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J Lough

Publications and source records attributed to J Lough.

At least 37 records · Page 2Linked to original sources
PubMed1996

Detection of Helicobacter pylori infection by saliva IgG testing.

OBJECTIVES: Most currently available tests for the detection of Helicobacter pylori are invasive, time consuming, or impractical. We examined the test performance of a simple and rapidly administered salivary IgG assay kit in the diagnosis of Helicobacter pylori infection. METHODS: Patients referred to a tertiary care setting for upper gastrointestinal endoscopy were included in a prospective evaluation of the test performance of the Helisal Kit which uses an ELISA technique to determine IgG antibodies in saliva. The results of the salivary IgG assay were compared to those of the Helisal Serum Kit, and to gastric histology. Two by two contingency table analyses were performed, and 95% confidence intervals (CI) were determined. RESULTS: Upper gastrointestinal endoscopy was performed on 106 patients over a 3-month period. A statistically significant correlation was found between the blood and saliva IgG results (r = 0.60, p = 0.0001). When compared to serum IgG, the salivary assay test performance was: sensitivity 84% (CI: 70-93%), specificity 81% (CI: 69-90%), positive predictive value 76% (CI: 61-87%), negative predictive value 88% (CI: 76-95%), and diagnostic accuracy 88% (CI: 76-95%). Compared with gastric histology, the test performance of the salivary IgG assay decreased to: sensitivity 66% (CI: 52-79%), specificity 74% (CI: 60-85%), positive predictive value 71% (CI: 57-83%), negative predictive value 68% (CI: 55-80%), and diagnostic accuracy 70% (CI: 60-78%). More specifically, the salivary assay gave false-negative results in nine of 17 patients with duodenal ulcers. Results did not vary significantly when outcomes of the salivary and serum assays were combined. The incremental information obtained in the salivary test was greatest in the patient population exhibiting an intermediate pretest probability (30-70%) of being infected with Helicobacter. CONCLUSION: The salivary IgG results correlated significantly with the serum IgG titers but exhibited only modest test performance, compared with the results of gastric histology. This salivary test may be most useful in certain patient subpopulations or in specific clinical contexts.

Adult↗
PubMed1995

Developmental expression of fibroblast growth factor receptor-1 (cek-1; flg) during heart development.

Previous work in this laboratory has indicated that fibroblast growth factor-2 (FGF-2; bFGF) regulates the initial stages of avian heart development in paracrine and autocrine fashion (Parlow et al. [1991] Dev. Biol. 146:139-147; Sugi et al. [1993] Dev. Biol. 157:28-37). Because these findings inferred the presence of a functional receptor for fibroblast growth factor (FGFR), we have immunochemically assessed the appearance of FGFR-1 (cek-1; flg) during development. Using a peptide-generated antibody, Western blots of total embryonic proteins revealed that FGFR-1 was barely detectable at pre-heart stages, followed by sequential increases in relative abundance that peaked at stage 24, followed by a decline at days 7-14. Western blots of proteins from isolated embryonic hearts demonstrated a similar developmental pattern, except that FGFR-1 expression was not decreased at later stages. The presence of FGFR-1 mRNA was verified by reverse transcription/polymerase chain reaction (RT/PCR) amplification. Immunohistochemical examination revealed punctate deposits of FGFR-1 in the precardiac endoderm at stage 6, followed by detection in the endoderm, foregut, and pre-cardiac splanchnic mesoderm at stage 8 and in the newly formed myocardium at the heart tube stage (9/10). By stage 13, FGFR-1 staining was observed only in the myocardium, a pattern which persisted at least until stage 30 (day 7), after which only isolated hearts were examined. After stage 30, staining was diminished in the ventricle, but not in the atrium. Staining of cardiac endothelial cells was not observed at any stage. A functional role for FGFR-1 was indicated by experiments in which anti-FGFR-1, but not pre-absorbed antiserum, retarded proliferation and multilayering of cardiogenic cells in an in vitro model of cardiac morphogenesis.

Animals↗
PubMed1995

Activin-A and FGF-2 mimic the inductive effects of anterior endoderm on terminal cardiac myogenesis in vitro.

