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Biomedical subjects

M Johnson

Publications and source records attributed to M Johnson.

At least 91 records · Page 5Linked to original sources
PubMed2001

Arbutamine stimulation detects viable myocardium 4 weeks after coronary occlusion.

At low doses, dobutamine has potent inotropic, but limited chronotropic, effects-properties that may be necessary for detection of hibernating myocardium. The efficacy of other catecholamines, which have more closely coupled inotropic and chronotropic effects, for the detection of viable myocardium is unknown. This study evaluated the efficacy of arbutamine, a catecholamine with potent chrono-tropic effects, for the detection of viable myocardium in a canine model of hibernating myocardium. Contractile reserve was assessed during stepwise arbutamine infusion (dosages of 2.5, 5, 10, 50, and 100 ng/kg/min) at 3 days (early) and 4 weeks (late) after coronary ligation. Segment shortening, wall thickening, and segmental wall motion were assessed by sonomicrometry and echocardiography. After 4 weeks of occlusion, functional recovery was assessed after revascularization. During the early arbutamine study, the sensitivity for predicting functional recovery was highest at a dosage of 50 ng/kg/min, which also produced tachycardia. The sensitivity was 50% for segment shortening, 20% for wall thickening, and 75% for wall motion score. The late arbutamine study had improved sensitivity. The sensitivity was 100% for segment shortening, 80% for wall thickening, and 90% for wall motion score at a dosage of 50 ng/kg/min. At the late arbutamine study, myocardial perfusion reserve in the ischemic zone of dogs with functional recovery was only mildly reduced (2.0 versus 2.6 in nonischemic zones, P =.53). After coronary occlusion, viable myocardium can be detected with high doses of arbutamine that produce tachycardia. However, the sensitivity of arbutamine stimulation for predicting functional recovery is low early after occlusion, but it is improved by 4 weeks after occlusion with adequate perfusion reserve.

Animals↗
PubMed2001

Preoperative risk factors for 30-day mortality after elective surgery for vascular disease in Department of Veterans Affairs hospitals: is race important?

PURPOSE: Racial variation in health care outcomes is an important topic. Risk-adjustment models have not been developed for elective abdominal aortic aneurysm repair (AAA), lower extremity bypass revascularization (LEB), or lower extremity amputation (AMP). Earlier studies examining racial variation in mortality and morbidity from AAA, LEB, or AMP were limited to administrative data. This study determined risk factors for mortality after surgery for vascular disease and determined whether race is an important risk factor. METHODS: Data in this prospective observational study were obtained from the Department of Veterans Affairs (VA) National Surgical Quality Improvement Program. Detailed demographic and clinical data were collected prospectively from patients' medical records by trained nurse reviewers. Eligible patients were those 18 years and older who underwent elective AAA, LEB, or AMP at one of 44 VA medical centers performing both vascular and cardiac surgery (phase I; October 1991 to December 1993) and at one of these 44 or 79 additional VA medical centers performing vascular but not cardiac surgery (phase II; January 1994 to August 1995). The independent association of several preoperative factors with the 30-day postoperative mortality rate was examined with stepwise logistic regression analysis for AAA, LEB, and AMP. Models were developed in the combined 44 VA medical centers and validated in the 79 VA medical centers. The independent association of race with the 30-day postoperative mortality rate was examined after controlling for important preoperative risk factors for each operation. RESULTS: More than 10,000 surgical operations were examined, and 5, 3, and 10 independent preoperative predictors of 30-day mortality rate were identified for AAA, LEB, and AMP, respectively. The observed mortality rate for patients undergoing AAA was higher (7.2% vs 3.2%; P =.02) in African American patients than in white patients in the 44 VA medical centers, although the differences were not significant in LEB and AMP or at the additional 79 hospitals. After important preoperative risk factors were controlled, there was no difference in 30-day mortality rates between African American patients and white patients. CONCLUSION: We identified several important preoperative risk factors for 30-day mortality rate in three vascular operations. From the results of this study, race was determined not to be an independent predictor of mortality.

