Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice.

Transgenic mice overexpressing the human growth hormone gene develop mammary carcinomas. Since human growth hormone gene can activate both the growth hormone receptor (GHR) and the prolactin (PRL) receptor (PRLR), it is not clear which receptor system is responsible for the malignant transformation. To clarify the receptor specificity, we created transgenic mice with two different genes: (a) transgenic mice overexpressing the bovine growth hormone (bGH) gene having high levels of bGH only activating the GHR and also high serum levels of IGF-I; and (b) transgenic mice overexpressing the rat PRL (rPRL) gene that have elevated levels of PRL (one line 150 ng/ml and one line 13 ng/ml) only binding to the PRLR and with normal IGF-I levels. When analyzed histologically, all of the PRL transgenic female mice developed mammary carcinomas at 11-15 mo of age. Only normal mammary tissue was observed among the bGH transgenic animals and the controls. Cell lines established from a tumor produced rPRL and expressed PRLR. In organ culture experiments, an auto/paracrine effect of rPRL was demonstrated. In conclusion, activation of the PRLR is sufficient for induction of mammary carcinomas in mice, while activation of the GHR is not sufficient for mammary tumor formation.

Localization of ornithine decarboxylase in mouse teeth. An in vitro and in vivo study.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, and thus in tissue growth and development, has been localized in mouse dental tissues, in vivo as well as in vitro by light and electron microscopic autoradiography with radiolabeled alpha-difluoromethylornithine ([3H]DFMO). Mandibular first molar germs from day-18 fetuses were incubated in vitro in the presence of [3H]DFMO and processed for autoradiography. For ODC localization in vivo, 3-day old puppies received [3H]DFMO by injection. As controls, puppies were injected either with unlabeled DFMO, or with cycloheximide before administration of isotope. Kidneys and mandibles were excised and processed for autoradiography. In vitro, labeling was found in all cell types of the tooth germ, but with a more intense labeling in ameloblasts and odontoblasts. In both these, radioactivity decreased from the tip of the cusps to the cervical loop. In vivo the binding of [3H]DFMO in cells of the ameloblast and odontoblast lineages, respectively, showed a gradual increase form the posterior end of the incisor to its anterior end. The distribution of radioactivity in the kidney was in accordance with findings by others. Both the kidney and tooth cell labeling decreased strongly after cycloheximide treatment. The results show that ODC is expressed in tooth-forming cells, and that ODC is not only present in differentiating cells but occurs at higher amounts in mature, secreting cells. The findings suggest that polyamines have a central role in tooth development.

Polyamine depletion-mediated effects on murine odontogenesis are dependent on tooth developmental stage and culture conditions.

Polyamines are known to play a central role in processes such as growth and development. Virtually nothing is known about their importance in tooth development, an attractive and frequently used experimental model for studies of developmental processes. A polyamine-depleted state was created in tooth cells in an organ culture system. First lower molar germs from 16-and 17.5-day old mouse fetuses were used. alpha-difluoromethylornithine (DFMO) and methylglyoxal bis-(guanylhydrazone) (MGBG) were used to deplete the cells from their polyamine content. Polyamine interconversion and catabolism were prevented by aminoguanidine sulfate (AG). In day-16 germs cultured in serum-containing medium, DFMO reduced the frequency of cycling cells as shown by [3H]thymidine incorporation, and induced a delay of odontoblast differentiation of about 24 h. Under the same conditions, MGBG induced an arrest of histo-morphogenesis, correlated to a significant decrease in the rate of cell proliferation. Addition of polyamines prevented DFMO- and MGBG-induced delay of tooth differentiation. Interestingly, MGBG did not delay the terminal differentiation of odontoblasts and ameloblasts in cultured day-17.5 molars; in these, cells at the tip of the cusps are only a few hours before their withdrawal from the cell cycle. In serum-deprived medium, dental cytodifferentiations did not occur. Addition of putrescine or spermidine to serum-free media, however, allowed for tooth morphogenesis and cytodifferentiation. Tooth explants in a serum-deprived medium reacted to DFMO in a cytocidal fashion, whereas MGBG showed only a mild toxicity in some cell types. Addition of putrescine to DFMO-containing medium prevented its cytotoxic effect. Addition of spermidine to MGBG-containing medium not only prevented its mild toxicity but also allowed for predentin secretion by differentiated odontoblasts. The results are discussed with regard to the well-established developmental events of tooth germs cultured in vitro and with respect to present knowledge of polyamine metabolism and their involvement in cellular processes.

