Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells

Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency.Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo.Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1

Role of human nucleoside transporters in the cellular uptake of two inhibitors of IMP dehydrogenase, tiazofurin and benzamide

Further studies are warranted to determine if this finding can be generalized to other cell types

Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase)

Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, L-[4.14C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted L-[4-14C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [14C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADP---MgCl2 or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [14C]SAICAR, L-[4-14C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme

Studies on the mechanism of resistance of selected murine tumors to L-alanosine

Sublines of P388 and L1210 leukemia were rendered resistant to L-alanosine [L-2-amino-3-(N-hydroxy-N-nitrosamino) propionic acid] and designated P388/LAL and L1210/LAL. Assessments were made of certain biochemical and pharmacological determinants of the sensitivity or resistance to L-alanosine of these sensitive and resistant lines. It was observed that the antibiotic strongly inhibited adenylosuccinate synthetase and DNA synthesis only in the parent or sensitive lines; moreover, after a therapeutic dose of the drug, the concentration of L-alanosyl-AICOR (L-alanosyl-5-amino-4-imidazole carboxylic acid ribonucleotide), the putative active anabolite of L-alanosine, was dramatically higher in these parent lines as compared with the resistant variants. Enzymologic studies established that, in P388/LAL, the specific activity of the enzyme SAICAR synthetase (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase), which is believed to conjugate L-alanosine with the nascent purine AICOR (5-amino-4-imidazole carboxylic acid ribonucleotide), was depressed significantly; the same was not true for L1210/LAL. In both resistant lines, however, the enzymes of purine salvage were present at levels about 200 per cent higher than those measured in the native strains. These studies establish that resistance to L-alanosine is very likely pluricausal, but that the ability of susceptible cells to synthesize or retain L-alanosyl-AICOR is an element important to the process

Regulators of the metabolism of l-asparagine: A search for endogenous inhibitors

1. 1. A study of over 400 intermediary metabolites and naturally occurring macromolecules has been carried out in an attempt to identify molecules capable of regulating the metabolism of l-asparagine. 2. 2. Test systems consisting of mouse hepatic and testicular l-asparaginase, as well as, mouse pancreatic and tumoral l-asparagine synthetase, were used. 3. 3. Selective inhibitors have been categorized as-trace elements; amino acids, analogs and peptides; nucleic acid bases, nucleosides and nucleotides; aldehydes, lipids, porphyrins and pigments; vitamins and coenzymes; proteinaceous materials; citric acid cycle intermediates and related molecules; carbohydrates; and miscellaneous agents. 4. 4. An attempt has been made to extrapolate these in vitro studies to the regulation of the metabolism of l-asparagine in vivo. © 1980

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H2O2, 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H2O2 markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-ζ, LKB1, and AMPK caused by exposure to H2O2. This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H2O2-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-ζ and LKB1 phosphorylation and the activation of PP2A

Characterization of human brain nicotinamide 5'-mononucleotide adenylyltransferase-2 and expression in human pancreas.

NMNAT (nicotinamide 5'-mononucleotide adenylyltransferase; EC 2.7.7.1) catalyses the transfer of the adenylyl group from ATP to NMN to form NAD. We have cloned a novel human NMNAT cDNA, designated hNMNAT-2, from human brain. The cDNA contains a 924 bp open reading frame that encodes a 307 amino acid peptide that was expressed as a histidine-patch-containing thioredoxin fusion protein. Expressed hNMNAT-2 shared only 35% amino acid sequence homology with the human NMNAT enzyme (hNMNAT-1), but possessed enzymic activity comparable with hNMNAT-1. Using human genomic databases, hNMNAT-2 was localized to chromosome 1q25 within a 171 kb gene, whereas hNMNAT-1 is on chromosome 1p32-35. Northern blot analysis revealed highly restricted expression of hNMNAT-2 to brain, heart and muscle tissues, which contrasts with the wide tissue expression of hNMNAT-1; different regions of the brain exhibited differential expression of hNMNAT-2. Substitution mutations of either of two invariant residues, His-24 or Trp-92, abolished enzyme activity. Anti-peptide antibody to a unique epitope within hNMNAT-2 was produced, and immunohistochemical analysis of sections of normal adult human pancreas revealed that hNMNAT-2 protein was markedly expressed in the islets of Langerhans. However, the pancreatic exocrine cells exhibited weak expression of hNMNAT-2 protein. Sections of pancreas from insulinoma patients showed strong expression of hNMNAT-2 protein in the insulin-producing tumour cells, whereas acinar cells exhibited relatively low expression of hNMNAT-2 protein. These data suggest that the unique tissue-expression patterns of hNMNAT-2 reflect distinct functions for the isoforms in the regulation of NAD metabolism

