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Biomedical subjects

A F Taylor

Publications and source records attributed to A F Taylor.

At least 37 records · Page 2Linked to original sources
PubMed1995

The initiation and control of homologous recombination in Escherichia coli.

The chromosome of Escherichia coli recombines at low frequency when it is an intact circle but recombines at high frequency when it is broken, for example by X-rays, or when a linear DNA fragment is introduced into the cell during conjugation or transduction. The high recombinogenicity of double-strand (ds) DNA ends is attributable to RecBCD enzyme, which acts on ds DNA ends and is essential for recombination and ds DNA break repair. RecBCD enzyme initiates DNA unwinding at ds DNA ends, and its nuclease activity is controlled by Chi sites (5' G-C-T-G-G-T-G-G 3') in such a way that the enzyme produces a potent single-stranded DNA substrate for homologous pairing by RecA and single-stranded DNA binding proteins. We discuss a unifying model for recombination and ds DNA break repair, based upon the enzymic activities of these and other proteins and upon the behaviour of E. coli mutants altered in these proteins.

Escherichia coli↗
PubMed1995

Effect of ductal patency on organ blood flow and pulmonary function in the preterm baboon with hyaline membrane disease.

OBJECTIVE: To examine the effect of early ductal ligation vs. maintenance of ductal patency on vital organ perfusion and pulmonary function in premature baboons with hyaline membrane disease. DESIGN: Randomized, controlled interventional study to compare early ligation with formalin infiltration of the ductus arteriosus. SETTING: Animal care facility at a dedicated research foundation. SUBJECTS: Eighteen premature baboons delivered by hysterotomy at 140 +/- 2 day gestation. INTERVENTION: Nine premature baboons underwent formalin infiltration of the ductus arteriosus (group 1), and nine underwent ductal ligation (group 2). Surgical ligation or formalin infiltration was performed at 2 to 4 hrs of age. Animals were maintained on mechanical ventilation and ventilator parameters were adjusted to maintain PaO2 and PaCO2 within the physiologic range. MAIN OUTCOME MEASURES: Left ventricular output indexed to body weight and vital organ perfusion were measured at 24 hrs of age by the radiolabeled microsphere method. Lung mechanics, including lung wet/dry weight ratio, total lung water, static compliance and functional residual capacity were measured immediately following euthanasia. RESULTS: Total pulmonary blood flow was significantly lower (p = .0001) in group 2 (mean = 94 mL/min/kg), compared with group 1 (mean = 287 mL/min/kg). Systemic blood flow and effective pulmonary blood flow were higher in group 1 (p = .07). No significant difference between groups in absolute organ blood flow was noted, although flow as a percent of left ventricular index was significantly higher in all organs except the kidney in group 2. There was no difference in arterial blood gas values, parameters of mechanical ventilation, percent lung water, or postmortem measurement of lung mechanics between groups. CONCLUSION: Early ductal ligation did not result in improved cardiac output, increased organ blood flow, or improved pulmonary function. We postulate that gradual constriction of the ductus arteriosus may play an important role in successful cardiovascular adaptation in the premature infant. While it is clear that premature infants with symptomatic patent ductus arteriosus often benefit from ductal closure, we question the practice of prophylactic early ductal closure.

Adrenal Glands↗
PubMed1995

Pemphigus foliaceus and pemphigus vulgaris autoantibodies react with the extracellular domain of desmoglein-1.

Pemphigus foliaceus is associated with an autoimmune response against desmoglein-1; however, the fine specificity of these autoantibodies and the role that they play in pathogenesis have not yet been elucidated. In an attempt to develop a system to facilitate the detection and characterization of this antigen/antibody system, recombinant human desmoglein-1 was expressed in COS-1 cells, a mammalian epithelial cell line. The desmoglein-1 transgene product was shown to be expressed on the surface of the COS-1 cells in the appropriate transmembrane orientation. All pemphigus foliaceus sera (endemic form, n = 24; nonendemic form, n = 7) reacted strongly with nonpermeabilized desmoglein-1-transfected cells, exhibiting a punctate cell-surface staining pattern. This reactivity against the desmoglein-1 ectodomain was predominantly an IgG4-restricted response and was calcium dependent. Ten of 18 pemphigus vulgaris sera also reacted with the extra-cellular domain of recombinant desmoglein-1. Use of this eukaryotic expression system should greatly facilitate further characterization of the anti-desmoglein-1 autoimmune response associated with pemphigus foliaceus and pemphigus vulgaris and may aid in determining its pathogenic relevance.

