Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Phosphorylation of Chromosomal Proteins in Resting and Wounded Potato Tuber Tissues

Wounding of quiescent white potato tuber tissue enhances chromatin-boundprotein phosphokinase activity, which exhibits two distinctphases during wound-healing. A moderate activation of the enzymesup to 20 hr after injury is followed by a dramatic increasein activity with a peak at 50 hr. This time-course resemblesthat of chromatinbound DNA-dependent RNA polymerase with a peakin activity at about 48 hr after wounding. The kinases phosphorylateendogenous proteins as well as added histones, phosvitin andcasein. The incorporated phosphate is stable under standardassay conditions, indicating the absence of protein phosphatases.Sensitivity of the incorporated phosphate toward trypsin andalkali, but not DNase, RNase, hydroxylamine or succinic acidpoints to seryl- and threonyl-bonds and proteins as acceptormolecules. Kinases from resting tissues are only weakly stimulatedeven by 100 mM MgCl₂, those from wounded tissues exhibit pronouncedMg^$$-optima at 5–10 mM with endogenous proteins, phosvitinand casein and 50 mM MgCl₂ with histones. Wounding also increasesthe sensitivity of the kinases toward p-hydroxymercuribenzoate. Chromatin preparations from both resting and wounded tissuescontain about 40 protein bands after polyacrylamide disc gelelectrophoresis. In vitro phosphorylation of these proteinsin chromatin from quiescent tissues is comparably low and uniform.Wounding induces changes in the protein and phosphorylationpattern with a general enhancement of phosphorylative capacityand preferential phosphorylation of low molecular weight proteins. (Received August 10, 1981; Accepted November 18, 1981)     

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-l-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.Abbreviations BMT S-adenosyl-l-methionine: bergaptol O-methyltransferase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Insulin regulation of insulin-like growth factor action in rat hepatoma cells

We have reported previously that insulin causes a complete but reversible desensitization to insulin action in rat hepatoma HTC cells in tissue culture, and that this insulin resistance is mediated by postbinding mechanisms rather than receptor down-regulation (Heaton, J. H., and Gelehrter, T. D. (1981) J. Biol. Chem. 256, 12257-12262). We report here that insulin causes a similar desensitization to the induction of tyrosine aminotransferase by the insulin-like growth factors IGF-I and IGF-II isolated from human plasma, and by multiplication-stimulating activity, the rat homologue of IGF-II. The results of both competition-binding studies and affinity cross-linking experiments indicate that insulin-like growth factors (IGFs) bind primarily to IGF receptors rather than to insulin receptors. The low concentrations at which these factors induce transaminase is consistent with their acting primarily via IGF receptors. This is confirmed by experiments utilizing anti-insulin receptor antibody which both inhibits 125I-insulin binding and shifts the concentration dependence of insulin induction of tyrosine aminotransferase to the right. This same immunoglobulin does not inhibit 125I-multiplication-stimulating activity binding and only minimally inhibits 125I-IGF-I binding. Anti-insulin receptor antibody also does not significantly shift the concentration dependence for the IGFs, suggesting that IGFs induce transaminase by acting via IGF receptors. Although insulin down regulates insulin receptors, it does not decrease IGF-I or IGF-II binding. We conclude that insulin causes desensitization of HTC cells to IGFs by affecting a postbinding step in IGF action, which may be common to the actions of both insulin and insulin-like growth factors.     

Das Bewegungsverhalten der Coelomzellen vonPsammechinus miliaris bei der Wundheilung (Echinodermata)

