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Biomedical subjects

J Paul

Publications and source records attributed to J Paul.

At least 217 records · Page 12Linked to original sources
PubMed1984

Differential response of multiple epsilon-globin cap sites to cis- and trans-acting controls.

The human epsilon-globin gene has a number of alternative transcription-initiation sites located upstream of the canonical mRNA cap site. In three nonerythroid cell lines, "leaky" epsilon-globin transcription occurs exclusively from one of these upstream sites, the -200 cap site. Using a transient expression assay, we have shown that transcription initiation from the -200 cap site and the major cap site can be independently regulated in response to plasmid replication, SV40 enhancer sequences in cis, and the adenovirus E1A gene in trans. The -200 cap site is located within a region of S1 hypersensitivity in the supercoiled plasmid, and in the absence of viral enhancer sequences it is the main initiation site following transfection into a number of cell lines. We suggest that the -200 cap site acts as a polymerase entry site by virtue of its accessible chromatin structure. The efficiency of polymerase binding at this site may be altered by trans-acting regulatory molecules.

Adenoviruses, Human↗
PubMed1984

Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.

The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo.

Base Sequence↗
PubMed1984

Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.

To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.

Base Sequence↗
PubMed1983

[Venous grafts as arterial substitutes. Immediate and medium-term development].

This study, based on the authors' experiments in dogs and on data from the literature, describes the immediate and mediumterm histological development of autologous vein grafts. Dissection alone results in extensive endothelial cell desquamation, which may be aggravated by distension and preservation of the graft in an inappropriate medium. Immediately after re-establishment of the arterial blood flow the inner surface of the graft is covered by a fibrinoerythrocytic membrane. Endothelialization begins 10-15 days later but remains incomplete in dogs after 9 months; it is not limited to the suture lines but proceeds by islets disseminated over the surface. As early as the 15th post-operative day, a myo-intimal layer of myofibroblastic cells develops; the origin and control of these cells are still unknown. A better knowledge of the histological development of vein grafts and underlying mechanisms appears to be essential to improvements in surgical procedures and subsequent long-term functional performance of the grafts.

Animals↗
PubMed1983

Ab initio calculations of electron distributions in heme-CO models.

We have, by the use of ab initio calculations, found a back-bonding state of pi symmetry close to the Fermi level for CO bound to FeN5C14. We thus find it likely that small shifts of the redox potential magnitude of EF - EV magnitude of will cause relatively large changes of the CO vibrational frequency. The separation of Fe 3d orbitals in our heme model is found to agree with what is predicted by ligand field theory for Oh symmetry. This paper presents nonrelativistic Hartree-Fock-Slater calculations of the 5 sigma bonding and 2 pi back-bonding between CO and Fe. The effects of up to 19 additional atoms are discussed for models of heme (COFe to COFeN5C14). The filled back-bonding state is found to be strongly influenced by second nearest neighbor atoms. By use of symmetry orbitals we have resolved the Fe 3d orbitals into the T2g and Eg representations of the Oh point group and find the former states to be occupied whereas the latter are unoccupied. The difference in occupancy is reduced when the CO ligand is removed which also causes an increased density of states at the Fermi level, i.e., the highest occupied and lowest unoccupied orbitals. Possible correlations between our data and experimental results are discussed for heme proteins as well as for metal surfaces.

Carbon Monoxide↗
PubMed1983

Multiple origins of transcription in the 4.5 Kb upstream of the epsilon-globin gene.

By a combination of primer extension and S1 mapping, we have identified and characterized at least nine epsilon-globin initiation sites in the 4.5 kb upstream of the canonical mRNA cap site. These occur in four regions, -65 to -250, -900, -1480, and -4500 bp from the translation initiation codon ATG. Transcripts from eight sites yield sufficient material for direct RNA analysis and in all of these instances the molecules are capped. Approximately 10%-15% of epsilon-globin transcripts originate from these upstream sites in both an erythroleukemic cell line and purified erythroblasts from first-trimester human embryos. In three established cell lines derived from adult non-erythroid tissue, low levels of transcription of the epsilon-globin gene occur, but the RNA molecules originate exclusively from one of the upstream sites identified in erythroid cells. These findings suggest modification of the current models of transcription of globin genes.

Cell Line↗
PubMed1983

Effects of bradykinin and bovine serum albumin on arachidonic acid and prostaglandin release from perfused rat heart.

The isolated perfused rat heart was used to study if arachidonic acid (AA) release affected prostaglandin (PG) production. The perfused heart avidly absorbed and esterified exogenous AA, mostly into polar lipids. The administration of bradykinin (BK) after prelabeling with 1-C14-AA caused a two-fold increase in the amount of AA released. Bovine serum albumin (BSA) stimulated release 2.5-fold. Perfusion with AA saturated BSA enhanced the release of 1-14C-AA 23-fold indicating the presence of an active deacylation/reacylation system in rat heart. BK caused a four-fold increase whereas BSA had no effect on the amount of prostaglandins released from perfused heart indicating that BK acted on a specific AA pool available to cyclooxygenase whereas the BSA stimulated a separate pool of AA which apparently was not available to the cyclooxygenase.

Animals↗
PubMed1983

Immediate and long-term evolution of the blood-venous graft interface. Experimental study in dogs.

