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Background We previously reported that the level of protein-conjugated acrolein (PC-Acro), a marker of cell or tissue damage, was increased in saliva from patients with primary Sjögren's syndrome (pSS), and that the level of PC-Acro was well correlated with the severity of pSS. Methods Acrolein-conjugated immunoglobulins were measured in saliva from pSS patients. Results The activities of autoantibodies recognizing Sjögren's syndrome SSA (Ro) and SSB (La) proteins in saliva from pSS patients were approximately 3- to 5-fold higher than those from control subjects. We also found that autoantibody activities recognizing SSA (Ro) and SSB (La) proteins increased after acrolein treatment of saliva from control subjects. When an antibody against human serum albumin was treated with acrolein, the ability to recognize albumin was reduced but the ability to recognize other proteins was increased. Twenty-four and eleven kinds of acrolein-conjugated amino acids were found at the variable and constant regions of peptides, respectively, obtained from the immunoglobulins in saliva from pSS patients. Conclusion The altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially involved in autoimmune diseases. [ABSTRACT FROM AUTHOR]

N -Methyl- d -aspartate receptors (NMDA receptors) are known to be permeable to Na + and Ca 2+ ions. In this study, we tested whether polyamines (putrescine, spermidine, spermine), organic cations found in cells, can permeate NMDA receptors expressed in Xenopus laevis oocytes and HEK293 cells. It was found that polyamines, especially spermidine, can permeate NMDA channels expressed from GluN1/GluN2A or GluN1/GluN2B activated by glycine and glutamate. Furthermore, spermidine and Ca 2+ influx through NMDA receptors was observed in the presence of Mg 2+ , although Na + influx was strongly inhibited by Mg 2+ . The Km values for spermidine influx through GluN1/GluN2A and GluN1/GluN2B were 2.2 mM and 2.7 mM, respectively in the presence of isotonic extracellular ion solutions. Spermidine uptake by NMDA receptors was dependent on the presence of glycine and glutamate, and inhibited by Ca 2+ and by memantine, an NMDA receptor channel blocker. The Km values for Ca 2+ influx through GluN1/GluN2A and GluN1/GluN2B were 4.6 mM and 3.3 mM, respectively, under the same ionic conditions. The results indicate that spermidine and Ca 2+ , but not Na + , can permeate NMDA receptors in the presence of Mg 2+ . Spermidine, if released locally from presynaptic terminals (where its concentration is high in synaptosomes and synaptic vesicles) could permeate NMDA receptors and play a role in synaptic plasticity mediated by NMDA receptors together with Ca 2+ . [ABSTRACT FROM AUTHOR]

The basidiomycetous mushroom Phellinus igniarius (L.) Quel. has been used as traditional medicine in various Asian countries for many years. Although many reports exist on its anti-oxidative and anti-inflammatory activities and therapeutic effects against various diseases, our current knowledge of its effect on stroke is very limited. Stroke is a neurodegenerative disorder in which oxidative stress is a key hallmark. Following the 2005 discovery by Igarashi’s group that acrolein produced from polyamines in vivo is a major cause of cell damage by oxidative stress, we now describe the effects of anti-oxidative extracts from P. igniarius on symptoms of experimentally induced stroke in mice. The toxicity of acrolein was compared with that of hydrogen peroxide in a mouse mammary carcinoma cell line (FM3A). We found that the complete inhibition of FM3A cell growth by 5 μM acrolein could be prevented by crude ethanol extract of P. igniarius at 0.5 μg/ml. Seven polyphenol compounds named 3,4-dihydroxybenzaldehyde, 4-(3,4-dihydroxyphenyl-3-buten-2one, inonoblin C, phelligridin D, inoscavin C, phelligridin C and interfungin B were identified from this ethanolic extract by LCMS and 1H NMR. Polyphenol-containing extracts of P. igniarius were then used to prevent acrolein toxicity in a mouse neuroblastoma (Neuro-2a) cell line. The results suggested that Neuro-2a cells were protected from acrolein toxicity at 2 and 5 μM by this polyphenol extract at 0.5 and 2 μg/ml, respectively. Furthermore, in mice with experimentally induced stroke, intraperitoneal treatment with P. igniarius polyphenol extract at 20 μg/kg caused a reduction of the infarction volume by 62.2% compared to untreated mice. These observations suggest that the polyphenol extract of P. igniarius could serve to prevent ischemic stroke. [ABSTRACT FROM AUTHOR]

Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. [ABSTRACT FROM AUTHOR]

Abstract: The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1–4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-d-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at −70mV, only compound 2 (0.4mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke. [Copyright &y& Elsevier]

Abstract: Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from spermine by spermine oxidase. Production of acrolein from spermine by spermine oxidase was clarified using spermine synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which spermine content is negligible or spermidine/spermine N 1-acetyltransferase activity is elevated. [Copyright &y& Elsevier]

We have recently reported that the altered recognition patterns of immunoglobulins due to acrolein conjugation are at least partially responsible for autoimmune diseases in patients with primary Sjögren’s syndrome (pSS). In the current study, it was found that the specific activity (activity/ng protein) of metalloproteinase-9 (MMP-9) in saliva was elevated about 2.4-fold in pSS patients. Accordingly, it was examined whether MMP-9 is activated by acrolein. It was found that the MMP-9 with 92 kDa molecular weight was activated by acrolein. Under the conditions studied, Cys99, located in the propeptide, was conjugated with acrolein together with Cys230, 244, 302, 314, 329, 347, 361, 373, 388 and 516, which are located in fibronectin repeats and glycosyl domains, but not on the active site of MMP-9. In addition, 82 and 68 kDa constructs of MMP-9s, lacking the NH 2 -terminal domain that contains Cys99, were not activated by acrolein. The results suggest that acrolein conjugation at Cys99 caused the active site of MMP-9 to be exposed. Activation of MMP-9 by acrolein was inhibited by cysteine, and slightly by lysine, because these amino acids inhibited acrolein conjugation with MMP-9. Conversely, MMP-9 activity in the presence of 50 μM acrolein was enhanced by 100 μM histidine. This was due to the inhibition of acrolein conjugation with His405 and 411 located at the Zn 2+ binding site of MMP-9. These results suggest that activation of 92 kDa MMP-9 by acrolein is involved in tissue damage in pSS patients and is regulated by cysteine and histidine, and slightly by lysine. Activated 82 and 68 kDa MMP-9 s were not detected in saliva of pSS patients by Western blotting. [ABSTRACT FROM AUTHOR]

Background and purpose N -Methyl- d -aspartate (NMDA) receptors have a high permeability to Ca 2+ , contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca 2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. Methods A unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. Results When the size of brain infarction was decreased by N 1 , N 4 , N 8 -tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca 2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca 2+ , the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca 2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca 2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca 2+ increase. Conclusion The results indicate that toxicity of Ca 2+ during brain infarction is correlated with the increase of acrolein. [ABSTRACT FROM AUTHOR]

Abstract: Background: We found previously that the amyloid β40/42 (Aβ40/42) ratio and the level of protein-conjugated acrolein (PC-Acro) in plasma were increased in mild cognitive impairment (MCI) and Alzheimer's disease (AD) subjects. We determined whether MCI and AD subjects can be differentiated based on the levels of Aβ40, Aβ42, and PC-Acro in cerebrospinal fluid (CSF). Methods: Aβ40, Aβ42, PC-Acro, Tau and phosphorylated Tau in CSF were measured by ELISA. Results: Median values of Aβ40, Aβ40/PC-Acro and Aβ40/42 in CSF were significantly lower in 54 AD subjects than those in 40 MCI subjects. Severity of VOI (volume of interest) atrophy was most intensely correlated with the decrease in Aβ40/PC-Acro and then that in Aβ40 and Aβ42/PC-Acro. MMSE was most intensely correlated with the decrease in Aβ42 and Aβ40, and then that in Aβ42/PC-Acro and Aβ40/PC-Acro. Conclusion: A decrease in Aβ40/PC-Acro in CSF is well correlated with brain damage, and a decrease in Aβ42 and Aβ40 is well correlated with cognitive ability. Measurement of PC-Acro together with Aβ40 and Aβ42 provides a more precise evaluation of severity of AD subjects. [Copyright &y& Elsevier]