Animals↗
PubMed1994

Development of an ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis.

Autoantibodies associated with the subepidermal blistering disorders bullous pemphigoid and herpes gestationis react with a 180-kD transmembrane hemidesmosomal protein, designated BP180. The BP180 ectodomain is composed of a series of interrupted collagen triple helical domains. Located on one of the noncollagenous extracellular segments of this protein is an immunodominant epitope, designated MCW-1, recognized by patient autoantibodies. In this investigation we have developed an enzyme-linked immunosorbent assay system to detect antibody reactivity against the MCW-1 epitope with the use of a bacterial fusion protein containing the BP180 autoantibody-reactive site. The following sera were assayed for reactivity with this recombinant protein: bullous pemphigoid (n = 62), herpes gestationis (n = 28), endemic pemphigus foliaceus (n = 17), lupus erythematosus (n = 15), and normal human sera (n = 22). This enzyme-linked immunosorbent assay-based protocol was shown to be highly specific (98.3%) in detecting autoantibody activity in bullous pemphigoid and herpes gestationis patients. Fifty-three percent of bullous pemphigoid sera and 71% of herpes gestations sera, but none of the control sera, yielded positive results in this assay. Of the patient sera that were known to react with full-length BP180, almost all showed reactivity with the MCW-1 antigenic site of this protein. Autoantibodies detected in this assay were predominantly of the immunoglobulin G class. The results presented here lend support to the hypothesis that this well-defined antigen/antibody system may be relevant in pathogenesis.

Autoantibodies↗
PubMed1993

Hormonal response of the premature primate to operative stress.

There are few data on the hormonal response to operation in the premature infant. Studies examining the response of newborn human infants have been performed on patients beyond the first few days of life, where some adaptation to postnatal life has occurred. This study evaluated the response of the newly born premature primate to surgical stress. Premature baboons (75% gestation) were intubated, mechanically ventilated and underwent thoracotomy at 2 hours of life with exposure of the ductus arteriosus (PDA). In group 1, formalin was infiltrated to keep the ductus patent. In group 2, the PDA was ligated. Controls had no operation. Blood was drawn at 0, 6, 24, 48, 72, and 96 hours of age. Echocardiograms were performed to confirm patency or closure of the ductus and to monitor cardiac function. Epinephrine, norepinephrine, renin, and cortisol levels were measured. Cortisol levels rose in all groups. Operation stimulated a marked increase in catecholamine and renin levels in both operative groups, which was more marked in the group with PDA ligation at 24 hours. These data reflect expected pathophysiology since early PDA ligation exerts additional hemodynamic demand on the heart. In conclusion, the premature primate is able to mount a significant and severity-dependent endocrine response to stress.

Animals↗
PubMed1993

A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.

Subepidermal blistering associated with the human skin diseases bullous pemphigoid and herpes gestationis has been thought to be an IgG autoantibody-mediated process; however, previous attempts to demonstrate the pathogenicity of patient autoantibodies have been unsuccessful. An immunodominant and potentially pathogenic epitope associated with these blistering diseases has recently been mapped to the extracellular domain of a human epidermal antigen, BP180. Patient autoantibodies that react with this well-defined antigenic site failed to crossreact with the murine form of this autoantigen and thus could not be assayed for pathogenicity in a conventional passive transfer mouse model. As an alternative, rabbit polyclonal antibodies were generated against a segment of the murine BP180 protein homologous with the human BP180 autoantibody-reactive site and were passively transferred into neonatal BALB/c mice. The injected animals developed a subepidermal blistering disease that closely mimicked bullous pemphigoid and herpes gestationis at the clinical, histological, and immunological levels. Autoantibodies that recognize the human BP180 ectodomain are therefore likely to play an initiatory role in the pathogenesis of bullous pemphigoid and herpes gestationis.

Amino Acid Sequence↗
PubMed1992

RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.

During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.

Bacteriophage lambda↗
PubMed1991

Cardiac output by Doppler echocardiography in the premature baboon: comparison with radiolabeled microspheres.

Pulsed-Doppler echocardiography (PDE) is a useful noninvasive method for determining left ventricular output (LVO). However, despite increasingly widespread use in neonatal intensive care units, validation studies in prematures with cardiopulmonary disease are lacking. The purpose of this study was to compare radiolabeled microsphere (RLM) and PDE measurements of LVO, using the critically ill premature baboon as a model of the human neonate. Twenty-two paired RLM and PDE measurements of LVO were obtained in 14 animals between 3 and 24 h of age. Average PDE LVO was 152 ml/min/kg (range, 40-258 ml/min/kg) compared to 158 ml/min/kg (range, 67-278 ml/min/kg) measured by RLM. Linear regression analysis of the paired measurements showed good correlation with a slope near unity (gamma = 0.94x + 4.20, r = 0.91, SEE = 25.7 ml). We conclude that PDE determinations of LVO compare well with those measured by RLM in the premature baboon. PDE appears to provide a valid estimate of LVO and should be useful in human prematures with cardiopulmonary distress.

