Induction by folate and folate analogs of extracellular and membrane-bound phosphodiesterase from Dictyostelium discoideum

Folate stimulation is known to enhance Dictyostelium discoideum differentiation. During early differentiation, D. discoideum cells possess two classes of folate receptors which can be distinguished by their difference in specificity (R. J. W. de Wit, FEBS Lett. 150, 445-448, 1982). We investigated the type of receptor by which folate affects cell differentiation. Two independently regulated developmental markers were used: the extracellular phosphodiesterase-inhibitor system and cell-surface phosphodiesterase activity. Our results indicate that the major effect of folate on development is mediated by the folate-specific receptor. The nonspecific folate receptor was only involved in a minor, transient enhancement of the extracellular phosphodiesterase activity very early in development.     

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.     

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.     

Functional aspects of the postovulatory follicle in the ovary of the African catfish,Clarias gariepinus,after induced ovulation

Summary In captive African catfish, Clarias gariepinus, ovulation was induced with human chorionic gonadotropin (HCG) 4 I.U./g body weight to study the function of postovulatory follicles (POFs). Ultrastructural and enzyme-histochemical data indicate that, apart from special theca cells, the granulosa of relative young POFs (i.e., from 16 h and 28 h after HCG-injection) is capable of producing steroids. Possible functions of the synthesized steroids are discussed. Histological comparison of POFs from stripped and from unstripped fish, as well as histochemical investigation of the contents of ovulated ova and granulosa of POFs at 48 h after HCG-injection, showed that the latter structure is involved in phagocytosis of the disintegrating ovulated eggs. The polysaccharide-lipid-protein material, initially taken up by heterophagolysosomes of the granulosa cells, subsequently undergoes fatty degeneration. The granulosa cells of the POFs showed strong acid phosphatase activity and abundant granular endoplasmic reticulum from 16 h after HCG-injection onward; heterophagolysosomes appeared at 32 h. These results indicate that after ovulation the phagocytotic function of the granulosa develops progressively. Autophagolysosomes, responsible for the final disintegration of POFs, become increasingly evident in the granulosa cells with increasing time after ovulation.     

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

Summary A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974).Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.This word was performed while one of us (Dr. Andrä) was in receipt of a visitor grant from the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Human lens γ-crystallin sequences are located in the p12-qter region of chromosome 2

Summary The human -crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human -crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human -crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.