Highlights: [•] The role of polyamines for the cell cycle was studied using synchronized HeLa cells. [•] Polyamine deficiency led to enhanced synthesis of p27Kip1 at G1/S boundary. [•] Unusual distribution of actin and α-tubulin at G2/M phase by polyamine deficiency. [•] Structural change of chromosome at G2/M was not observed by polyamine deficiency. [•] Involvement of polyamines at G2/M phase was less than that at G1/S boundary. [Copyright &y& Elsevier]

Abstract: Objective: Acrolein-conjugated lysine residues in proteins are present in human atherosclerotic lesions, and are detected in human low-density lipoprotein (LDL). These findings suggest that acrolein may contribute to macrophage foam cell formation and atherogenesis through modification of LDL. The purpose of this study is to determine whether acrolein-conjugated LDL (Acro-LDL) induces macrophage conversion to form foam cells. Methods: Acro-LDL was prepared by incubation of LDL with acrolein. Characteristics of Acro-LDL were examined by agarose gel electrophoresis and western blotting. Cholesterol contents of THP-1 macrophages incubated with Acro-LDL were determined by enzymatic method. Pathway of Acro-LDL uptake by THP-1 macrophages was determined using neutralizing antibody against scavenger receptors. Delivery of Acro-LDL into lysosome and formation of lipid droplet by incubation with Acro-LDL were demonstrated by confocal microscopy. Results: The mobility of Acro-LDL determined by agarose gel electrophoresis was increased by modification with acrolein, and the shift of mobility was dependent on the concentration of acrolein. Acrolein interacted with apolipoprotein B in LDL and Acro-LDL uptake by THP-1 macrophage was a more effective inducer of cholesterol accumulation than oxidized LDL uptake. Acro-LDL uptake was mediated by scavenger receptor class A type 1 (SR-A1), but not by CD36. As a result of Acro-LDL uptake, cholesterol ester accumulated in lipid droplets of macrophages, converting them to foam cells. Conclusions: The results show that Acro-LDL uptake via SR-A1 receptors can mediate macrophage foam cell formation. [Copyright &y& Elsevier]

Abstract: Background: We have recently found that the median relative risk value (RRV) (0–1) of brain infarction estimated by protein-conjugated acrolein (PC-Acro), IL-6 and CRP together with age was in the order silent brain infarction (SBI) (0.80)>carotid atherosclerosis (CA) (0.76)>white matter hyperintensity (WMH) (0.46)>control (0.14). We clarified how metabolic disorders [hypertension (HT), hyperlipidemia (HL) and hyperglycemia (HG)] are correlated with RRV. Methods: The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography. Results: The median RRV of metabolic disorders was in the order HT+HG (0.84)>HT+HL (0.73)>HT (0.65)≈HG (0.65)>HL (0.61)>HL+HG (0.48)>no metabolic disorder (0.24)>normal (0.11). Correlation with SBI was in the order HT+HG (52%)>HT+HL (42%)>HT (40%)>HG (34%)≈HL(33%)>HL+HG (14%)≈no metabolic disorder (14%). Conclusion: The results indicate that HT is the most strongly associated factor with SBI among metabolic disorders and that the seriousness of metabolic disorder estimated by RRV was well correlated with SBI. [Copyright &y& Elsevier]

