Hormonal control of vitellogenin synthesis in avian liver

Estradiol induces the synthesis of vitellogenin in the avian liver. We describe the precursor-product relationship between vitellogenin and the yolk proteins phosvitin and lipovitellin. The high rate of vitellogenin synthesis is a consequence of the accumulation of a stable messenger RNA. We suggest that estradiol acts at the level of the genome by opening a hitherto non-transcribed gene.     

Immunochemical isolation and characterization of vitellogenin mRNA from liver of estradiol-treated chicks

Several commercially available samples of galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) were found to contain high proteolytic activity on proteins such as fibrinogen, transferrin, albumin and casein. A simple, efficient method was devised for the purification of galactose oxidase which relies on the affinity of the enzyme for agarose (Sepharose 6B). The purified galactose oxidase was recovered in high yield free from proteolytic activity. The enzymic affinity for Sepharose and Sephadex was investigated to clarify the absorption mechanism.     

Increased messenger RNA from protein synthesis inhibited human fibroblasts

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as "superinduction" and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.     

Size, complexity and abundance of a specific poly(A)-containing RNA of liver from male Xenopus induced to vitellogenin synthesis by estrogen

Estrogen treatment of Xenopus males leads to the appearance of a new species of poly (A)-containing RNA in the liver, at a stage when large amounts of the estrogen-induced yolk precursor protein, vitellogenin, is produced. This estrogen-induced RNA sediments at 28 S and migrates on gels in aqueous solution with an apparent molecular weight of 2.0 X 10(6). Contour length measurements under denaturing conditions in the electron microscope reveal a molecular weight of 2.34 X 10(6) compared to the mouse 28-S rRNA. Labeling experiments show that the estrogen-induced RNA has a stability than the average liver poly(A)-containing RNA and represents 10-20% of the poly(a)-containing RNA in the cytoplasm after 24 h of labeling. Hybridization of complementary DNA, synthesized on the isolated estrogen-induced RNA, with its template suggests a complexity corresponding to a single species of poly(A)-containing RNA of such a high molecular weight. Hybridization of the complementary DNA with cytoplasmic poly (A)-containing RNA from estrogen-treated Xenopus males and control toads show that the estrogen-induced RNA constitutes 12-15% of all cytoplasmic poly(A)-containing RNA, and is at least 2000-fold less abundant in untreated males. Size, complexity and abundance of the estrogen-induced RNA are characteristics expected for a mRNA coding for vitellogenin.     

The structural organization of nuclear messenger RNA precursor. I. Reassociation and hybridization properties of double-stranded hairpin-like loops in messenger RNA precursor

The hybridization and renaturation properties of double-stranded hairpin-like loops isolated from giant nuclear messenger RNA precursor of mouse liver or ascites carcinoma cells were studied. About half of the hairpins in messenger RNA precursor appear to contain similar sequences, as indicated by the very fast kinetics of renaturation of the denatured double-stranded RNA sequences. These sequences have no tissue specificity. About one third of the hairpin sequences can hybridize to messenger RNA. It is suggested that the long hairpins in messenger RNA precursor play the role of sequences separating messenger RNA sequences from non-informative sequences and that these hairpins are recognized by processing enzymes.     

Isolation and translation of non-crystallin messenger RNA from calf lens

Affinith chromatography of lens polyribosomal RNA on oligo(dT)-cellulose yields three fractions. As arule the second fraction has been neglected in other studies reported in the literature. According to our investigations this fraction in particular contains the messengers for the non-crystallin lens proteins.     

Globin messenger RNA from anaemic rabbit spleen. Size of its polyadenylate segment

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and T1 ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly (A) segments of spleen globin mRNA were found to be 25-90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10-30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.     

Effect of preparations of messenger RNA from the spleens of immunized rats on antibody synthesis in rat lymphosarcoma

Messenger RNA was isolated from spleen polysomes of rats immunized with sheep red cells, two methods being employed: a sorption of mRNA on on membrane filters, and poly(U)-cellulose affinity chromatography. The rat transplantable lymphosarcoma cells treated with this RNA started hemolysine synthesis which persisted throughout many cell generations in transplantation of the tumor. The probability is discussed of m-RNA not only functioning as a template for antibody synthesis but also deblocating appropriate genes in recipient cells.     

Biotin synthase from Arabidopsis thaliana. cDNA isolation and characterization of gene expression

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli.     