Animals↗
PubMed1991

Ductal shunting and effective systemic blood flow following single dose surfactant treatment in the premature baboon with hyaline membrane disease.

We studied the hemodynamic effects of using natural surfactant in premature baboons with hyaline membrane disease (HMD). Study animals (n = 5) received a single dose of surfactant immediately after delivery and control animals (n = 8) did not. Using microspheres at 3, 8, and 23 h we found no significant differences in left ventricular output, effective systemic flow, systemic-to-pulmonary patent ductus arteriosus (PDA) shunting, or in cerebral or renal organ blood flow. However, both groups had large PDA shunts (fraction of LVO to lungs greater than 0.40-0.55 at 3 and 8 h) resulting in low systemic perfusion (less than 80 ml/min/kg). Single dose surfactant did not improve the myocardial dysfunction and low cerebral and renal blood flow which occur during treatment for HMD.

Animals↗
PubMed1991

High-frequency oscillatory ventilation versus intermittent mandatory ventilation: early hemodynamic effects in the premature baboon with hyaline membrane disease.

We studied the hemodynamic consequences during the first 24 h of life in premature baboons (140 d) with hyaline membrane disease that were treated with high-frequency oscillatory ventilation (HFOV) or conventional intermittent mandatory ventilation (IMV). Cardiac output and organ blood flow were measured at three time-points using the radiolabeled microsphere technique. Seven of seven HFOV and six of eight IMV animals survived the 24-h period. By design, initial mean airway pressure (Paw) was higher in the HFOV group (p less than 0.01). HFOV Paw was progressively reduced during the study period because of improving oxygenation as measured by the arterial to alveolar oxygen ratio. In contrast, it was necessary to increase Paw in the IMV animals to maintain the arterial to alveolar oxygen ratio. By 23 h, the IMV group required higher Paw than the HFOV group (p less than 0.05) and had a lower arterial to alveolar oxygen ratio (p less than 0.05). We found no significant differences in left ventricular output, effective systemic flow, organ blood flow, or central venous pressure between the two groups at 3, 8, or 23 h. The HFOV strategy used in our study resulted in significant improvement in oxygenation during the initial 24 h of treatment without adverse effect on left ventricular output, cerebral blood flow, or central venous pressure. We conclude that when appropriate changes in Paw are made during HFOV in response to improvement in arterial oxygenation and changes in lung inflation as assessed by chest radiographs HFOV can be achieved without depressing cardiovascular dynamics more than during conventional therapy with IMV.

Animals↗
PubMed1990

Action of RecBCD enzyme on cruciform DNA.

We tested the hypothesis that RecBCD enzyme of Escherichia coli resolves pre-existing Holliday recombination intermediates by examining the action of the purified enzyme on an open-ended DNA cruciform with limited ability to branch migrate. The enzyme cleaved two strands of the cruciform near its base to produce "recombinant" products, with a marked bias in the direction of cleavage. The two nicks necessary to cleave the cruciform were made separately. Cruciforms whose four termini were blocked by synthetic hairpin-shaped oligonucleotides were not detectably nicked by the enzyme. With one terminus open the enzyme made a nick at the base of the cruciform but not a double-strand cut. With two or more termini open the enzyme made double-strand cuts. We infer that RecBCD enzyme molecules must enter the termini of duplex DNA and approach the cruciform from more than one direction in order to cleave it into recombinant products. Previous results on RecBCD-mediated recombination between phage lambda and lambda dv imply that intracellular RecBCD enzyme can approach pre-existing Holliday junctions from only one direction. We infer that intracellular RecBCD enzyme cannot cleave pre-existing Holliday junctions into recombinants and suggest that the enzyme may cleave Holliday junctions in whose formation it participates.

Base Sequence↗
PubMed1990

Left ventricular dysfunction following ligation of the ductus arteriosus in the preterm baboon.