N -Methyl- d -aspartate (NMDA) receptors have been implicated in learning and memory, and may also play a central role in various conditions leading to neuronal degradation. NMDA receptor antagonists could therefore be of therapeutic benefit for a number of neurological disorders. We have designed hybrid compounds of polyamines and memantine, both of which function as NMDA channel blockers. The triamine derivative with a guanidine moiety showed more potent antagonistic activity than memantine. [ABSTRACT FROM AUTHOR]

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain. [ABSTRACT FROM AUTHOR]

Background We previously reported that the level of urinary 3-hydroxypropyl mercapturic acid (3-HPMA)/creatinine (Cre) was reduced following stroke. The aim of this study was to determine whether the level of 3-HPMA/Cre in urine was reduced in subjects with dementia. Methods The level of 3-HPMA was measured by LC–MS/MS, and that of amino acid conjugated acrolein (AC-Acro) was by ELISA. The study included 128 elderly subjects divided into 74 non-demented (control), 22 mild cognitive impairment (MCI) and 32 Alzheimer's disease (AD) subjects. Results The urinary 3-HPMA/Cre and AC-Acro/Cre in MCI plus AD subjects were significantly lower than those in control subjects. In addition, urinary Cre in AD subjects was significantly higher than that in MCI subjects, and 3-HPMA/Cre and AC-Acro/Cre in AD subjects were significantly lower than that in MCI subjects. Among these three markers, the lower 3-HPMA/Cre ratio was most strongly correlated with the decline of MMSE (Mini-Mental State Examination) and the increase in CDRsob (Clinical Dementia Rating Scale Sum of Boxes Scores). Furthermore, reduction in 3-HPMA/Cre in urine was well correlated with increase in Aβ 40/42 in plasma in demented subjects. Conclusion The results indicate that 3-HPMA/Cre in urine is the most reliable biochemical marker to distinguish AD subjects from MCI subjects among three markers. [ABSTRACT FROM AUTHOR]

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues. [ABSTRACT FROM AUTHOR]

Abstract: We have recently reported that acrolein is more toxic than reactive oxygen species. Thus, the mechanism of cell toxicity by acrolein was studied using mouse mammary carcinoma FM3A cells. Acrolein-conjugated proteins were separated by gel electrophoresis with subsequent determination of their amino acid sequence, and it was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the major acrolein-conjugated proteins in cells. Acrolein interacted with cysteine-150 at the active site of GAPDH, and also with cysteine-282. When cells were treated with 8μM acrolein, the activity of acrolein-conjugated GAPDH was greatly reduced, and the ATP content in cells was thus significantly reduced. In addition, it was shown that acrolein-conjugated GAPDH translocated to the nucleus, and the level of acetylated GAPDH and the number of TUNEL positive cells was increased, indicating that cell death is enhanced by acrolein-conjugated GAPDH. Inhibition of cell growth by acrolein was partially reversed when the cDNA encoding GAPDH was transformed into cells. These results indicate that inactivation of GAPDH is one mechanism that underlies cell toxicity caused by acrolein. [Copyright &y& Elsevier]

The objective of this study was to determine whether plasma levels of acrolein, a compound that causes cell damage, and amyloid-β (Aβ) are useful biochemical markers for Alzheimer's disease (AD). The study included 221 elderly subjects divided into 101 non-demented [33 healthy control and 68 non-demented subjects with white matter hyperintensity (nd-WMH)], 50 mild cognitive impairment (MCI), and 70 AD. Increases in both protein-conjugated acrolein (PC-Acro) and Aβ40/42 ratio were observed in MCI and AD patients compared with values in control subjects. When the combined measurements of PC-Acro and Aβ40/42 ratio were evaluated using the median value of the relative risk value for dementia, they were in the order AD (0.98) ≥ MCI (0.97) > nd-WMH (0.83) > control (0.35). The results indicate that measurements of PC-Acro and Aβ40/42 ratio not only detect MCI and AD patients but also nd-WMH subjects. Furthermore, both PC-Acro and Aβ40/42 ratio in plasma for 120 MCI and AD patients were significantly higher than those for 101 control and nd-WMH subjects, indicating that both values become useful biochemical markers for MCI and AD subjects. [ABSTRACT FROM AUTHOR]

