Tentacle morphogenesis in hydra

Summary Hydra oligactis exposed to 3 g/ml actinomycin D for 24 hours regenerated only the first pair of tentacles (the mid-laterals). If left uncut, actinomycin D treated animals underwent a reduction of the normal number of tentacles to two or less.Inductive activity was retained in the 2-tentacled hypostomes. However, the tentacles present exhibited reduced capacities to capture and manipulate prey.Histological studies showed that the tentacles of actinomycin D treated hydra were morphologically identical to those of the controls. The interstitial cell (I-cell) population of the treated animals, however, became depleted. Replacing the hypostome of an actinomycin D treated hydra with a normal hypostome reversed the cellular effects of actinomycin D treatment.The modifications in tentacle morphogenesis occurring after actinomycin D treatment are consistent with an impairment of hypostomal function in the animal. It is suggested that the morphological site of this malfunction may be in the nervous system.Research supported by American Cancer Society Institutional Grant No. 342-9157, USPHS Institutional Grant No. 342-9241 and by a grant from Research Corporation.Part of this work was completed while L.H. was an undergraduate research student supported by the National Science Foundation.     

Biosynthetic events of hydrid regeneration

Summary The results of a combined morphological and biochemical study of the role of DNA synthesis during distal regeneration inHydra oligactis revealed that a burst of³H-thymidine incorporation into DNA preceded the elaboration of each of the initial three tentacles. In addition, the relative level of each burst of precursor incorporation relfected the number of tentacles formed at that time. Cytological localization of concentrated amounts of labeled material in nuclei of the hypostome and tentacle regions provided corroborative evidence for the biochemical findings.Evidence that the increased DNA specific activity levels described above are associated with tentacle initiation derived from studies in which regenerating hydra were cultured in hydroxyurea and studies in which hydra regenerated proximally rather than distally. Hydra regenerating in 8 mg/ml (0.105 M) hydroxyurea developed morphologically recognizable hypostomes but no tentacles, and incorporated³H-thymidine into DNA at a level distinctly below that exhibited by uncut, untreated animals. Similarly, hydra regenerated a normal, functional basal disc in the absence of any increased DNA specific activity. Therefore, it is suggested that tentacle initiation inH. oligactis requires concomitant DNA synthesis and, as such, represents an epimorphic phenomenon.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata: I. Head-body proportion regulation

The precision with which an almost uniform sheet of hydra cells develops into a complete animal was measured quantitatively. Pieces of tissue of varying dimensions were cut from the body column of an adult hydra and allowed to regenerate. The regenerated animals were assayed for number of heads (hypostomes plus tentacle rings), head attempts (body tentacles), and basal discs. To ascertain whether the head and body were reformed in normal proportions, the average number of epithelial cells in the heads and bodies was measured. Pieces of tissue, from $\text{1}\text{2}$ to $\text{1}\text{20}$ an adult in size, formed heads that were a constant fraction of the regenerate. Thus, over a 10-fold size range, a proportioning mechanism was operating to divide the tissue into head area and body area quite precisely, but appeared to reach limits at the extremes of the range. However, the regenerates were not all normal miniatures with one hypostome and one basal disc. As the width-length ratio of the cut piece was increased beyond the circumference-length ratio of the intact body column, the incidence of extra hypostomes in the “head” and body tentacles and extra basal discs in the “body” rose dramatically. A proportioning mechanism based on the Gierer-Meinhardt model for pattern formation is presented to explain the results.     

OBSERVATIONS ON SUPERNUMERARY HEAD FORMATION INDUCED BY LITHIUM CHLORIDE TREATMENT IN THE REGENERATING HYDRA, PELMATOHYDRA ROBUSTA

Lithium chloride treatment of hydras cut just proximal to the tentacle circle and just distal to the budding region induces a supernumerary head at the proximal cut surface. Such a supernumerary head does not appear in the normal course of regeneration. The bipolar hydra thus formed persists for several weeks and later separates to form two normal individuals. The supernumerary head is not formed at the proximal cut surface when the hydra is transected just distal to the budding zone and the distal portion is allowed to regenerate in the Li-containing medium. LiCl has a slight inhibitory effect on the regeneration of hypostomes or tentacles when the animal is cut at the base of the hypostome.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Acute activation of NHE3 by dexamethasone correlates with activation of SGK1 and requires a functional glucocorticoid receptor

Glucocorticoids stimulate the intestinal absorption of Na⁺ and water partly by regulation of the Na⁺/H⁺ exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner. Na⁺/H⁺ exchanger 3; serum and glucocorticoid-inducible kinase 1     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA : II. Ultrastructure

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.     

Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells.

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell-based experiments, we used SAA (normal hEGFR-overexpressed NIH3T3) cells and B104-1-1 (neu*-transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D-treated cell lysates with polyclonal anti-Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti-Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose-dependent manner in both B104-1-1 and EGF-stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104-1-1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras-MAP kinase signaling pathway. These results suggest that actinomycin D could be a non-phosphorylated natural and cellular membrane-permeable SH2 domain antagonist.     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Interaction of protein phosphatase 1 delta with nucleolin in human osteoblastic cells.

We examined the expression and cytolocalization of the protein phosphatase type 1 delta (PP1delta) isoform and nucleolin in human osteoblastic MG63 and Saos-2 cells. Cellular fractionation of MG63 cells was done and protein was prepared from each fraction. Anti-nucleolin antibody interacted with the 100- and 95-kD proteins present in the whole-cell lysate. The 100-kD protein was detected in nuclear and nucleolar fractions. The 95-kD protein was detected in cytosolic and nucleoplasmic fractions. PP1delta and nucleolin were co-localized in the nucleolus in MG63 and Saos-2 cells revealed by an immunofluorescence method. PP1delta and nucleolin were also co-immunoprecipitated with anti-nucleolin and anti-PP1delta antibodies. In the actinomycin D-treated cells, the subcellular localization of PP1delta and nucleolin was changed. Expression of PP1delta was upregulated with actinomycin D treatment. The level of 100-kD protein did not change in the actinomycin D-treated cells. However, the level of the 95-kD band increased with actinomycin D treatment. These results indicate that PP1delta was associated with nucleolin in the nucleolus of MG63 and Saos-2 cells and that nucleolin is a possible candidate substrate for PP1delta.     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Regulation in the numbers of tentacles of aggregated hydra cells

Head formation was investigated during regeneration of dissociated and aggregated cells of Hydra magnipapillata. The surface area measured at the hollowing stage was found to be a useful quantity for characterizing the size of an aggregate. Four kinds of aggregates were examined, using tissue originating from (1) whole animals, (2) apical halves, (3) decapitated animals, and (4) decapitated animals allowed to regenerate for several hours before dissociation. For aggregate types (1), (2), and (4), not all the tentacles observed at an intermediate stage of the regeneration process were localized around hypostomes: the number of such body tentacles at the intermediate stage was comparable to that of the hypostomal tentacles and was approximately proportional to the surface area. These results and others suggest that the formation of body tentacles takes place independently of hypostome formation. However, for aggregate type (3), most of the tentacles appearing at the intermediate stage were hypostomal. The correlation between the surface area and the number of tentacles at the steady state apparently resulted from a regulation process by which body tentacles decreased and hypostomal tentacles increased. It is considered that the number of body tentacles appearing at an intermediate stage of regeneration would depend on the initial level of head-activation potential and that body tentacles are formed by the local fluctuation of head-activation potential.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Achromosomal Division of Early Starfish Embryos Cultured in the Presence of Actinomycin D

Embryos of the starfish Asterina pectinifera were examined for their ability to undergo the early events of embryonic development in the presence of actinomycin D, a most widely used inhibitor of RNA synthesis. Fertilized eggs continued to divide eight or nine times in the presence of 25 μg ml⁻¹actinomycin D, although delay of development was observed. Chromatin disintegrated in the blastomeres of actinomycin D-treated embryos specifically at the 32-cell stage and the nucleus was undetectable at later stages. Before the 32-cell stage, RNA synthesis was not affected by the presence of actinomycin D whereas DNA synthesis was severely inhibited. The stage when achromosomal divisions cease and embryos begin to die corresponds to the period just before onset of blastulation, suggesting that the presence of the nucleus and chromosomes is a prerequisite for blastula formation and development beyond the 512-cell stage in this species.     

