Partial purification and characterization of a factor from a cloned thymic epithelium cell line

The culture supernatant from a cloned line of thymic epithelium (TEPI) is shown to enhance the response of thymocytes to alloantigen as measured by cell-mediated lympholysis. The supernatant has no effect on the spleen cell response to alloantigen as measured by cell-mediated lysis and does not contain interleukin 1, interleukin 2, interleukin 3, or interferon-gamma activity. The activity is shown to have an apparent m.w. of 160,000 by Sephacryl S-200 gel permeation chromatography, to have an isoelectric point of 6.5, and to elute from DEAE-Sepharose at 0.07 M NaCl.     

Two or more copies of Drosophila heat shock consensus sequence serve to activate transcription in yeast

A synthetic oligonucleotide bearing the Drosophila heat shock consensus sequence confers heat inducibility on a CYC1-lacZ gene in Saccharomyces cerevisiae. This sequence CTGGAATTTTCTAGA was inserted in place of the upstream activation sites of the CYC1 promoter adjacent to CYC1 TATA boxes. These constructs were transformed into yeast and found to be heat-inducible when two or more inserts were present. The level of inducibility seemed to increase with the number of inserted sequences: however, the orientations of these sequences relative to each other did not have much effect.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Effect of oxygen pressure on glycogen synthesis by rat-liver slices

1. Glycogen synthesized by rat-liver slices 0.5mm. thick incubated at 1atm. oxygen pressure in Hastings medium with glucose was localized in the cells of the periphery of the slice. Cells of the interior of this slice do not synthesize glycogen. 2. Inner cells of thin slices (about 0.3mm. thick) can synthesize glycogen when such slices are incubated under the same conditions, but oxygen pressures higher than 1atm. are required if inner cells of slices 0.5mm. or more thick are to be able to synthesize glycogen. 3. Localization of newly synthesized glycogen in rat-liver slices incubated in Hastings medium with glucose does not depend on glucose concentration. 4. Calculation of the minimum oxygen pressure required to synthesize glycogen gives values between 0.09 and 0.17atm. 5. The advantages of high oxygen pressures for the study of the synthesis of glycogen and other compounds that require ATP are discussed.     

Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 by the conscious marine toad, Bufo marinus.

Tissue distribution, elimination, and metabolism of 3H-labelled leukotriene (LT) C4 were studied in ureter-catheterized conscious marine toads, Bufo marinus. Six and 24 h after injection, organs containing the highest percent of injected radioactivity were small intestine, liver, and kidney. Radioactivity declined in these organs at 24 h by approximately threefold. Peak elimination time for radioactivity in the urine was between 2 and 4 h after the injection. During the 24-h collection period, 55.2 +/- 0.2% of the injected radioactivity was eliminated in the urine. Polar metabolites represented 40.3 +/- 1.1, 57.3 +/- 5.6, and 62.8 +/- 1.6% of the radioactivity at 2, 4, and 6 h, respectively. The primary urinary polar metabolite was 20-carboxy-LTE4, with 18-carboxydinor-LTE4 and 20-hydroxy-LTE4 also present. [3H]LTE4 decreased from 37.2 +/- 1.8% at 2 h to 15.8 +/- 3.3 and 15.0 +/- 2.1% of the radioactivity at 4 and 6 h, respectively. Bile radioactivity was low. N-Acetyl-LTE4 was not detected in urine or bile samples. Radioactivity in the pan water was 14.3 +/- 2.4 and 15.8 +/- 2.5% of the injected radioactivity, at 6 and 24 h, respectively, suggesting that the skin was a route for excretion of leukotrienes. The marine toad is an interesting model demonstrating both similarities and differences from mammals in distribution, elimination, and metabolism of peptide leukotrienes.     

Short-term stimulation of cellular autophagy by furosemide in the thick ascending limb of Henle's loop in the rat kidney

Summary In an effort to investigate the functional relationship between cell-specific work and intracellular degradative processes, the effect of furosemide on cellular autophagy was investigated in two different portions of the nephron, namely, the thick ascending limb of Henle's loop (TAL), which is a main target of this drug, and the proximal convoluted tubule (PCT) as a reference structure. Eight male adult rats were treated with furosemide (60 mg/kg body weight, s.c.). Eight control animals received physiological saline. 1 to 4 h after the injections the animals were killed by perfusion fixation. Small specimens of kidney tissue from the inner stripe of the outer medulla and from the outer cortex were processed for electron microscopy; they were investigated morphometrically for volume fraction and numerical density of autophagic vacuoles (AVs). A significant increase of both parameters (volume fraction: 0.42 × 10^-4 to 1.09 × 10^-4; numerical density: 4.2 × 10⁵/mm³ to 15.5 × 10⁵/mm³) was seen under the influence of furosemide in TAL cells, whereas PCT cells did not show a significant increase in volume fraction or any increase in numerical density of AVs. These data suggest that the functional unloading of TAL, via blocking of the Na⁺- 2Cl^- — K⁺ co-transport by furosemide, results in adaptative structural unloading, i.e., an increased sequestration of cytoplasmic components into AVs, within a short-time interval.