Biomedical subjects

J Lough

Publications and source records attributed to J Lough.

At least 55 records · Page 3Linked to original sources

PubMed1989

Affinity isolation of transcriptionally active murine erythroleukemia cell DNA using a cleavable biotinylated nucleotide analog.

We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.

Acetamides↗

PubMed1989

Cancer sweepstakes.

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Financial Management↗

PubMed1989

Histones synthesized at different stages of myogenesis are differentially degraded in myotube cells.

We recently reported that cultures of terminally differentiating myotube cells synthesize histones in reduced but significant amounts in comparison with proliferating myoblasts (Wunsch et al., 1987, Dev. Biol., 119: 85-93). In this study, the stability of myotube histone has been determined, comparing the degradation of de novo-synthesized histones in nascent (day 3) and maturing (day 4) myotubes with histones in the same cells that had been previously made during myoblast proliferation (day 1). Histones synthesized in proliferating myoblasts and myotubes were pulse-labeled with 3H-lysine and chased up to seven days, followed by determinations of radioactivity remaining in histone bands using fluorography of one- and two-dimensional polyacrylamide gels. Considered in aggregate, core histones synthesized de novo in nascent (day 3) myotubes were degraded most rapidly, followed by myotube histones that had been previously made during the proliferative phase (day 1) of myogenesis. De novo-synthesized histones in maturing (day 4) myotubes were relatively stable. Individual histone classes were degraded in the following order of increasing half-life, regardless of the differentiative stage at which they were synthesized: H2A.Z, H2A, H2B, H3(.2, day 1; .3, days 3 and 4), H4.

Animals↗

PubMed1989

ATH-3, a new gene for atherosclerosis in the mouse.

Mice derived from resistant A/J (A) and susceptible C57BL/6J (B) strains have been used to study the genetics of diet-induced atherosclerosis. A comparison of lesion scores between the parental strains, the F1 offspring of A x B and B x A matings and the offspring of (B x A)F1 hybrids backcrossed to either parent strain, indicates that a single major gene with alleles for resistance and susceptibility is responsible for the difference in response of A/J and C57BL/6J mice to a high fat, high cholesterol diet. By comparing the strain distribution pattern of susceptibility with known genetic markers in 30 A x B and B x A recombinant inbred (RI) strains, this gene, designated Ath-3, has been mapped close to a coat colour gene, c, on chromosome 7. Although a single gene may be primarily responsible for the difference in susceptibility to diet-induced atherosclerosis in these mice, the results of the breeding experiments indicate that its expression is probably modified by one or more additional genes.

Animals↗

PubMed1988

Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation.

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.

Animals↗

PubMed1988

The effects of electrical stimulation on denervated muscle using implantable electrodes.

This experimental study investigated the effects of continuous electrical stimulation on denervated muscle. The canine peroneal nerve was severed and repaired microsurgically, and the denervated extensor muscle group of the leg was stimulated continuously with an implantable electrode and pulse generator. EMG study, muscle force measurement, muscle weight measurement, histology, and histochemistry were performed to study the effect at eight weeks after the operation. Continuous electrical stimulation (pulse frequency 130 pps, burst rate approximately 1 train/min) was effective in decreasing muscle atrophy and in improving muscle force. These findings may have broader clinical applications.

Animals↗

PubMed1988

Genetically determined susceptibility and resistance to diet-induced atherosclerosis in inbred strains of mice.

To determine whether recombinant inbred strains derived from C57BL/6J and A/J mice would provide a good model in which to study the genetics of diet-induced atherosclerosis, male mice of the parent strains were compared in a number of experiments designed to correlate various biochemical changes with susceptibility or resistance to the disease. In both strains fed an atherogenic diet containing 27% coconut oil and 4.5% cholesterol, there was a significant rise in serum very low-density plus low-density lipoprotein cholesterol levels, but only C57BL/6J mice developed discernible fatty lesions in the aortic wall. In A/J mice a significant rise in high-density lipoprotein cholesterol was also observed, which corresponded to the appearance of a second species of high-density lipoprotein in the serum, but in C57BL/6J mice there was a fall. In susceptible C57BL/6J mice, free cholesterol is secreted into bile, which becomes supersaturated, leading to the formation of gallstones. In the resistant strain, however, dietary cholesterol accumulates in the liver. A difference in hepatic cholesterol metabolism between the two strains may thus be a factor in determining their different susceptibilities to diet-induced atherosclerosis.

Animals↗

PubMed1987

Synthesis and ubiquitination of histones during myogenesis.

One and two-dimensional polyacrylamide gel electrophoresis have revealed that cultures of postmitotic (G0) chicken skeletal myotube cells synthesize significant but reduced quantities of histone proteins as compared to their proliferating myoblast precursors. In addition, modulation of variant synthesis within the histone H2A and H3 classes may accompany myotube formation. That the histone bands contain no nonhistone contaminants was shown by exclusion of [3H]tryptophan. It is unlikely that these results reflect synthesis of histone by contaminating replicating cells, since a single treatment with cytosine arabinoside at the time of fusion effectively removed unfused cells while suppressing synthesis of DNA in the myotube cultures. The relatively sparse incorporation of label by major variants of the H2A class in dividing myoblasts was shown to be caused by heterogeneity due to phosphorylation and extensive ubiquitination, which decline at the time of myotube formation. As determined by quantitative Western-blotting, dividing myoblasts and myotubes contain an average of 1.0 and 0.4 molecules of ubiquitinated H2A (uH2A), respectively, per 10 nucleosomes.