Endothelial damage and repair in autogenous saphenous vein grafts was evaluated serially from 30 s to 9 months after implantation into the femoral arteries of 25 dogs. Light and scanning electron microscopy, quantitative morphometry and cytochemistry with specific rabbit antidog factor VIII-related antigen were used. These studies demonstrate: (1) early complete endothelial cell desquamation both on the venous graft and adjacent artery apparently related to dissection and suture; (2) differing early platelet activation on the venous and adjacent arterial wall, platelet aggregates were noted on the adjacent artery, whereas only adhesive platelets are noted on the exposed vein graft; (3) endothelial reconstitution being initiated after 10-15 days but not reaching completion even 9 months after implantation; (4) a mediointimal proliferation which is a constant feature. A unique cell possessing both fibroblastic and myocytic characteristics takes part in the process.

Animals↗
PubMed1983

Diminished osmotic and chemically induced haemolysis of human erythrocytes following exposure to contrast media molecules.

Radiocontrast molecules (RCM) used in coronaroangiography and/or urography diminished their osmotic fragility when they were incubated for 30 min with human erythrocytes. The shift of the osmotic fragility curve towards lower NaCl concentration is related to the hypertonicity of RCM, but in addition, at a given osmolality (100 +/- 5 mosm/kg), RCM at the concentration of 4-10% v/v increase the resistance to osmotic lysis or even suppress it. Similar protection is observed towards erythrocyte lysis induced by a detergent (saponin), polyenic antibiotic (filipin) or non-polyenic cholesterol-specific agent (digitonin). The effect is (1) proportional to the amount of RCM present, (2) independent of hypertonicity of the molecule, and (3) related to the nature of acidic molecules. A weak insertion of RCM into the erythrocyte membrane is suggested since it was suppressed by a single washing of the cells.

Contrast Media↗
PubMed1983

Increased circulation of T lymphocytes bearing surface thymosin alpha 1 in patients with myasthenia gravis: effect of thymectomy.

We studied the interaction of the thymic hormone thymosin alpha 1 with peripheral blood B and T lymphocytes in patients with myasthenia gravis (MG), using antibodies against thymosin alpha 1 in an immunofluorescence technique. Eleven of 16 patients with symptomatic MG had an increased number of T lymphocytes bearing surface thymosin alpha 1 (T alpha 1); 5 patients with asymptomatic disease had normal levels of T alpha 1. In six young adults with symptomatic MG who subsequently responded to thymectomy, the number of T alpha 1 cells returned to normal 1 month after thymectomy. Because levels of T alpha 1 correlated with symptoms and thymosin alpha 1 specifically recruits helper T cells, our findings suggest that T alpha 1 may play an immunoregulatory role in the pathogenesis of MG. Determination of T alpha 1 levels may prove to be helpful in assessing residual thymic activity after thymectomy.

Fluorescent Antibody Technique↗
PubMed1982

Contrast agents used for excretory urography impair platelet function in human patients.

The platelet function was investigated in 65 patients submitted to diagnostic excretion urography (injection of 60 ml contrast medium). Blood sampling was performed before (T0) 90 seconds after (T1) and 30 minutes after (T2) injection of the radiocontrast molecule (RCM). Five RCM of different osmolality ionicity and nature of the lateral chain have been tested. Platelet aggregation, platelet release of ATP, osmolality, total calcemia and ionized calcium level were determined on each plasma sample as well as RCM concentration at T1 and T2. Decrease (20 - 40%) of platelet aggregation occurred at T1, whichever platelet aggregating agent (ADP, collagen, Ristocetin or thrombin) were used (significant after Iopamidol 300 and Na Meg Ioxaglate). Platelet release of ATP induced by collagen was also decreased or delayed. These changes were rapidly reversible and a tendency to improvement was observed at T2. Among the five RCM tested, one (Na Diatrizoate) might be a proaggregating agent. No changes of osmolality occurred in the plasma and no correlation could be established between RCM concentration and platelet inhibition. A pathogenic hypothesis is suggested by the significant fall of total and ionized calcium level after RCM injection.

Adenosine Triphosphate↗
PubMed1982

Epsilon globin gene transcripts originating upstream of the mRNA cap site in K562 cells and normal human embryos.

RNA transcribed from the human epsilon globin gene was studied in K562 cells and human embryos of 5-10 weeks gestation. Using both primer extension and S1 analysis RNA molecules were found which extend 53 +/- 1 bp, 55 +/- 1 bp and 270 +/- 1 bp upstream from the first coding base and are colinear with the gene. The first pair of molecules represent transcripts initiating at the canonical cap site but the other species represents a transcript over 200 nucleotides longer which initiates upstream of the CAAT and ATAA boxes.

Base Composition↗
PubMed1982

Transfer of human globin genes to erythroleukemic mouse cells.

Thymidine kinase negative (TK-) Friend cells were transformed with recombinant molecules carrying human globin genes and the thymidine kinase gene of herpes simplex virus type 1 DNA. Transformation frequencies of 1 transformant/microgram donor DNA/1 x 10(6) cells were obtained by standard procedures and this was increased 20- to 30-fold by treating recipient cells with dimethyl sulfoxide or glycerol. Transformed cell lines expressed thymidine kinase activity of viral origin as determined by its insensitivity to 0.2 mM dTTP and electrophoretic mobility in polyacrylamide gels. The physical status of donor DNA in the transformed cells was examined in Hirt precipitates and supernatants by Southern blot hybridization and spot hybridization techniques. This analysis showed that most donor sequences were present in a circular or concatenate configuration, but also was suggestive of some donor sequences being integrated into high molecular weight DNA. Expression of human globin genes and particularly the epsilon-globin gene in the transformed Friend cells was studied by Northern blot hybridization analysis.

Animals↗