, induced only a delayed and gradual contractile response, which reached a maximum at 6 to 8 hours. With 10(-6) mol/L ouabain, the delayed response of 1.6 +/- 0.2 g peaked at 7.3 +/- 1.1 hours and was sustained for 16 hours. The time course was similar to that for change in [Na+] but somewhat later. Ouabain at 10(-5) and 10(-4) mol/L induced a delayed response that was identical in magnitude but also induced an early rapid contractile response, which was prevented by reserpine or phentolamine pretreatment. These agents did not influence the delayed response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