Search PubMed⌕ Search

Biomedical subjects

J J Harrington

Publications and source records attributed to J J Harrington.

15 recordsLinked to original sources
PubMed1995

DNA structural elements required for FEN-1 binding.

In eukaryotic cells, a 5'-flap DNA endonuclease and a double-stranded DNA 5'-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)'-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the Fadj strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the Fadj strand using double flap structures reveals that the Fadj strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.

Base Sequence↗
PubMed1995

Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human.

We recently purified and cloned the gene for a DNA structure-specific endonuclease, FEN-1, from murine cells. The murine protein recognizes 5' DNA flap structures that have been proposed in DNA replication, repair, and recombination. Here, we report the sequence of the human FEN1 gene. The translated sequence is identical to peptide sequence obtained from maturation factor-1, which is 1 of the 10 essential proteins for cell-free DNA replication. The human protein has the same structure-specific DNA endonuclease activity as the murine protein. Two human chromosomal hybridization signals, 11q12 and 1p22.2, were observed by FISH analysis using human genomic clones homologous to the mouse Fen-1 gene. The localization on human 11q12 was confirmed using radiation-reduced hybrids. The mouse Fen-1 gene is assigned to chromosome 19 based on somatic cell hybrids. The significance of these FEN1 gene localizations in human and mouse is discussed.

Amino Acid Sequence↗
PubMed1994

Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.

Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair. Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures. Here, we describe the cloning of the murine FEN-1 gene. The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2. We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases. The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair. The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.

Amino Acid Sequence↗
PubMed1994

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.

Animals↗
PubMed1990

Chemically sensitized erythrocytes for hemagglutination reactions.

The use of chemically modified indicator erythrocytes for hemagglutination reactions can result in increased sensitivity. Treatment of erythrocytes with polyvinylpyrrolidone (PVP) or dextran T40 (10% weight/volume) induces changes in the cell surface in the form of extensions and blebbing, thereby increasing the surface area. These sensitized cells can be used in forensic science when detection or quantitation of erythrocyte surface reacting antibodies is important. The effect of altering membrane lipid fluidity on erythrocyte surface antigens has also been investigated. Treatment of cells with a reagent that increases the membrane ratio of cholesterol to phospholipid results in enhanced hemagglutination capacity despite the lack of extensive spiculation.

ABO Blood-Group System↗
PubMed1988

Detection and use of salivary hemagglutinins for forensic blood grouping.

A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. The method utilizes a bromelin treated erythrocyte suspension as indicator cells and includes a simple procedure to concentrate these hemagglutinins. Antiserum directed against immunoglobulin A enhances the hemagglutination assay. We find that these salivary hemagglutinins are present in over 90% of the population and that their titer remains stable over a period of two months. These hemagglutinins can be used to blood type the donor of a saliva sample and can be used in a confirmatory test that complements the commonly used absorption-inhibition test which is used to detect salivary blood group agglutinogens. In preliminary studies we have determined that hemagglutinins can be successfully isolated and analyzed from dried saliva stains.

Agglutination Tests↗
PubMed1988

Detection of hemagglutinins in dried saliva stains and their potential use in blood typing.

Since 1928, hemagglutinins have been known to exist in saliva; however, they have not been utilized as evidence in criminal investigations because in the past, techniques for measuring them have not been sufficiently sensitive. In this paper we describe improved techniques for detecting salivary hemagglutinins and report initial results obtained with these methods. The stability of salivary hemagglutinins at several different temperatures was examined in liquid samples and in dried stains on filter paper, cigarette butts, and envelope flaps. Our observations indicate that salivary hemagglutinins may be sufficiently stable, over periods of one to several days at ambient room temperatures, to be of value to forensic science investigators. The results of the hemagglutinin assay are not affected by the age or sex of the sample donor. Because salivary hemagglutinins can be used to determine ABO blood type, analyses of this kind can serve as an important confirmatory test which the forensic serologist can use in conjunction with salivary agglutinogen determinations.

Blood Grouping and Crossmatching↗
PubMed1977

Pseudoaneurysm of the left ventricle: an unusual echocardiographic presentation. Review of the literature.

Echocardiography showed a large anterior chamber communicating with the left ventricle cavity through the interventricular septum in a patient with a previous left ventricular aneurysmectomy. At postmortem examination this chamber proved to be an 11-cm diameter pseudoaneurysm that opened into the left ventricle through a 3-cm orifice. A review of the literature showed 67 cases of histologically proven left ventricular pseudoaneurysm, most of which occurred after myocardial infarction and cardiac surgery. Twenty-six of 32 left ventricular pseudoaneurysms were successfully operated upon. Among 35 patients with pseudoaneurysms not operated upon, rupture was a cause of death in 11.

Autopsy↗
PubMed1976

The influence of left ventricular stroke volume on aortic root motion: an echocardiographic study.

Aortic root motion was studied in 24 normal volunteers at rest and during the Valsalva maneuver, isometric exercise, and amyl nitrite inhalation. In addition root motion was correlated with the stroke volumes determined at cardiac catheterization in 24 patients. The root has distinct systolic movement, the amplitude and duration of which were easily measured both at rest and during the interventions. At rest,the mean (+/-1 SE) systolic amplitude of the anterior aortic wall was 11.2 +/- 0.5 mm and that of the posterior wall 9.5 +/- 0.3 mm. During the strain phase of the Valsalva maneuver anterior wall amplitude fell to 8.2 +/- 0.4 mm and the posterior wall to 7.3 +/- 0.5 mm (P less than 0.001). With release, anterior wall amplitude rebounded to 12.5 +/- 0.8 mm and the posterior wall to 10.8 +/- o.5 mm, values greater than control (P less than 0.01). With isometric exercise there was no change in amplitude compared to rest; however, amyl nitrite caused an increase in the anterior wall to 13.5 +/-0.8 mm and posterior wall to 11.9 +/-0.6 mm (P less than 0.01). In the patient group the amplitude of posterior wall motion correlated weakly with cardiac index (r = 0.63) and stronger with stroke index (r = 0.78). This study quantifies the echocardiographic pattern of normal aortic root motion. The findings indicate that the aortic root motion is an index of stroke volume; they further suggest that root motion is acutely sensitive to variations in stroke volume since its amplitude changed in accord with the documented effects of the employed maneuvers on stroke volume.

Adult↗
PubMed1976

Echocardiographic evaluation of the Valsalva Maneuver in healthy subjects and patients with and without heart failure.

The Valsalva maneuver was evaluated by echocardiography in three groups: A) 10 normal volunteers, B) 10 patients with no history of heart failure and normal ejection fractions, and C) 10 patients with heart failure and depressed ejection fractions. Groups A and B had a significant fall in left ventricular internal dimensions and calculated stroke volume by end strain which returned rapidly to baseline in recovery without significant overshoot. Arterial pressure showed a signoidal strain pattern with a normal overshoot in early recovery in all group B patients. In group C ventricular dimensions did not diminish during strain; arterial pressures showed a "square wave" pressure elevation during strain without an overshoot in recovery. Echocardiography allows a new approach to evaluate further the left ventricular response to the Valsalva maneuver. Patients with severely depressed ejection fractions, unlike those with normal ventricular function, are unable to alter stroke output in response to acutely increased intrathoracic pressure. A square wave pressure response is a likely consequence of a fixed stroke output during the strain maneuver.

Adult↗