Cycloleucine Research Articles

Cycloleucine induces neural tube defects by reducing Pax3 expression and impairing the balance of proliferation and apoptosis in early neurulation

S-adenosylmethionine (SAM) plays a critical role in the development of neural tube defects (NTDs). Studies have shown that the paired box 3 (Pax3) gene is involved in neural tube closure. However, the exact mechanism between Pax3 and NTDs induced by SAM deficiency remains unclear. Here, The NTD mouse model was induced using cycloleucine (CL), an inhibitor of SAM biosynthesis, to determine the effect of Pax3 on NTDs. The effect of CL on NTD occurrence was assessed by 5-ethynyl-2′-deoxyuridine (EdU) staining, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and Western blot in NTD embryonic brain tissues and immortalized hippocampal neuron cells (HT-22). A high incidence of NTDs was observed when CL was administered at a dose of 200 mg/kg body weight. The levels of SAM and Pax3 were significantly reduced in NTD embryonic brain tissues and HT-22 cells after CL exposure. Decreased proliferation and excessive apoptosis were observed in neuroepithelial cells of NTD embryos and HT-22 cells under SAM deficiency, but these effects were reversed by overexpression of Pax3. These results suggest that decreased expression of Pax3 impairs the dynamic balance between cellular proliferation and apoptosis, contributing to NTDs induced by SAM deficiency, which would provide new insights for clarifying the underlying mechanism of NTDs.

Cycloleucine negatively regulates porcine oocyte maturation and embryo development by modulating N6-methyladenosine and histone modifications

Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.

Combination Methionine-methylation-axis Blockade: A Novel Approach to Target the Methionine Addiction of Cancer.

Cancers are selectively sensitive to methionine (MET) restriction (MR) due to their addiction to MET which is overused for elevated methylation reactions. MET addiction of cancer was discovered by us 45 years ago. MR of cancer results in depletion of S-adenosylmethionine (SAM) for transmethylation reactions, resulting in selective cancer-growth arrest in the late S/G2-phase of the cell cycle. The aim of the present study was to determine if blockade of the MET-methylation axis is a highly-effective strategy for cancer chemotherapy. In the present study, we demonstrated the efficacy of MET-methylation-axis blockade using MR by oral-recombinant methioninase (o-rMETase) combined with decitabine (DAC), an inhibitor of DNA methylation, and an inhibitor of SAM synthesis, cycloleucine (CL). We determined a proof-of-concept of the efficacy of the MET-methylation-axis blockade on a recalcitrant undifferentiated/unclassified soft-tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) mouse model. The o-rMETase-CL-DAC combination regressed the USTS PDOX with extensive cancer necrosis. The new concept of combination MET-methylation-axis blockade is effective and can now be tested on many types of recalcitrant cancer.

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.

Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe)

Interleukin-6 (IL-6) is a multifunctional cytokine having primarily anti-apoptotic and anti-inflammatory effects. Recent reports have documented that IL-6 plays a key role in liver regeneration. Intracellular deficiency of S-adenosylmethionine (SAMe) is a hallmark of toxin-induced liver injury. Although the administration of exogenous SAMe attenuates liver injury, its mechanisms of action are not fully understood. Here we investigated the effects of exogenous SAMe on IL-6 production in monocytes and Kupffer cells. RAW 264.7 cells, a murine monocyte cell line, and isolated rat Kupffer cells were stimulated with lipopolysaccharide (LPS) in the absence or presence of exogenous SAMe. IL-6 production was assayed by ELISA and intracellular SAMe concentrations were measured by HPLC. We have found that exogenous SAMe administration enhanced both IL-6 protein production and gene expression in LPS-stimulated monocytes and Kupffer cells. Cycloleucine (CL), an inhibitor for extrahepatic methionine adenosyltransferases (MAT), inhibited LPS-stimulated IL-6 production. The enhancement of LPS-stimulated IL-6 production by SAMe was inhibited by ZM241385, a specific antagonist of adenosine (A 2) receptor. Our results demonstrate that SAMe administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-6 production.

MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE ‐INDUCED DEFICIENCY OF METHYL DONORS

We used cycloleucine (CL) — which prevents methionine conversion to S‐adenosyl‐methionine (SAMe) by inhibiting ATP‐L‐methionine‐adenosyl‐transferase (MAT) — to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35–50%. SAMe p‐toluensulphonate (SAMe‐SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL‐treated animals killed at day 18 after birth, starting from labelled substrates ([3H]‐choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL‐treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30–40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL.Among proteins, in sciatic nerves of CL‐treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe‐SD4 treatment counteracted, and in some cases normalized, these changes.In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe‐SD4 administration.The financial support of Telethon‐Italy (grant No. D 51) is gratefully acknowledged.

Biochemical Analysis of Myelin Lipids and Proteins in a Model of Methyl Donor Pathway Deficit: Effect of S-Adenosylmethionine

S-Adenosylmethionine (SAMe) is the methyl donor to numerous acceptor molecules. We used cycloleucine (CL), which prevents the conversion of methionine to SAMe by inhibiting ATP–l-methionine–adenosyltransferase (MAT), to characterize the lipid and protein changes induced in peripheral nerve and brain myelin in rats during development. We also investigated the effect of exogenous SAMe by administering SAMe-1,4-butane disulfonate (SAMe-SD4). CL was given on days 7, 8, 12, and 13 and SAMe-SD4 was given daily from day 7; the animals were killed on day 18. CL accumulates in the brain reaching a concentration within 24 h compatible with its ID50in vitro and interacting with methionine metabolism; brain MAT activity and SAMe levels were lower and methionine levels higher than in controls. CL significantly reduced brain and nerve weight gains, brain myelin content, proteins, phospholipids, and galactolipids. Among phospholipids in nerve and brain, only sphingomyelin was significantly increased, by 35–50%. Sciatic nerve protein analyses showed some significant changes: protein zero in sciatic nerve remained unchanged but the 14.0- and 18.5-kDa isoforms of myelin basic protein showed a dramatic increase. Among the main proteins, in purified brain myelin, the proteolipid protein and dimer-20 isoform decreased after CL. SAMe-SD4 highlights some sensitive parameters by counteracting, at least partially, some alterations of PL—particularly galactolipids and sphingomyelins—and proteins induced by CL. The partial beneficial effects might also be explained by the age-related limited bioavailability of exogenous SAMe, a finding, to our knowledge, not yet reported elsewhere. This study demonstrates that availability of methyl donors is closely related to the formation of myelin components.

The evolution of an experimental distal motor axonopathy. Physiological studies of changes in neuromuscular transmission caused by cycloleucine, an inhibitor of methionine adenosyltransferase.

Cycloleucine (CL), a synthetic amino acid is known to cause degeneration of motor nerve terminals. This paper describes the changes in neuromuscular transmission, the morphology of motor end-plates and the responses of muscle spindles after a single dose of CL was administered to weanling and adult mice. Animals were allowed to survive for between 12 h and 7 days. Twitch and tetanic responses of muscles stimulated through their nerves fell dramatically within 24 h in both young and adult mice and intracellular recordings revealed that a large proportion of end-plates in calf muscles became denervated, whilst at other end-plates intermittent failure of transmission and end-plate potentials (EPPs) with prolonged latency were demonstrated. End-plates with an abnormally high frequency of miniature end-plate potentials (mEPPs) were found in young mice at 12 h, and in the adult mice at 24 h. Morphological abnormalities in intramuscular nerves and nerve terminals included areas of electron lucent axoplasm, swollen degenerative mitochondria and loss of synaptic vesicles. Over the next 2-3 days further reductions occurred in the number of end-plates at which mEPPs or EPPs could be demonstrated. At 7 days a limited recovery of function occurred in distal muscles but proximal muscles, hitherto unaffected, now began to show abnormalities of transmission. Muscle spindles were found to be both functionally and structurally intact. It is suggested that this acute distal motor axonopathy is due to abnormalities in phospholipid composition of the axolemma of motor nerve terminals resulting from the failure of methyl-transfer pathways. These abnormalities in phospholipid composition might cause an increase in microviscosity of the axolemma and hence a decrease in efficiency of ion channels and pumps responsible for maintaining electrochemical gradients, essential for the structural and functional integrity of the neuromuscular junction.

Distal motor axonopathy and central nervous system myelin vacuolation caused by cycloleucine, an inhibitor of methionine adenosyltransferase.