Enhanced spontaneous locomotor activity in bovine GH transgenic mice involves peripheral mechanisms.

Clinical and experimental studies indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. Recently we showed that transgenic mice with general overexpression of bovine GH display increased spontaneous locomotor activity. In the present study, we investigated whether this behavioral change is owing to a direct action of GH in the central nervous system or to peripheral GH actions. A transgenic construct, containing the glial fibrillary acidic protein promoter directing specific expression of bovine GH to the central nervous system, was designed. The central nervous system-specific expression of bovine GH in the glial fibrillary acidic protein-bovine GH transgenic mice was confirmed, but no effect on spontaneous locomotor activity was observed. Serum bovine GH levels were increased in glial fibrillary acidic protein-bovine GH transgenic mice but clearly lower than in transgenic mice with general overexpression of bovine GH. In contrast to the transgenic mice with general overexpression of bovine GH, glial fibrillary acidic protein-bovine GH mice did not display any difference in serum IGF-I levels. The levels of free T(3) and the conversion of the free T(4) to free T(3) were only increased in transgenic mice with general overexpression of bovine GH, but serum corticosterone levels were similarly increased in both transgenic models. These results suggest that free T(3) and/or IGF-I, affecting dopamine and serotonin systems in the central nervous system, may mediate the enhanced locomotor activity observed in transgenic mice with general overexpression of bovine GH.

Effects of chronically elevated growth hormone levels on polyamine metabolism in elderly transgenic mice.

The polyamines are ubiquitous, multifunctional aliphatic amines with roles in cell growth, proliferation, differentiation, and malignant development. After growth stimulation, rapid and transient changes occur in polyamine regulatory enzymes. In this respect, acute effects of growth hormone (GH) injection on polyamine metabolic enzymes have earlier been shown. The present investigation comprises studies of the effects on polyamine metabolism of constitutively elevated levels of circulating GH in elderly transgenic (tg+) mice, overexpressing bovine GH. Polyamine levels were found to be constitutively altered in the liver and kidney of tg+ mice. Less changes were found in the spleen and none in the brain. The cellular uptake of polyamines in the liver from tg+ mice showed an increase and considerable changes were observed in the activity of ornithine decarboxylase (ODC) in the liver and kidney and S-adenosylmethionine decarboxylase (AdoMetDC) in the liver. A conspicuous finding was the distribution pattern of ODC protein in the liver and both tg- and tg+ animals. The results show that the effects of chronically elevated GH levels are organ-dependent and complex, and differ from acute GH effects. Despite high ODC activity and polyamine levels in liver, these mice did not display any malignant transformation even at an advanced age, indicating that high ODC activity is not sufficient to induce tumorigenesis in vivo.

Healing of mandibular defects with different biodegradable and non-biodegradable membranes: an experimental study in rats.

Membranes, clinically used to improve bone regeneration according to the osteopromotion principle, have primarily been made of expanded polytetrafluoroethylene (Gore-Tex Membrane). Recently, different types of biodegradable membranes have become available. This investigation explored the osteopromotive potential of 10 different biodegradable and non-biodegradable membrane materials. Scanning electron microscopy revealed quite different surface configurations of these membranes, even though some of them were chemically closely related. Standardized, transosseous, critical size mandibular defects were made bilaterally in adult rats and were randomly covered with the different types of membrane. After 6 wk of healing, evaluation was performed by light microscopy according to a histological scoring system. Varying degrees of bone healing were seen beneath the different membranes. Some of the membranes (such as Gore-Tex Augmentation Material, Millipore and Resolut 'long term') revealed a good osteopromotive effect, whereas others had little or no beneficial effects on bone healing. Certain membrane materials caused a pronounced inflammatory response in the surrounding soft tissue, while others displayed a low inflammatory reaction. The study shows that different membranes differ strongly in osteopromotive efficacy, even if seemingly chemically closely related. Furthermore, the study demonstrates that membranes developed primarily for periodontal regeneration purposes may not be adequate to promote bone healing.

Extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca2+ second messenger response in osteoblastic cells.

