Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-d -glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.     

The influence of phosphorus,zinc and manganese on absorption and translocation of iron in watercress

Summary Absorption and translocation of iron by intact watercress plants (Rorippa nasturtium-aquaticum (L) Hayek) was studied in short period uptake experiments utilising ⁵⁹Fe labelled ferric chloride. Total translocation of iron was inhibited by increasing levels of phosphorus, zinc and manganese in the nutrient medium; the elevated phosphorus and zinc concentrations enhanced iron absorption into roots, but increased retention of absorbed iron in translocating portions of the plant. High levels of manganese in the medium reduced the initial absorption of iron into the root system.     

Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection.

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86-258) and a small peptide (324-329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.     

Transformation of iron and manganese in rice soils under different moisture regimes and organic matter applications

Summary Transformation of iron and manganese under three different moisture regimes,viz continuous waterlogged (W₁), continuous saturated (W₂) and alternate waterlogged and saturated (W₃) and three levels of organic matterviz 0, 0.5 and 1.0% in all possible combinations was studied in four soils. The results showed that under waterlogged moisture regime there was a sharp increase in the content of water soluble plus exchangeable manganese accompanied by significant decrease in the content of reducible manganese in all the soils excepting the acidic soil which was very poor in active manganese content. The increase in respect of iron in similar form was, however, very small. The increase in the content of water soluble plus exchangeable manganese as well as iron under the continuous saturated and alternate waterlogged and saturated moisture regimes was always much lower as compared to that under the continuous waterlogged condition. Application of organic matter brought about an increase in the content of water soluble plus exchangeable manganese in all the soils excepting the lateritic one irrespective of moisture regimes but did not cause any change in the content of iron and manganese in insoluble complex. The content of water soluble plus exchangeable iron and of insoluble ferrous iron although recorded some increase due to organic matter application, the increase was not so marked in any of the soils.     

Plasmodium falciparum: inhibition in vitro with lactoferrin, desferriferrithiocin, and desferricrocin

The microbial iron chelators desferriferrithiocin and desferricrocin as well as human lactoferrin were tested in vitro against Plasmodium falciparum. The microbial chelators inhibit the growth of P. falciparum in a dose dependent way. Parasite multiplication is stopped at 25-30 microM desferriferrithiocin, whereas 60-90 microM desferricrocin are needed to exhibit the same effect. After iron saturation, the microbial chelators are ineffective. Human lactoferrin (30 microM), both iron free and iron saturated, inhibits P. falciparum. A 3-day preincubation of host erythrocytes with iron free and iron saturated lactoferrin prior to infection enhances this effect, which is therefore attributed to lactoferrin bound iron. It has been suggested that the lactoferrin/iron complex generates oxygen free radicals, which may cause membrane damage of both erythrocyte and parasite. This process can be considered to lead to growth inhibition of the parasite.     

Growth and nutrient uptake of peanut inoculated with the mycorrhizal fungusGlomus fasciculatum compared with non-inoculated ones

Summary In a greenhouse study, inoculation with the mycorrhizal fungusGlomus fasciculatum enhanced peanut growth and increased its dry matter more than 2-fold compared with the non-inoculated control, in both sterilized and non-sterilized soil. It also significantly increased uptake of phosphorus and micronutrients such as zinc, copper, manganese and iron.Present address: International Crops Research Institute for the semi-arid tropics (ICRIAST), Patancheru 502 324, A.P. India.     

FERRIC CHELATE REDUCTASE ACTIVITY AS AFFECTED BY THE IRON‐LIMITED GROWTH RATE IN FOUR SPECIES OF UNICELLULAR GREEN ALGAE (CHLOROPHYTA)1

Four species of green algae (Chlorella kessleri Fott et Nováková, Chlorococcum macrostigmatum Starr, Haematococcus lacustris[Girod‐Chantrans] Rostaf., Stichococcus bacillaris Näg.) were grown in iron‐limited chemostats and under phosphate limitation and iron (nutrient) sufficiency. For all four species, steady‐state culture density declined with decreasing degree of iron limitation (increasing iron‐limited growth rate), whereas chl per cell or biovolume increased. Plasma membrane ferric chelate reductase activity was enhanced by iron limitation in all species and suppressed by phosphate limitation and iron sufficiency. These results confirm previous work that C. kessleri uses a reductive mechanism of iron acquisition and also suggest that the other three species use the same mechanism. Although imposition of iron limitation led to enhanced activities of ferric chelate reductase in all species, the relationship between ferric chelate reductase activity and degree of iron limitation varied. Ferric chelate reductase activity in C. macrostigmatum and S. bacillaris was an inverse function of the degree of iron limitation, with the most rapidly growing iron‐limited cells exhibiting the highest ferric chelate reductase activity. In contrast, ferric chelate reductase activity was only weakly affected by the degree of iron limitation in C. kessleri and H. lacustris. Calculation of ferric reductase activity per unit chl allowed a clear differentiation between iron‐limited and iron‐sufficient cells. The possible extension of the ferric chelate reductase assay to investigate the absence or presence of iron limitation in natural waters may be feasible, but it is unlikely that the assay could be used to estimate the degree of iron limitation.     

