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Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin... more
Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to r...
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ABSTRACT
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Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and theaflavin-3,3'-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth,... more
Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and theaflavin-3,3'-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 microM) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC50s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 microM. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Amon...
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Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there occurs a transient increase in ornithine decarboxylase (EC4.1.1.17) (ODC) activity. The addition of 10 to 20 mM Na+, K+ or Mg++ completely... more
Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there occurs a transient increase in ornithine decarboxylase (EC4.1.1.17) (ODC) activity. The addition of 10 to 20 mM Na+, K+ or Mg++ completely inhibits this induction of ODC activity with no effect on cell growth. These cations also inhibit the increase of ODC activity in neuroblastoma cells and in H-35 cells which is induced by prostaglandin E1 plus 3-isobutyl-1-methyl-xanthine and by 15% fetal calf serum respectively. This inhibitory effect of low levels of cations on the induction of ODC activity in different cell lines suggests that the intracellular function of ODC, and of the products of the reaction it catalyzes (putrescine, spermidine and spermine), may be intimately involved with changes in cation pools.
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Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent protein serine/threonine kinase that phosphorylates many different nuclear proteins, including high mobility group (HMG) proteins,... more
Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent protein serine/threonine kinase that phosphorylates many different nuclear proteins, including high mobility group (HMG) proteins, heterogeneous nuclear ribonuleoprotein (hnRNP) fractions, and nuclear matrix proteins, but not histones. The enzyme appears to be essential in growth regulation. However, it is not clear how the enzyme is regulated in vivo. To understand the regulation of this enzyme, we have searched for possible effectors for this enzyme. Spermine, at physiological concentrations, significantly stimlulates nuclear protein phosphorylation catalyzed by nuclear kinase II (NII kinase). Using various subnuclear fractions as substrates, we showed that the stimulatory effect of spermine was confined only to nuclear matrix proteins. Thus, spermine at 1 mM stimulated a >5-fold increase in nuclear matrix phosphorylation, but had little or no effect on the phosphorylation of HMG and ...
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Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated... more
Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system. The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects. Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action. On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered. Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT.
Research Interests: Enzyme Inhibitors, Gene expression, Cell Division, Antioxidants, Cell line, and 15 moreCell morphology, Humans, Clinical Sciences, Histones, Histone acetyltransferases, Acetylation, Growth rate, NAD, Life Span, Histone Acetylation, Biochemistry and cell biology, Histone deacetylases, Cell Size, fibroblasts, and Niacin
Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This... more
Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.
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The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in N-18 neuroblastoma cells in tissue culture was studied by the covalent incorporation of 8-azido-cyclic adenosine... more
The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in N-18 neuroblastoma cells in tissue culture was studied by the covalent incorporation of 8-azido-cyclic adenosine 3':5'-[32P]monophosphate, together with the techniques of sodium dodecyl sulfate:polyacrylamide gel electrophoresis and autoradiography. Greater than 95% of the total cAMP binding activity of N-18 neuroblastoma cells was identified as being regulatory subunits of the type I (RI) and type II (RII) species, with RI being the predominant form of the two (RI:RII = 3:1). The specific activity of RI but not of RII increased 3-fold when cells were grown in medium containing 1% rather than 10% fetal calf serum. Under the same conditions, the specific activity of acetylcholinesterase increased 3- to 5-fold. The increase in RI was inversely related to the serum concentration in the medium and was specific for cells at the stationary phase of growth. An increase in intracellular cAMP, concomitant with the increase in RI, was also observed. Morphological examination of stationary-phase neuroblastoma cells maintained in medium containing 1% fetal calf serum suggested the presence of a high proportion of highly-differentiated cells. It is proposed that the regulatory control of RI cAMP-binding protein by serum may involve modulation of intracellular cAMP and that the expression RI may be used as a biochemical index of differentiation in mouse neuroblastoma cells.
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Page 1. Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis PABLO HERNA´ NDEZ-ROMANO1, ROBERTO HERNA´ NDEZ1, ROSSANA ARROYO2, JOHN F. ALDERETE3 ...
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ABSTRACT
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Resveratrol (R-3), a trihydroxy trans-stilbene from grape, inhibits multistage carcinogenesis in animal models. A resveratrol derivative 3,4,5,4'-tetrahydroxystilbene (R-4) exhibits potent growth inhibitory effect against transformed... more
Resveratrol (R-3), a trihydroxy trans-stilbene from grape, inhibits multistage carcinogenesis in animal models. A resveratrol derivative 3,4,5,4'-tetrahydroxystilbene (R-4) exhibits potent growth inhibitory effect against transformed human cells. Here we report that 3,4,5,4'-tetramethoxystilbene (MR-4), converted from R-4, was more potent against cancer cell lines (WI38VA, IMR-90SV, HeLa, LNCaP, HT-29, and HepG2), but had almost no inhibitory effect on the growth of normal cells (WI38, IMR-90, BJ-T) at the concentrations tested. The IC50 value of MR-4 on the growth inhibition of transformed WI38VA human cells was 0.5 microM, as compared to the value of greater than 50 microM for the normal WI38 cells. Resveratrol, however, did not exhibit such clear differential effect and the IC50 value of R-3 for WI38VA cells was about 50 microM. The growth inhibitory effect of MR-4 correlated with the induction of apoptosis in the transformed cells. When normal WI38 cells and transformed ...
