Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K⁺ acetate.Abbreviations CMT cytomatrix - CSK cytoskeleton - DOC sodium deoxycholate - DTT dithiothreitol - EGTA ethylenglycolbis (-Ether)-N,N-N-N-Tetraacetic Acid - GFAP glial fibrillary acidic protein - PR polyribosome - PRCMC polyribosomes-cytomatrix complex     

Distribution of golgi apparatus-associated polyribosomes across the polarity axis of dictyosomes of wild carrot (Daucus carota L.)

Summary Endoplasmic reticulum-polyribosome-Golgi apparatus associations were a general feature of cells of suspension cultures of wild carrot (Daucus carota L.). Free polyribosomes occurred within the Golgi apparatus zone for all dictyosomes and with equal frequency at all levels within the stack including the most mature or trans face. When evaluated and quantified from electron micrographs, approximately 60% of the dictyosome profiles were characterized by a system of transition elements consisting of part smooth-part rough endoplasmic reticulum. These were encountered most frequently in the immediate vicinity of the immature, forming or cis face, usually toward the periphery of the stacked cisternae. Analysis of serial sections showed that those dictyosome profiles not exhibiting this characteristic did so primarily because of an unfavorable plane of sectioning. All dictyosomes examined in 5 or more serial sections revealed some type of close association with endoplasmic reticulum. Some of the associations were so close that direct connections between Golgi apparatus and endoplasmic reticulum tubules could not be excluded. Also present, especially at the forming or cis face, were small 600 nm transition vesicles with nap-like surface coats on nearly 90% of the dictyosomes examined. More than 50% exhibited spiny (clathrin-)coated vesicles at the mature or trans face.     

Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation

The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.     

Partial characterisation of and the effect of insect growth hormones on the ribosomes and polyribosomes of the nematode, Panagrellus redivivus

An investigation of some of the properties of the ribosomes and polyribosomes of Panagrellus redivivus revealed that: the polyribosomal RNA was resolvable into eight species, four of which possessed typical S-values and M.W.s and closely resembled those of Aphelenchus avenae; the estimated S-value of the ribosomes was 92; the polyribosomes were mainly free and not membrane-bound: and, the polyribosomes showed a low level of activity in in vitro amino-acid incorporation. The polysomes (double-labelled or unlabelled) revealed no effect of synthetic juvenile hormone or ß-ecdysone (1 × 10^?5M) on their polyribosomal profile, at intervals up to and including 19 h of incubation.     

Requirements for extraction of polyribosomes from lyophilised peel tissue of climacteric pear

Profiles of polyribosomes have been obtained from lyophilised peel tissue of climacteric pear (Pyrus communis cv Passe-Crassane) isolated in various buffers. Messenger RNA chains bearing up to 7 ribosomes (heptamers) were resolved and exhibited the highest absorption peak. High vacuolar concentrations of phenolics and acids, which are major obstacles in extracting fruit polyribosomes, were circumvented with the use of polyethylene glycol, insoluble polyvinylpyrrolidone (Polyclar AT), extraction at low temperature and high ionic strength buffer. Addition of Ca²⁺ to the extracting medium precipitated polysomes but ethyleneglycol bis(2-aminoethylether) tetra-acetic acid (EGTA), a divalent cation chelator with a high affinity for Ca²⁺, increases the proportion of polyribosomes.     

Synthesis of frog virus 3 proteins occurs on intermediate filament-bound polyribosomes

Immunogold labeling and biochemical methods were used to localize the site of viral protein synthesis in frog virus 3 (FV3)-infected baby hamster kidney (BHK) cells. Immunogold labeling studies of Triton-extracted (cytoskeletons), FV3-infected BHK cells with antivimentin antibodies showed that the major components of the detergent-resistant residue are the intermediate filaments (IF) and polyribosomes. Double immunogold labeling studies with anti-FV3 and antivimentin antibodies revealed that FV3 proteins are associated with polyribosomes that are bound to the IF network. Biochemical studies of the cytoskeletons prepared from FV3-infected cells showed that a substantial fraction of all newly synthesized FV3 proteins associate with the cytoskeleton and that the association is not disrupted by colchicine (microtubule-disrupting drug) or cytochalasin B (microfilament-disrupting drug). Together the above studies suggest that IF may provide the scaffold for the attachment of polyribosomes that are active in viral protein synthesis.     

TOR and S6K1 promote translation reinitiation of uORF‐containing mRNAs via phosphorylation of eIF3h

Mammalian target‐of‐rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF‐mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin‐1. Torin‐1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1—eIF3h—is phosphorylated and detected in polysomes in response to auxin. In TOR‐deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF‐mRNAs and eIF3h was impaired. Transient expression of eIF3h‐S178D in plant protoplasts specifically upregulates uORF‐mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.     