Animals↗

PubMed1987

Modulation of histone H3 variant synthesis during the myoblast-myotube transition of chicken myogenesis.

We have previously reported that nucleosomal histones are synthesized by cultured, postmitotic myotube cells at 9-29% of the rate in their dividing myoblast precursors (A. M. Wunsch, A. L. Haas, and J. Lough, 1987, Dev. Biol. 119, 85-93). In that study, histones were separated by two-dimensional polyacrylamide gels containing 8 M urea in the first-dimension to optimally separate variants of the H2A class. To separate and compare synthesis of variants in the H2B and H3 classes during myogenesis, 5.75 M urea has been used in the first dimension. Although no changes in the H2B variant pattern were discerned, a dramatic change in H3 variant synthesis was detected, in which a predominance of H3.2 synthesis in dividing myoblasts was almost completely replaced by a lower level of H3.3 synthesis after myotube formation. With increasing differentiation, H3.2 synthesis became undetectable, while H3.3 synthesis continued. Control experiments indicated that these results were not mediated by replicating cells in the myotube cultures, the effects of cytosine arabinoside, or contaminating non-histone proteins. These results suggest that histone H3.2 is replaced by histone H3.3 in nucleosomes during skeletal muscle maturation.

Animals↗

PubMed1987

Kupffer cell depletion associated with capillarization of liver sinusoids in carbon tetrachloride-induced rat liver cirrhosis.

Rats were made cirrhotic by carbon tetrachloride inhalation associated with phenobarbital in the drinking water over 10 weeks. After a 1-week recovery period a 99mTc sulphur colloid radioisotope scan of the liver was carried out on each animal following bolus injection into the iliac vein. Kupffer cells were then histologically identified by one of three methods: colloidal carbon uptake, iron staining after ferritin-dextran, or endogenous peroxidase staining. The degree of liver injury was classified into four pathological groups and these were correlated with the phagocytic capacity of each liver as recorded on the isotope scan. All three histological markers demonstrated that in normal liver, Kupffer cells are more plentiful in periportal areas. In cirrhotic liver, there were very few Kupffer cells in nodular regenerative areas, where continuous capillaries are found, but Kupffer cells were present in the remaining more normal trabecular-sinusoidal areas. Morphometric counting of carbon- and ferritin-labelled Kupffer cells demonstrated a significant decrease in cirrhotic livers. A close correlation was also found between increasing degree of liver injury and diminished hepatic phagocytic capacity, as demonstrated by the radioisotope scan. The study demonstrates that where regenerative liver is capillarized, with replacement of fenestrated sinusoids, Kupffer cells are absent.

Animals↗

PubMed1986

Affinity isolation of transcriptionally active DNA.

Chicken erythrocyte nuclei were nick translated with the chemically cleavable biotinylated nucleotide, Bio-12-SS-dUTP. DNA was purified, digested with restriction endonucleases, and applied to an avidin-agarose affinity column. Seventy percent of the nick translated DNA bound to the column. This DNA was recovered from the column by chemical cleavage of the linker arm joining biotin to the DNA. Dot hybridization analysis of this DNA revealed a significant enrichment of the alpha-D-globin gene. This result suggests an approach to isolate transcriptionally active genes.

Animals↗

PubMed1986

Interferon-mediated inhibition of differentiation in a murine myoblast cell line.

The effects of highly purified (greater than 5 X 10(7) IU/mg) murine beta-interferon (IFN) on a mouse myoblast line (MM14DZ) have been investigated to confirm and extend the previous observation that partially purified chicken interferon inhibits differentiation of cultured avian myoblasts (Lough et al., Biochem Biophys Res Commun 109:92, 1982). Cultures treated with 20-2,000 lU IFN/ml medium for 5 days exhibited dose-dependent 1) inhibition of differentiation, as indicated by reduced myotube formation and creatine kinase (CK) activity and 2) increases in DNA content, suggesting that the inhibitory effect was accompanied by continued proliferation of myoblasts. Mock-IFN had no such effects. Based on findings in other systems that IFN inhibits activity of ornithine decarboxylase (ODC), the polyamine products of which are required for myogenesis, the hypothesis that inhibition of differentiation was mediated by an effect of IFN on polyamine metabolism was tested. However, observations that 1) IFN-treated myoblasts retained control levels of ODC activity and 2) exogenous polyamines did not prevent IFN-inhibition did not indicate such a mechanism of action. On the other hand, treatment of control cultures with polyamines alone resulted in potentiation of myogenesis as revealed by precocious myotube formation and a marked increase in CK activity.