Cycloleucine (CL), an inhibitor of methionine adenosyltransferase, has previously been used to produce an experimental model of subacute combined degeneration of the spinal cord. A re-investigation of its effects on the morphology of the nervous system and on brain concentrations of methionine and S-adenosylmethionine (SAM) was undertaken. Cycloleucine was administered as a single dose intraperitoneally (2 mg/g body weight) to young mice aged 21 d and adults aged 6 or 10 wks. The 21-day-old mice showed clinical evidence of toxicity within 24 h and thereafter developed progressive muscle weakness and ataxia. Animals did not survive longer than 1 wk. Light and electron microscopic examination of the central and peripheral nervous systems showed that intramyelinic vacuolation developed in the white matter of brain and cord within 12 h. The intramyelinic vacuolation in the white matter of brain and cord became more severe with longer survival, vacuoles coalescing and secondary axonal degeneration becoming evident. There was no myelin vacuolation in peripheral nerves. Axonal lesions occurred in the distal parts of motor nerves within 12-24 h resulting in degeneration of intramuscular nerve fibres and terminals. Later there was evidence of axonal degeneration in tibial and sciatic nerves. Many dorsal root ganglion cells became vacuolated or necrotic and numerous degenerated fibres were noted in the white matter of the spinal cord, particularly in the gracile funiculus. The optic nerves were not affected at any stage. In adult mice the pathology consisted of distal motor axonal degeneration which developed at 1-2 d. Little or no intramyelinic vacuolation in white matter was noted. Brain concentrations of SAM were reduced and levels of methionine became greatly elevated. The morphological effects of CL are considered to be the result of SAM deficiency impairing transmethylation processes known to be important in the formation and stabilization of myelin through the methylation of myelin basic protein. The immature developing central nervous system is much more vulnerable than the fully myelinated adult brain and spinal cord. The distal, predominantly motor axonopathy is a new observation and may be a reflection of the importance of transmethylation processes in the maintenance of axonal terminal membranes and the mechanisms of release of acetylcholine at the neuromuscular junction.

Effects of nicotine and nicotine/ethanol on human placental amino acids transfer

Ethanol abuse and smoking during pregnancy both result in decreased offspring weight. One explanation for this may be impaired placental nutrient transport. This study assessed this possibility utilizing the 4-hr perfused human placental system and human placental vesicles exposed to “physiological”, 0.2 μM and large (about 20 μM) nicotine concentrations alone, as well as nicotine combined with ethanol, 200 or 400 mg/ml, for up to 48 hr. Two nonmetabolizable amino acids, alpha-aminoisobutyric acid (AIB) and cycloleucine (CLEU) were used as probes. Nicotine was measured by gas chromatography in the placental perfusion system and vesicles and verified as to concentration. There was no statistically significant evidence of decreased transport of these amino acids with exposure to nicotine alone or nicotine and ethanol together in either test system. Thus, brief exposure to nicotine and ethanol does not impair amino acid transport by the human placenta.

Effect of ethanol on human placental transport of model amino acids and glucose.

Prior studies in rodents, sheep, and subhuman primates have shown that ethanol, especially after chronic exposure, inhibits the transport of amino acids by the placenta. A small decrease in glucose transport by rat placenta chronically exposed to ethanol has also been noted. With human placental slices, however, only pharmacological (high) concentrations of ethanol impaired uptake of amino acids, and there are no data on glucose transport. In the present study, the effect of brief exposure to ethanol on human placental transport of model amino acids and glucose was studied by two techniques not previously jointly employed for this--the perfused human placental cotyledon and human placental vesicle systems. The nonmetabolizable amino acids, alpha-aminoisobutyric (AIB) acid and cycloleucine (CLEU), as well as D-glucose, and nonmetabolized glucose (3-O-methyl-D-glucose), were used as probes. AIB and CLEU are transferred normally by active transport and D-glucose by facilitated transport from maternal to fetal compartments. The perfused placental system was exposed to ethanol (300-500 mg%) for 2-4 hr and the vesicles to 200-400 mg% ethanol for times varying from 10 min to 48 hr. There was no impairment of AIB, D-glucose, or 3-O-methyl-D-glucose transfer by ethanol using these techniques. Normally, about 60% of AIB transport by human placenta is sodium dependent. This component (using the vesicle system) was also not impaired by ethanol. Ethanol caused a very small decrease of CLEU clearance by the perfused human placenta (p = 0.05) but not using vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in 2-aminoisobutyric acid and cycloleucine uptake produced by 2,4-dichlorophenoxyacetic acid in chinese hamster ovary cells

