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Biomedical subjects

A M Healy

Publications and source records attributed to A M Healy.

30 records · Page 2Linked to original sources
PubMed1995

Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system.

We have targeted the murine thrombomodulin (TM) gene in embryonic stem cells and generated embryos as well as mice with TM deficiency. The heterozygous TM-deficient (TM+/-) mice as compared to wild-type (TM+/+) littermates exhibit 50% reductions in the levels of TM mRNA and TM protein. However, TM+/- mice appear normal and are free of thrombotic complications. The homozygous TM-deficient (TM-/-) embryos die before embryonic day 9.5. An overall retardation in growth and development of TM-/- embryos is first evident on embryonic day 8.5 (8-12 somite pairs). However, no specific pathologic abnormalities are observed. These initial changes take place at a time when TM is normally expressed in the parietal yolk sac. The removal of embryonic day 7.5 TM-/- embryos from maternal decidua and their subsequent culture in vitro allow development to proceed to stages not observed in vivo (13-20 somite pairs) with the appearance of normal blood vessels in the visceral yolk sac and embryo. The results of our studies suggest that the failure of TM-/- embryos to survive at mid-gestation is a consequence of dysfunctional maternal-embryonic interactions caused by the absence of TM in the parietal yolk sac and demonstrate that the receptor is necessary for normal embryonic development in utero.

Animals↗
PubMed1994

Induction of plasminogen activator inhibitor-1 in HepG2 human hepatoma cells by mediators of the acute phase response.

The liver plays a central role in the systemic acute phase response of an organism to injury. Plasminogen activator inhibitor-1 (PAI-1), a major regulator of fibrinolysis, is an important component of the acute phase response in humans. The source of plasma PAI-1 has been a matter of controversy, but recent in situ hybridization experiments have demonstrated that human hepatocytes express the PAI-1 gene in vivo. However, little is known about regulation of human hepatic PAI-1 gene expression by mediators of the acute phase response. We have analyzed the regulation of PAI-1 mRNA accumulation by interleukin (IL)-1, IL-6, and dexamethasone, known mediators of the acute phase response, in HepG2 cells, a highly differentiated human hepatoma cell line that produces a broad spectrum of acute phase proteins including PAI-1. Incubation of HepG2 cells with IL-1 resulted in a rapid and transient 40-fold induction of the 3.2-kilobase PAI-1 mRNA and a 30-fold induction of the 2.2-kilobase PAI-1 mRNA. Although IL-6 alone had only a modest effect on PAI-1 expression, in combination with IL-1, it caused a synergistic induction of PAI-1 mRNA accumulation. Dexamethasone alone did not increase PAI-1 mRNA accumulation but enhanced it in combination with IL-1. Using nuclear run-on experiments, we determined that the mechanism by which IL-1 alone, or in combination with IL-6, induced PAI-1 mRNA accumulation was to cause a 10-15-fold, transient stimulation of PAI-1 gene transcription. We found no evidence of an effect of these cytokines on PAI-1 mRNA stability. These data demonstrate that mediators of the acute phase response induce the accumulation of PAI-1 mRNA in human hepatoma cells by rapidly and transiently increasing the transcription of the PAI-1 gene.

Acute-Phase Reaction↗
PubMed1992

Preparation of fluorescent basic fibroblast growth factor: localization in living retinal microvascular endothelial cells.

A biologically active fluorescent derivative of recombinant human basic fibroblast growth factor (bFGF) was prepared by immobilization on heparin-Sepharose 4B (HS) and derivatization with the fluorophore, Texas Red (TR). TR-bFGF was separated from free dye and carrier protein by elution from HS using 1.5 M NaCl. TR-bFGF contained an average of two dye molecules bound per bFGF, retained its mitogenic activity and was visible using a fluorescence microscope equipped with silicon intensified target camera (SIT). TR-bFGF stimulated the growth of bovine aortic endothelial cells (BAEC), microvessel endothelial cells (MVEC) and BHK-21 cells grown in culture. BAEC, MVEC and BHK-21 cells treated with 20 ng ml-1 (1 nM) TR-bFGF for 72 hr were stimulated over serum controls by 87, 26 and 6%, respectively. TR-bFGF stimulated EC growth was inhibited in a dose-dependent fashion when cells were coincubated with microM chloroquine. When EC were treated with TR-bFGF at 4 degrees C and then monitored at 37 degrees C, bright, focal, cytoplasmic spots were observed, which accumulated as punctate, perinuclear fluorescence. EC internalization of TR-bFGF was inhibited 80% by the addition of 100-fold molar excess unlabeled bFGF or by maintaining cultures at 4 degrees C. TR-bFGF colocalized with an EC lysosomal marker, but TR-bFGF was not detected in the nucleus. Results of these localization studies suggest that TR-bFGF stimulates EC proliferation without entering the nucleus.