Animals↗

PubMed1986

Cholangiocarcinoma as a late complication of choledochoenteric anastomoses.

Two cases of cholangiocarcinoma developing several years after choledochoenteric anastomoses are reported. It is speculated that these may represent heretofore unrecognized late complications of this procedure. The intermittent chronic obstruction with bile stasis and recurrent inflammation may be causative factors in the development of cholangiocarcinoma after choledochoenterostomy.

Adenoma, Bile Duct↗

PubMed1986

Gastric carcinoma: intestinal metaplasia and tumor growth patterns as indicators of prognosis.

We have reviewed 200 cases of gastric carcinoma treated between 1970 and 1980 to assess the value of intestinal metaplasia in the stomach and tumor growth patterns in determining prognosis. Intestinal metaplasia was found to be more frequently associated with early gastric tumors, expanding-type tumors, and tumors located in the antrum. The survival rate was 53% with intestinal metaplasia and 34% without. Sixty-three percent of expanding tumors with metaplasia survived. If the lymph nodes were not involved, the survival rate with metaplasia was 81%. We conclude that intestinal metaplasia and growth patterns are valuable in predicting outcome. Preoperative evaluation of gastric tumors should include multiple endoscopic mucosal biopsy specimens. If intestinal metaplasia is present, the improved possibility of survival should influence the surgeon in the choice of operative treatment.

Female↗

PubMed1985

The inlet patch: heterotopic gastric mucosa in the upper esophagus.

Careful endoscopic survey of the upper esophagus indicates that a visually recognizable patch of gastric mucosa is grossly visible much more commonly than is generally recognized. Sixteen instances were found in 420 sequential endoscopies, an incidence of 3.8%. The gastric mucosal rest presented as a velvety red patch with a distinct border, varying from a few millimeters in diameter to complete encirclement of the esophagus, was occasionally paired, and was found either at or just below the upper esophageal sphincter. In all instances except one the patch consisted of gastric-type mucosa containing both parietal and chief cells; in the single exception, it consisted of gastric-type mucous glands without parietal cells. Although larger patches can be shown to function under stimulation (they produce a local drop in pH with pentagastrin stimulation), there are usually no apparent associated clinical symptoms.

Adult↗

PubMed1985

Differential sensitivity of chicken MM-creatine kinase to trypsin and proteinase-K.

Under several conditions of SDS-PAGE, the chicken MM-creatine kinase (MM-CK) monomer migrated as a approximately 50,000 dalton polypeptide, approx 25% larger than usually reported. Characterization by sedimentation equilibrium indicated that the anomalous molecular weight was an artifact of electrophoresis. Digestion with trypsin caused only moderate reductions in CK activity, despite extensive degradation of the denatured enzyme revealed by SDS-PAGE. Characterization of trypsinized MM-CK under non-denaturing conditions of electrophoresis and HPLC revealed no fragmentation of the native enzyme, suggesting that MM-CK quaternary structure was maintained despite extensive tryptic nicking. In contrast, much lower concentrations of proteinase-K generated only a single fragment in SDS-PAGE while causing a nearly total loss of enzyme activity.

Amino Acids↗

PubMed1985

Transforming skeletal muscle for myocardial assist: a feasibility study.

Canine experiments were undertaken to determine the feasibility of transforming skeletal muscle for myocardial assistance. Initially, a rectus-muscle pedicle flap was used to power a ventricular assist conduit. A specially designed "pulse-train" stimulator produced skeletal muscle contractions capable of augmenting myocardial function. Statistically significant increases in either systolic or diastolic pressures were achieved by appropriate synchronization and signal delays of the stimulator. Then, the left rectus muscle was conditioned by stimulating it at low frequencies (2 to 10 Hz) for 6 to 12 weeks. The degree of transformation from type II (fast) to type I (slow) fibres was examined and the resultant tolerance to fatigue studied. Preliminary data show that a greater proportion of type I fibres is associated with increased resistance to fatigue. Such transformed skeletal muscle should therefore be more suited to power the cardiac assist devices.

Animals↗

PubMed1984

Spermine-induced phosphorylation of myotube histones by endogenous nuclear protein kinases.

The effects of spermine on phosphorylation of nuclear proteins in isolated nuclei from proliferation and myotube stage cells during differentiation of cultured chicken myoblasts have been investigated. Incorporation of phosphate from 32P-gamma-ATP was assessed by incubating nuclei with and without 2 mM spermine, which caused an approx. 1.5-fold increase in phosphorylation of total nuclear proteins in both cell types. Modification of individual proteins was assessed by extracting basic proteins in dilute acid, followed by SDS-electrophoresis on 18% acrylamide gels and radioautography. Results indicated that whereas most phosphoproteins in both cell types were increased 1.5-2.0-fold, phosphorylation of a 31 000 D band increased several-fold. Most strikingly, myotube nuclei displayed selective 3.5- and 9-fold increases in specific radioactivity of histones Hla and H3, respectively, which normally exhibit little, if any, phosphorylation.

Animals↗