Electron microscopy and histochemistry of oncospheral hook formation by the cestode Catenotaenia pusilla

Swiderski Z. 1973. Electron microscopy and histochemistry of oncospheral hook formation by the cestode Catenotaenia pusilla. International Journal for Parasitology3: 27–33. Ultrastructure and histochemistry of oncospheral hook development in the cestode C. pusilla are described. The six anlagen of embryonal hooks appear in six specialized hook forming cells (oncoblasts) in the advanced phase of the preoncosphere.Electron microscopy shows a close connection of hook primordium with an abundance of free ribosomes, extended Golgi regions, and mitochondrial aggregations, evidently engaged in the hook morphogenesis. The shank completion occurs simultaneously with the progressive degeneration of hook forming cells, which completely disappears in the last phase of hook development. The mature oncospheral hook is a heterogeneous, bipartite structure composed of a dense outer sheath or cortex and a less dense inner core.Histochemistry shows evident changes in the reactivity for —SH, and —S—S— groups through the consecutive stages of hook development. The early hook anlage shows strongly positive reaction for sulfhydryl (—SH) groups (unconsolidated prekeratin), which remains in the subsequent stages mainly in the zone of keratinization, undergoing continuous displacement toward the base during hook maturation.The sulfhydryl groups of prokeratin through the oxidation process form bisulfite (—S—S—) links of mature hook keratin; reactivity for —SH groups completely disappears. The difference in electron density between the outer and inner part of the hook corresponds to a different reactivity for —S—S— links; the outer sheath shows evidently stronger reaction than the inner core.     

Elektronenmikroskopische Untersuchung der quergestreiften Muskelfasern im Corpus pineale von Wistar-Ratten

Zusammenfassung Unter 35 Pinealdrüsen von erwachsenen Wistar-Ratten wurden 8 mit quergestreiften Muskelfasern gefunden. Diese liegen in der Peripherie des Organs und in der Nähe des Pinealstiels. In 3 Fällen wurden direkte Kontakte zwischen Muskelfasern und Kapillaren gesehen.Die Pinealmuskelfasern besitzen ein Sarkolemma, das aus einer Basalmembran und einem retikulären Fibrillengitter aufgebaut ist. Mehrere Merkmale, die für embryonale Muskelfasern charakteristisch sind, werden in den Pinealmuskelfasern gefunden: Reichtum am Sarkoplasma, Armut an Myofibrillen, helikoidale Polyribosomen, unterschiedlich lokalisierte und häufig longitudinale Triaden, Übergang von granulärem in agranuläres sarkoplasmatisches Retikulum, Anzeichen mikropinozytotischer Prozesse, ungeordnete Myofilamente und primitiv gebaute A-Bänder. Die A- und I-Bänder sind in Längsschnitten gut sichtbar, während H-Zonen und M-Streifen meistens fehlen. Die Z-Streifen, gut ausgebildet nach jeder Fixation, sind nie reglemäßig und geradlinig wie in der Skelettmuskulatur. Die Länge einer Sarkomere beträgt 1,25–1,4 m nach OsO₄-Fixation und 2,4–3,1 m nach Aldehydvorfixation. Soweit elektronenmikroskopische Befunde ein Urteil erlauben, dürften die Pinealmuskelfasern — obwohl embryonalen Charakters — kontraktionsfähig sein.In einem Fall wurde ein wahrscheinlich (neuro)endokrino-muskulärer Kontakt zwischen einer Muskelfaser und einem Pinealzellausläufer gefunden.

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Electron microscopic investigation of the striated muscle fibers in the pineal body of wistar rats

Summary 8 of 35 pineal bodies obtained from adult male Wistar rats were found to contain striated muscle fibers. These are located at the periphery of the pineal body or in close proximity to the pineal stalk. In 3 cases a direct contact between capillaries and muscle fibers was observed.The pineal muscle fibers possess a sarcolemma which is formed by the basal membrane and a network of reticular fibers. Several features characteristic of embryonic muscle fibers are found in the fibers of the pineal body: abundance of sarcoplasm, scarce myofibrils, helicoidal polyribosomes, triads variously located and frequently longitudinally oriented, transition of rough- to smooth-surfaced endoplasmic reticulum, presence of micropinocytotic vesicles and primitively formed A-bands. A- and I-bands are well visible in longitudinal sections, while H- and M-bands are mostly lacking. The Z-lines, well formed after every fixation, are never regular and straight as seen in skeletal muscles. After fixation in OsO₄ the length of a sarcomere is 1,25–1,4 m, after aldehyde prefixation it is 2,4–3,1 m. According to electron microscopic findings the pineal muscle fibers, although being of embryonic character, seem to have the ability to contract.In one case a possibly (neuro)endocrino-muscular contact between a muscle fiber and a pineal cell process has been observed.

     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.