Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Ca- and Mg-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca²⁺- and Mg²⁺-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca²⁺. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca²⁺-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 μg/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Interaction of flavonoids with rabbit muscle phosphorylase kinase

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Isolation and Identification of Eutypa lata from Pistacia vera in Greece

The fungus Eutypa lata (syn. E. armeniacae), known as the causal agent of the death of many different woody plants, was found on dead branches of pistachio (Pistacia vera L.) in Greece. Isolations from diseased branches yielded consistently typical colonies of the asexual stage of the fungus (Libertella blepharis, syn. Cytosporina sp.), which proved to be undistinguishable from other cultures of the pathogen obtained from 15 different hosts. Furthermore, all isolates from pistachio tested for pathogenicity on apricot were pathogenic and yielded characteristic cankers.     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Immune network behavior—II. From oscillations to chaos and stationary states

Two types of behavior have been previously reported in models of immune networks. The typical behavior of simple models, which involve B cells only, is stationary behavior involving several steady states. Finite amplitude perturbations may cause the model to switch between different equilibria. The typical behavior of more realistic models, which involve both B cells and antibody, consists of autonomous oscillations and/or chaos. While stationary behavior leads to easy interpretations in terms of idiotypic memory, oscillatory behavior seems to be in better agreement with experimental data obtained in unimmunized animals. Here we study a series of models of the idiotypic interaction between two B cell clones. The models differ with respect to the incorporation of antibodies, B cell maturation and compartmentalization. The most complicated model in the series has two realistic parameter regimes in which the behavior is respectively stationary and chaotic. The stability of the equilibrium states and the structure and interactions of the stable and unstable manifolds of the saddle-type equilibria turn out to be factors influencing the model's behavior. Whether or not the model is able to attain any form of sustained oscillatory behavior, i.e. limit cycles or chaos, seems to be determined by (global) bifurcations involving the stable and unstable manifolds of the equilibrium states. We attempt to determine whether such behavior should be expected to be attained from reasonable initial conditions by incorporating an immune response to an antigen in the model. A comparison of the behavior of the model with experimental data from the literature provides suggestions for the parameter regime in which the immune system is operating.     

Physical mapping of the human T-cell receptor beta gene complex,using yeast artificial chromosomes

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.