Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation

Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.

Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2−: Role of nitrogen dioxide free radical

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O2•−) to O2 and H2O2. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H2O2, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of l-α-lecithin liposomes was enhanced greatly during the SOD/H2O2 reaction in the presence of nitrite anion (NO2−) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO2− also greatly enhanced α-tocopherol (α-TH) oxidation by SOD/H2O2 in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was α-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing γ-tocopherol (γ-TH) were incubated with SOD/H2O2/NO2−, the major product identified was 5-NO2-γ-TH. Nitrone spin traps significantly inhibited the formation of α-tocopheryl quinone and 5-NO2-γ-TH. NO2− inhibited H2O2-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO2− by an SOD-bound oxidant to the nitrogen dioxide radical (•NO2). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO2− is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1−/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

A PII-like protein in Arabidopsis: Putative role in nitrogen sensing

PII is a protein allosteric effector in Escherichia coli and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (GLN) and asparagine synthetase (ASN) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (GLB1) in two higher plants, Arabidopsis thaliana and Ricinus communis (Castor bean). Each plant PII-like protein has high overall identity to E. coli PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of GLB1 mRNA are affected by light and metabolites. To initiate studies of the in vivo function of the Arabidopsis PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.

Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4′-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(−) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard–phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard–DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

Inhibitory sites in enzymes: Zinc removal and reactivation by thionein

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1–80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97–354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97–354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

Substratal idiothetic navigation of rats is impaired by removal or devaluation of extramaze and intramaze cues

The spatial orientation of vertebrates is implemented by two complementary mechanisms: allothesis, processing the information about spatial relationships between the animal and perceptible landmarks, and idiothesis, processing the substratal and inertial information produced by the animal's active or passive movement through the environment. Both systems allow the animal to compute its position with respect to perceptible landmarks and to the already traversed portion of the path. In the present study, we examined the properties of substratal idiothesis deprived of relevant exteroceptive information. Rats searching for food pellets in an arena formed by a movable inner disk and a peripheral immobile belt were trained in darkness to avoid a 60° sector; rats that entered this sector received a mild foot shock. The punished sector was defined in the substratal idiothetic frame, and the rats had to determine the location of the shock sector with the use of substratal idiothesis only, because all putative intramaze cues were made irrelevant by angular displacements of the disk relative to the belt. Striking impairment of place avoidance by this “shuffling procedure” indicates that effective substratal idiothesis must be updated by exteroceptive intramaze cues.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.