Cell Division↗
PubMed1992

Density-dependent accumulation of basic fibroblast growth factor in the subendothelial matrix.

Recent evidence indicates that basic fibroblast growth factor (bFGF), which lacks a conventional signal recognition sequence, is a component of the subendothelial matrix. However, the molecular mechanisms regulating its cellular release and subsequent matrix deposition remain equivocal. To examine the cellular and subcellular mechanisms regulating bFGF release and subendothelial sequestration, we generated polyclonal antibodies against a chemically cross-linked bFGF. We then used anti-bFGF IgG in conjunction with 3T3 cell [3H]thymidine incorporation assays, enzyme immunoassays and immunofluorescence to learn whether bFGF accumulation in the subendothelial matrix is dependent upon endothelial cell (EC)-cell contact, which coincides with growth arrest. In contrast to subconfluent cultures, which lacked any detectable extracellular matrix bFGF localization, bovine aortic and microvascular EC plated at confluent densities displayed a punctate extracellular staining pattern that was abolished when EC were pretreated with 10 micrograms/ml cycloheximide. Additionally, when EC were treated with either 1 mM beta-D xyloside, an inhibitor of proteoglycan assembly, or 100 micrograms/ml heparin, there was a 40% reduction in matrix-associated bFGF (quantified by image analysis of antibody stained cultures). 3T3 [3H]thymidine incorporation assays indicated that the beta-D xyloside-induced reduction of matrix-associated bFGF coincided with a significant increase in bFGF activity in the conditioned media. Neither sparsely-plated nor confluent EC cultures possessed specific bFGF localization of the nuclear compartment when cells were fixed using cold methanol; however, when EC were fixed in formaldehyde and lysed in isotonic buffers containing 0.1% Triton X-100 or absolute acetone, there was a marked decrease in anti-bFGF staining of the postconfluent extracellular matrix and a concomitant increase in nuclear fluorescence. Because bFGF-stimulated vascular cell growth has been implicated in controlling neointimal cell proliferation, we screened normal and atherosclerotic coronary blood vessels for bFGF, but we were unable to detect it either in lesioned or normal intima. In contrast, significant bFGF levels were observed in association with the EC and mesangial cells of the renal corpuscle, where heparan sulfate accumulates within the glomerular basement membrane. Our in vitro results suggest that bFGF accumulates within the proteoglycan-containing subendothelial matrix concomitant with the formation of cell-cell contacts. In situ, the composition of the microvascular matrix and the cellular phenotype may facilitate the selective accumulation of bFGF that we observed. This, in turn, may influence vascular morphogenesis and remodeling during angiogenesis.

3T3 Cells↗
PubMed1991

CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase.

Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Amino Acid Sequence↗
PubMed1990

A sequence that directs transcriptional initiation in yeast.

While RNA polymerase II of the yeast Saccharomyces cerevisiae initiates transcription at discrete sites, these sites are located over a wide range of distances from the TATA box for different genes. This variability has led to a number of proposals for consensus sequences located at the initiation site which, in conjunction with the TATA box, would direct initiation. We tested this hypothesis via oligonucleotide-directed mutagenesis, by placing the sequence CAAG, a member of one of these consensus sequences, upstream of the coding sequence of the CYC7 gene at a site at which initiation does not occur. The distance between the TATA sequence and this putative initiation site was varied by inserting it into the wild-type gene and three deletion mutants. The results demonstrated that this sequence can serve as an initiation site when located 49, 77, or 106 bp from the TATA sequence, but not when located 30 bp away.

Base Sequence↗
PubMed1987

Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes.

