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Biomedical subjects

J Flynn

Publications and source records attributed to J Flynn.

At least 19 recordsLinked to original sources
PubMed1995

Purification and stabilization of mouse DNA methyltransferase.

Cytosine methylation within DNA has been implicated in genetic imprinting, X-chromosome inactivation, regulation of tissue-specific gene expression, aging, and cancer. Unfortunately, DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) from various mammalian sources have been difficult to isolate and stabilize, precluding investigations of these critical enzymes. We describe a novel FPLC purification of the 190,000 Mr DNA methyltransferase from mouse Friend erythroleukemia cells. The homogeneous 190 kD Mr form of the enzyme is the only polypeptide detected at various stages of cell growth and has not undergone detectable N-terminal proteolysis.

Amino Acid Sequence↗
PubMed1995

Evaluation of a PCR based method for identification of mycobacterial isolates.

In recent years an increasing number of mycobacterial species have been described as causing disease in humans. Identification of isolates to the species level is essential for evaluation of the significance of an isolate. Conventional methods for identification based on selective inhibitors of growth and biochemical reactions are slow. We have evaluated a novel method, PCR restriction enzyme pattern analysis (PRA), for identification of mycobacterial isolates. Fifty three cultures including a mixed culture of M. tuberculosis and M. avium and four mycobacterial cultures contaminated with rapidly growing organisms of other genera were studied. The method permits identification of most isolates within 1 to 2 days, including some contaminated cultures, and results were generally in accordance with those obtained by conventional methods.

Bacteriological Techniques↗
PubMed1995

Major influences on maternal responsiveness to infants.

Thirty healthy, middle-class mothers were interviewed during their postpartum hospital stay to identify major influences on mothers' responses to their infants. Sullivan's theorem of tenderness served as a guide for exploring maternal responsiveness. Three major categories, infant tenderness needs, maternal perception, and maternal needs, and their related properties, were described. Support persons were viewed as very important. Mothers also believed that some infant needs were more important than others. Finally, mothers felt that they were constantly faced with priorities, and that the emotional state of the mother and infant often influenced their interaction.

Adult↗
PubMed1995

Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis.

We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and NG-monomethyl-L-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis.

Amino Acid Oxidoreductases↗
PubMed1995

Multiplex PCR for identifying mycobacterial isolates.

AIMS: To develop a multiplex polymerase chain reaction (PCR) method to facilitate identification of mycobacterial isolates. METHODS: Type strains of 14 species of mycobacteria and 56 clinical isolates were lysed by boiling in TE Triton. The lysate (5 microliters) was used directly in a PCR reaction incorporating three pairs of PCR primers expected to amplify fragments from the genome of (a) all mycobacteria, (b) Mycobacterium tuberculosis complex only and (c) M avium only. PCR products were visualised by electrophoresis on agarose gels. RESULTS: Multiplex PCR applied to 14 type strains yielded patterns on electrophoresis which permitted identification of the mycobacterial isolates as M tuberculosis complex, M avium or as mycobacteria other than the former. The identification of 56 clinical isolates by multiplex PCR was consistent with other methods and was accomplished in less than one working day. CONCLUSIONS: This method may facilitate rapid and convenient identification of most clinical isolates of mycobacteria by PCR and gel electrophoresis. Further evaluation is warranted.

Base Sequence↗
PubMed1994

PCR based fingerprinting of Enterobacter cloacae.

An outbreak of lower respiratory tract infection with Enterobacter cloacae occurred in an intensive care unit in a university teaching hospital. Random amplification of polymorphic DNA (RAPD) was used to assist in the investigation of the outbreak. The technique was readily applied to this organism and permitted differentiation between strains which had identical biochemical profiles and antibiograms. The versatility of this technique makes it attractive for use in hospitals where fingerprinting of any one of the many Gram-negative rods associated with nosocomial infection may be required from time to time.

Cross Infection↗
PubMed1994

Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection.

It has, for many years, been widely assumed that the fundamental mechanism of protection in tuberculosis infection is a CD4 T cell response producing lymphokines that activate macrophages to kill or restrict the intracellular growth of M. tuberculosis. Just as certain cytokines, e.g. IFN-gamma, are currently perceived to be important for protection, others, particularly tumor necrosis factor (TNF), are thought to be responsible for much of the tissue destruction associated with the disease. Yet there are remarkably few critical experimental or clinical data that have defined the immunological requirements for protection and pathogenesis. One of the initial stimuli to the work we have undertaken has been careful reflection on the results of the many prospective trials of BCG against tuberculosis. Two aspects have impelled us to reconsider conventional wisdom. The first, of course, is the wide discrepancy in the degree of protection imparted, ranging from 0% in South India to 77% in the British MRC trial (1, 2). The second is that, in all trials that examined them, skin test conversions to tuberculin positivity were 85% or greater, indicating a disparity between the presence of delayed hypersensitivity to tuberculin and protection. We and others have argued (1, 2) that there are multiple possible explanations for this discrepancy, the principal one being protection caused by infection with environmental mycobacteria. But, the general point raised is whether cell mediated immunity as manifested by CD4+ cell production of lymphokines and macrophage activation is a sufficient mechanism for protection against M. tuberculosis infection.

