Optimum feeding and growth in preterm neonates.

Approximately 10% of all babies worldwide are born preterm, and preterm birth is the leading cause of perinatal mortality in developed countries. Although preterm birth is associated with adverse short- and long-term health outcomes, it is not yet clear whether this relationship is causal. Rather, there is evidence that reduced foetal growth, preterm birth and the long-term health effects of both of these may all arise from a suboptimal intrauterine environment. Further, most infants born preterm also experience suboptimal postnatal growth, with potential adverse effects on long-term health and development. A number of interventions are used widely in the neonatal period to optimise postnatal growth and development. These commonly include supplementation with macronutrients and/or micronutrients, all of which have potential short-term risks and benefits for the preterm infant, whereas the long-term health consequences are largely unknown. Importantly, more rapid postnatal growth trajectory (and the interventions required to achieve this) may result in improved neurological outcomes at the expense of increased cardiovascular risk in later life.

Absence of selenoprotein P but not selenocysteine lyase results in severe neurological dysfunction.

Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all the known selenoproteins are found in brain, where expression is facilitated by selenocysteine (Sec)-laden selenoprotein P. The brain also expresses selenocysteine lyase (Scly), an enzyme that putatively salvages Sec and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of Scly to selenoprotein P (Sepp1) knockout mice for similarity of neurological impairments and whether dietary selenium modulates these parameters. We report that Scly knockout mice do not display neurological dysfunction comparable to Sepp1 knockout mice. Feeding a low-selenium diet to Scly knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of Sepp1 is exacerbated in male vs. female mice. These findings indicate that Sec recycling via Scly becomes limiting under selenium deficiency and suggest the presence of a complementary mechanism for processing Sec. Our studies illuminate the interaction between Sepp1 and Scly in the distribution and turnover of body and brain selenium and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology.

Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis.

Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Structure-expression relationships of the 15-kDa selenoprotein gene. Possible role of the protein in cancer etiology.

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3'-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3'-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.

Regulation of human thioredoxin reductase expression and activity by 3'-untranslated region selenocysteine insertion sequence and mRNA instability elements.

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3'-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.

Exercise training and chronic obstructive pulmonary disease: past and future research directions.

Chronic obstructive pulmonary disease (COPD) is a major cause of death and disability in the United States. It is characterized by symptoms of breathlessness that result in sedentary lifestyle, physical deconditioning, and reduced quality of life. Previous research has shown that exercise training in patients with COPD will improve physical function and may help improve the quality of life. Unfortunately, the majority of these previous studies have not been pursued with adequate scientific rigor and the conclusions regarding the efficacy of exercise as an adjunct in the treatment of COPD are equivocal. The purpose of this article is to review the previous research that has focused on the effects of exercise training on individuals with COPD, to examine the problems with this previous research, and to emphasize the need and identify topics for further outcome-based research.

Ultraviolet-induced cell death blocked by a selenoprotein from a human dermatotropic poxvirus.

Selenium, an essential trace element, is a component of prokaryotic and eukaryotic antioxidant proteins. A candidate selenoprotein homologous to glutathione peroxidase was deduced from the sequence of molluscum contagiosum, a poxvirus that causes persistent skin neoplasms in children and acquired immunodeficiency syndrome (AIDS) patients. Selenium was incorporated into this protein during biosynthesis, and a characteristic stem-loop structure near the end of the messenger RNA was required for alternative selenocysteine decoding of a potential UGA stop codon within the open reading frame. The selenoprotein protected human keratinocytes against cytotoxic effects of ultraviolet irradiation and hydrogen peroxide, providing a mechanism for a virus to defend itself against environmental stress.

Knowing when not to stop: selenocysteine incorporation in eukaryotes.

The regulation of translation frequently involves protein-RNA interactions. An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine. Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS element) and a unique selenocysteyl-tRNA, are required for this process. In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding. Whether eukaryotes use a similar mechanism is currently the subject of intense investigation.

Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis.

Selenocysteine is co-translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons. However, the only component of the eukaryotic selenocysteine incorporation machinery identified to date is the selenocysteine-specific tRNA(Sec). In prokaryotes, selenocysteine is synthesized from seryl-tRNA(Sec) and the active selenium donor, selenophosphate. Selenophosphate is synthesized from selenide and ATP by the selD gene product, selenophosphate synthetase, and is required for selenocysteine synthesis and incorporation into bacterial selenoproteins. We have now cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase. Despite significant differences between the mechanisms of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring selenium incorporation into bacterial selenoproteins. Human selenophosphate synthetase has only 32% homology with the bacterial protein, although a highly homologous region that has similarity to a consensus ATP/GTP binding domain has been identified. Point mutations within this region result in decreased incorporation of selenium into type 1 iodothyronine deiodinase in all but one case. Further analysis revealed that reduced selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases.

Nutritional and hormonal regulation of thyroid hormone deiodinases.