We recently reported that the differentiation of cultured embryonic precardiac myocytes is specifically promoted by anterior lateral plate endoderm from Hamburger-Hamilton stage 6 chick embryos. Polypeptide growth factors are probable mediators of cardiogenesis during embryonic development. It was previously noted that activin-A is a major secretory product of endoderm cultured from chicken embryos. Also, fibroblast growth factor-like proteins are present in anterior endoderm of stage 6 chick embryos. Therefore, we have examined the cardiogenic effects of these growth factors on cultured precardiac mesoderm cells explanted from stage 6 embryos. Similar to the effects of anterior endoderm, low concentrations of activin-A, FGF-2 (bFGF), or insulin significantly increased the incidence of explants that exhibited synchronous contractions and expressed cardiac alpha-actin mRNA. By contrast, explants treated with transferrin, bovine serum albumin, or nerve growth factor never contracted and contained only cytoplasmic beta-actin transcripts. These results provide additional evidence that endoderm-secreted activin-A, FGF-2, and perhaps insulin participate in regulating terminal cardiac differentiation in the embryo.

Actins↗
PubMed1995

Uptake of 75-Selenium into the central nervous system of the rat.

These experiments have investigated selenium movement between blood and the CNS in anaesthetized rats. Each animal was anaesthetized and the left femoral blood vessels cannulated for blood withdrawal and solute infusion. Each rat received 75-Se as sodium selenite infused in normal saline and experiments lasted between 5 minutes and 5 hours during which blood samples were periodically taken. At termination, the CNS was removed, dissected and analysed with the plasma samples for 75-Se radioactivity by gamma-counting. Data were analyzed by multiple-time uptake analysis. Results showed unidirectional uptake of 75-Se into the CNS and some regional differences were found. On average the CNS influx rate constant (Kin) was about 7 +/- 1 x 10(-5) ml/min/g. This indicates that the 75-Se most likely entered the CNS in a protein-bound form.

Animals↗
PubMed1994

Anterior endoderm is a specific effector of terminal cardiac myocyte differentiation of cells from the embryonic heart forming region.

The ability of anterior lateral plate mesoderm cells in the heart-forming region (HFR) of stage 6 chicken embryos to respond to cardiogenic stimuli from cells in adjacent germ layers has been investigated using explants cultured under defined conditions. Two types of explantation were evaluated: those in which two germ layers were explanted in contiguity, and those in which germ layers were isolated and co-cultured. Two parameters--contractility and expression of sarcomeric alpha-actin--were monitored to evaluate the terminal differentiation of cardiac myocytes. Contiguously explanted anterior endoderm/mesoderm became multilayered and underwent terminal differentiation within 2 days. By contrast, although contiguous anterior ectoderm/mesoderm or posterior endoderm/mesoderm co-explants also became multilayered, these explants did not differentiate, up to 5 days. To ascertain the cardiogenic potential of cells from different regions of the embryo, individual germ layers were isolated and co-cultured by placing the explants in separate areas of the culture chamber. These determinations demonstrated that anterior, but not posterior, endoderm effected differentiation of anterior mesoderm. As before, mesoderm in both types of co-culture survived and became multilayered; by contrast, mesoderm did not survive when cultured in isolation. These experiments provide evidence that anterior endoderm regulates the terminal differentiation, as opposed to growth, of presumptive cardiac myocytes in mesoderm cells from the anterior lateral plate. Finally, anterior endoderm was co-cultured with mesoderm from the posterior half of the embryo, which does not contain an HFR. The failure of these co-cultured explants to differentiate infers that pre-cardiac myoblasts in stage 6 anterior mesoderm are previously specified to respond to the terminal cardiogenic effects of endoderm.

Actins↗
PubMed1993

Inhibition of precardiac mesoderm cell proliferation by antisense oligodeoxynucleotide complementary to fibroblast growth factor-2 (FGF-2).