Aged↗
PubMed2001

Circadian variation of serum leptin in healthy and diabetic men.

Leptin, from the Greek leptos, meaning thin (in reference to its ability to reduce body fat stores), is a hormone secreted primarily by adipocytes. At one time, leptin was portrayed as a potential means of combating obesity. Recently, leptin has been identified as a potent inhibitor of bone formation, acting through the central nervous system. Since numerous studies clearly show that bone remodeling is circadian rhythmic with peak activity during sleep, it is of interest to explore circadian variability in serum leptin. Accordingly, circadian characteristics of serum leptin were examined in 7 clinically healthy men and 4 obese men with type II diabetes. Blood samples were collected for 24 h at 3 h intervals beginning at 19:00. The dark (sleep) phase of the light-dark cycle extended from 22:30 to 06:30, with brief awakening for sampling at 01:00 and 04:00. Subjects consumed general hospital meals (2400 calories) at 16:30, 07:30, and 13:30. Serum leptin levels were determined by a R&D Systems enzyme immunoassay technique. Data were analyzed by linear least-squares estimation using the population multiple components method. A statistically significant (P < .018) circadian rhythm modeled by a single 24 h cosine curve characterized the data of each group. The 24 h mean leptin level was statistically greater (P < .001) in the obese diabetic men than in the healthy men (9.47 +/- 0.66 ng/mL vs. 24.07 +/- 1.71 ng/mL, respectively). Higher leptin levels occurred between midnight and roughly 02:30, and lowest leptin levels occurred between noon and the early afternoon. The phasing of this rhythm is similar to the circadian rhythm in bone remodeling previously described. Our results suggest the findings from a single morning blood sampling for leptin may be misleading since it may underestimate the mean 24 h and peak concentrations of the hormone.

Adult↗
PubMed2001

Interactions between progesterone receptor isoforms in myometrial cells in human labour.

Progesterone acts to maintain uterine quiescence during pregnancy. In contrast to many other species, no decrease in maternal serum levels of progesterone can be observed in humans before the onset of labour. Therefore, a 'functional' progesterone withdrawal in association with labour has been proposed. In humans the progesterone receptor (PR) exists in two isoforms, PR-A and PR-B. While PR-B generally mediates the effects of progesterone upon gene transcription, the role of PR-A during pregnancy, and in parturition, is unknown. In this study, term myometrium cells cultured before the onset of labour were transiently transfected with expression vectors for either PR-A or PR-B. Only those cells expressing PR-B significantly increased expression of a progesterone-sensitive reporter when stimulated with progesterone. Co-transfection of both isoforms of PR demonstrated that PR-A is a dominant repressor of transactivation in these cells. Western blot analysis showed that PR-A is present in human myometrium samples taken only after, but not before, the onset of labour. These data suggest that increased expression of PR-A in human myometrium may contribute to 'functional' progesterone withdrawal and the initiation of human labour.

Cells, Cultured↗
PubMed2001

Are concerns over right laparoscopic donor nephrectomy unwarranted?

OBJECTIVE: To examine the ability of several large, experienced transplantation centers to perform right-sided laparoscopic donor nephrectomy safely with equivalent long-term renal allograft function. SUMMARY BACKGROUND DATA: Early reports noted a higher incidence of renal vein thrombosis and eventual graft loss. However, exclusion of right-sided donors would deprive a significant proportion of donors a laparoscopically harvested graft. METHODS: A retrospective review was performed among 97 patients from seven centers performing right-sided laparoscopic donor nephrectomy. Surgical and postoperative demographic factors were evaluated. Complications were identified and long-term renal allograft function was compared with historical left-sided laparoscopic donor nephrectomy cohorts. RESULTS: Right laparoscopic donor nephrectomy was performed for varying reasons, including multiple left renal arteries or veins, smaller right kidney, or cystic right renal mass. Mean surgical time was 235.0 +/- 66.7 minutes, with a mean blood loss of 139 +/- 165.8 mL. Conversion was required in three patients secondary to bleeding or anatomical anomalies. Mean warm ischemic time was limited at 238 +/- 112 seconds. Return to diet was achieved on average after 7.5 +/- 2.3 hours, with mean discharge at 54.6 +/- 22.8 hours. Two grafts were lost during the early experience of these centers to renal vein thrombosis. Both surgical and postoperative complications were limited, with few long-term adverse effects. Mean serum creatinine levels were higher than open and left laparoscopic donor nephrectomy on postoperative day 1, but at all remaining intervals the right laparoscopic donors had equivalent creatinine values. CONCLUSIONS: These results confirm that right laparoscopic donor nephrectomy provides similar patient benefits, including early return to diet and discharge. Long-term creatinine values were no higher than in traditional open donor or left laparoscopic donor cohorts. These results establish that early concerns about high thrombosis rates are not supported by a multiinstitutional review of laparoscopic right donor nephrectomies.