The initiation of growth in isolated lima bean axes. Physiological and fine structural effects of actinomycin D, cycloheximide and chloramphenicol.

Excised lima bean axes were germinated for 24 hr in water, variousconcentrations of CHI, CAP or actinomycin D. None of the inhibitorsaffected water uptake before onset of growth. Growth was completelyinhibited by CHI and CAP, but only partly by actinomycin D.Rate of ³H-leucine incorporation rose during the lag phase inwater uptake and increased further during growth. The incorporationwas inhibited by CAP and CHI at all stages, but by actinomycinD only during growth. Actinomycin D prevented developmentalfine structural changes. In CHI treated axes number of mitochondriaincreased, O₂2 uptake was reduced, and higher cytochrome oxidaseactivity induced. CAP caused changes in the appearance of proteinvacuoles, but apparently prevented loss of storage material.The relation between the physiological and structural effectsof the inhibitors is discussed. (Received June 13, 1970; )     

Physiology of rat-liver polysomes: Protein synthesis by stable polysomes

Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.     

Changes in the Synthesis of RNA and Protein during Tracheary Element Differentiation in Single Cells Isolated from the Mesophyll of Zinnia elegans

Cycloheximide (CH) prevented tracheary element (TE) differentiationand cell division in a culture of single cells isolated fromthe mesophyll of Zinnia elegans at the concentrations whichinhibited incorporation of [¹⁴C]-leucine into protein. Whenthe cells were pulse-treated with this inhibitor for 12 h atvarious times of culture, TE formation was inhibited most stronglyby the treatments made between 24 and 60 h of culture. Incorporationof [¹⁴C]-leucine into protein showed a high level during thisperiod. The inhibitory effect of actinomycin D (Act-D) on TEdifferentiation was also marked when it was administered from24 to 60 h of culture when incorporation of [¹⁴C]-uridine intonucleic acid was at a high level. These results indicate thatRNA and protein syntheses are prerequisites for cytodifferentiationto TE and that the syntheses between 24 and 60 h of cultureare closely associated with cytodifferentiation. Studies of qualitative changes in proteins using two-dimensionalelectrophoresis revealed that approximately 400 polypeptidesextracted from [³⁵S]-methionine-labeled cells could be reproduciblyresolved and that most of them were synthesized in both differentiatingand non-differentiating cells. During TE differentiation, however,the synthesis of two polypeptides was shut off and two otherpolypeptides were newly synthesized between 48 and 60 h of culture,preceding the morphological changes. The relationship betweenTE differentiation and the synthesis of RNA and protein is discussed. (Received November 20, 1982; Accepted February 18, 1983)     

Two types of surface amine receptors modulating the feeding response inHydra japonica: the depressing action of dopamine and related amines

The effect of amines on the feeding response of Hydra japonicawas determined using the tentacle ball formation assay. Manyaliphatic and aromatic amines depressed the response immediatelyupon addition to the bathing medium at final concentrationsranging from 1 nmol/l to 100 µmol/l. Examining the depressingeffect of various amines, we concluded that two kinds of aminereceptors were located on the surface of Hydra. One perceiveddopamine and D-noradrenaline, and depressed only the responseelicited by weak stimuli. Dopamine depressed the response at1 nmol/l. D,L-Propranolol was a competitive antagonist againstthis receptor. The other perceived tyramine and most of theamines examined, and depressed the response elicited by bothweak and strong stimuli. Tyramine depressed the response at1 µmol/l. D,L-Propranolol did not block its action. Afterthe animals were treated with dibenamine, both dopamine andtyramine became ineffective in depressing the response withoutinfluence on the response of the animal elicited by S-methylglutathione. ^*Present address: Tokyo Laboratory of Japan Synthetic RubberCo. Kawasaki 215, Japan     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.