DNA SYNTHESIS IN RANA PIPIENS TADPOLE LIVER DURING TRIIODOTHYRONINE-INDUCED METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during Ts-induced metamorphosis of R. pipiens liver was investigated. Rates of DNA synthesis were estimated by rates of 3 H-thymidine incorporation into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. During T3 -induced metamorphosis, periods of DNA synthesis and fluctuations in DNA content preceded expression of biochemical differentiation as measured by the enzyme arginase, and fluctuations in synthesis rates preceded corresponding fluctuations in content. The earliest response to T3- , was a 50% decrease in liver DNA, followed by increases in thymidine incorporation at 16 hr, 2 days, and 5-8 days. The size of the endogenous thymine pool was not significantly altered by T3 These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthetic rates and content during metamorphosis. Expression of thyroxin-induced development of the tadpole liver appears to be associated with both proliferation and cellular death, and metamorphosis of the liver cannot be occurring in a “fixed population of cells.”     

Effects of acute CS2 intoxication on liver protein and drug metabolism

Rats pretreated with phenobarbitone and their controls were exposed to 0.15% atmospheric CS₂ for 2 h. Liver protein metabolism and microsomal drug metabolizing enzyme activities were analyzed 1, 4 and 46 h later. In the pretreated rats the [¹⁴C]leucine uptake was at first inhibited, then liver RNA tended to increase. This increase was followed by a decline in the [¹⁴C]leucine uptake, while RNA content diminished to the control level. In the control rats (not pretreated with phenobarbital) the effects of CS₂ on liver protein metabolism were less; only at 4 h after the exposure was liver RNA increased and [¹⁴C]leucine uptake slightly stimulated. In the pretreated rats CS₂ had decreased microsomal P-450 by about 50% at 1 and 4 h, and the activity of 7-O-dealkylase of ethoxycoumarin had decreased even more. The measurable UDPglucuronosyltransferase of the liver microsomes of the pretreated rats had increased by 26% at 1 h and by 80% at 4 h after the CS₂ exposure. The in vivo activation of microsomal UDPglucuronosyltransferase may result from a stimulated lipid peroxidation of reticuloendothelial membranes by CS₂ metabolites in vivo, as suggested by the diene conjugation spectra. In control rats CS₂ depressed only the ethoxycoumarin deethylase activity. All the microsomal changes caused by CS₂ exposure were restored within 46 h.It is suggested that the different action of CS₂ on the liver protein metabolism of rats pretreated with phenobarbitone and controls results from the different stage of protein turnover in the two groups, i.e. in the barbituratetreated animals there is a phase of increased protein synthesis and accordingly the protein turnover is more sensitive to the action of CS₂.     

Rapid and direct stimulation of hepatic gluconeogenesis by L-triiodothyronine (T3) in the isolated-perfused rat liver

T3 injection into the portal vein of the isolated hypo- and euthyroid liver rapidly stimulated alanine and ¹⁴C-α-amino-isobutyric acid uptake, O₂-consumption, glucose and urea production, and increased the overall, cytoplasmic and mitochondrial energy state. The concentration of the effector molecules long chain acyl CoA, acetyl-CoA, citrat and AMP remained unchanged. After T3 application no alteration in hepatic cAMP content, protein kinase activation, and in the tissue levels of the intermediates of the Embden-Meyerhof-pathway were observed. Our results indicate that T3 rapidly stimulates hepatic glucose production independently of cAMP by increasing amino acid uptake and mitochondrial ATP regeneration and translocation in the cytoplasmic compartment, thus providing both the precursor and energy for gluconeogenesis.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Insulin-like growth factor 1 regulation of proliferation and differentiation of <Emphasis Type="Italic">Xenopus laevis</Emphasis> myogenic cells in vitro

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1’s cell differentiation promotion, suggesting the possibility that IGF-1’s differentiation-promotion effect is an indirect effect via IGF-1’s cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100–500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-l-thyronine (T₃) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T₃. In larval tail cells, cell count was 76% lower in the presence of T₃, and IGF-1 did not promote proliferation and differentiation in T₃-containing medium. In larval dorsal cells, cell count was also lower in the presence of T₃, but IGF-1 enhanced proliferation and differentiation in T₃-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T₃ and helps accelerate dorsal muscle remodeling during metamorphosis.     

