Enhancement of embryo yield from isolated microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> by early iron starvation

Induction of embryogenesis in isolated microspores of Brassica napus requires stress conditions to trigger the developmental instead of the gametophytic pathway. To obtain further insight into the involvement of different ions in this process, a comparison has been made between embryo yields obtained with standard NLN-13 medium and the same medium without cobalt, copper or iron. It was confirmed that iron was essential to control embryo development, but not cobalt and copper. For the latter two ions, the concentration is probably too low to play a significant role in microspore embryogenesis. With the timing of iron application, as well as its chemical form, embryo yield could be improved or reduced. In media that exhibited iron deficiency, microspores initiated embryogenesis and the number of observed divided microspores increased 6 days after isolation. However, embryo development was not achieved. Addition of iron ions chelated with EDTA at day 3, leading to the doubling of embryo yield. Some of the putative role(s) of Fe-EDTA in the early events of embryogenesis is discussed.     

Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.     

Importance of mitochondria in survival of Cryptococcus neoformans under low oxygen conditions and tolerance to cobalt chloride

Cryptococcus neoformans is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in C. neoformans, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl(2))-sensitive mutants. All the CoCl(2)-sensitive mutants had a growth defect under low oxygen conditions at 37 degrees C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl(2) as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.     

Transformation of Fe,Mn, Co,and Ni compounds in humic podzols at different moisture

Incubation of humic podzol at soil moisture of 60 and 100% field capacity (FC) and after addition of peat and glucose increased the content of nickel and cobalt compounds in the water-soluble, exchangeable, organic matter-bound, and amorphous iron-bound fractions. At the same time, the content of elements bound to crystallized iron compounds decreased twofold and fourfold at 60 and 100% FC, respectively. The content of cobalt and nickel decreased in the residual fraction by 25 and 50%, respectively. The transformation of cobalt and nickel in soil is closely related to the transformation of iron and manganese compounds as well as to redox processes. The lowest pH and redox potential (RP) as well as the highest increase in the mobility of the elements was observed after soil incubation with glucose at 100% FC.     

Cobalt targets multiple metabolic processes in Salmonella enterica

Cobalt is essential for growth of Salmonella enterica and other organisms, yet this metal can be toxic when present in excess. Wild-type Salmonella exhibits several metabolic defects when grown in the presence of cobalt, some of which generate visible growth consequences. Work herein identifies sulfur assimilation, iron homeostasis, and Fe-S cluster metabolism as targets for cobalt toxicity. In each case it is proposed that cobalt exerts its effect by one of two mechanisms: direct competition with iron or indirectly through a mechanism that involves the status of reduced thiols in the cell. Cobalt toxicity results in decreased siroheme production, increased expression of the Fur regulon, and decreased activity of Fe-S cluster proteins. The consequences of reduced sulfite reductase activity in particular are exacerbated by the need for glutathione in cobalt resistance. Significantly, independent metabolic perturbations could be detected at cobalt concentrations below those required to generate a detectable growth defect.     

Cobalt toxicity and iron metabolism in Neurospora crassa

1. Increasing concentrations of cobalt in the medium result in increased production of an iron-binding compound and a corresponding fall in catalase activity of Neurospora crassa. 2. Cobalt rapidly depletes the medium of iron by enhancing the rate of iron uptake by the mycelium. 3. With toxic amounts of cobalt there is a fall in bound (59)Fe and haem (59)Fe as well as a decreased incorporation of [2-(14)C]glycine into the mycelial haem fraction. The production of the iron-binding compound precedes the fall in the iron-dependent systems mentioned. 4. The (59)Fe bound to the iron-binding compound acts as a better iron source for haem synthesis in cell-free extracts as compared with (59)FeSO(4). 5. Cobalt inhibits iron incorporation into protoporphyrin in cell-free extracts but is not itself incorporated to an appreciable extent.     

Effect of metal oxides on the growth,hemolytic and serologic properties of Klebsiella pneumoniae

Silicon, dysprosium, germanium, yttrium, iron, cobalt, samarium, lutecium oxides, as well as the mixture of 8 metal oxides, at a concentration of 20 g/l were found to produce a stimulating or inhibiting effect on the growth of K. pneumoniae strains 204 and K-9. Silicon, dysprosium, germanium and yttrium oxides were shown to stimulate the growth of K. pneumoniae strain 204. Iron, cobalt, samarium and lutecium oxides, as well as the mixtures of all oxides under study, inhibited the growth of this strain. Silicon, samarium and lutecium oxides produced no effect on the growth of K. pneumoniae strain K-9; at the same time germanium and yttrium oxides stimulated the growth of these bacteria, while dysprosium, iron, cobalt oxides, as well as the mixture of all oxides, inhibited their growth. The presence of metal oxides did not change the serological activity of the cultures of both strains growing old, i.e. by 24 hours of their growth. The addition of silicon, germanium and iron oxides to the culture medium increased the hemolytic activity of K. pneumoniae strain K-9 seven to ninefold in comparison with the control grown in a synthetic nutrient medium without metal oxides. The comparison of these two strains (K-9 and 204) revealed that K. pneumoniae strain K-9 possessed greater hemolytic activity.     