Expression of thyroid hormone receptors alpha and beta-1 messenger RNAs in human endothelial cells. The T3 hormone stimulates the synthesis of the messenger RNA of the intercellular adhesion molecule-1

A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.     

Intrastriatal infusion of nerve growth factor after quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA

Excitotoxic striatal lesions induced by quinolinic acid, a model for Huntington's disease, were used to test for neuroprotective actions of nerve growth factor on striatal cholinergic and GABAergic neurons. Expressions of the trkA receptor for nerve growth factor, choline acetyltransferase and glutamate decarboxylase were analysed by messenger RNA in situ hybridization in adult rats following quinolinic acid lesion (150 nmol) and daily striatal administration of nerve growth factor (1 microgram) or control protein (cytochrome C) for one week. One week after toxin administration, the numbers of cells expressing trkA or choline acetyltransferase messenger RNAs were decreased when compared with unlesioned animals. Moreover, the surviving cells showed a strong down-regulation of these messenger RNAs as deduced from grain count analysis of sections processed for emulsion autoradiography. Daily intrastriatal nerve growth factor administration for one week completely prevented the reduction in the number of cells expressing either of the two markers. Nerve growth factor treatment increased the cellular expression of choline acetyltransferase messenger RNA three times above control levels and restored the levels of trk A messenger RNA expression to control levels. In contrast to the protective effects on cholinergic cells, nerve growth factor treatment failed to attenuate the quinolinic acid-induced decrease in glutamate decarboxylase messenger RNA levels. Optical density measurements of the entire striatum on autoradiographs of brain sections from quinolinic acid-lesioned animals revealed a reduction of the glutamate decarboxylase messenger RNA-specific hybridization signal, which was unaltered by infusion of nerve growth factor or control protein. Our findings strongly suggest that in both the intact and the quinolinic acid-lesioned adult rat striatum, nerve growth factor action is confined to trk A-expressing cholinergic neurons. Striatal glutamate decarboxylase messenger RNA-expressing GABAergic neurons which degenerate in Huntington's disease are not responsive to nerve growth factor.     

Separate isolation of cells from nodules and surrounding parenchyma of the same precancerous rat liver: biochemical and cytochemical characterization

Various enzyme and metabolic alterations have been observed in the hyperplastic nodules which appear during the hepatocarcinogenesis. These alterations have been mainly specified by histochemical observations. In this report, a technique of hepatocyte isolation is described which enables the separation of 2 cellular fractions, respectively, from the nodules and from the surrounding parenchyma of the same liver of a rat previously treated with a hepatocarcinogen. Such a technique allowed parallel analysis of both cellular populations by biochemical and cytochemical techniques.     

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.     

Further characterization of rat liver mitochondrial fractions. Lipid composition and synthesis, and protein profiles

Although large hemoglobin inclusions are observed in intraerythrocytic Babesia microti parasites, they are absent from parasites freed of hamster red cells by immune lysis with anti-hamster erythrocyte serum. Babesia microti has no cytostome. This parasite, therefore, does not appear to feed by phagocytosis of large boluses of hemoglobin, as does Plasmodium. To determine whether Babesia can pinocytose protein, free parasites were fed ferritin in an in vitro system. Ferritin was taken up from the entire cell surface into narrow channels within 15 min at 37 C. Only merozoites, with their pellicular complex, failed to take up the protein. By 60 min, the ferritin was highly concentrated in many channels and vesicles, which formed interconnecting stacks. The ferritin-containing channels became associated with membrane whorls of the multimembranous structure. Membrane whorls were also observed in the process of extrusion in samples incubated for longer times. These events may represent steps in the digestion and excretion of the pinocytosed protein. Empty channels formed when Babesia was fed albumin. The diaminobenzidine reaction for hemoprotein was positive for the channels in both free and intraerythrocytic babesias. The staining reaction was completely inhibited by cyanide, but not at all by aminotriazole. These results further suggest that Babesia pinocytoses hemoglobin in vivo. Plasmodium lophurae parasites freed of red cells by immune lysis are surrounded by 2 membranes and apparently can ingest ferritin only through the cytostome. Extracellular cytostomal feeding involves both membranes, as it does in vivo. Ferritin was found in food vacuoles, some of which contained hemoglobin ingested before parasite isolation, connected to or near the cytostome. In both Plasmodium and Babesia low temperature inhibited ferritin uptake.