Surgical removal of the acid-producing part of the stomach (oxyntic mucosa) reduces bone mass through mechanisms not yet fully understood. The existence of an osteotropic hormone produced by the so-called ECL cells has been suggested. These cells, which are numerous in the oxyntic mucosa, operate under the control of circulating gastrin. Both gastrin and an extract of the oxyntic mucosa decrease blood calcium and stimulate Ca2+ uptake into bone. Conceivably, gastrin lowers blood calcium indirectly by releasing a hypothetical hormone from the ECL cells. The present study investigated, by means of fura-2 fluorometry, the effect of extracts of preparations enriched in ECL cell granules/vesicles from rat oxyntic mucosa on mobilization of intracellular Ca2+ in three osteoblast-like cell lines, UMR-106.01, MC3T3-E1 and Saos-2, and of extracts of isolated ECL cells in UMR-106.01 cells. The extracts were found to induce a dose-related rapid increase in intracellular Ca2+ concentrations in the osteoblast-like cells. The response was not due to histamine or pancreastatin, known ECL cell constituents, and could be abolished by pre-digesting the extracts with exo-aminopeptidase. The results show that the increase in [Ca2+](i) reflects a mobilization of Ca2+ from the endoplasmic reticulum. The observation of an increase in [Ca2+](i) also in murine embryonic fibroblasts show that the response is not limited to osteoblastic cells. The finding that the extracts evoked a typical Ca2+ -mediated second messenger response in osteoblastic cells provides evidence for the existence of a novel osteotropic peptide hormone (gastrocalcin), produced in the ECL cells, and supports the view that gastrectomy-induced osteopathy may reflect a lack of this hormone.

Opposing effects of suramin and DL-alpha-difluoromethylornithine on polyamine metabolism contribute to a synergistic action on B16 melanoma cell growth in vitro.

Polyamines are crucial for normal and neoplastic cell growth. Treatment with the polyanionic drug suramin has pronounced antigrowth activity in several tumor cell lines, but its clinical use has been hampered by its toxicity. We have earlier shown that suramin affects cellular polyamine metabolism and transport, and that these effects were, in some respects, opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor to ornithine decarboxylase, a key metabolic enzyme for polyamines. DFMO has been used in anticancer trials, although with limited success. Combinations of suramin and DFMO were, hence, evaluated in vitro and were found to strongly inhibit B16 melanoma cell proliferation. DFMO alone induced melanoma cell differentiation, and suramin used in combination with DFMO did not abrogate this DFMO-induced differentiation. Synergy analysis demonstrated a pronounced growth-inhibitory synergism between suramin and DFMO. The results suggest that the efficacy of combinations of DFMO with suramin or its analogues should be further explored, especially in cells requiring high levels of polyamines for their growth.

Effects of suramin on polyamine metabolism in B16 murine melanoma cells.

Polyamines and their biosynthetic enzymes, such as ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are crucial for normal and neoplastic cell growth and differentiation. Suramin inhibits the growth of several tumor cells by affecting various intracellular targets, but its effects on polyamines are not known. In this study, the effects of suramin on some parameters of polyamine metabolism in B16 melanoma cells were investigated in vitro. Suramin increased cellular ODC activity and ODC mRNA levels, whereas the drug was directly inhibitory to the enzyme. AdoMetDC was not affected. Cellular putrescine levels were enhanced by suramin, whereas spermidine and spermine pools were unaltered. Cells cultured in the presence of suramin showed decreased cellular polyamine transport, but no direct inhibitory effect on the polyamine transporter could be found. Fluorescence spectroscopy demonstrated a direct interaction between suramin and spermine. It may be concluded that suramin affects polyamine metabolism, and that its effects in some respects are opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor of ODC.

Evc regulates a symmetrical response to Shh signaling in molar development.

Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.

Morphological alterations in dental and periodontal tissues in murine mucopolysaccharidosis type VII.