Adolescent↗
PubMed2001

Cancer pain education: a structured clinical instruction module for hospice nurses.

The learning experience with the Cancer Pain Structured Clinical Instruction Module (SCIM), a highly structured skills training course for medical students, has been reported favorably. The purpose of this study was to present the Cancer Pain SCIM to registered nurses employed in a hospice setting. The goal of the study was to pilot test a structured cancer pain educational program for hospice nurses and to determine the perceived effectiveness of this course on the participants' cancer pain assessment and management skills. A multidisciplinary Cancer Pain SCIM was presented to 25 hospice nurses to improve their understanding of the management of cancer pain. The development group identified essential aspects of cancer pain management and then developed checklists defining specific station content. During the 2-hour Cancer Pain SCIM, nurses rotated through 8 stations in groups of 3, spending 15 minutes at each station. Eight instructors and 6 standardized patients, 5 of whom were survivors of cancer, participated in the course. All participants (students, instructors, and patients) evaluated the course, using a 5-point Likert scale (1 = strongly disagree; 5 = strongly agree). Nurses provided self-assessments of their perceived competence on important aspects of cancer pain management both before and after the SCIM. The self-assessment items used a 5-point scale ranging from 1 (not competent) to 5 (very competent). Twenty-five hospice nurses, averaging 4.1 years (range 1-30 years) postgraduation, participated in the Cancer Pain SCIM. Overall, nurses agreed that they improved on each of the 8 teaching items (P < 0.001). The average (SD) pretest score of 2.8 (0.72) improved to 3.8 (0.58) on the post-test (P < 0.001). Nurses believed that their mastery of specific clinical skills, taught in all 8 stations, improved as a result of participation in the course. Nurses strongly agreed (mean +/- SD) that it was beneficial to use patients with cancer in the course (4.6 +/- 0.82). Faculty members enjoyed participating in the course (4.9 +/- 0.35) and indicated a willingness to participate in future courses (4.7 +/- 0.49). Significant perceived learning among hospice nurses took place in all aspects of the Cancer Pain SCIM. Participating nurses, instructors, and patients with cancer appreciated the SCIM format. Nurses and faculty considered the participation of actual patients with cancer highly beneficial. The SCIM format has great potential to improve the quality of cancer pain education.

Education, Nursing↗
PubMed2001

Screening for high utilizing somatizing patients using a prediction rule derived from the management information system of an HMO: a preliminary study.