Quantification and Comparison of Soil Elements in the Tibetan Plateau Kaschin-Beck Disease Area

The prevalence rate of Kaschin-Beck disease (KBD) in most parts of China is currently decreasing, but the disease is still active and severe on the Tibetan Plateau. Soil samples including the surface layer (0–20 cm) and the subsurface layer (20–40 cm) in the cultivated and natural soil profiles were collected, and the mechanical composite and total concentration of arsenic, cobalt, copper, iron, mercury, manganese, molybdenum, selenium, and zinc were determined. Concentrations of arsenic, iron, manganese, copper, and selenium in the surface soil were lower than those in the subsurface soil. The same was true of physical clay in the soil profiles. However, there were no significant differences between the different soil layers. The concentrations of selenium, molybdenum, and mercury were somewhat lower compared with the average concentrations of soils in China. Deficiencies of molybdenum and selenium both play a critical role in occurrence of KBD, but whether or not soil mercury at a low level contributes to KBD is still unclear. A correlation analysis of soil elements showed that there is a positive correlation between iron, cobalt, and manganese due to their similar chemical characteristics. Selenium concentrations in soil as well as the physical clay and iron descend with the deterioration of KBD, but mercury concentrations in soil ascend with the aggravation of the disease. The results show that selenium deficiencies greatly influence the disease, and a deficiency of molybdenum is likely another important factor in inducing KBD. Moreover, determining whether low levels of soil mercury contribute to KBD should be investigated in the future.     

Properties of membrane bound ferrochelatase purified from baboon liver mitochondria

1. Baboon ferrochelatase was purified to apparent homogeneity. 2. The pH optimum was 7.85 and the pI 5.3. 3. The estimated molecular weight was 205 K made up by two 50 + 60 K heterodimers. 4. The Km values for proto- and mesoporphyrin were 18.5 and 10.8 microM with iron as co-substrate. With cobalt as co-substrate the Km values were 34.5 and 10.4 microM, respectively. The mean Km value for iron was 2.2 microM while cobalt acted as a complete inhibitor. 5. Lead played a dual role that of both pseudo substrate and inhibitor. As shown by inhibitor kinetics, Pb acted as a two-step two-site parabolic competitive inhibitor. The mean Ki value at low Pb levels was 0.65 mM and at high levels 0.17 mM. 6. Substrate inhibition occurred above 36 microM for proto- and 44 microM for mesoporphyrin with iron as co-substrate. For iron, with mesoporphyrin as co-substrate it occurred above 29 microM.     

Structural studies of iron and cobalt tetrasulfonated phthalocyanine-globin complexes.

The structure of the complexes of iron and cobalt tetrasulfonated phthalocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis. Electrophoretic investigations and the molecular weight estimation indicates that the model complexes in the solutions are dimers. It is evident from the results of CD measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohemoglobin significantly increases the helical structure of the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of Fe(III)L-globin are observed which suggest an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(III)L-globin are in two low electronic states. Electronic structures of the cobalt ions in Co(II)L-globin and oxyCo(II)L-globin are similar to those of coboglobin and oxycoboglobin, respectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(II)L-globin can be described as a superoxide ion corrdinated to a formally cobaltic phthalocyanine compound.     

Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Cobalt-substituted hemoglobin Zürich (alpha 2 beta 263His leads Arg). Oxygen equilibria and EPR spectra.

Cobalt hemoglobin Zürich (alpha 2 beta 263His leads to Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl-. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl- affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H2O to 2H2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal beta subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

Induction of iron and cobalt dependent acrylonitrile hydratases inArthrobacter sp. IPCB-3

Summary ACN-hydratase inArthrobacter sp. IPCB-3 has been found to be induced by acetonitrile and urea and repressed by glucose. When acetonitrile was used as an inducer the synthesis of enzyme increased to about 2 folds and 4.5 folds on addition of iron and cobalt to the medium, respectively. However, when urea was used as an inducer only cobalt stimulated the enzyme synthesis and gave maximum activity (70 units/mg dry cells). In contrast to the stimulation of iron containing ACN-hydratase, yeast extract failed to stimulate further the synthesis of cobalt containing enzyme irrespective of the inducer present in the medium.     

The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.