A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

Evidence for incorporation of n-acetylglucosamine in endogenous nuclear acceptors during the nuclear matrix preparation

• 1.1. The presence of glycoproteins within the nucleus of cell is now well established and the question arises on the nature of the nuclear glycosylation and the site of their glycosylation.
• 2.2. In order to study endogenous nuclear proteins acceptors, we have isolated a subnuclear fraction: nuclear matrix characterized by DNA, RNA, phospholipids and proteins content. Nuclear matrix acceptors were obtained from nuclei incubated with UDP-N-acetyl [¹⁴C]glucosamine.
• 3.3. In this report we describe the presence of three major glycoproteins labeled with N-acetyl [¹⁴C]glucosamine in the nuclear matrix fraction. We obtained gP 32, gP 67 and gP70 with pI values around 6.2, 6.5 and 8.2.

     

31P NMR studies on kidney intracellular pH in acute renal acidosis

• 1.1. Phosphorus nuclear magnetic resonance (³¹P NMR) provides a non-destructive method for the quantitative observation of the most abundant phosphate-containing metabolites in isolated perfused kidney and in kidneys inside an anaesthetised animal.
• 2.2. In this paper, the NMR method is introduced and the intracellular pH changes following acute renal acidosis are measured (from the resonance position of P_i) for the isolated perfused rat kidney.
• 3.3. The results are discussed in relation to the biochemical data obtained by freeze extraction.

     

Pest sequences in nuclear proteins

• 1.1. Most of proteins which are rapidly degraded inside eukaryotic cells have been found to contain amino acid sequences (PEST sequences) enriched in proline, acidic residues (glutamic acid and/or aspartic acid) and hydrophilic residues (serine and threonine) (Rogers et al. (1986) Science234, 364–368).
• 2.2. This correlation was tested on nuclear proteins and a close relationship was found between nuclear protein stability and the presence of PEST regions.
• 3.3. Nuclear proteins with structural functions which can be considered as stable components of cell nuclei generally lack PEST sequences.
• 4.4. In contrast, regulatory nuclear factors which have specific and transient functions generally possess at least one PEST sequence.

     

Prostaglandin E1 and analogs of prostacyclin influencing superoxide production by the human neutrophil nadph oxidase system

• 1.1. The effects of prostaglandin (PG) E₁, and I₂ analogs (OP-41483 and OP-2507) on the Superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated.
• 2.2. In a whole-cell system, OP-2507 inhibited the Superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action.
• 3.3. The concentration of the drug required for 50% inhibition of the oxidase (IC₅₀) was 21 μM.
• 4.4. In a cell-free system, however, the drug in concentrations of < 100 μM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent.
• 5.5. Although PGE₁ and OP-41483 did not inhibit the Superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC₅₀ values of 44 and 170 μM, respectively.
• 6.6. In addition, in the cell-free system, the K_m value for NADPH of the oxidase was unchanged by PGE₁.
• 7.7. The results suggest that the PGI₂ analog, OP-2507, is a possible superoxide-scavenger and that PGE₁ inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

     

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

• 1.1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments.
• 2.2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long.
• 3.3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction.
• 4.4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

The haemocytes and the activities of leucine aminopeptidase and alkaline phosphatase during development of Ceratitis capitata: Specific secretion of a leucine aminopeptidase isozyme

• 1.1. The types of haemocytes during larval development were studied.
• 2.2. The developmental profile of leucine aminopeptidase and alkaline phosphatase was studied. The maximum LAP activity was found to be in early larval development, while the maximum alkaline phosphatase during the white pupal stage.
• 3.3. These activities were compared with those determined in cell-free haemolymph.
• 4.4. Both hydrolytic enzymes have been found histochemically in the prohaemocytes and in the plasmatocytes.
• 5.5. In cultured haemocytes experiments it was found that 64% of the total LAP activity was secreted into the incubation medium, while electrophoretic analysis of released LAP activity demonstrated that only LAP A isozyme was secreted.
• 6.6. Based on the above results we suggest that both hydrolytic enzymes are functionally important throughout larval development.

