Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

Uptake of Ga-67 into rat cerebral hemisphere and cerebellum. Comparison with Fe-59.

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K) were four times slower than those for 59Fe. Kin for Ga when infused with citrate were 0.88 ± 0.24 and 0.94 ± 0.39 x 10 ml gh for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of Ga into brain, particularly when infused with citrate; mean increases in uptake of Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

Hypothesis: Iron chelation plays a vital role in neutrophilic inflammation

Neutrophil influx into tissues occurs in many diverse diseases and can be associated with both beneficial and injurious effects. We hypothesize that the stimulus for certain neutrophilic inflammatory responses can be reduced to a series of competing reactions for iron, with either a labile or reactive coordination site available, between host chelators and chelators not indigenous to that specific living system. The iron focuses the transport of host phagocytic cells through a metal catalyzed generation of oxidant sensitive mediators including cytokines and eicosanoids. Many of these products are chemotactic for neutrophils. We also postulate that the iron increases the activity of the phagocyte associated NADPH oxidoreductase in the neutrophil. The function of this enzyme is likely to be the generation of superoxide in the hostÕs attempt to chemically reduce and dislodge the iron from its chelate complex. After the reoxidation of Fe in an aerobic environment, Fe will be coordinated by host lactoferrin released by the neutrophil. When complexed by this glycoprotein, the metal does not readily undergo oxidation/reduction and is safely transported to the macrophages of the reticuloendothelial system where it is stored in ferritin. Finally, we propose that the neutrophil will attempt to destroy the chelator not indigenous to the host by releasing granular contents other than lactoferrin. Inability to eliminate the chelator allows this sequence to repeat itself, which can lead to tissue injury. Such persistence of a metal chelate in the host may be associated with biomineralization, fibrosis, and cancer.     

Microbial adaptation to iron: a possible role of phosphatidylethanolamine in iron mineral deposition

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with iron(III) (5 mm) complexed to citrate, the sole source of carbon, with no apparent diminution in cellular mass. Atomic absorption studies of different cellular fractions and supernatant at various growth intervals revealed that the trivalent metal was initially internalized. At approximately 41 h of incubation, the soluble cellular extract contained 9.5% of the iron originally found in the growth medium. However, as bacterial multiplication progressed, most of the metal was deposited as an extracellular insoluble gelatinous residue. Phosphatidylethanolamine appeared to be an important organic constituent of this precipitate. X-ray fluorescence and diffraction studies revealed that iron(III) was deposited as amorphous hydrated oxide. Scanning electron microscopy and energy dispersive X-ray microanalysis of the pellet aided in the identification of irregular shaped bodies rich in iron and oxygen that were associated with carbon-containing elongated structures. Examination of the bacterial cells by a transmission electron microscope equipped with an electron energy loss spectrometer indicated the deposition of iron within the cells.     

Binding characterization of the iron transport receptor from the outer membrane of Escherichia coli (FepA): differentiation between FepA and FecA

The dissociation constants for the binding of ferric enterobactin with FepA and FecA are quantitated with displacement experiments. It is found that K _d for FepA is 12 times lower than the one for FecA. This indicates that FepA is an high-affinity receptor while FecA binds ferric enterobactin with a lower affinity. Monoclonal antibodies specific for binding epitopes of FepA inhibit the binding of ferric enterobactin with purified FepA. These same antibodies do not inhibit the binding of ferric enterobactin with purified FecA. This indicates that the binding epitopes in FecA and FepA are different.     

The nutritional ecology of larvae of Alsophila pometaria and Anisota senatoria feeding on early- and late-season oak foliage

