Structure of a eukaryotic decoding region A-site RNA.

The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects.

Structural origins of aminoglycoside specificity for prokaryotic ribosomes.

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.

The potential impact of iron supplementation during adolescence on iron status in pregnancy.

Iron deficiency anemia (IDA) during pregnancy is associated with significant morbidity for mothers and infants. Over 50% of pregnant women in developing countries suffer from IDA. It is also prevalent among adolescent girls because the growth spurt and onset of menstruation increase iron requirements. Women who conceive during or shortly after adolescence are likely to enter pregnancy with low or absent iron stores or IDA. Iron supplementation during adolescence is one of the new strategies advocated to improve iron balance in pregnancy. However, iron requirements are highest in the second and third trimesters and the model described here indicates that iron balance at this stage depends more on adequate intakes of bioavailable iron than on the size of the iron stores at conception. Furthermore, although supplementation will correct anemia and increase iron stores in girls, the positive effect on iron status will be temporary if their diets do not contain adequate bioavailable iron. Although iron status in early pregnancy may be improved if the period of supplementation continues up to the time of conception, supplementation before pregnancy should be viewed as an additional strategy to supplementation during the second and third trimesters.

The effect of calcium on iron absorption.

The experimental and epidemiological evidence demonstrating that Ca inhibits Fe absorption was reviewed, with the objectives of estimating the potential impact of variations in Ca intake on dietary Fe bioavailability and of providing some guidelines for predicting the effects on Fe status of recent recommendations for higher dietary Ca intake. In animal models Ca salts reduced both haem- and non-haem-Fe absorption, the effect being dependent on the amount of Ca administered rather than the Ca:Fe molar ratio; dairy products had a variable effect; factors other than Ca may have been important. In single-meal human absorption studies, both haem- and non-haem-Fe absorption was inhibited by Ca supplements and by dairy products, the effect depending on the simultaneous presence of Ca and Fe in the lumen of the upper small intestine and also occurring when Ca and Fe were given in the fasting state. The quantitative effect, although dose dependent, was modified by the form in which Ca was administered and by other dietary constituents (such as phosphate, phytate and ascorbic acid) known to affect Fe bioavailability. The mechanism by which Ca influences Fe absorption has not been elucidated. The effects of factors that modulate Fe bioavailability are known to be exaggerated in single-meal studies, and measurements based on several meals are more likely to reflect the true nutritional impact. The results of most multiple-meal human studies suggest that Ca supplementation will have only a small effect on Fe absorption unless habitual Ca consumption is very low. Outcome analyses showed that Ca supplements had no effect on Fe status in infants fed Fe-fortified formula, lactating women, adolescent girls and adult men and women. However it should be noted that the subjects studied had adequate intakes of bioavailable Fe and, except in one study, had relatively high habitual Ca intakes. Although cross-sectional analyses in Europe have shown a significant inverse correlation between Ca intake (derived primarily from dairy foods) and Fe stores, the quantitative effect was relatively small. The general conclusion is that dietary Ca supplements are unlikely to have a biologically significant impact on Fe balance in Western societies unless Ca consumption is habitually very low; however, increased consumption of dairy products may have a small negative effect that could be functionally important in pregnancy if Fe supplements are not taken. It is uncertain whether the inverse relationship between consumption of dairy products and Fe status is due entirely to increased Ca intake; substitution of milk proteins for meat may also have negative effects on Fe balance.

Violence in the lives of pregnant teenage women: associations with multiple substance use.

Using data from a consecutive sample of approximately 700 prenatal patients aged 12 to 19, the extent of violence and substance use in the lives of these women was examined, as were associations between violence and substance use. The findings show that 29% of the study participants had been victims of violence, with 15% experiencing physical violence only, 5% sexual violence only, and 9% both physical and sexual violence. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression analyses showed that, after controlling for confounding factors, victims were more likely than nonvictims to use cigarettes, alcohol, and illicit drugs, with victims of both physical and sexual violence being the most likely to use each type of substance. Ordinal logistic regression analysis found that, compared to nonvictims, violence victims were more likely to have more severe patterns of substance use (use multiple types of substances), with victims of both physical and sexual violence being the most likely to be multiple substance users. These findings underline the importance of both violence and substance use as health concerns among our youth.

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.

Interaction of iron with other nutrients.