Triiodothyronine but Not Thyroxine Accelerates Myofibrillar Proteolysis via ATP Production in Cultured Muscle Cells

These experiments were done to clarify that the differential effects of thyroxine (T₄) and triiodothyronine (T₃) on skeletal muscle protein turnover are caused by their roles on ATP production. Primary cultured chick muscle cells were treated with a physiological level of T₄ (60 ng/ml), T₃ (12 ng/ml), or ATP (0.5 mM) for 6 days and the protein content, ATP production, proteasome activity, and myofibrillar protein breakdown were measured. The protein content measured as an index of cell growth was not affected by T₄, T₃, or ATP. The cellular ATP level was increased by T₃ and ATP, but not by T₄. Proteasome activity and N ^τ-methylhistidine (MeHis) release measured as an index of myofiblillar protein breakdown was also increased by T₃ and ATP, but not by T₄. These results indicate that T₃ but not T₄ increases ATP production followed by an increase in proteasome activity, and thus stimulates myofibrillar proteolysis.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.     

Roles of macrophages in programmed cell death and remodeling of tail and body muscle of Xenopus laevis during metamorphosis

 Examination was made of the involvement of macrophage phagocytosis in programmed cell death of tail and body muscle of the frog, Xenopus laevis, during metamorphosis by electron microscopy and immunohistochemical analysis. Electron microscopic observation revealed that macrophages were often found to be present in body and tail muscles at the most active stage of metamorphosis and to actively phagocytose apoptotic muscle fragments. Developmental changes in macrophages were examined using the macrophage-specific antibody, HAM56. Macrophages initially appeared in the early climax stage (stage 59), when the triiodothyronine (T₃) level was high, increased rapidly during the process of muscle cell death, and assumed their greatest number at the late climax stage (stage 63/64). They decreased after stage 65/66, with a decrease in T₃. Distribution and change in the number of macrophages were the same as those of muscle apoptotic bodies (sarcolytes) during metamorphosis, which suggests an interactive mechanism between macrophages and dying muscle cells. For clarification of this, study was made of the expression of HAM 56 antigens that were X. laevis homologs of mouse attachmin, non-specific adhesion proteins in macrophages. The expression of HAM56 antigens in macrophages was found to increase with macrophage phagocytosis at the late climax stage, thus, macrophage differentiation would appear to take place during metamorphosis and HAM56 antigens may be essential for macrophage–dying muscle cell interactions. Accepted: 29 May 1997     

Role and Metabolism of Free Leucine in Skeletal Muscle in Protein Sparing Action of Dietary Carbohydrate and Fat

Feeding rats with either a carbohydrate meal or a fat meal to the previously fasted rats caused significant decrease in urinary output of urea and total nitrogen. The content of free leucine in skeletal muscle decreased in the rats fed either a carbohydrate meal or a fat meal. Feeding of either a carbohydrate meal or a fat meal stimulated incoiporation of l-leucine-1–¹⁴C into protein fraction of skeletal muscle and reduced its oxidation to ¹⁴CO₂.

These results suggest that the metabolism of leucine is under nutritional regulation and that the decrease in content of free leucine in skeletal muscle might be caused by enhanced reutilization of leucine into protein by the feeding of a carbohydrate meal or a fat meal. The role of free leucine in skeletal muscle as a regulator of protein turnover in the tissue are discussed in relation to the metabolism of this branched chain amino acid.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Trypanosoma cruzi: inhibition of protein synthesis by nitrofuran SQ 18,506

SQ 18,506 is a nitrofuran compound related to the trypanocide Lampit. In vitro, radiolabeled leucine, uridine, and thymidine were incorporated into macromolecular protein, RNA, and DNA in order to study growth inhibition of Trypanosoma cruzi. Our findings suggest that the primary effect of the drug is on protein synthesis and not mediated solely by inhibition of RNA synthesis as indicated by prior studies. The drug was also found to reduce markedly the uptake of uridine into the nucleotide precursor pool but to affect only slightly the formation of aminoacyl-tRNA.     

