Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF.

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.     

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.     

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.     

Immunocytochemical diagnosis of acute leukemia with pleural involvement

Cytologic examination of the pleural effusion from a patient with acute leukemia, leukocytosis and bleeding revealed the presence of many leukemic cells, "lymphocytes" and erythrocytes. The significance of these cellular changes was investigated by simultaneous study of blood and effusion leukocytes by morphologic, cytochemical and immunochemical methods. Both the leukemic blasts and the "lymphocytes" in the effusion and the blood were found to be neoplastic and contained antigens characteristic of both myeloid cells (OKM-1) and lymphoblasts (C-ALLA, common acute lymphoblastic leukemia antigen). These results, when analyzed in the context of the clinical findings, were indicative of acute leukemia with pleural involvement. Such a clinically oriented approach may further enhance the potential of cytodiagnosis in patients with serous effusions.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

Metabolic inhibition of size-fractionated marine plankton radiolabeled with amino acids,glucose, bicarbonate,and phosphate in the light and dark

The effects of various metabolic inhibitors (dichlorophenyl dimethylurea, chloramphenicol, cycloheximide, carbonyl cyanide m-chlorophenyl hydrazone) on the accumulation of radiolabeled substrates (amino acids, glucose, bicarbonate, phosphate) by size-fractionated marine microbial plankton from the Sargasso Sea and the eastern Canadian arctic were studied in time-course fashion during experimental incubations either exposed to or shielded from ambient solar radiation. Picoplankton accounted for 65% of the organic substrates and phosphate accumulated by the assemblages. The rate of organic substrate accumulation was stimulated by solar radiation in some cases but inhibited in other cases. Presumably, stimulation and inhibition co-occur and the measured response is the net result arising from these counteracting tendencies. Approximately 40% of H¹⁴ CO₃ ^– accumulation in the Sargasso Sea was associated with the picoplankton. The insensitivity of picoplankton¹⁴C-labeling to cycloheximide suggested active prokaryotic photosynthesis rather than heterotrophic assimilation of¹⁴C-labeled algal photosynthates as the route of labeling. The usefulness of some inhibitors was restricted in this study because of inconsistent correlations between the intended primary metabolic effect and the measured ecological response within the duration of the experiment.     

T and B cells induce macrophages which suppress proliferation but not lymphokine secretion

In vitro culture of mouse spleen cells for 2 days or more leads to the production of adherent, phagocytic, Thy-1-, Ia+, Lyt-2- cells ("suppressor macrophages") which strongly inhibit the proliferative response of T and B lymphocytes to a variety of stimuli: mitogens, specific antigens, and antigen-nonspecific growth factors. Suppressive activity fails to develop, however, in cultured spleen cells from which nonadherent cells have been removed before the initial 48-hr incubation, and only partial suppression is obtained from cell suspensions from which T cells have been depleted before culture. We find that the requirement for nonadherent cells can be replaced by graded doses of lymphocytes. Lyt-2- and Lyt-2+ T cells are about equally potent in inducing suppressive activity in nonadherent cells. Surprisingly, B cells (containing fewer than 0.1% contaminating T cells) are also able to induce suppression in this system. The suppression induced includes both indomethacin-sensitive and indomethacin-resistant components. Interestingly, not all stages of mitogen-induced T-cell activation are blocked by these adherent cells: proliferation is inhibited, but production of interleukin 2 (IL-2) and interleukin 3 (IL-3) is unaffected.     

Effect of physico-chemical factors on anion-sensitive ATPase activity

Effects of temperature, pH and anions on the ATPase activity of submitochondrial particles of rat liver, rat heart, mouse liver, of red blood cell membranes, and of soluble enzyme of rat liver, mouse liver mitochondria were studied. The temperature relationships of membrane-bound and soluble ATPases have the breaks at 18-21 degrees C and 30-32 degrees C. These breaks were not shifted by sulfite, thiocyanate, methanol, glycerol and GTP. The pH changes from 6.0 to 8.5 produced no effect on the temperature relationships of ATPase activities but, strongly influenced the rate of ATPase reaction. The conformity between the obtained data and earlier proposed mechanism of anion control over anion-sensitive ATPase activity was discussed.     

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.