Abstract: Background: We found previously that increases in plasma levels of protein-conjugated acrolein and polyamine oxidases, enzymes that produce acrolein, are good biomarkers for stroke. The aim of this study was to test whether 3-hydroxypropyl mercapturic acid (3-HPMA), an acrolein-glutathione metabolite, was increased in the urine of stroke patients. Methods: The level of 3-HPMA in urine was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Stroke (78 subjects) was divided into 52 cerebral infarction (CI) and 26 cerebral hemorrhage (CH) on the basis of clinical information including brain imaging. Results: A major acrolein derivative in urine is 3-HPMA. Being different from the results of PC-Acro in plasma, 3-HPMA in urine decreased following stroke. The median value of μmol 3-HPMA/g creatinine (Cre) for 90 control subjects was 2.83, while that for 78 stroke patients was 1.56. The degree of the decrease in 3-HPMA was similar in both CI and CH patients. Furthermore, the median value of μmol 3-HPMA/g Cre in 56 patients with lesions ≥1cm in diameter (1.39) was significantly lower than that in 20 patients with lesion <1cm in diameter (2.16). Conclusion: Inverse correlation between stroke and urinary 3-HPMA was observed. The results suggest that stroke is aggravated when nervous system tissues have a reduced level of glutathione. [Copyright &y& Elsevier]

The objective of this study was to determine whether plasma levels of acrolein, a compound that causes cell damage, and amyloid-β (Aβ) are useful biochemical markers for Alzheimer's disease (AD). The study included 221 elderly subjects divided into 101 non-demented [33 healthy control and 68 non-demented subjects with white matter hyperintensity (nd-WMH)], 50 mild cognitive impairment (MCI), and 70 AD. Increases in both protein-conjugated acrolein (PC-Acro) and Aβ40/42 ratio were observed in MCI and AD patients compared with values in control subjects. When the combined measurements of PC-Acro and Aβ40/42 ratio were evaluated using the median value of the relative risk value for dementia, they were in the order AD (0.98) ≥ MCI (0.97) > nd-WMH (0.83) > control (0.35). The results indicate that measurements of PC-Acro and Aβ40/42 ratio not only detect MCI and AD patients but also nd-WMH subjects. Furthermore, both PC-Acro and Aβ40/42 ratio in plasma for 120 MCI and AD patients were significantly higher than those for 101 control and nd-WMH subjects, indicating that both values become useful biochemical markers for MCI and AD subjects. [ABSTRACT FROM AUTHOR]

The gene expression networks governing embryonic stem cell (ESC) pluripotency are complex and finely regulated during differentiation toward specific lineages. We describe a new role for Amd1 (adenosyl methionine decarboxylase), a key enzyme in the polyamine synthesis pathway, in regulating both ESC self-renewal and differentiation to the neural lineage. Amd1 is highly expressed in ESCs and is translationally down-regulated by the neural precursor cell (NPC)-enriched microRNA miR-762 during NPC differentiation. Overexpression of Amd1 or addition of the polyamine spermine blocks ESC-to-NPC conversion, suggesting Amd1 must be down-regulated to decrease the levels of inhibitory spermine during differentiation. In addition, we demonstrate that high levels of Amd1 are required for maintenance of the ESC state. We show that forced overexpression of Amd1 in ESCs results in maintenance of high Myc levels and a delay in differentiation on removal of LIF. We propose that Amd1 is a major regulator of ESC self-renewal and that its essential role lies in its regulation of Myc levels within the cell. [ABSTRACT FROM AUTHOR]