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis

 The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T₃ action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T₃ and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59–60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T₃ (10^-8 M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occured specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T₃ action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T₃ and 32 times as much by the action of T₃ plus HC, indicating the important action of T₃–HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T₃–HC synergism on adult erythroblast proliferation and T₃-mediated programmed death of larval precursor cells. Accepted: 14 January 1999     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Energy source utilization by embryos and larvae of the muricid snail Chorus giganteus (Lesson, 1829)

The muricacean snail Chorus giganteus presents intracapsular development and the occurrence of nurse eggs that are ingested by the early encapsulated embryos indicate both that these snails develop through a lecitotrophic type of development and that reserves would be sufficient to support settlement and metamorphosis. In order to get more information about the use of energy resources, the dynamics of biochemical components throughout development at three temperatures (9, 12 and 15 °C) and the energetic cost of free-swimming life and metamorphosis are described. The uptake of ³H-alanine, as representative of dissolved organic matter, by embryo and larval stages is also investigated. While protein levels increased at all temperature conditions after ingestion of nurse eggs, lipids only increased when embryo and larvae were reared at 15 °C, and no change in carbohydrate levels was detected at any of the temperatures. The RNA/DNA indexes showed no significant differences with temperature at any stage of development but decreased along with the development of individuals. After hatching, organic matter and energy content of juveniles steadily decreased. Individuals at any of the developmental stage showed to be able to uptake alanine from seawater; the aminoacid uptake capacity increased along with intracapsular development. Uptake of alanine showed to be an active process and to follow Michaelis-Menten kinetics. This would be the first report about dissolved organic matter uptake by encapsulated development stages of any marine invertebrate species and let conclude that these larvae have the ability to obtain exogenous food in a dissolved form and to incorporate it into metabolizable compounds.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Effects of exogenous triiodothyronine on fast axonal transport during tadpole metamorphosis

Bullfrog tadpoles at metamorphic stages V, X and XVIII were immersed in 25 nM triiodothyronine (T₃) to assess whether the 4–5 fold increase in fast axonal transport (FAxT) previously observed during this span of spontaneous metamorphosis (1) could be mimicked by precocious application of thyroid hormone. The trend initially observed was for T₃ to stimulate [³⁵S]methionine incorporation into lumbar DRG and inhibit incorporation in tail DRG. Both effects, however, appeared to be exerted primarily on satellite cells rather than neurons since most of the T₃-induced changes in DRG were of a similar magnitude to those in the respective nerve trunks. Findings consistent with this observation resulted from use of the retrogradely transported lectin, ricin₁₂₀, to determine the proportion of DRG incorporation occurring in neurons. When incorporation of [³⁵S]methionine in lumbar DRG neurons was examined, T₃ had no stimulatory effect at any of the metamorphic stages examined. When FAxT was assessed as [³⁵S]protein accumulating proximal to a nerve trunk ligature, and expressed as a percentage of newly-synthesized protein in lumbar DRG neurons, no stimulatory effect of T₃ was detected. The question remains whether the changes in FAxT in peripheral neurons observed during spontaneous metamorphosis may be induced by circulating hormones other than T₃ or are secondary to changes in the target tissues. Special issue dedicated to Dr. Marion E. Smith.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Decreased cyclin-dependent kinase activity promotes thyroid hormone-dependent tail regression in <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

The thyroid hormone (TH), 3,5,3′-triiodothyronine (T₃), is an important regulator of diverse cellular processes including cell proliferation, differentiation, and apoptosis, with increasing evidence that the modulation of the phosphoproteome is an important factor in the TH-mediated response. However, little is understood regarding the mechanisms whereby phosphorylation may contribute to T₃-mediated cellular outcomes during development. The cyclin-dependent kinases (Cdks) and mitogen-activated protein kinases (MAPK/ERK) have been implicated in TH signaling in mammalian cells. In this study, we have investigated, in frogs, the possible role that these kinases may have in the promotion of tail regression during tadpole metamorphosis, an important postembryonic process that is completely TH-dependent. Cdk2 steady state levels and activity increase in the tail concurrent with progression through the growth phase of metamorphosis, followed by a precipitous decrease coinciding with tail regression. Cyclin-A-associated kinase activity also follows a similar trend except that its associated kinase activity is maintained longer before a decrease in activity. Protein steady state levels of ERK1 and ERK2 remain relatively constant, and their kinase activities do not decrease until much later during tail regression. Tail tips cultured in serum-free medium in the presence of T₃ undergo regression, which is accelerated by coincubation with a specific Cdk2 inhibitor. Coincubation with PD098059, a MAPK inhibitor, has no effect. Thus, T₃-dependent tail regression does not require MAPKs, but a decrease in Cdk2 activity promotes tail regression. This work was supported by a NSERC operating grant, NSERC University Faculty Award, and Michael Smith Foundationfor Health Research Scholar Award.