The larvae of Alsophila pometaria (Harr.), feeding on the young foliage of oak, has a higher relative growth rate (RGR) and relative nitrogen accumulation rate (RNAR) than the larvae of Anisota senatoria (J. E. Smith), feeding on the mature foliage of oak. Although the young oak foliage is more efficiently digested by A. pometaria (higher AD's), it is not more efficiently assimilated and used for growth (no difference in ECI's). Thus, the higher growth rate of A. pometaria is due entirely to a higher consumption rate (RCR and RNCR). Young foliage is significantly higher in nitrogen and water than mature foliage, but phenol and tannin levels are comparable in young and old foliage. A. pometaria consumes the foliage of different oak species at the same rate, independent of nitrogen content, while A. senatoria increases its consumption rate in response to decreased nitrogen levels. As a result, the growth rate of A. pometaria is directly related to leaf nitrogen content, while the growth rate of A. senatoria is independent of leaf nitrogen. The two species of insects have digestive systems that are very similar biochemically, and that are well-designed for effective protein digestion. Tannins and phenols do not influence the nutrional indices of either species. We suggest that the major benefit of spring feeding is the availability of succulent, high-nitrogen foliage, and not the avoidance of high-tannin foliage. The spring feeder appears to have a feeding strategy that favors rapid growth at the expense of efficiency, while the late summer feeder has a strategy that favors efficiency over rate.

---

Résumé Alimentées sur feuillage jeune de chêne, les chenilles d'Alsophila pometaria avaient un taux relatif de croissance (RGR) et un taux relatif d'accumulation d'azote (RNAR) plus élevés que les chenilles d'Anisota senatoria alimentées sur feuillage mûr de chêne. Bien que le jeune feuillage soit plus efficacement digéré par A. pometaria (AD plus élevé), il n'est pas assimilé et utilisé pour la croissance avec de meilleurs rendements (les ECI ne sont pas différents). Ainsi le taux de croissance plus élevé d'A. pometaria est dû entièrement à un taux de consommation plus important (RCR et RNCR). Le feuillage jeune est significativement plus riche en azote et en eau que le feuillage mûr, mais les niveaux de phénol et de tanins sont les mêmes. A pometaria consomme les feuilles de différentes espèces de chênes au même taux, indépendamment de la teneur en azote, tandis que A. senatoria accroît sa consommation en réponse à une diminution de la teneur en azote. Il en résulte que le taux de croissance d'A. pometaria dépend directement de la teneur en azote des feuilles, tandis que celui d'A. senatoria en est indépendant. Les systèmes digestifs des deux insectes sont biochimiquement semblables et sont efficaces pour la digestion des protéines. Les tanins et les phénols n'influent pas sur les indices nutritionnels de ces deux espèces. Nous estimons que le principal intérêt de l'alimentation printanière est la disponibilité en feuillage succulent, riche en azote, et non l'absence de feuilles à haute teneur en tanin. L'alimentation printanière semble correspondre à une strategie alimentaire qui favorise la croissance aux dépens de l'efficacité tandis que l'alimentation en fin d'été est une stratégie qui favorise l'efficacité sur la rapidité.

     

Molecular cloning of the Escherichia coli K-12 entACGBE genes

Abstract A 7-kb piece of Escherichia coli DNA that contains five genes ( entA, C, G, B and E ) required for the biosynthesis of the iron transport molecule enterochelin was isolated. A restriction map was constructed and proteins specified by the E. coli DNA were identified in mini- and maxicell systems. Plasmids containing portions of the entACGBE DNA generated by BAL31 digestion or restriction enzyme treatment were constructed; complementation studies done with these indicated that the five genes constitute an operon. The approximate site of the promoter was determined and the product of entE was tentatively identified as an M _r 63000 polypeptide.     

EFFECTS OF IRON DEFICIENCY ON ISOCHRYSIS GALBANA (CHRYSOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of Isochrysis galbana Parks and Phaeodactylum tricornutum Bohlin were grown in iron-limited chemostats. With increasing iron deficiency, photosynthetic rate per cell and assimilation number decreased. The pattern of photosynthesis was also altered; in Fe deficient cells the proportion of ¹⁴C fixed in glycine and serine decreased with an accompanying increase into alanine after 3 min assimilation. Although there was no significant effect of Fe deficiency on the proportion of ¹⁴C incorporated into total amino acids and amides, the percentage of total ¹⁴C fixed in protein increased with increasing Fe deficiency. Cellular levels of chlorophyll a, carotenoids, cytochromes and protein also decreased with increasing Fe deficiency. However, the reduction in chlorophyll a/cell was not as great as that of cytochrorne f₁ and Fe deficient cells therefore showed a marked increase in chlorophyll a:cytochrorne f₁ ratio.