BACKGROUND: Somatization is a common, costly problem with great morbidity, but there has been no effective screening method to identify these patients and target them for treatment. OBJECTIVES: We tested a hypothesis that we could identify high utilizing somatizing patients from a management information system (MIS) by total number of visits and what we termed "somatization potential," the percentage of visits for which ICD-9 primary diagnosis codes represented disorders in the musculoskeletal, nervous, or gastrointestinal systems or ill-defined complaints. METHODS: We identified 883 high users from the MIS of a large staff model HMO as those having six or more visits during the year studied (65th percentile). A physician rater, without knowledge of hypotheses and predictors, then reviewed the medical records of these patients and identified somatizing patients (n = 122) and nonsomatizing patients (n = 761). In two-thirds of the population (the derivation set), we used logistic regression to refine our hypothesis and identify predictors of somatization available from the MIS: demographic data, all medical encounters, and primary diagnoses made by usual care physicians (ICD-9 codes). We then tested our prediction model in the remaining one-third of the population (the validation set) to validate its usefulness. RESULTS: The derivation set contained the following significant correlates of somatization: gender, total number of visits, and percent of visits with somatization potential. The c-statistic, equivalent to the area under the ROC curve, was 0.90. In the validation set, the explanatory power was less with a still impressive c-statistic of 0.78. A predicted probability of 0.04 identified almost all somatizers, whereas a predicted probability of 0.40 identified about half of all somatizers but produced few false positives. CONCLUSIONS: We have developed and validated a prediction model from the MIS that helps to distinguish chronic somatizing patients from other high utilizing patients. Our method requires corroboration but carries the promise of providing clinicians and health plan directors with an inexpensive, simple approach for identifying the common somatizing patient and, in turn, targeting them for treatment. The screener does not require clinicians' time.

Adult↗
PubMed2001

Creatine supplementation and health variables: a retrospective study.

PURPOSE: Long-term safety of creatine supplementation has been questioned. This retrospective study was performed to examine markers related to health, the incidence of reported side effects and the perceived training benefits in athletes supplementing with creatine monohydrate. METHODS: Twenty-six athletes (18 M and 8 F, 24.7 +/- 9.2 y; 82.4 +/- 20.0 kg; 176.5 +/- 8.8 cm) from various sports were used as subjects. Blood was collected between 7:00 and 8:30 a.m. after a 12-h fast. Standard clinical examination was performed for CBC and 27 blood chemistries. Testosterone, cortisol, and growth hormone were analyzed using an ELISA. Subjects answered a questionnaire on dietary habits, creatine supplementation, medical history, training history, and perceived effects of supplementation. Body mass was measured using a medical scale, body composition was estimated using skinfolds, and resting heart rate and blood pressure were recorded. Subjects were grouped by supplementation length or no use: Gp1 (control) = no use (N = 7; 3 F, 4 M); Gp2 = 0.8-1.0 yr (N = 9; 2 F, 7 M); and Gp3 = 1(+) (N = 10; 3 F, 7 M). RESULTS: Creatine supplementation ranged from 0.8--4 yr. Mean loading dose for Gp2 and Gp3 was 13.7 +/- 10.0 and the maintenance dose was 9.7 +/- 5.7 g.d(-)1. Group differences were analyzed using one-way ANOVA. CONCLUSIONS: Expected gender differences were observed. Of the comparisons made among supplementation groups, only two differences for creatinine and total protein (P < 0.05) were noted. All group means fell within normal clinical ranges. There were no differences in the reported incidence of muscle injury, cramps, or other side effects. These data suggest that long-term creatine supplementation does not result in adverse health effects.

Adolescent↗
PubMed2001

How does nonpenetrating glaucoma surgery work? Aqueous outflow resistance and glaucoma surgery.

Histologic, experimental, and theoretical studies of the aqueous outflow pathways point toward the juxtacanalicular region and inner wall of Schlemm's canal as the likely site of aqueous outflow resistance in the normal eye. At least 50% of the aqueous outflow resistance in the normal eye and the bulk of the pathologically increased resistance in the glaucomatous eye resides in the trabecular meshwork and the inner wall of Schlemm's canal. The uveoscleral, or uveovortex, pathway, which accounts for perhaps 10% of the aqueous drainage in the healthy aged human eye, can become a major accessory route for aqueous drainage after pharmacologic treatment. Surgeries designed to incise or remove the abnormal trabecular meshwork of glaucoma address the pathologic problem of the disease. Surgeries that unroof Schlemm's canal or expand the canal, such as viscocanalostomy, probably cause inadvertent ruptures of the inner wall and juxtacanalicular tissue, thus relieving the abnormal outflow resistance of glaucoma. This review is a summary of current thought on the pathophysiology of aqueous outflow resistance in glaucoma and, in light of this, provides an interpretation of the mechanism of pressure reduction created by these new surgeries.

Anterior Eye Segment↗
PubMed2001

Genetic counseling outcomes validation by genetics nurses in the UK and US.