We evaluated the effect of early closure of the patent ductus arteriosus (PDA) on cardiac performance in a preterm baboon model of hyaline membrane disease. Eleven baboon fetuses were delivered at 75% gestation, resuscitated, and treated with conventional ventilation. The animals were divided into two groups, and underwent left thoracotomy at 2 hr of age. Group 1 (N = 5) had ductal infiltration with formalin to maintain ductal patency. Group 2 (N = 6) had ductal ligation. Hemodynamic parameters were compared between groups before and after surgical intervention. Examination of cardiovascular function was performed at intervals over a 24-hr period using two-dimensional, M-mode, and pulsed Doppler echocardiography. Ventricular performance was significantly worse in the ligation animals as demonstrated by reduced left and right ventricular outputs and reduced left ventricular shortening. However, pulmonary disease and arterial blood gases were not different. Early ligation was also associated with significantly increased systemic vascular resistance. We conclude that early ductal ligation leads to impaired ventricular performance in the premature primate. We hypothesize that the PDA may protect the immature ventricle by preventing pressure overload during adaptation to postnatal life.

Animals↗
PubMed1990

Proximal, tracheal, and alveolar pressures during high-frequency oscillatory ventilation in a normal rabbit model.

To study the effect of different high-frequency oscillatory ventilation parameters on airway pressure, we measured oscillatory pressure amplitude ([Paw[) and mean airway pressure (Paw) at three sites in open-chested normal rabbits: proximal, trachea, and alveolus. Five animals were studied to test a new pleural capsule design, which was then used in two groups of animals to measure right upper (n = 4) or middle (n = 5) lobe alveolar pressures. Animals were randomly sequenced through combinations of frequency (10, 15, and 20 Hz) and fractional inspiratory time (Ti) (0.3 and 0.5) while normoxic and eucapnic. During capsule testing, we noted that alveolar pressures increased (p less than 0.05) with increasing capsule mass, suggesting that compressive forces from the capsule may alter the capsule measurement. We thus used a low-mass (430 mg) transducer system in the rabbit high-frequency oscillatory ventilation experiments. Using multifactorial analysis of variance, we found significant main effects of Ti on Paw, and of measurement site on both [Paw[ and Paw (all p less than 0.009). Frequency did not influence variations in either [Paw[ or Paw. For both Ti settings, alveolar upper lobe Paw was lower compared with that of the middle lobe (p less than 0.0005). Lengthening Ti (0.3 to 0.5) increased tracheal Paw in each capsule group (p less than 0.0005). At Ti = 0.5, tracheal Paw exceeded Paw measured proximally (p less than 0.05). Our data support in vivo alveolar Paw inhomogeneity and demonstrate significant changes in pressures within the lung related to Ti during high-frequency oscillatory ventilation.

Analysis of Variance↗
PubMed1988

Heterosexual and perinatal transmission of human immunodeficiency virus in a low prevalence community.

Six previously unidentified cases of human immunodeficiency virus (HIV) infection were found during an investigation and follow-up of three clusters of heterosexually and perinatally transmitted HIV infections. The investigations were to identify those infected, to interrupt the transmission of HIV, to attempt to reduce the likelihood of the development of the acquired immunodeficiency syndrome among those found to be seropositive, and to show the usefulness of traditional disease control strategies during this epidemic. Our findings indicate the value of contact tracing for HIV infections and the need to provide and evaluate educational activities regarding the transmission of HIV in low prevalence populations.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Flow cytometric analysis of lymphocyte subset phenotypes comparing normal children and adults.

The use of monoclonal antibodies and flow cytometry to characterize lymphocyte subsets has become an essential part of immunological research. In this study, we determined the normal ranges and absolute numbers of cells for several lymphocyte subsets in the peripheral blood of children and adolescents (ages 4-16 years). These were compared to the same subsets found in adults. Several differences were observed among the T and NK cell subsets. The total number of T cells, especially the CD4+ subset, were higher in children while two CD8+ subpopulations (CD8+CD11+ and CD8+Leu 7+) were found to be lower. Children had approximately half the absolute numbers of natural killer (NK) cells (CD16+), including a lower percentage of the CD16+CD8+ and CD16+Leu 7+ subsets. Monocytes and B-cell values were not significantly different between the two groups.

Adolescent↗
PubMed1986

recD: the gene for an essential third subunit of exonuclease V.

Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes. We report here that the enzyme contains in addition a third polypeptide (alpha) with a molecular mass of about 58 kDa. The alpha polypeptide is not synthesized by a class of mutants (previously designated recB) lacking the nuclease activity of exonuclease V but retaining recombination proficiency. The gene, recD, coding for the alpha polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-argA on the E. coli chromosome. The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone. In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.

Bacterial Proteins↗