Animals↗
PubMed1994

A complex bilateral polysyndactyly disease locus maps to chromosome 7q36.

We demonstrated that the gene responsible for a congenital limb deformity (polysyndactyly) maps to chromosome 7q36 in a large family. Pre- and postaxial anomalies of the extremities are inherited in this family as an autosomal dominant trait. The disease locus is closely linked to D7S550 (maximum lod score = 6.85, theta = 0). This region is homologous to a segment of mouse chromosome 5, where the mutations hammer toe (HM) and hemimelic extra toes (HX) have been mapped. These data suggest that human chromosome 7q36 and the homologous region of mouse chromosome 5 contain genes involved in limb pattern formation.

Base Sequence↗
PubMed1994

Left ventricular dysfunction and acute lung injury induced by continuous administration of endotoxin in sheep.

Sixteen sheep were surgically prepared for chronic study. Seven days later, Escherichia coli endotoxin (10 ng/kg/min, lipopolysaccharide (LPS) group, n = 10) or an equivalent amount of 0.9% NaCl (Control group n = 6) was administered. Between 1 and 8 h post-LPS, there was a hypodynamic state with low cardiac index (CI, LPS 5.0 +/- 0.2; sham 6.3 +/- 0.4 liters/min/m2 at 4 h). During this period, the left ventricular end-systolic pressure-diameter relationship (ESPDR), a sensitive index of myocardial contractility, was also lower (LPS 10.4 +/- 1.2; sham 17.2 +/- 0.8 mmHg/mm). Mean pulmonary arterial pressure (PAP) and pulmonary vascular resistance index (PVRI) were remarkably increased 1 h after the administration of LPS (PAP:LPS 37.5 +/- 1.9; sham 21.8 +/- 0.9 mmHg, PVRI: LPS 600 +/- 58; sham 158 +/- 23 dynes x s x cm-5 x m2). The early changes in cardiopulmonary function occurred concomitantly with an elevation in tumor necrosis factor (LPS 1221 +/- 520; sham 0 +/- 0 pg/ml) and thromboxane B2 (LPS 1382 +/- 266; baseline 82 +/- 20 pg/ml) in arterial blood. Following this first phase, the sheep presented a persistent hyperdynamic state characterized by a significant increase in CI. The ESPDR continued to fall. By 24 h post-LPS the CI was 10.1 +/- 0.5 liters/min/m2 (sham, 6.3 +/- 0.3) but the ESPDR had fallen to 8.2 +/- 2.3 mmHg/mm (sham 16.0 +/- 3.0). The pulmonary hypertension was maintained for the duration of the LPS infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Molecular detection of Bacteroides forsythus in human periodontitis.

The usefulness of a digoxigenin-labeled genomic DNA probe for the detection of subgingival Bacteroides forsythus was examined. In addition, the arbitrarily primed polymerase chain reaction (AP-PCR) was used to delineate the genetic diversity of B. forsythus periodontal isolates. The DNA probe detected 10(3) B. forsythus cells and yielded a strong signal at 10(4) cells. It reacted with B. forsythus ATCC 43037T and 44 clinical isolates and showed no detectable reactivity with 75 strains of 24 other oral microbial species. In comparison to culture, the DNA probe in a dot-blot method demonstrated a sensitivity of 88.8% and a specificity of 38.4% (accuracy, 72.5%). By colony-blotting on primary plates, a sensitivity of 98.1% and a specificity of 53.8% (accuracy, 82.5%) were obtained. B. forsythus was detected in 449 (73.1%) of 614 periodontitis patients. The occurrence of the organism was closely associated with Porphyromonas gingivalis, both species being present in 54.8% and absent in 22.2% of 270 study samples. AP-PCR identified 24 B. forsythus genotypes among 27 test strains. This study demonstrated the utility of a non-radioactive genomic probe for direct detection of B. forsythus in subgingival specimens. The species showed a considerable degree of genetic diversity. DNA analysis may help to determine the role of B. forsythus in periodontal disease and its mode of transmission among exposed individuals.

Adolescent↗
PubMed1994

Gender, race, and perception of environmental health risks.