The effect of dichlorophenoxyacetic acid on the transport of two non-metabolizable amino acids, 2-aminoisobutyric acid (AIB) and cycloleucine (CL) was studied in Chinese hamster ovary (CHO) cells. The herbicide did not exert any direct effect on the AIB transport. However, when the pesticide was in contact with the cells for 24 h an inhibition of the uptake was observed. Removal of the pesticide from the culture medium restored the influx of the amino acids which reached maximum values 1 h before cell division. The transport kinetics showed changes in V max but no variations in K m . These results may indicate that 2,4-dichlorophenoxyacetic acid produces a decrease in the carrier number but without modification of the affinity.

Two types of inhibitor of phospholipid methylation in MOPC-31C cells with modified membrane phospholipids

Phosphatidyl- N-monomethylethanolamine and phosphatidyl- N, N'-dimethylethanolamine, which are intermediates in the methylation pathway of phosphatidylcholine biosynthesis from phosphatidylethanolamine, increased in the membranes of MOPC-31C cells on addition of N-monomethylethanolamine and N, N'-dimethylethanolamine, respectively, to the culture medium. Phospholipid methylation in these modified cells both in vivo and in vitro was 10–25-times that of choline-treated control cells. The phospholipids synthesized via the methylation pathway in choline- and N-monomethylethanolamine-treated cells were mainly phosphatidyl- N, N'-dimethylethanolamine and phosphatidylcholine, whereas in N, N'-dimethylethanolamine-treated cells phosphatidylcholine was synthesized. The enhanced methylation in these modified cells could be ascribed to stimulation of the second and third steps in the methylation pathway. Similar enhancement was observed with choline-treated cell membranes when these intermediate phospholipids were added exogenously. S-Benzyl O, O'-diisopropyl phosphorothiolate inhibited phospholipid methylation both in vivo and in vitro. However, its extents of inhibition of choline-, N, N'-dimethylethanolamine- and N-monomethylethanolamine-treated cells were different: it inhibited the methylation most in choline-treated cells and less in the modified cells both in vivo and in membranes in vitro. The production of phosphatidyl- N, N'-dimethylethanolamine in N-monomethylethanolamine-treated cells was stimulated by low concentrations of the drug. Inhibition of the methylation of S-benzyl O, O'-disopropyl phosphorothiolate was irreversible both in vivo and in vitro, whereas inhibition of phospholipid methylation by cy cloleucine was reversible and observed only in vivo. The effects of these inhibitors on the three methylation steps of phosphatidylcholine synthesis from phosphatidylethanolamine were discussed.

Enhancement by glucocorticoid deficiency of the increase in cycloleucine accumulation induced in rat thymocytes by triiodothyronine.

In previous studies, we have demonstrated that 3,5,3'-triiodothyronine (T3), either added to suspended media in vitro or injected acutely in vivo, increases the in vitro accumulation of the non-metabolized amino acid cycloleucine (CLE) by thymocytes harvested from weanling rats. We now report that this reponse is greatly enhanced by prior adrenalectomy of the donor rat. In vitro, a significant increase in CLE accumulation in thymocytes from adrenalectomized rats was induced by T3 at a concentration of 1 x 10-10 M, while a concentration of 1 x 10-6 M was required to produce a similar and significant effect in thymocytes from intact animals. In adrenalectomized animals, a single iv dose of T3 (0.5 microgram/100 G bw) significantly increased the in vitro accumulation of CLE in thymocytes harvested two hours later. In contrast, ten-times that dose was ineffective in control animals. Increased sensitivity to T3 was abolished by physiological replacement doses of hydrocortisone. The data are consistent with the well-known opposing effects of physiological levels of thyroid and glucocorticoid hormones on the growth and function of lymphoid tissue in vivo and, together with other findings, suggest that thyroid hormones modulate the cellular accumulation of amino acids in the intact animal.

Beta-adrenergic potentiation of the increased in vitro accumulation of cycloleucine by rat thymocytes induced by triiodothyronine.