     

Poly(d(A—T)) dependent RNA polymerase activity after treatment of nuclei with micrococcus nuclease

• 1.1. Rat liver nuclei were incubated with or without 20 units micrococcus nuclease (EC3.1.4.7)/mg nuclear DNA.
• 2.2. The soluble poly(d(A—T)) dependent RNA polymerases were reduced in activity to 15–20% that of the controls after treatment with micrococcus nuclease.
• 3.3. RNA polymerases I plus III activities were completely, RNA polymerase II activity partially reversible on removal of the DNA released into the soluble fraction by treatment of nuclei with micrococcus nuclease.
• 4.4. Inhibitory constants obtained with the solubilized DNA were 17.1 μM and 20.7 μM nucleotide-DNA for RNA polymerases I plus III and RNA polymerase II, respectively. The corresponding inhibitory constants obtained with native salmon DNA were 23.0 μM and 34.4 μM nucleotide-DNA.

     

A comparative study of the basic nuclear proteins from sperm of bivalve molluscs

• 1.1. Basic nuclear proteins from spermatozoa of the three mollusc species belonging to the class Bivalvia have been analyzed using one- and two-dimensional electrophoresis.
• 2.2. Four nuclear basic proteins have been purified and their amino acid compositions determined.
• 3.3. In these spermatozoa histone-type proteins coexist with protamine-like proteins.
• 4.4. The protamine-like proteins that have been studied show different electrophoretic behavior but in general are similar, with a high content of lysine, arginine, alanine and serine.
• 5.5. Interspecific variability has been found for the H1-like histone.

     

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Poly(adp-ribosylation) in N,N-diethylnitrosamine-treated mice

• 1.1. Liver nuclei isolated from male mice treated with the carcinogen N,N-diethylnitrosamine were examined for the homopolymer poly(adenosine diphosphate ribose) and for the activity of the conjugate polymerase.
• 2.2. At all levels of the carcinogen tested, a concomitant increase in both poly(adenosine diphosphate ribose) content and activity of the enzyme were found.
• 3.3. Both responses were transitory and dose dependent.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Ultrahistopathology of enchytraeid oligochaetes (annelida) after exposure to pesticides—A means of identification of sublethal effects?

• 1.1. The effects of the commonly used insecticides E605f (parathion), Ripcord 10 (cypermethrin) and the fungicide DuPont Benomyl on chloragocytes of an enchytraeid species were studied by transmission electron microscopy.
• 2.2. Animals were exposed to concentrations varying between 0.1 × and 100 × the minimum concentration recommended for agricultural application.
• 3.3. Ultrastructural changes regarding nuclei, mitochondria and other cytoplasmic compartments were obvious and were correlated to concentration and exposition time of the pesticides.
• 4.4. The importance of ultrahistopathology for terrestrial bioassays and monitoring is discussed.

     

The characterization of the linoleic acid desaturation and elongation system in ovine placental tissue

• 1.1. The desaturation and elongation of linoleic acid has been studied in homogenates and in subfractions of ovine placental tissue.
• 2.2. The reaction was characterized in terms of pH and temperature optima, time course and protein concentration.
• 3.3. Activity was found to be confined to the 11,000g supernatant fraction of the tissue and the results suggest that the enzymes are membrane bound.
• 4.4. The cytosolic fraction and ATP were required for full activity and the reaction was inhibited by cyanide.
• 5.5. The properties of the reaction are compared with those of other desaturation systems and their implications with regard to possible reaction mechanisms are discussed.

     

The kinetics of an enzyme catalyzed reaction in the presence of an unstable,irreversible modifier

• 1.1. The chemical modifications of enzymes is a widely used technique to gain information about the number, type and location of aminoacid residues which are essential for activity. A number of these modifiers are unstable in aqueous solutions and act irreversibly.
• 2.2. The kinetics of such systems have previously been studied under assumptions, some of which are either unnecessary or too restrictive.
• 3.3. We replace these assumptions by others which are more realistic and less stringent.
• 4.4. These assumptions allow us to derive analytical expressions for the evolution of all the species involved in the reaction.
• 5.5. From the analytical expression for the time course of the product formation an experimental design and a kinetic data analysis allowing the easy characterization of these systems are suggested.

     

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

• 1.1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated.
• 2.2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor.
• 3.3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities.
• 4.4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

     

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Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.