Biomedical subjects

M L Fitzgerald

Publications and source records attributed to M L Fitzgerald.

27 records · Page 2Linked to original sources

PubMed1993

Targeted deletion of beta 1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expression.

The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the beta 1 integrin gene through homologous recombination, we have assessed the role of the beta 1 integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the beta 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (beta 1 TKO). Elimination of the beta 1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of beta 1 integrins did not promote significant compensatory upregulation of either beta 3 or beta 5 subunits, both of which are known to act as vitronectin receptors when associated with alpha v. The loss of beta 1 integrins severely affected morphological differentiation when the beta 1-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from beta 1-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from beta 1-deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a non-contiguous layer containing clumps of disorganized cells was observed. However, loss of beta 1 integrins did not interfere with induction by differentiating agents of tissue-specific gene products for either visceral or parietal endoderm. These results suggest that beta 1 integrins mediate morphological differentiation (migration and epithelial formation) but not tissue-specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system.

Amino Acid Sequence↗

PubMed1993

Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.

Avian Myeloblastosis Virus↗

PubMed1993

Debriefing: from military origin to therapeutic application.

1. The objective of the debriefing process is the prevention of post-traumatic stress disorder (PTSD). Risk factors for PTSD can be classified into three categories: premorbid personality, the environment, and the trauma itself. 2. Nurses are in a position to intervene using debriefing at primary, secondary, and tertiary levels of prevention. 3. Debriefing, individually and in groups, is an effective intervention in preventing the adverse reactions incurred by a traumatic stressor. The use of a debriefing model counteracts the tendency to suppress the powerfully affective component of the traumatic experience.

Adaptation, Psychological↗

PubMed1992

Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase.

Concerted integration of retroviral DNA termini, which produces a characteristic duplication of sequences at the integration site and formation of the proviral state, is a necessary step of the retroviral life cycle. We investigated the pairwise integration reaction catalyzed by purified avian retrovirus integrase by measuring the response to solution parameters and how the sequences of the viral termini, which comprise the avian imperfect inverted repeat, affect the reaction. When we optimized the reaction, an efficiency was achieved which approached that measured in systems using cytoplasmic extracts from virus-infected cells. The response of purified avian integrase to solution parameters was similar to that of the integration activity derived from cellular extracts. For strand transfer, the U3 viral terminal sequences were preferred to those of the U5 termini, a result we previously showed for the trimming reaction. That the sequence preference was the same for trimming and strand transfer may be further evidence that only one catalytic site is used for both reactions. A significant number of integration sites were sequenced. Interesting trends were found for the fidelity of the host duplications to the avian 6-bp duplication size, the clustering of the integration sites in the nonessential region of the lambda host DNA, and the sequence characteristics of the duplication sites.

Avian Myeloblastosis Virus↗

PubMed1992

Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway, in vivo.

Development of the human embryo depends on the ability of first trimester cytotrophoblastic stem cells to differentiate and invade the uterus. In this process, transient expression of an invasive phenotype is part of normal cytotrophoblast differentiation. Morphologically, this process begins when polarized chorionic villus cytotrophoblasts form multilayered columns of nonpolarized cells, and invade the uterus. Using immunocytochemistry, we compared the presence of adhesion receptors and extracellular matrix ligands on cytotrophoblasts in villi, cell columns, and the uterine wall. Villus cytotrophoblasts, anchored to basement membrane, stained for alpha 6 and beta 4 integrin subunits and both merosin and A-chain-containing laminin. Nonpolarized cytotrophoblasts in columns expressed primarily alpha 5 and beta 1 integrin subunits and a fibronectin-rich matrix. Cytotrophoblast clusters in the uterine wall stained for alpha 1, alpha 5, and beta 1 integrins, but not for most extracellular matrix antigens, suggesting that they interact primarily with maternal cells and matrices. Tenascin staining was restricted to stroma at sites of transition in cytotrophoblast morphology, suggesting that tenascin influences cytotrophoblast differentiation. Our results suggest that regulation of adhesion molecule expression contributes to acquisition of an invasive phenotype by cytotrophoblasts and provide a foundation for studying pathological conditions in which insufficient or excessive trophoblast invasion occurs, such as preeclampsia or choriocarcinoma.

Basement Membrane↗

PubMed1991

Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity.

A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.

Avian Myeloblastosis Virus↗

PubMed1991

92-kD type IV collagenase mediates invasion of human cytotrophoblasts.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.

Antibody Specificity↗

PubMed1990

Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein.

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.

Avian Myeloblastosis Virus↗

PubMedDate not supplied

Sexual abuse, alcohol and other drug use, and suicidal behaviors in homeless adolescents.

Previous research has shown that homeless youth have high rates of suicidal ideation, sexual abuse, and abuse of alcohol and other drugs. However, little is known about how these rates differ by gender and ethnicity. Our objective was to describe patterns of sexual abuse, alcohol and other drug use, and indicators of suicidal behaviors in homeless adolescents and to determine gender and ethnic differences in these factors. We used secondary data analysis of data from surveys completed by 96 homeless youth whose average age was 17.9 years. Over 60% of the sample reported a history of sexual abuse; the majority were under the age of 12 years when they first tried alcohol, marijuana, and cocaine; 56.3% had injected drugs, and 46.9% had tried inhalants. During the past 12 months, 35.1% had seriously considered suicide and 12.3% had actually attempted suicide at least once. Significantly more Hispanics than Whites had considered suicide (chi 2 = 4.31, p = .038). A disproportionate number of Hispanics (95% of the sample) reported a history of sexual abuse. Participants with a history of sexual abuse were significantly more likely than those who did not have a history of sexual abuse to have used alcohol and/or marijuana (chi 2 = 9.93, p < .01) and to have considered suicide in the past 12 months (F = 14.93, p < .001). We found that sexual abuse history is greater in this sample than in the general population and is particularly prevalent among Hispanic/Latino subjects. As in other studies, sexual abuse was more common among females than among males. High prevalence of sexual abuse, alcohol and other drug use, and suicidal behaviors in this sample of homeless youth underscores the need to develop and test community-based interventions to improve their health status.

Adolescent↗