Selenocysteine has been identified in the active center of types 1 and 3 iodothyronine deiodinases, two important enzymes regulating the formation and degradation of the active thyroid hormone, 3,5,3'-triiodothyronine (T3). Selenium is thus required for such complex processes as normal growth, brain development, and metamorphosis, all of which are thyroid hormone dependent. Structural and functional analyses of the type 1 deiodinase mRNA allowed identification of the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the mRNA. SECIS elements with conserved sequence and structural features are also present in the 3' untranslated regions of the mRNAs encoding selenoprotein P and the glutathione peroxidase family of selenoproteins. These elements are necessary and sufficient for directing selenocysteine incorporation into the deiodinases and the other mammalian selenoproteins.

Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recording UGA requires specific signals in the corresponding mRNAs. In eukaryotes, stem-loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient. To investigate the reason for this, we examined the effects of the amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are contransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.

Identification of critical amino acids for 3,5,3'-triiodothyronine deiodination by human type 1 deiodinase based on comparative functional-structural analyses of the human, dog, and rat enzymes.

The selenoenzyme, type 1 iodothyronine deiodinase (type 1 DI), catalyzes the activation of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) but 3,3',5'-triiodothyronine (rT3) is the preferred substrate for the human enzyme. Since the dog type 1 DI has a significantly lower affinity for rT3, we cloned the dog type 1 DI to identify amino acids critical for rT3 binding. The Km of the transiently expressed dog enzyme for rT3 5'-deiodination is 25-fold higher than that of the human enzyme. However, the Ki of T4 for rT3 deiodination by dog type 1 DI is only 3-fold higher than that for the human, suggesting that the differences between the two proteins affect binding of rT3 more than that of T4. Comparative competition studies in which rT3 or T4 is used to block covalent bromoacetyl T3 binding to the two proteins support this. Mutational studies showed that the critical differences between the dog (D) and human (H) enzymes are Asn (D) 45 Gly (H), Gly (D) 46 Glu (H), and Leu (D) 60:Phe (H) 65. Substitution of the human residues for those of the dog at these positions causes the predicted changes in the Km (rT3) and vice versa. A Phe65 to Leu mutation alone in the human enzyme increases the Km (rT3) 10-fold. We speculate that Phe65 is especially important for rT3 binding due to an interaction between the tyrosyl ring of rT3 and the aromatic ring of Phe65.

Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase.

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3',5-triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID-I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID-I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar Km and Vmax values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID-I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region.

Selenocysteine is incorporated cotranslationally at UGA codons, normally read as stop codons, in several bacterial proteins and in the mammalian proteins glutathione peroxidase (GPX), selenoprotein P and Type I iodothyronine 5' deiodinase (5'DI). Previous analyses in bacteria have suggested that a stem-loop structure involving the UGA codon and adjacent sequences is necessary and sufficient for selenocysteine incorporation into formate dehydrogenase and glycine reductase. We used the recently cloned 5'DI to investigate selenoprotein synthesis in eukaryotes. We show that successful incorporation of selenocysteine into this enzyme requires a specific 3' untranslated (3'ut) segment of about 200 nucleotides, which is found in both rat and human 5'DI messenger RNAs. These sequences are not required for expression of a cysteine-mutant deiodinase. Although there is little primary sequence similarity between the 3'ut regions of these mRNAs and those encoding GPX, the 3'ut sequences of rat GPX can substitute for the 5'DI sequences in directing selenocysteine insertion. Computer analyses predict similar stem-loop structures in the 3'ut regions of the 5'DI and GPX mRNAs. Limited mutations in these structures reduce or eliminate their capacity to permit 5'DI translation. These results identify a 'selenocysteine-insertion sequence' motif in the 3'ut region of these mRNAs that is essential for successful translation of 5'DI, presumably GPX, and possibly other eukaryotic selenocysteine-containing proteins.

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.

Evidence that cysteine, not selenocysteine, is in the catalytic site of type II iodothyronine deiodinase.

Recent cloning of the cDNA for Type I iodothyronine deiodinase revealed that the mRNA contains a UGA codon encoding the amino acid selenocysteine. Mutagenesis of the selenocysteine codon to a cysteine codon produced a protein with lower deiodinase activity. The presence or absence of selenocysteine in Type II deiodinase, which differs from the Type I enzyme in a number of parameters, has not been determined. Gold inhibits the activity of both the Type I deiodinase and the only other known eukaryotic selenocysteine-enzyme, glutathione peroxidase. Substitution of cysteine for selenocysteine in Type I deiodinase reduced its sensitivity to inhibition by gold 500-fold. We found that gold thioglucose was a competitive inhibitor with respect to the iodothyronine substrate of both deiodinases. However, the Type II enzyme from brown fat and pituitary was 100 to 1000-fold less sensitive to gold than was Type I activity in liver and pituitary, similar to the results with the cysteine-substituted Type I enzyme. This suggests that Type II deiodinase contains cysteine instead of selenocysteine in the active site.