Mucopolysaccharidoses (MPSs) in humans are frequently associated with tooth and periodontal aberrations. Although the cause is known, namely, enzyme deficiency, the pathophysiology of these alterations is not well defined. A murine MPS VII (beta-glucuronidase deficiency) model has earlier been identified with morphological, genetic, and biochemical characteristics that closely mimic those of human MPS VII. The present investigation describes the histopathological alterations in dental and periodontal tissues from such mutant mice. Homozygous animals were identified by external phenotypical features and as being beta-glucuronidase deficient by a fluorometric assay of liver samples. In the incisor and the periodontium, abnormalities were evident in both cells and the extracellular matrices. Mesenchyme-derived cells were more aberrant than epithelial cells. Moreover, undifferentiated cells appeared unaffected, whereas actively synthesizing and resorbing cells were distended by virtually empty or granular material-containing vacuoles, the content presumably being glycosaminoglycans. The cells most affected were those in which macromolecular turnover is normally the highest, namely, odontoblasts, postsecretory ameloblasts, and periodontal ligament fibroblasts. Extracellularly, predentin displayed abnormal collagen fibrils, whereas mineralization defects occurred in both dentin and enamel. This murine model of MPS VII provides a good tool for understanding the pathophysiology of this disease in bone, periodontium, and teeth.

Localization of S-adenosylmethionine decarboxylase in murine tissues by immunohistochemistry.

S-adenosylmethionine decarboxylase (AdoMetDC), one of three key enzymes in polyamine biosynthesis, is present in minute concentrations in adult tissues, whereas increased AdoMetDC activity occurs in conjunction with differentiation and growth. The occurrence of AdoMetDC in tissues has earlier been studied by biochemical technique only. In this work, an antiserum against recombinant human AdoMetDC was used to investigate the localization of the enzyme in different mouse tissues. In all tissues studied, immunolabeling was seen in cytoplasm, while cell nuclei were negative. In the kidney, AdoMetDC immunoreactivity was confined to the inner part of the cortex; the outer part of the cortex and the medulla displayed only a weak AdoMetDC immunoreaction. In the cortex, renal tubules were strongly reactive, while renal corpuscules were weakly immunolabeled. In developing teeth, AdoMetDC immunoreactivity displayed a gradient of distribution, the immunolabeling intensity being increased from the cervical region to the tip of the cusps. In the incisor, post-secretory ameloblasts were strongly labeled. In the oral mucosa, epithelial cells demonstrated stronger immunolabeling than did cells of the subjacent connective tissue. As for muscle cells, immunoreactive material was confined to the periphery of the fibers. In vitro, treatment with DL-alpha-difluoromethylornithine (DFMO) or methylglyoxal bis-(guanylhydrazone) (MGBG) led to an increase in AdoMetDC. It can be concluded that antibodies to recombinant human AdoMetDC provide a tool for the immunohistochemical localization of AdoMetDC, and that the distribution of the enzyme in the tissues studied gives further support to the importance of polyamines in the development and functions of these organs.

Skin fibroblasts from spermine synthase-deficient hemizygous gyro male (Gy/Y) mice overproduce spermidine and exhibit increased resistance to oxidative stress but decreased resistance to UV irradiation.

Hemizygous gyro male (Gy/Y) mice are a model for X-linked hypophosphataemic rickets. As in humans, the disease is caused by deletions in the Phex gene, a phosphate-regulating gene having homologies with endopeptidases on the X chromosome. Some phenotypic abnormalities in Gy/Y mice have recently been attributed to the fact that the Gy deletion also includes the neighbouring spermine synthase gene, resulting in spermine deficiency. Spermine and its precursors spermidine and putrescine are essential for cell growth and differentiation. As a novel method for studying the function of spermine, we established primary cultures of skin fibroblasts from hemizygous Gy/Y mice. The Gy/Y cells contained no detectable spermine. In view of the fact that spermine is a free-radical scavenger in vitro, we were surprised to find that Gy/Y cells were more resistant to oxidative stress than their normal (X/Y) counterparts. However, our finding that spermidine accumulates markedly in the spermine-deficient Gy/Y cells can probably explain this increased resistance. It is the first indication that spermidine can serve as a free-radical scavenger in vivo and not only in vitro. When subjecting the Gy/Y cells to UV-C irradiation we made another interesting finding: the mutant cells were more sensitive than the normal X/Y cells. This finding indicates that spermine, probably because of its high-affinity binding to DNA, is important in protection against chromatin damage.

Na+/Ca2+ exchanger isoforms of rat odontoblasts and osteoblasts.