This laboratory recently reported the occurrence of concentrated deposits of fibroblast growth factor-2 (FGF-2; basic FGF)-like proteins in the sarcoplasm of embryonic cardiac myocytes from the earliest stage of avian heart development (M. H. Parlow, D. L. Bolender, N. P. Kokan-Moore, and J. Lough, Dev. Biol. 146, 139, 1991). To determine the role, if any, of FGF-2 during embryonic cardiogenesis, the proliferative and functional effects of treating cultured anterior lateral plate mesoderm from Hamburger-Hamilton stage 6 embryos with antisense oligodeoxynucleotide (ODN) complementary to FGF-2 mRNA were determined. Within 2 days of culture in defined medium, the explanted monolayer normally differentiates into a multilayer of proliferative cells that express sarcomeric alpha-actin and exhibit rhythmic contractions. The inclusion of 25 microM ODN that is complementary to the second exon of chicken FGF-2 mRNA caused a 50% inhibition in these cells' proliferative ability as judged by incorporation of 5'-bromodeoxyuridine; contractility was similarly inhibited. These effects were prevented by including recombinant human FGF-2 protein in the medium. Treatment with sense ODN did not cause inhibition. Inhibition of FGF-2 protein synthesis in the explanted tissue by antisense ODN was verified by immunoprecipitation analysis. These results point to a critical role for FGF-2 in the autocrine regulation of proliferation, and perhaps differentiative function, of embryonic cardiac myocytes.

Amino Acid Sequence↗
PubMed1992

Onset of expression and regional deposition of alpha-smooth and sarcomeric actin during avian heart development.

The sequential appearance of mRNAs for smooth, cardiac, and skeletal alpha-actin has been described during development of the chicken heart (Ruzicka, D.L., and R.J. Schwartz 1988 J. Cell Biol., 107:2575-2586). To assess whether this reflects the deposition of corresponding isoproteins, we have immunocytochemically localized smooth and sarcomeric (cardiac and skeletal) alpha-actin in Hamburger-Hamilton (H-H) stage 7-18 embryos using monoclonal antibodies. Within the developing embryo at stage 9-, smooth muscle alpha-actin was exclusively detected in the developing heart, upon fusion of the endocardial tubes; sarcomeric alpha-actin was observed later (stage 9). By the onset of contraction at stage 10+, intense immunostaining of both smooth and sarcomeric isoproteins was observed in the ventricle; at this time smooth muscle alpha-actin was also detected in splanchnic mesoderm of the pre-vitelline area, in a cellular layer adjacent to the only embryonic cells that exhibited factor VIII (von Willebrand factor) antigens. Double immunostaining of the myocardium at stage 11, at which time striations were first detected, revealed the co-existence of smooth and sarcomeric actin in developing sarcomeres. Intense expression of sarcomeric actin continued in the heart after stage 11, whereas smooth muscle alpha-actin was down-regulated in the ventricle and became regionalized to the inflow and outflow tracts. As expected, smooth muscle alpha-actin was detected around intra- and extra-embryonic vascular structures at later developmental stages, while sarcomeric actin was observed in somites.

Actins↗
PubMed1992

Hepatic sinusoidal ectasia.

Oral contraceptive-associated sinusoidal ectasia is a rare lesion of unknown pathogenesis. We describe the case of a 31-year-old woman who had used oral contraceptives for 10 years and was found to have abnormal liver function tests on "routine" examination. A single 9 x 5 x 4 cm hypervascular lesion was demonstrated radiographically. The hepatic immuno-diacetic acid and liver-spleen scans were normal. One subcapsular lesion was identified in the resected right hepatic lobe; it was characterized by focal marked dilatation of the sinusoidal spaces associated with rare hepatocyte necrosis and early intrasinusoidal fibrosis. The subcapsular location and the vascular nature of this wedge-shaped lesion suggest it may represent the telangiectatic precursor of a centrally scarred focal nodular hyperplasia.

Adult↗
PubMed1991

Normal transitions in synthesis of replacement histones H2A.Z and H3.3 during differentiation of dystrophic myotube cells. A brief note.