PURPOSE: To validate genetic counseling outcomes with a sample of genetics nurses from the United Kingdom (UK), and to compare elements of genetic counseling outcomes with those from a sample of genetics nurses from the United States (US). DESIGN: Descriptive-comparative survey. METHODS: Concept analysis and literature review were used to designate outcomes, and genetics nurses were surveyed to validate the outcomes. A revision of Fehring's 1987 methodology for assessing content validity was used to estimate content validity and sensitivity of the genetic counseling outcomes. Data are reported on a convenience sample of 50 UK nurse members of the Association of Genetic Nurses and Counsellors. Findings were correlated with prior data from a convenience sample of 92 U.S. nurse members of the International Society of Nurses in Genetics, Inc., and data were compared between groups. FINDINGS: A significant positive correlation was found between samples of U.K. and U.S. nurses regarding components of outcomes of the genetic counseling process and between groups regarding extent of contribution of nurses to the outcomes. Strength of nursing contributions to knowledge of disease and indicators of coping varied according to country. CONCLUSIONS: Genetics nurses in the UK and US had similar definitions of outcomes of genetic counseling, but priorities of indicators differed between countries. Terminology used in measures to identify outcomes of the process of genetic counseling must be consistent with cultural norms.

Genetic Counseling↗
PubMed2001

Dynamic behavior of lung surfactant.

We have previously developed an adsorption-limited model to describe the exchange of lung surfactant and its fractions to and from an air-liquid interface in oscillatory surfactometers. Here we extend this model to allow for diffusion in the liquid phase. Use of the model in conjunction with experimental data in the literature shows that diffusion-limited transport i.s important for characterizing the transient period from the start of oscillations to the achievement of steady-state conditions. Matching previous data shows that upon high levels of film compression, large changes occur in adsorption rate, desorption rate, and diffusion constant, consistent with what one might expect if the subsurface region was greatly enriched in DPPC. Collapse of the surfactant film that occurs during compression leads to a .significant elevation of surfactant concentration immediately heneath the interface, consistent with the subsurface depot of surfactant that has heen postulated by other investigators. Modeling studies also uncovered a phenomenon of surfactant behavior in which the interfacial tension remains constant at its minimum equilibrium value while the film is compressed, hut without collapse of the film. The phenomenon was due to desorption of surfactant from the interface and termed "pseudo-film collapse.'' The new model also gave improved agreement with steady-state oscillatory cycling in a pulsating bubble surfactometer.

Adsorption↗
PubMed2001

A biphasic, anisotropic model of the aortic wall.

A biphasic, anisotropic elastic model of the aortict wall is developed and compared to literature values of experimental measurements of vessel wall radii, thickness, and hvdraulic conductivity as a function of intraluminal pressure. The model gives good predictions using a constant wall modulus for pressures less than 60 mmHg, but requires a strain-dependent modulus for pressures greater than this. In both bovine and rabbit aorta, the tangential modulus is found to be approximately 20 times greater than the radial modulus. These moduli lead to predictions that, when perfused in a cylindrical geometry, the aortic volume and its specific hydraulic coonductivity are relatively independent of perfusion pressure, in agreement with experimental measurements. M, the parameter that relates specific hydraulic conductivy, to tissue dilation, is found to be a positive quantity correcting a previous error in the literature.

Animals↗
PubMed2001

Specific hydraulic conductivity of corneal stroma as seen by quick-freeze/deep-etch.

Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.

Animals↗
PubMed2001

Multiple-dose pharmacokinetics and tolerability of gemifloxacin administered orally to healthy volunteers.