Iron is essential for oxygen transport, oxidative metabolism, and cellular growth. Interactions between iron and other dietary factors play a significant role in determining the adequacy of iron nutrition and have important implications for food fortification in developing countries. Vitamin A and vitamin C deficiency states may affect iron transport, metabolism, and storage within the body.

Bovine ferritin iron bioavailability in man.

The bioavailability of ferritin iron was evaluated in human subjects using radiolabelled [55Fe]ferritin isolated from bovine spleen and liver. Preliminary studies with bovine spleen ferritin labelled in vitro demonstrated an inappropriately high absorption compared with ferritin labelled in vivo, and the latter was therefore used in all subsequent absorption studies. In 10 subjects, geometric mean absorption from 5 mg of ferritin iron was 3.8% when taken without and 3.2% when taken with food (P > 0.05). These values were significantly lower than absorption from the same dose of iron given as ferrous sulphate, which averaged 24.1% without and 8.2% with food. When the iron dose was increased 10-fold, absorption of ferritin iron averaged only 0.6-0.7% with or without food as compared with 7.9% without and 2.6% with food when the iron was given as ferrous sulphate. In a further study, mean absorption from bovine spleen ferritin of 4.0% did not differ significantly from the mean of 2.7% observed with bovine liver ferritin. These findings confirm previous studies indicating that ferritin iron is poorly absorbed. Furthermore, its use as a pharmaceutical iron preparation cannot be advocated.

Deliberations and evaluations of the approaches, endpoints and paradigms for iron dietary recommendations.

Iron deficiency severe enough to cause anemia is associated with significant morbidity while uncontrolled iron absorption which occurs in disorders such as hereditary hemochromatosis causes multiorgan failure and early death. Preliminary data from the Third National Health and Nutrition Examination Survey demonstrate that the prevalence of iron deficiency anemia in the United States is now very low. This implies that the current iron consumption is adequate for most individuals. An important unresolved question relates to the necessity for further reducing the prevalence of iron deficiency without anemia. More information is required to determine whether this lesser degree of iron deficiency is harmful. Recent survey data indicate that concomitantly with the reduced prevalence of iron deficiency there has been a rise in serum ferritin concentrations in American men and postmenopausal women. These findings have led to concern about the effectiveness of the physiological mechanisms for limiting storage accumulation in normal individuals and carriers of the hemochromatosis gene when dietary iron content is high. Furthermore, recent epidemiological observations suggest that a modest increase in iron stores (in a range previously considered safe) is a possible risk factor for ischemic heart disease and cancer; however, a causal relationship remains to be proven. Nonetheless, because there is no known benefit of high iron storage status, it seems prudent to avoid further increases in and possibly to reduce the dietary iron intake of men and postmenopausal women. Mean intake in these groups exceeds the current RDA by a significant margin. Therefore, the sources of dietary iron as well as other factors contributing to high serum ferritin values have to be defined. Also, efforts should be made to increase the awareness of professionals and the public about the possible risks of excessive dietary iron. The complexity of the Western diet and an incomplete understanding of all of the factors affecting serum ferritin concentrations make it very difficult to specify a safe upper range for daily iron intake at the present time.

Iron overload: prevalence and impact on health.

Clinical manifestations attributable to the damaging effects of iron occur when the total body iron is 5-10 times higher than normal. In adults this degree of iron overload occurs in hereditary hemochromatosis, a human leukocyte antigen (HLA)-linked autosomal recessive disorder, as well as in secondary iron-overload conditions.

Inhibitory effect of a soybean-protein--related moiety on iron absorption in humans.

The inhibitory effect of soybean protein isolates on nonheme-iron absorption was studied in 34 human subjects. Iron absorption was measured by using an extrinsic radioiron label in liquid-formula meals containing hydrolyzed corn starch, corn oil, and either egg white or a series of soybean-protein derivatives. The unmodified soybean-protein isolate markedly inhibited iron absorption. Percentage absorption was 19-fold higher when an extensively enzyme-hydrolyzed preparation with very little phytate was used as the protein source. Both the glycinin (11S) and conglycinin (7S) fractions of soybean protein were inhibitory to iron absorption. Dephytinization removed the inhibitory effect of the glycinin but not of the conglycinin fraction. We conclude that there are two major inhibitors of iron absorption in soybean-protein isolates, phytic acid and a protein-related moiety contained in the conglycinin (7S) fraction.