We previously reported that differentiating G0 myotube cells cultured from normal chicken embryos exhibit a histone synthesis pattern that is highlighted by transitions in the expression of the minor replacement variants H3.3 and perhaps H2A.Z (Wunsch and Lough, Dev. Biol. 119 (1987) 94-99). Because these proteins may be synthesized to maintain chromatin structure during the differentiation and maturation of the skeletal muscle fiber, it was of interest to determine whether they are made at normal levels during the differentiation of dystrophic muscle. To this end, the synthesis of histone proteins in cultured myoblasts and myotubes from normal and dystrophic avian embryos has been characterized by two-dimensional polyacrylamide gel electrophoresis and fluorography. Proliferating myoblasts (day 1) as well as two stages of differentiating myotubes (days 3, 4) exhibited histone synthesis patterns that were indistinguishable when comparing normal and dystrophic cells. It is noteworthy that this study also revealed that, in both cell types, the change in H2A.Z synthesis during the myoblast/myotube transition was remarkable, increasing from approximately 20% of the non-ubiquitinated H2As in myoblasts to 80% in myotubes. Also, gel staining patterns and immunoblotting detected no differences in the degree of histone ubiquitination between normal and dystrophic cells. These findings indicate that, up to this point in dystrophic differentiation, neither the synthesis nor ubiquitination of histones are perturbed.

Animals↗
PubMed1991

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.

Blotting, Western↗
PubMed1991

Localization of bFGF-like proteins as punctate inclusions in the preseptation myocardium of the chicken embryo.

Immunocytochemistry has been employed to map the appearance of bFGF-like proteins in precardiac and preseptation myocardial cells between stages 6 and 15 of chicken embryogenesis. Stage 6 embryos exhibited no staining, with the exception of a subtle signal in endoderm cells. At subsequent stages, staining was observed only in cells of the developing myocardium, first appearing at the time of heart tube fusion (stage 9+) as punctate cytoplasmic aggregates. While the expression of bFGF-like antigen was temporally similar to that of myosin heavy chain, their staining patterns differed in that bFGF-like proteins were nonsarcomeric and did not extend into the inflow or outflow tracts. Western blotting of heparin agarose affinity-isolated proteins from stage 15 hearts revealed an antigen migrating at approximately 19 kDa. In contrast with the unique localization of bFGF-like proteins in myocardial cells, FGF receptor (FGFR) staining was widely distributed in the embryo; however, concentrated deposits of FGFR were detected in endothelial and myocardial cells, which diminished in the myocardium but not in the endothelium by stage 15. These results suggest that FGF-like proteins may have autocrine and/or paracrine functions during early cardiac morphogenesis.

Animals↗
PubMed1991

Secretion of inhibin beta A by endoderm cultured from early embryonic chicken.

Although several reports have indicated a role for endoderm in the regulation of heart development, the mechanism remains unknown. To begin characterization of endoderm-secreted proteins, explants from postgastrulation (Hamburger-Hamilton stage 5/6-8) chicken embryos were cultured in defined medium. Fluorography of SDS-PAGE gels revealed a pattern of synthesized, secreted proteins that was independent of time in culture or embryonic stage when explants were removed. Approximately 10 labeled bands were detected, the most prominent of which migrated at 17, 25, and 200 kDa. ELISA analysis revealed that while acidic and basic fibroblast growth factor-like antigens were barely detectable, fibronectin and inhibin beta A were very reactive. Western blot analysis verified the presence of fibronectin and, most remarkably, inhibin beta A, activin dimers of which have recently been implicated in Xenopus mesoderm induction (Smith, Price, Van Nimmen, and Huylebrock (1990). Nature 345, 729.)

Activins↗
PubMed1991

Pathology of atherosclerosis in cholesterol-fed, susceptible mice.

In recent years the C57BL/6J mouse has gained popularity as a model for studying the genetics of diet-induced atherosclerosis. After 10-20 weeks of consuming a diet enriched with saturated fat and cholesterol, it develops fatty streak-like lesions in the valve sinus region of the ascending aorta. The current study shows that continuing the atherogenic diet for a further 15 weeks leads to the development of fibro-fatty lesions which have many of the characteristics of human atheromatous plaques. This finding lends credence to the use of C57BL/6J mice for studying the pathogenesis of atherosclerosis.

Animals↗
PubMed1991

The effect of a high-fat diet on murine macrophage activity.

The phagocytic, oxygen free radical generating and cytotoxic activities of macrophages from C57BL/6J mice fed either a normal or an atherogenic high-fat diet have been investigated. Phagocytosis of aggregated low density lipoprotein (LDL) was only slightly inhibited by the high-fat diet although phorbol myristate acetate (PMA)-induced hydrogen peroxide (H2O2) and superoxide anion (O2-) production was significantly reduced. Activation of tumoricidal activity against L929 target cells by lipopolysaccharide (LPS) or lymphocyte-derived macrophage-activating factor (MAF), but not N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), was also significantly reduced in macrophages from mice fed the high-fat diet. These results indicate that an atherogenic diet is capable of significantly affecting the responsiveness of macrophages to a number of stimulatory agents which act via specific membrane receptors.