Gemifloxacin mesylate (SB-265805-S, LB-20304a) is a potent, novel fluoroquinolone agent with a broad spectrum of antibacterial activity. The pharmacokinetics and tolerability of oral gemifloxacin were characterized in two parallel group studies in healthy male volunteers after doses of 160, 320, 480, and 640 mg once daily for 7 days. Multiple serum or plasma and urine samples were collected on days 1 and 7 and were analyzed for gemifloxacin by high-performance liquid chromatography (HPLC)-fluorescence (study 1) or HPLC-mass spectrometry (study 2). Safety assessments included vital signs, 12-lead electrocardiogram (ECG) readings, hematology, clinical chemistry, urinalysis, and adverse experience monitoring. Gemifloxacin was rapidly absorbed, with a time to maximum concentration of approximately 1 h after dosing followed by a biexponential decline in concentration. Generally, maximum concentration and area under the concentration-time curve (AUC) increased linearly with dose after either single or repeat doses. Mean +/- standard deviation values of AUC(0-tau) on day 7 were 4.92 +/- 1.08, 9.06 +/- 2.20, 12.2 +/- 3.69, and 20.1 +/- 3.67 microg x h/ml following 160-, 320-, 480-, and 640-mg doses, respectively. The terminal-phase half-life was approximately 7 to 8 h, independent of dose, and was similar following single and repeated administrations. There was minimal accumulation of gemifloxacin after multiple dosing. Approximately 20 to 30% of the administered dose was excreted unchanged in the urine. The renal clearance was 160 ml/min on average after single and multiple doses, which was slightly greater than the accepted glomerular filtration rate (approximately 120 ml/min). These data show that the pharmacokinetics of gemifloxacin are linear and independent of dose. Gemifloxacin was generally well tolerated, although one subject was withdrawn from the study after 6 days at 640 mg for mild, transient elevations of alanine aminotransferase and aspartate aminotransferase not associated with any clinical signs or symptoms. There were no other significant changes in clinical chemistry, hematology or urinalysis parameters, vital signs, or ECG readings. In conclusion, the results of these studies, combined with the antibacterial spectrum and potency, support the further investigation of once-daily administration of gemifloxacin for indications such as respiratory tract and urinary tract infections.

Adult↗
PubMed2001

Mechanisms involved in stimulation of human immunodeficiency virus type 1 replication by aminooxypentane RANTES.

Aminooxypentane (AOP)-RANTES is a potent inhibitor of nonsyncytium-inducing (NSI), CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1) isolates. Although classical chemotactic responses are not induced in primary leukocytes by AOP-RANTES, recent studies suggest that a remnant of cell signaling occurs upon binding of receptor to this compound. We have detected a breakthrough of NSI/R5 replication from the inhibitory effects of high AOP-RANTES concentrations (<100 nM). A stimulation of different primary syncytium-inducing (SI), CXCR4-tropic (X4) HIV-1 isolates was also observed in the presence of AOP-RANTES. This stimulation was also observed after 110 h in PCR and RT-PCR for minus-strand strong-stop DNA and unspliced and multiply spliced RNA, respectively. However, there was significant variability between different SI/X4 or NSI/R5 HIV-1 isolates with regard to this AOP-RANTES-mediated stimulation or breakthrough, respectively. To further define the mechanism(s) responsible for this AOP-RANTES effect, we performed detailed retroviral replication studies with an NSI/R5 (B-92BR021) and SI/X4 (D-92UG021) HIV-1 isolate in the presence of the drug. Treatment of peripheral blood mononuclear cells with 125 nM AOP-RANTES and virus did not alter coreceptor expression, HIV-1 entry, reverse transcription, or mRNA transcription from the long terminal repeat, but it did result in increased HIV-1 integration. This AOP-RANTES-mediated increase in HIV-1 integration was diminished by treatment with pertussis toxin. Phosphorylation of the mitogen-activated protein kinase (MAPK) isoforms, extracellular signal-regulated kinase 1 (ERK1) and ERK2, was increased in a CD4(+) CCR5(+) U87 cell line treated with AOP-RANTES or with an NSI/R5 HIV-1 isolate. These findings suggest that AOP-RANTES may induce a MAPK/ERK signal transduction pathway upon binding to a G-protein-coupled receptor. MAPK/ERK1 and -2 appear to phosphorylate the HIV-1 preintegration complex, a step necessary for nuclear translocation and successful integration.

Anti-HIV Agents↗
PubMed2001

Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro.

We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.

Avian Sarcoma Viruses↗