Resonance Raman spectroscopy of the cytochrome c oxidase from Paracoccus denitrificans.

Resonance Raman spectra are reported for the fully reduced unliganded and cyanide-bound mixed-valence forms of the cytochrome c oxidases from bovine heart and Paracoccus denitrificans in both detergent-solubilized forms and within their natural membrane environments. Comparison of the vibrational patterns observed for these enzymes indicates that overall the heme environments are similar for both. The only major differences seen between the spectra of these two enzymes are for vibrations associated with the low-spin bis(histidine)-coordinated heme cytochrome a. The data reported here serve to further illustrate the close structural and functional relationship between these evolutionarily distant enzymes. However, the data also demonstrate specific differences between the nature of the heme-protein interactions in the cytochrome a binding pocket which may be of mechanistic importance with regard to intramolecular electron transfer in these enzymes.

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.

Soy protein, phytate, and iron absorption in humans.

The effect of reducing the phytate in soy-protein isolates on nonheme-iron absorption was examined in 32 human subjects. Iron absorption was measured by using an extrinsic radioiron label in liquid-formula meals containing hydrolyzed corn starch, corn oil, and either egg white or one of a series of soy-protein isolates with different phytate contents. Iron absorption increased four- to fivefold when phytic acid was reduced from its native amount of 4.9-8.4 to less than 0.01 mg/g of isolate. Even relatively small quantities of residual phytate were strongly inhibitory and phytic acid had to be reduced to less than 0.3 mg/g of isolate (corresponding to less than 10 mg phytic acid/meal) before a meaningful increase in iron absorption was observed. However, even after removal of virtually all the phytic acid, iron absorption from the soy-protein meal was still only half that of the egg white control. It is concluded that phytic acid is a major inhibitory factor of iron absorption in soy-protein isolates but that other factors contribute to the poor bioavailability of iron from these products.

In vitro growth modulation by L-ascorbic acid of colony-forming cells from bone marrow of patients with myelodysplastic syndromes.

In vitro colony growth was studied on bone marrow cells from 51 patients with myelodysplastic syndromes (MDS), using a cell culture method with the unique feature of daily feeding, in an effort to gain insight into the pathophysiology of MDS and to assess the clinical utility of this cell culture assay. The colony growth pattern of MDS marrow cells is remarkably similar to that of acute myeloid leukemia but quite dissimilar from that of normal marrow, in support of a common pathophysiological mechanism for these two disorders. In particular, L-ascorbic acid (LAA) enhanced colony growth in 30% and suppressed growth in 16% of cases, a finding also similar to that in acute myeloid leukemia, indicating a unique growth requirement which may be explored for therapeutic purposes. Further, these LAA effects have prognostic value, with LAA-sensitive (both LAA-enhanced and LAA-suppressed) cases displaying shorter survivals than LAA-insensitive cases (median survival of 5 months versus 18 months; P = 0.011). This prognostic value is independent of, and more powerful than, bone marrow blasts; the median survival was 18 months for less than 5% bone marrow blasts and 8 months for greater than 5% bone marrow blasts (P = 0.044). These two risk factors can be used together to identify patients with an extremely good or an extremely poor prognosis. This study establishes the clinical usefulness of the LAA effect in MDS as a prognostic factor and provides a new lead to explore in understanding differential biochemical/molecular events and, possibly, a new therapeutic approach to the management of MDS.

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmström (Malmström, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.

Assessment of the role of nonheme-iron availability in iron balance.

To assess the nutritional relevance of absorption studies that use extrinsically labeled single meals, we developed a method for measuring nonheme-iron absorption from the diet and compared the results with absorption from single meals. When subjects consumed their usual diet, there was good agreement between dietary absorption (6.4%) and representative single meals fed in the laboratory (6.1%). Nonheme-iron availability, as estimated by a model that incorporated the effect of both enhancers and inhibitors, correlated significantly with absorption from single meals but not with dietary absorption. When the diet was modified to promote iron absorption maximally, dietary absorption increased only slightly (8.0%) and remained significantly lower than it was from single meals (13.5%). With an inhibitory diet, the decrease in absorption from single meals was similarly exaggerated. These results indicate that in the context of a varied Western diet, nonheme-iron bioavailability is less important than absorption studies with single meals would suggest.