Animals↗
PubMed1990

Enrichment of ubiquitinated histone H2A in a low salt extract of micrococcal nuclease-digested myotube nuclei.

We recently demonstrated that ubiquitinated histone H2A (uH2A) declines 2.5-fold during terminal skeletal muscle differentiation, coincident with reductions in transcriptional activity (Wunsch, A. M., Haas, A. L., and Lough, J. (1987) Dev. Biol. 119, 85-93). To assess whether this indicates an association of uH2A with transcriptionally active genes, we have used micrococcal nuclease digestion and salt extraction to fractionate myotube nuclei. An oligonucleosomal fraction obtained by micrococcal nuclease digestion and extraction in low salt (100 mM NaCl) comprising only 25% of the nuclear DNA contained 90% of the total uH2A, as revealed by Western blotting. Further fractionation of this 100 mM salt extract by sucrose gradient centrifugation revealed that virtually all of the uH2A was localized in monomer to heptamer-sized oligonucleosomes. A second ubiquitinated species of 57-kDa (u57) was also localized in the 100 mM salt extract. In contrast, an 18-kD band (u18) was associated with fractions that were resistant to micrococcal nuclease digestion and salt extraction. Although micrococcal nuclease recognized a unique structural feature of active myotube chromatin, as evidenced by the appearance of hybridized skeletal alpha-actin sequences as a smear rather than the nucleosomal ladder exhibited by inactive and bulk sequences, neither the skeletal alpha-actin gene nor the inactive alpha D-globin gene was exclusively localized in the 100 mM salt fraction. Moreover, further fractionation of the 100 mM salt extract on a sucrose gradient failed to separate active from inactive genes. These findings suggest that uH2A is localized in a fraction of myotube chromatin which, although nuclease-sensitive and relatively soluble, is not enriched in active or inactive genes.

Animals↗
PubMed1990

Arabinosylcytosine-induced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation.

This laboratory has recently reported the occurrence of DNA nicking at the onset of terminal skeletal myogenesis by using the technique of in situ nick translation (Dawson and Lough: Dev. Biol., 127:362-367, 1988). Because 1-beta-D-arabinofuranosylcytosine (araC), a cytocidal agent that is routinely used to removed dividing fibroblasts from myogenic cultures, inhibits DNA repair, it was of interest to determine whether araC treatment resulted in an accumulation of the endogenously created nicks. Thus, we have assessed the accumulation of DNA nicks in myotube cells during a 20 hour araC treatment period at the onset of terminal myogenesis (44-64 hours in vitro) by using three techniques: alkaline sucrose gradient density centrifugation, kinetic in situ nick translation, and cellular in situ nick translation. Although alkaline sucrose gradient centrifugation revealed no detectable nicking after 20 hours, kinetic in situ nick translation analysis revealed subtle but significant increases in DNA nicks caused by araC within 7 hours of drug application, and a 1.5-fold increase in DNA repair sites after 20 hours of drug treatment. That these observations reflected nicking specifically in myotube nuclei was determined by immunocytochemical localization of nicked sites after repair with a biotinylated nucleotide analog (biotin-11-dUTP). The effects of araC were only incompletely reversible, whether or not the drug was removed from the cultures, within 2 days of the treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Histone variant patterns during vertebrate embryogenesis and limb development.

Two-dimensional gel electrophoresis was used to examine the relative content of core histone variants during early chicken embryogenesis and at selected stages of hindlimb development. Nuclei from stage 19 limb buds displayed a pattern similar to whole embryos at stage 1, at which time all of the known avian histone variants, including the minor isoprotein H3.3, were detected. Variant ratios did not change during limb development, up to stage 29. However, the portion of H2A variants migrating as ubiquitinated conjugates increased more than twofold during limb development, advancing from 4.5% of the total H2A proteins at stage 19 to 12% at stage 29.

Animals↗