This paper reports the results of a national survey in which perceptions of environmental health risks were measured for 1275 white and 214 nonwhite persons. The results showed that white women perceived risks to be much higher than did white men, a result that is consistent with previous studies. However, this gender difference was not true of nonwhite women and men, whose perceptions of risk were quite similar. Most striking was the finding that white males tended to differ from everyone else in their attitudes and perceptions--on average, they perceived risks as much smaller and much more acceptable than did other people. These results suggest that socio-political factors such as power, status, alienation, and trust are strong determiners of people's perception and acceptance of risks.

Data Collection↗
PubMed1992

Tuberculosis in the west of Ireland 1986-1990.

A review of mycobacteria isolated from clinical samples from the Western Health Board Area (WHBA) for the years 1986 to 1990 was performed to establish the pattern of mycobacterial infection. The incidence of microbiologically proven cases of tuberculosis (13.3/100,000) and of sputum smear positive cases (6.2/100,000) was determined and correlated with notification data. M. bovis accounts for 6.3% of cases of microbiologically confirmed tuberculosis in this area. Resistance to one or more of the first line anti-tuberculous drugs was noted in 5.3% of isolates (excluding pyrazinamide resistance in M. bovis). Clinically significant isolates of mycobacteria other than tuberculosis (MOTT) were rare throughout the five year period. The overall incidence of microbiologically proven cases of tuberculosis is relatively high and M. bovis and drug resistant isolates of M. tuberculosis are relatively common.

Drug Resistance, Microbial↗
PubMed1992

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

AIMS: To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. METHODS: Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. RESULTS: M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. CONCLUSIONS: The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Base Sequence↗
PubMed1992

Pulmonary hemodynamics and tissue damage after one lung infusion of Pseudomonas aeruginosa in sheep.

The relative roles of hematogenous mediators and direct bacterial toxicity due to phagocytosis by pulmonary intravascular macrophages were determined by selective bacterial infusion into the left pulmonary artery and comparison of right and left lungs at 24 h. Chronically instrumented sheep received 15-min pulmonary arterial infusions of live Pseudomonas aeruginosa (0.35-2.9 x 10(9), n = 6) or saline (n = 5). The saline group demonstrated stable cardiopulmonary function over time. Left lung blood flow, measured by Doppler flow probe, decreased 15 min into the bacterial infusion, with a concomitant sevenfold increase in left lung pulmonary vascular resistance index. The right lung pulmonary vascular resistance index doubled at 1 h, in association with increased plasma thromboxane B2 levels. An increase in cardiac index and decrease in systemic vascular resistance occurred at 12 h. The wet-to-dry weight ratio of the Pseudomonas-infused left lung was increased compared with that of the sham-infused lung. The tissue count of neutrophils in the lungs was doubled in both sides, but neutrophils on the left were more degranulated. The left lung tissue damage was caused by direct bacterial toxicity, including activation of phagocytic cells. Hematogenous mediators induced pulmonary and systemic hemodynamic changes and right lung neutrophil sequestration, but they did not damage the noninfused lung.

Animals↗
PubMed1992

Effect of cyclooxygenase inhibition on retinal and choroidal blood flow during hypercarbia in newborn piglets.

The effect of the cyclooxygenase inhibitor, indomethacin, on choroidal (ChBF) and retinal (RBF) blood flow during hypercarbia was examined in 16 paralyzed and mechanically ventilated piglets less than 8 d old. The animals were randomly assigned to a control group (mean +/- SEM: wt, 1.66 +/- 0.1 kg; n = 8) that received a placebo infusion or to an indomethacin treatment group (wt, 1.68 +/- 0.2 kg; n = 8) that received an infusion of indomethacin (5 mg/kg i.v. over 30 min). Baseline ChBF and RBF were measured using radiolabeled microspheres in room air before and 15 min after the administration of placebo or indomethacin. Animals were then exposed to 30 min of hypercarbia (6-7% CO2, arterial CO2 pressure 8-10 kPa) and measurements were repeated. There were no significant differences in RBF between control (40 +/- 3 mL/min/100 g) and indomethacin-treated animals (40 +/- 3 mL/min/100 g) before administration of placebo or indomethacin. However, RBF decreased significantly in the indomethacin-treated animals (28 +/- 2 mL/min/100 g) compared to the control group (42 +/- 4 mL/min/100 g) 15 min after administration of placebo or indomethacin. Furthermore, an increase in RBF occurred during hypercarbia in the control group (86 +/- 6 mL/min/100 g), but this change was blunted in the indomethacin-treated animals (33 +/- 5 mL/min/100 g) (p less than 0.001). In contrast, ChBF did not differ significantly between the control and indomethacin groups during the periods studied. These results suggest that the increase in RBF during hypercarbia is at least partially mediated by cyclooxygenase by-products of arachidonic acid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