In odontoblasts as well as osteoblasts, a number of mechanisms for the inflow and extrusion of Ca2+ have been demonstrated. The entrance of Ca2+ ions into odontoblasts occurs mainly through voltage-gated calcium channels. Extrusion of Ca2+ is found to be an ATP-dependent process and, in addition, Na+/Ca2+-antiports exist, which are provoked by extracellular Na+. The aim of this study was to identify the Na+/Ca2+-antiport isoforms expressed in dentinogenically active rat incisor odontoblasts and to make a comparison with different osteoblastic cells. Using RT-PCR and RNAse protection assay, we demonstrated the expression of three different isoforms, NaCa 3, 7, and 10, of the NCX1-encoded antiport in odontoblasts and osteoblastic cells. When incubated in the presence of Na+, dissected rat incisor odontoblasts as well as the osteoblastic cells extruded Ca2+ ions, as detected by chlorotetracycline and Fura-2 fluorometry, thus supporting a physiological role for the detected isoform expression. Odontoblasts and rat calvarial osteoblasts, as well as osteoblast-like cell lines UMR-106.01 and Saos-2, were shown to exhibit identical phenotypes of Na+/Ca2+-antiport isoform expression, different from the expression patterns of other tissues. The significance of this specific expression pattern is unknown, but there is a possibility that it is in some way related to the unique demands on these cell types to produce mineralized connective tissue.

Effects of suramin, a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro.

Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [3H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.

Studies of the regulation of the mouse carboxyl ester lipase gene in mammary gland.

The lactating mammary gland and pancreas of mouse constitute the main tissues for synthesis and secretion of a bile-salt-stimulated lipase called carboxyl ester lipase (CEL). In this paper we have analysed the endogenous CEL gene expression in mammary gland. It is shown that the gene is expressed at day 14 of pregnancy, which is synchronous with that of the whey acidic protein (WAP) gene. Even though the CEL and WAP genes are induced at the same time during mammary gland differentiation, their regulation is different with respect to dependence on lactogenic hormones. The high induction of the WAP gene expression due to the activation of signal transducer and activator of transcription (STAT)5 by prolactin has not been observed for the CEL gene, even though it has been demonstrated that both STAT5 isoforms interact with one of the gamma-interferon activation sequence sites in the promoter of the CEL gene. Hence we have demonstrated that the prolactin/STAT5 signal is not involved in a general and significant activation of 'milk genes'. Instead of a direct effect of the lactogenic hormones, the up-regulation of the CEL gene is correlated with an increase in the number of differentiated epithelial cells. Furthermore, promoter studies using the mammary-gland-derived cell line, HC11, show that a major positive element in the CEL gene promoter interacts with a member(s) of the CCAAT-binding transcription factor/nuclear factor 1 family, binding to a palindromic site. Binding of this factor(s) is important for the tissue-specific activation of the CEL gene in the mammary gland, because no activation by this factor(s) was seen in cells of pancreatic origin.

The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides.

Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) are members of the Hedgehog (Hh) family of signaling molecules known to be involved in embryonic patterning and morphogenesis. The Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, we detect signaling peptides in producing cells, but we also find that ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, we show that induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement.

Nuclear translocation of antizyme and expression of ornithine decarboxylase and antizyme are developmentally regulated.

The polyamines are important regulators of cell growth and differentiation. Cells acquire polyamines by energy-dependent transport and by synthesis where the highly regulated ornithine decarboxylase (ODC) catalyzes the first and rate-controlling step. Inactivation of ODC is mainly exerted by antizyme (AZ), a 20--25 kDa polyamine-induced protein that binds to ODC, inactivates it, and targets it for degradation by the 26S proteasome without ubiquitination. In the present study, we have performed a systematic analysis of the expression of ODC and AZ, at the mRNA and protein levels, during mouse development. The expression patterns for ODC and AZ were found to be developmentally regulated, suggesting important functions for the polyamines in early embryogenesis, axonogenesis, epithelial-mesenchymal interaction, and in apoptosis. In addition, AZ protein was found to translocate to the nucleus in a developmentally regulated manner. The nuclear localization is consistent with the fact that the amino acid sequence of AZ exhibits features that characterize nuclear proteins. Interestingly, we found that cultivation of mandibular components of the first branchial arch in the presence of a selective proteasome inhibitor caused ODC accumulation in the nucleus of a subset of cells, suggesting that the observed nuclear translocation of AZ is linked to proteasome-mediated ODC degradation in the nucleus. The presence of AZ in the nucleus may suggest that nuclear ODC activity is under tight control, and that polyamine production can be rapidly interrupted when those developmental events, which depend on access to nuclear polyamines, have been completed.