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Human diploid fibroblasts in tissue culture undergo replicative senescence after a finite number of divisions that is characterized by a permanent loss of their dividing potential. However, senescence-like phenotypes, including growth... more
Human diploid fibroblasts in tissue culture undergo replicative senescence after a finite number of divisions that is characterized by a permanent loss of their dividing potential. However, senescence-like phenotypes, including growth cessation, morphological changes, and appearance of senescence-associated beta-galactosidae (SA-gal) activity, can be induced by treating early passage cells with C(6)-ceramide, H(2)O(2), LY294002, or trichostatin A. While there is convincing evidence that telomere shortening is causally related to replicative senescence, the role of telomere shortening in the chemical-induced premature senescence is unclear. Here we employed a normal human BJ cell strain and its telomerase-transfected counterpart, termed BJ-T cells, to examine whether active telomerase in BJ-T can block or delay the premature senescence induced by various chemicals and, if not, whether telomere shortening still occurs. We found that, although all four chemicals tested could induce gro...
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NF-Y, a heterotrimeric CCAAT binding protein, may have a role in regulating some G1/S genes whose expressions are attenuated during replicative senescence [Matuoka and Chen (1999) Exp Cell Res 253: 365-371]. The hallmark of replicative... more
NF-Y, a heterotrimeric CCAAT binding protein, may have a role in regulating some G1/S genes whose expressions are attenuated during replicative senescence [Matuoka and Chen (1999) Exp Cell Res 253: 365-371]. The hallmark of replicative senescence is the loss of dividing potential. Hence, attenuation of G1/S gene expressions may be causally related to aging. To understand how NF-Y is involved in regulating G1/S genes during replicative senescence, we have examined the expressions of three NF-Y subunit genes in human IMR-90 cells over the entire course of their life-span. The mRNA levels of NF-YA, B, and C did not show any age-dependent change. In contrast, the protein level of NF-YA exhibited a significant and progressive decrease during cell senescence. Cross-linking experiments indicated that NF-Y may interact with proteins such as GCN5 and P/CAF. Co-transfection of cells with plasmid encoding NF-YA protein enhanced the expression of reporter gene fused with G1/S gene promoter that...
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The hallmark of cellular aging is the failure of senescent cells to initiate the DNA synthesis during the progression of cell cycle. Since most, if not all, of the G1/S genes exhibit a significant down-regulation during aging, an... more
The hallmark of cellular aging is the failure of senescent cells to initiate the DNA synthesis during the progression of cell cycle. Since most, if not all, of the G1/S genes exhibit a significant down-regulation during aging, an alteration of gene regulation at late G1/S boundary could be a major contributing factor for the loss of dividing potential during cell senescence. The underlying cause for the apparent global attenuation of gene expression at late G1/S boundary is not clear. Since we have shown that thymidine kinase (TK) and dihydrofolate reductase (DHFR) are transcriptionally regulated during aging, we suspect that a similar mechanism may be operative in the age-dependent down-regulation of other G1/S genes. DNA binding activities using Y-box containing sequence in TK promoter or E2F containing sequence in DHFR promoter show prominent serum-responsiveness in low passage cells and dramatic attenuation in senescent cells. Promoter analysis using GCG program reveals striking...