Zusammenfassung 1. Das Bewegungsverhalten der Coelomzellen des EchinoidenPsammechinus miliaris Gmel. wird an kleinen von Stacheln, Füßchen und Pedicellarien befreiten Stellen der Skelettoberfläche in Periproctnähe untersucht.2. Aus dem freiliegenden Bälkchenwerk treten Coelomzellen aus, von denen nur die rotbraunen Amoebocyten auf dem hellen Kalkuntergrund im Auflicht (Ultropak;E. Leitz) sichtbar sind.3. Nach einigen Stunden ist die Wundfläche mit einer dicken rötlichen Zellmasse bedeckt, dem primären Wundverschluß. Außer den Coelomzellen enthält der Wundverschluß noch verschieden große Kalkpartikel, die vom Abschleifen der Versuchsstelle herrühren.4. Bei direkter Beobachtung ist weder an den rotbraunen Amoebocyten noch am Wundverschluß die geringste Bewegung zu erkennen.5. Zeittransformation (Zeitraffung [Z.R.] auf 1/240 und 1/480) zeigt die mit erheblicher Ortsverlagerung und Metabolie verbundene Bewegung der allein wahrehmbaren rotbraunen Amoebocyten auf der Wundfläche. Im scheinbar in Ruhe befindlichen Wundverschluß findet eine ständig hin- und herwogende Bewegung der Zell-Kalkamsse statt.6. Bereits nach 6 bis 7 Stunden ist das Operationsfeld völlig geglättet; die Lücken im Kalkskelett sind kaum noch zu erkennen infolge der neu eingebauten Kalkelemente. Die eigentlichen Heilungsvorgänge (Wiederherstellung der Feinstruktur des Kalkskelettes) erfolgen unterhalb des primären Wundverschlusses, sind also nicht der Beobachtung zugängig.7. Wird der primäre Wundverschluß im ganzen vorsichtig abgehoben und zerzupft, so kann das Bewegungsverhalten der entstandenen kleinen und großen Aggregate im Durchlicht unter Z.R. untersucht werden.8. Die im zerriebenen Explantat erhaltenen Coelomzell-Aggregate aller Größen weisen erhebliche Ortsveränderungen auf; oft breiten sie sich langsam aus unter Auswanderung zehlreicher randlich liegender Zellen. An den Außenzonen mittlerer und großer Aggregate werden plasmatische Netze sichtbar, die ständig ihre Gestalt und Maschenweite ändern.9. Diese Plasma-Netze bilden die Grundlage der Aggregate; ihre Kontraktionen und Dilatationen bewirken die Ortsverlagerungen der Aggregate (Netzbildende Coelomzellen;Kuhl 1937).10. Wenigzellige Aggregate vereinigen sich in den allermeisten Fällen, sobald ein gewisser Abstand überbrückt ist. Mittlere und große Aggregate gehen häufig eine Verbindung ein; meist werden vorher lockere Coelomzell-Brücken hergestellt. In manchen Fällen gleiten die Aggregate aneinander vorbei.11. Im polarisierten Licht lassen sich bei gekreuzten Nicols die ersten kleinen neugebildeten Kalkkristalle in den skelettbildenden Coelomzellen (= netzbildenden Zellen) nachweisen.12. Der Verschluß kleiner Kratzwunden im noch dünnen primären Wundverschluß (die Kratzer dringen bis zur abgeschliffenen Skelettoberfläche vor) wird unter Z.R. im Ultropak-Auflicht untersucht. Die Ergebnisse am explantierten Wundverschluß im Durchlicht führen zum Verständnis der Bewegungsvorgänge im ungewohnten Auflicht.13. Im zunächst verwirrenden Bewegungsgeschehen (die auffälligen rotbraunen Amoebocyten haben bei der Wundheilung keine Funktion) fallen die durch die Operationsnadel herausgerissenen kleinen Kalktrümmer auf; sie werden passiv durch die Plasmanetze bewegt, gelangen auch zufällig in die Kratzer und werden an den Rändern durch neugebildetes Kalkmaterial festgelegt oder eingebaut. Aus der Tiefe der Kratzer können lose Kalkpartikel heraufbefördert werden; auch diese werden häufig eingebaut. Die entstehenden Kalkbrücken werden schließlich untereinander verbunden und dadurch die kleine Wunde verschlossen. Das eingebaute Kalkmaterial zeigt auch unter starker Z.R. keine passive Bewegung mehr.14. In seltenen Fällen kann der Vorgang des schubweisen Aufsteigens der skelettbildenden Zellen aus dem Panzer und ihre Zusammenballung im Z.R.-Laufbild beobachtet werden.15. Ob der Einbau von herausgerissenem Kalkmaterial temporär oder dauernd ist, muß noch geprüft werden.

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The locomotory behaviour of coelom cells ofPsammechinus miliaris (Echinodermata) during wound-healing

In the sea urchinP. miliaris application of time lapse photography allows a study of the very slow movements of coelom cells during the healing process of small wounds on the surface of the calcareous skeleton near the periproct. For observation and time lapse photography LEITZ-Ultropak objectives were used (incident light). Ambulacral feet, spines and pedicellaria were removed, and the animal was fixed in three places in a ring of plexiglass by means of three little screws, which touched the equator of skeleton. The rate of time transformation was 1/240 to 1/480. The film reveals the behaviour of coelom cells, which move out the skeleton to the surface of the small experimental region. Within several hours the white polished surface is covered with hundreds of red-brown amoebocytes; only these are visible on the white lime-ground; they have no function in the healing process, which takes place below the surface of the primäre Wundverschluß and therefore cannot be observed. There are three main types of coelom cells: red-brown amoebocytes, körnchenführende Zellen (white amoebocytes) and leucocytes (netzbildende or skelettbildende Zellen); the flagellated cells may be neglected here. In order to be able to study the behavior of the three main types of coelom cells, the primäre Wundverschluß, i. e. the total cell-covering of the wound, is removed and torn into microscopic fragments. These are studied (time lapse) under normal optical conditions (transmitted light). The slides show many aggregates of different sizes, single cells and little calcareous concrements torn off the skeleton. The aggregates, even the big ones, exhibit slow locomotion and change their positions considerably. If the distance of two aggregates becomes small enough, they fuse. In these cases a loose cell bridge between the two aggregates is formed. Sometimes no union occurs, although the distance is very small. Even big aggregates suddenly show considerable contractions if spreading has preceded. All movements and place changing of cell-aggregates are caused by contractions and dilatations of the plasmatic network which forms the cellular basis. Little wounds in the newly built Wundverschluß scratched with a lancet, heal within several hours. Time lapse shows passive movements of small calcareous fragments, which by chance sometimes enter the small wounds, where they help and accelerate the closing of the injury. The fragments are fixed on the edge of the wound by newly produced lime. Skeleton building coelom cells (netzbildende Coelomzellen) come up in batches from the depth of the sea urchin's skeleton; each cell contains lime crystals.