Metabolites of alanosine, an antitumor antibiotic

The metabolism of alanosine. DL-2-amino-3-(N-hydroxy, N-nitrosamino) propionic acid (NSC-143647), a new antitumor antibiotic, was studied in mice, rats, monkeys and dogs. Urine is the principal excretory vehicle for the drug in these four species. In rats, unchanged alanosine is the principal excretory product. In the other species, a second, more acidic component accounts for the major fraction of the drug-derived radioactivity in urine; this product retains the characteristic u.v. spectrum and both carbons, 1 and 3, of alanosine: it is chromatographically and spectrally indistinguishable from the compound generated by the action of NADH and malate dehydrogenase on the product resulting from the incubation of L -alanosine with L-glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) and α-ketoglutarate. On the basis of this evidence, this metabolite is concluded to be the α-hydroxy counterpart of L-alanosine. The antibiotic was susceptible to transamination in vitro by extract of organs rich in GOT; heart was pre-eminent in this regard, and α-ketoglutaric acid was found to be the preferred α-keto partner in the reaction. Crystalline GOT catalyzed an identical reaction in vitro, and the product, like oxaloacetic acid, was susceptible to enzymatic condensation with acetyl CoA, in the presence of citrate synthase. Inability to detect the -α- keto analogue of alanosine. 2-oxo-3-(N-hydroxy, N-nitrosamino) propionic acid, in tissues and excreta is attributed to the facile decomposition of this metabolite in vivo. In vitro, alanosine was susceptible to decarboxylation by homogenates of mouse brain and by purified L-glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli. No evidence could be adduced for denitrosation of the antibiotic nor for reduction of the nitroso function in a system containing hepatic microsomes and NADPH. However, L-amino acid oxidase (EC 1.4.3.2) and high concentrations of pyridoxal phosphate catalyzed the deamination of alanosine at alkaline pH. In confirmation of the observations of Hurlbert et al,| Proc. Am. Ass. Cancer Res. 18, 234,(1977)|. alanosine was found to be used by phosphoribosylaminoimi-dazole-succinocarboxamide synthetase (EC 6.3.2.6) as a fraudulent substrate. Also observed was the condensation of alanosine with IMP. catalyzed by a partially purified preparation of adenylosuccinate synthetase (EC 6.3.4.4) from rabbit muscle; this anabolite exhibited chromatographic properties quite similar to adenylosuccinic acid. In as much as a substantial percentage of the administered dose of alanosine was found to associate with carcass-macromolecules for protracted periods, attempts were made to determine the basis for this fate. Equivalent labeling was produced irrespective of whether DL-[1-14C] or DL-[3-14C] alanosine was the injectate, so that reutilization of metabolically generated [14C]O2 is not likely to explain macromolecular retention of the antibiotic. In vitro, no esterification of alanosine to tRNA was observed, but the drug did bind to tRNA in an ATP-independent reaction. In vivo, ten times more DL-[3-14C] alanosine was incorporated into the hemoglobin of animals recovering from phenylhydrazine anemia than was observed in their saline-treated counterparts. Isolated reticulocytes incorporated only minor amounts of purified DL-[I-14C] alanosine into their molecules; this process was insensitive to inhibition by cycloheximide. In vitro, alanosine (used in lieu of L-aspartic acid) neither supported nor inhibited globin synthesis by rabbit reticulocyte lysates. These results leave unsettled the question of whether macromolecular association of alanosine reflects incorporation or affiliation