We have previously demonstrated that 3,5,3'-triiodothyronine (T(3)), whether administered in vivo or added to suspending media in vitro, promptly stimulates the in vitro accumulation of the nonmetabolized amino acids, alpha-aminoisobutyric acid, and cycloleucine (CLE) by thymocytes isolated from weanling rats. In these studies, we have examined the in vitro interaction between catecholamines and T(3) with respect to this effect. The previously reported enhancement of CLE accumulation in thymocytes by T(3) in vitro (1 muM) was confirmed. When added alone in concentrations ranging between 10 nM and 0.1 mM, the adrenergic agonists, epinephrine and norepinephrine, had no effect on CLE accumulation. At a concentration of 1 muM, isoproterenol, terbutaline, and phenylephrine were also without effect. However, the effect of T(3) was clearly potentiated by the concomitant addition of epinephrine, norepinephrine, and possibly isoproterenol, whereas terbutaline and phenylephrine were without effect. Neither basal nor T(3)-enhanced CLE accumulation was affected by the addition alone of the adrenergic blocking agents, propranolol (0.1 mM), phentolamine (10 muM), or practolol (0.1 mM). Nevertheless, the beta(1)- and beta(2)-antagonist, propranol, and the beta(1)-antagonist, practolol, blocked the increment in CLE accumulation produced by epinephrine; the alpha-antagonist, phentolamine, was without effect. The enhancement of CLE accumulation that occurred in the presence of T(3), with or without epinephrine, was seen to be a result of an inhibition of CLE efflux, because T(3) alone inhibited CLE efflux, and this effect was increased when epinephrine was also present. On the other hand, neither T(3) alone nor T(3) plus epinephrine appreciably altered the rate of inward transport of CLE. As judged from studies of the ability of thymocytes to exclude trypan blue, neither T(3) alone nor T(3) plus epinephrine either enhanced or impaired viability of cells during 3-h periods of incubation. Cell water content, measured with [(3)H]urea, was unaffected by T(3), either alone or in the presence of epinephrine. In confirmation of previous results, the stimulatory effect of T(3) on CLE accumulation was unaffected by concentrations of puromycin sufficient to inhibit protein synthesis by at least 95%, and the potentiating action of epinephrine on the response to T(3) was similarly unaffected. From these findings, it is concluded that the effect of T(3) to increase CLE accumulation by thymocytes in vitro, though itself independent of adrenergic mediation, is potentiated by beta(1)-adrenergic stimulation. This interaction appears distinctly different from other thyroid hormone-catecholamine interactions, in which thyroid hormones enhance physiological responses to catecholamines. Its mechanism remains unclear, but the properties of the T(3) effect, and possibly the interaction itself, suggest that T(3) enhances CLE accumulation by an action at the level of the cell membrane.

The effect of chondrocyte growth factor on membrane transport by articular chondrocytes in monolayer culture.

Chondrocyte growth factor (CGF), a contaminant of pituitary glycoprotein hormones, stimulates growth of cultured lapine articular chondrocytes while depressing SO4-proteoglycan synthesis. To study its effect on membrane transport, NIH-bTSH and two other preparations with comparable CGF activity were employed. In early log phase (36 hr) cultures CGF (64 microgram/ml) did not alter thymidine (dThd) uptake during the first 5 min. By 15 min however, TCA-precipitable dThd was 4-fold greater than in controls while the TCA-soluble fraction remained the same. CGF increased deoxy-glucose (DG) uptake in 36-hr old cultures. At 66 hr, CGF reduced DG transport. The transport of cycloleucine (CL) and aminoisobutyric acid (AIB) was reduced by CGF in 36 and 66-hr old cultures. There was a dose dependency between CGF concentration, the lowered uptake of DG, CL and AIB, and cell protein content. The effect of CGF on DG transport and dThd incorporation into DNA was not immediate but required prior exposure of the cells to CGF. CGF did not alter DG transport in rabbit or mouse fibrocytes or Chang liver cells. This and the reported finding that pituitary fibroblast growth factor (FGF), increases amino acid transport in other cells suggests that the biological specificity of CGF may not be identical to that of FGF.

Cycloleucine inhibition of amino acid transport in human and rat kidney cortex.

Cycloleucine inhibition of amino acid transport in human and rat kidney cortex.