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Research Interests: Engineering, Physics, Chemistry, Protein Folding, Biology, and 15 moreMedicine, Multidisciplinary, Neurodegenerative Diseases, Humans, Promoter Analysis, Neurons, PLoS one, HeLa cells, Neurodegenerative Disease, Heat Shock, Heat Shock Protein, Cell Survival, DNA binding proteins, Gene knockout, and Reporter gene
An 18,000-dalton protein can be metabolically labeled by (TH)putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the... more
An 18,000-dalton protein can be metabolically labeled by (TH)putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of
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Expression of thymidine kinase gene in normal human diploid cells is both cell cycle- and age-dependent and appears to be transcriptionally regulated. Strong DNA protein binding with a 28-bp fragment (-91/-64) that contains the distal... more
Expression of thymidine kinase gene in normal human diploid cells is both cell cycle- and age-dependent and appears to be transcriptionally regulated. Strong DNA protein binding with a 28-bp fragment (-91/-64) that contains the distal inverted CCAAT box is observed in serum-stimulated young (low population doubling level) IMR-90 cells but not in senescent cells. This cell cycle- and age-dependent binding factor was termed CBP/ tk, indicating CCAAT binding protein for the thymidine kinase gene. Based on immunoshift assay and purification, it has been suggested that CBP/tk is equivalent to NF-Y, previously identified as the binding protein for the Y box within E alpha gene promoter. In this study, we examined the mRNA level and protein amount of NF-Y, proteins in young and old IMR-90 cells during serum stimulation by Northern and Western blot blot analyses. In addition, we compared (1) the turnover rate of NF-Y in IMR-90 cells with that of CBP/tk binding activity and (2) the levels of NF-Y and CBP/tk in normal and cancer cells. Both NF-YA and NF-YB were constitutively expressed at mRNA level in IMR-90 cells. However, expression of NF-YA, and to a lesser degree, NF-YB, at the protein level were clearly age-dependent. The half-life of NF-YA and NF-YB were, respectively, 4- and > 10-fold longer than that of CBP/tk binding activity in IMR-90 cells. In addition, we found that the amount of NF-Y did not correlate with the overexpression of CBP/tk binding activity in HeLa cells. Taken together, our results suggested that although CBP/tk may contain NF-YA or related proteins, NF-A and NF-YB alone may not account for all the characteristics of CBP/tk observed in normal and transformed human cells.
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DMF) . Acknowledgment. We thank Professor K. Barry Sharpless for useful discussions and preprinta concerning improved ligands for the asymmetric dihydroxylation re-action. We thank LH Li and TF DeKoning for the biological evaluation of 13... more
DMF) . Acknowledgment. We thank Professor K. Barry Sharpless for useful discussions and preprinta concerning improved ligands for the asymmetric dihydroxylation re-action. We thank LH Li and TF DeKoning for the biological evaluation of 13 and 17. ... Supplementary Material ...
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Page 1. JOURNAL OF CELLULAR PHYSIOLOGY 128:149-154 (1986) Increased Level of CAMP-Dependent Protein Kinase in Aging Human Lung Fibroblasts ALICE Y.-C. LIU,* ZEE-FEN CHANG, AND KUANG YU CHEN Departments ...
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The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) was found to have differential inhibitory effects on the expression of morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. PMA completely... more
The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) was found to have differential inhibitory effects on the expression of morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. PMA completely inhibited neurite extension and associated growth characteristics and partially inhibited the increased expression of R1 cAMP-binding protein; PMA had no effect on the induction of acetylcholinesterase activity in cells prompted to differentiate either by treatment with 1 mM dibutyryl cAMP or by serum deprivation. 4-alpha-Phorbol-12, 13-didecanoate, an inactive analogue of phorbol ester tumor promoter, was without effect. The implications of these findings concerning the mechanism of action of phorbol ester tumor promoters in the control of cell differentiation are discussed.
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We have previously reported that osmotic stress prominently induces the DNA binding activity of the heat shock transcription factor 1 (HSF1). In the present study, we examined the effects of medium osmolarity on both the activation of... more
We have previously reported that osmotic stress prominently induces the DNA binding activity of the heat shock transcription factor 1 (HSF1). In the present study, we examined the effects of medium osmolarity on both the activation of HSF1 and the programmed cell death in normal human fibroblasts during cellular senescence. The activation of HSF1 occurred rapidly in presenescent (early passage) IMR-90 cells when exposed to either hypo-osmotic or hyperosmotic stress. In contrast, the activation of HSF1 was significantly attenuated in senescent cells. Western blot analysis indicated that equal amounts of HSF1 were present as monomers in the cytoplasm of both presenescent and senescent cells in normal growth medium. Under either hypo-osmotic or hyperosmotic stress, trimerization and nuclear localization of HSF1 occurred in presenescent cells but not in senescent cells. More than 80% of HSF1 in senescent cells remained as monomers in the cytoplasm under osmotic stress, suggesting a defect in the signal transduction pathways that lead to HSF1 trimerization or a dysfunction in the HSF1 protein itself. Possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in the activation HSF1 was investigated by monitoring the activation of the three MAPKs, ERK1/2, JNK1/2, and p38, in cells exposed to hypo-osmotic or hyperosmotic stress. All three MAPKs were activated by hyperosmotic stress but not hypo-osmotic stress, suggesting that the MAPK signal transduction pathways may not be directly linked to the osmotic stress-induced activation of HSF1. In contrast to the rapid heat shock transcription factor (HSF) activation, apoptosis occurred only after long-term exposure to hypo-osmotic or hyperosmotic stress. Despite the prominent induction of HSF1 activation, the presenescent cells were more sensitive than the senescent cells to the osmotic stress-induced apoptosis.