A series of BAL 31 deletions were constructed in the upstream region of the Saccharomyces cerevisiae CYC7 gene to determine sequences required for transcriptional initiation. These deletions identified the TATA box as an alternating A-T sequence at -160 and the initiation sequences as well as the spatial relationship between them. The TATA box was necessary for wild-type levels of expression of the CYC7 gene. Decreasing the distance between the TATA sequence and the initiation site did not alter gene expression, but the site of transcription was shifted 3'-ward. In most cases, transcription initiated at a number of sites, the 5'-most of which was the first suitable site greater than 45 base pairs 3' of the TATA sequence, suggesting a spatial relationship between these sequences. Consensus sequences previously proposed for initiation sites were evaluated with respect to the start sites identified in this study as well as the start sites of other yeast genes.

Base Sequence↗
PubMed1987

Hormone 1 alpha,25-dihydroxyvitamin D3 modulates heat shock response in monocytes.

Monocytes, which play a central role in inflammatory reactions, are exposed to elevated temperatures in febrile states or to potential cellular toxins (e.g., reactive oxygen species, immune mediators). We analyzed the heat shock (HS) response in human peripheral blood monocytes and the monocytic line U937 compared with that in dermal fibroblasts. After HS, U937 cells and fibroblasts synthesized mainly the 70- and 83-kDa HS proteins (HSPs), whereas the spectrum of HSPs synthesized by monocytes varied over the range 41-45 degrees C. Incubation with hydrogen peroxide induced synthesis of the 70-kDa HSP in monocytes only. The hormone 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] that protects monocytes from thermal injury had similar effects on the HS response in U937 cells, increasing synthesis of HSPs and levels of HSP 70 mRNA and partially preventing the decrease in normal protein synthesis, whereas interferon-gamma had no effect. 1,25-(OH)2D3 did not modify the HS response in fibroblasts, although these cells contained cellular receptors for 1,25-(OH)2D3. 1,25-(OH)2D3 could play a specific role in modulating the HS response in monocytes and related cells.

Calcitriol↗
PubMed1987

1,25-Dihydroxyvitamin D3 induces collagen binding to the human monocyte line U937.

Interactions of cells with components of the extracellular matrix can modulate cellular functions. We measured binding of a major matrix protein to U937 cells, a human promonocytic line. Radioiodinated type I or type III human collagen was bound only to U937 cells differentiated to a more mature phenotype with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Binding was observed at 4 degrees C and was saturable; Scatchard analysis of the binding to 1,25-(OH)2D3-pretreated U937 cells indicated a single class of high-affinity binding sites. Preincubation of U937 cells with interferon gamma did not induce collagen binding. Collagen binding did not appear to be dependent on fibronectin binding. Surface proteins of U937 cells were 125I labeled and cell membrane proteins resolved by affinity chromatography on collagen-Sepharose. Major specifically labeled bands of 180, 155, and 125 kD were identified in membrane fractions from 1,25-(OH)2D3-pretreated U937 cells only. 1,25-(OH)2D3 appears to specifically regulate collagen binding to monocyte precursors.

Calcitriol↗
PubMed1986

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.

Calcitriol↗
PubMedDate not supplied

Morbidity and mortality in a large Irish feedlot; microbiological and serological findings in cattle with acute respiratory disease.

A survey of morbidity and mortality was undertaken in a slatted unit containing 6399 beef cattle over a 6 month period. The mortality rate was 0.78% and the morbidity rate was 12.7%. The interval from arrival to morbidity was 25.5 days +/- 0.9. Significantly more sick cattle were identified on either Mondays or Tuesdays than were seen on Saturdays or Sundays (P < 0.005). The mean interval to mortality (all diseases) was 48.5 days +/- 7.4. Respiratory disease was the most frequently recorded disease and was responsible for 62% of morbidity and 58% of mortality. Samples from 133 cattle, taken as they presented with acute onset respiratory disease, contained antibodies to Bovine Herpes Virus -1(BHV-1) (14.3%), Bovine Viral Diarrhoea Virus (BVDV) (78%), Bovine Respiratory Syncytial Virus (BRSV) (94%) and Parainfluenza type 1 Virus (P13V) (99%). When the same cattle were resampled 4 to 6 weeks later antibodies were found for BHV-1 (93.2%), BVDV (99.2%), BRSV (100%) and P13V (100%). Eleven of 27 bronchoalveolar lavage samples taken from the above group of cattle exhibited positive fluorescence for BHV-1 but all 27 samples were negative for BVDV, BRSV and P13V. Pasteurella multocida was isolated from eight of the 27 lavage samples and Pasteurella haemolytica from one sample.

Acute Disease↗