DNase I hypersensitivity and expression of the Shrunken-1 gene of maize

The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure.     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.     

Alteration of poly(adenosine diphosphoribose) metabolism by ethanol: mechanism of action

We present evidence that ethanol alters intracellular poly(adenosine diphosphoribose) metabolism and we further describe the mechanism by which ethanol exerts its effect on polymer synthesis. One percent ethanol stimulates polymer accumulation as much as 2.5-fold but does not alter polymer degradation in intact cells following DNA damage. Ethanol directly stimulates polymer synthesis following low doses of DNA damage induce by deoxyribonuclease I in a nucleotide-permeable cell system that does not possess a functional polymer turnover system. Ethanol has no measurable effect on polymer synthesis in undamaged nucleotide-permeable cells or in permeable cells treated with high doses of deoxyribonuclease I. Ethanol concentrations that stimulate poly(adenosine diphosphoribose) polymerase activity in vitro specifically lower KDNA without affecting KNAD or Vmax. The results clearly show that ethanol alters the binding of this enzyme to the DNA component of chromatin and that this altered binding is responsible for the activation of the enzyme. Altered affinity of poly(adenosine diphosphoribose) polymerase and perhaps other regulatory proteins for chromatin may play an important role in the pathology of alcohol.     

Joint report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus x Bos indicus, and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

The surface potential on the purple membrane measured using a modified bacteriorhodopsin chromophore as the spectroscopic probe

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13-CF₃ retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13-CF₃ chromophore is around 7.3. The apparent p_K of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy-Chapman equation. The surface charge density was found to be −1.65 ± 0.15 × 10⁻³ electronic charges per Ų or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light-induced proton release from the purple membrane.     

The effect of erythrocyte associated light scattering on membrane fluorescence polarization

Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 10⁷ cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 10⁶ cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells.     

Human renal clear cell carcinoma: Establishment and characterization of a new cell line (G-2101)

Summary A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simulataneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population. This work was supported in part by a grant from Triton Biosciences, Inc.