Research Interests: Transcription Factors, Apoptosis, Heat Shock Transcription Factor, Cell line, Humans, and 13 moreOsmotic pressure, Signal Transduction Pathway Models, Programmed cell death, Cellular Physiology, Medical Physiology, Mitogen Activated Protein Kinase, DNA binding, Western blot, Osmotic Stress, DNA binding proteins, Nuclear Localization, fibroblasts, and heat shock factor
Page 1. The Modulation of the Induction of Ornithine Decarboxylase by Spermine, Spermidine and Diamines JOHN S. HELLER, KUANG YU CHEN, DIMITRI A. KYRIAKIDIS, WANG F. FONG AND E. S. CANELLAKIS Department ...
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The possible functions of ornithine decarboxylase (ODC) and polyamines in the differentiation of mouse NB-15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiating and nondifferentiating... more
The possible functions of ornithine decarboxylase (ODC) and polyamines in the differentiation of mouse NB-15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiating and nondifferentiating NB-15 cells over a 5-day culture period. Differentiation of NB-15 cells was induced by the addition of dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine (1BMX) to the growth medium and was monitored by neurite outgrowth, increases of acetylcholinesterase (AChE), and RI cAMP-binding protein. Plating of NB-15 cells in fresh serum-containing growth medium was accompanied by rapid growth and a marked increase of ODC activity; this early increase of ODC activity was attenuated, both in duration and in magnitude, in the differentiating cells. The spermidine content of the differentiating neuroblastoma cells was significantly lower than that of the nondifferentiating cells. In the fully differentiated neuroblastoma cells, the ODC activity and spermidine content were lower than that of the undifferentiated cells by approximately 15-fold and five-fold, respectively. Based on these results it is proposed that changes of polyamine metabolism may be of significance in the differentiation of mouse neuroblastoma cells.
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When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an... more
When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.
Research Interests: Protein synthesis, Polyamines, Cell line, Humans, Connective tissue, and 15 moreCellular Physiology, Medical Physiology, Amino Acids, Lysine, Eflornithine, Respiratory System, Putrescine, Chemical Reaction, Metabolic pathway, Cell Survival, Diploidy, Organic Compound, Organic Acid, Enzyme Induction, and fibroblasts
Neurons have a limited capacity for heat shock protein (HSP) induction and are vulnerable to the pathogenic consequence of protein misfolding and aggregation as seen in age-related neurodegenerative diseases. Sirtuin 1 (SIRT1), an NAD(+)... more
Neurons have a limited capacity for heat shock protein (HSP) induction and are vulnerable to the pathogenic consequence of protein misfolding and aggregation as seen in age-related neurodegenerative diseases. Sirtuin 1 (SIRT1), an NAD(+) -dependent lysine deacetylase with important biological functions, has been shown to sustain the DNA-binding state of HSF1 for HSP induction. Here we show that differentiation and maturation of embryonic cortical neurons and N2a neuroprogenitor cells is associated with decreases in SIRT1 expression and heat shock-dependent induction of HSP70 protein. Tests of a pharmacological activator and an inhibitor of SIRT1 affirm the regulatory role of SIRT1 in HSP70 induction. Protein cross-linking studies show that nuclear SIRT1 and HSF1 form a co-migrating high molecular weight complex upon stress. The use of retroviral vectors to manipulate SIRT1 expression in N2a cells show that shRNA-mediated knock down of SIRT1 causes spontaneous neurite outgrowth coincident with reduced growth rate and decreased induction of hsp70-reporter gene, whereas SIRT1 over-expression blocks the induced neural differentiation of N2a cells. Our results suggest that decreased SIRT1 expression is conducive to neuronal differentiation and this decrease contributes to the attenuated induction of HSPs in neurons.
Research Interests: Neurogenesis, Transcription Factors, Signal Transduction, RNA interference, Cerebral Cortex, and 15 moreMice, Animals, Cellular Physiology, Medical Physiology, Neurons, Rats, Time Factors, Heat Shock Proteins, Transfection, Neural Stem Cells, Cell Proliferation, Gestational Age, Protein Binding, Sirtuin, and DNA binding proteins
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Research Interests: Oxidative Stress, Cell line, Cellular, Humans, Sequence alignment, and 14 moreAnimals, Cell Death, Cellular Physiology, Medical Physiology, Shell Half-Life, Caco-2 cells, Protease Inhibitors, Colon cancer, RNA-binding proteins, Base Sequence, Cell Survival, DNA fragmentation, Molecular Sequence Data, and fibroblasts
Research Interests: Flow Cytometry, Transcription Factors, Cellular, Humans, Transcription Factor, and 11 moreHydrogen Peroxide, Cell nucleus, Western blot, Protein Expression, Heat Shock, DNA binding proteins, Diploidy, Molecular and Cellular Biochemistry, Biochemistry and cell biology, fibroblasts, and heat shock factor
Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or... more
Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or deoxyhypusine synthase are essential for cell survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.