白念珠菌CaSPO75基因的敲除与功能研究

酿酒酵母中ScCsc1是一个最近鉴定的钙离子通透性压力门控阳离子通道蛋白,参与调控胞内离子稳态。ScSpo75是ScCsc1的一个同源蛋白,可能参与孢子壁的装配过程。在白念珠菌中存在一个与ScSPO75同源的基因CaSPO75,其功能尚不清楚,因此采用SAT1-flipper方法敲除CaSPO75的两个等位基因以研究其功能。通过表型筛选,发现该基因的缺失导致白念珠菌对特比萘芬(Teb)、荧光增白剂(CFW)、氯化锰和氯化锌耐受,对酮康唑(KCZ)敏感。因此,CaSPO75基因可能参与白念珠菌耐药性和细胞壁压力反应的调控,以及胞内锰离子和锌离子稳态的调控。     

Growth and nutrition of small Betula pendula plants at different relative addition rates of iron,manganese and zinc

In order to investigate the dependence of growth rate upon plant concentrations of iron, manganese and zinc, Betula pendula seedlings were cultivated in a hydroponic system. In three different experiments, all essential nutrient elements except iron, manganese or zinc, were titrated in non-growth limiting amounts at low external concentrations. The solution was continuously recirculated and sprayed on the roots. The micronutrients (Fe, Mn and Zn) were added as addition rates, R_A (day^-1), relative to the calculated internal amount in the plants. No chelates were added to the culture solution.At steady-state nutrition, plant relative growth rate showed a linear dependence upon the internal concentration of the limiting micronutrient. These data do not support the Steenbjerg effect where negative correlations between growth and plant nutrient concentrations have been reported. Steady-state nutrition was associated with very different growth responses to the different limiting nutrients.     

Concentrations of Trace Metals in Dominant Aquatic Plants of the Lake Provala (Vojvodina, Yugoslavia)

The trace metal (Fe, Mn, Zn, Cu, Ni, Pb, Cd, Sr, and Cr) contents in the most common submerged and floating aquatic plants Ceratophyllum demersum L., Myriophyllum spicatum L., and Nymphoides flava Hill. of Provala Lake were evaluated. Considerable higher contents of iron, manganese, zinc, nickel, lead and strontium were found in submerged species than in the floating ones. The presence of cadmium and lead in plant tissues points to a certain degree of lake water pollution.     

Effect of cations,including heavy metals,on cadmium uptake by Lemna polyrhiza L.

Cations, including calcium, magnesium, potassium, sodium, copper, iron, nickel and zinc, inhibited (up to 40%) extracellular binding and intracellular uptake of cadmium by Lemna polyrhiza in solution culture. Test plants showed a high capacity of extracellular cadmium binding which was competitively inhibited by copper, nickel and zinc; however, calcium, magnesium and potassium caused non-competitive inhibition. Iron and sodium increased K _m and decreased V _max, thereby causing mixed inhibition of extracellular binding. Intracellular cadmium uptake displayed Michaelis-Menten kinetics. It was competitively inhibited by calcium, magnesium, iron, nickel and zinc. Monovalent cations (sodium and potassium) caused non-competitive and copper caused mixed inhibition of intracellular cadmium uptake. Thus, high levels of cations and metals in the external environment should be expected to lower the cadmium accumulation efficiency of L. polyrhiza.     

Influence of soil waterlogging on subsequent plant growth and trace metal content

Summary The uptake of trace metals by two plant species (French bean and maize) has been measured on two soils subjected to various waterlogging regimes. Uptake of both manganese and iron was increased due to soil waterlogging, although reoxidation of the soil affected iron more than manganese. Zinc and copper uptake was influenced by a species factor; French bean (Phaseolus vulgaris) showed preferential uptake of zinc, whereas maize (Zea mays) took up copper preferentially. Uptake of cobalt by both species was increased due to waterlogging, following the pattern of manganese.The abilities of these species to take up trace metals from soil followed the pattern predicted by selective extraction of soil for manganese, iron and cobalt, but not for zinc and copper.     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

<Emphasis Type="Italic">Legionella</Emphasis>, Protozoa,and Biofilms: Interactions Within Complex Microbial Systems

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.     

<Emphasis Type="Italic">Synechocystis</Emphasis> ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP⁺ oxidoreductase (FNR _S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR _S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR _S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR _S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR _S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.     

Relationship of trace metals to the alcohol-effect in citric acid synthesis by Aspergillus fonsecaeus

Summary Synthesis of citric acid by Aspergillus fonsecaeus in a basal medium supplemented with trace metals (iron, zinc, copper, and manganese) and methanol has been investigated. Methanol was found to be stimulatory to acid synthesis when manganese or manganese and iron were present. No other mineral tested could be substituted for manganese in the presence of the alcohol. However, comparable yields of citric acid could be obtained if methanol were deleted, the concentration of iron increased and zinc substituted for manganese.This investigation was conducted under a contract with the United States Department of Agriculture, authorized by the Research and Marketing Act, and administered through the Northern Regional Research Laboratory.     

Metal complexes of bovine lactoferrin inhibit in vitro replication of herpes simplex virus type 1 and 2

The inhibitory effect of bovine lactoferrin (BLf) saturated with ferric, manganese or zinc ions, on the infection of Vero cells by human herpes simplex virus type 1 (HSV1) and 2 (HSV2) was investigated. Viral infectivity determined by intracellular antigen synthesis and plaque formation was efficiently inhibited by metal saturated lactoferrins in a dose-dependent manner. Effective BLf concentrations which reduced the infection by 50% ranged from 5.2 to 31 mug ml and were far below the cytotoxicity threshold. Fe BLf and Mn BLf exhibited selectivity indexes higher than Zn BLf and apoBLf for both viruses and the effect was mainly directed towards the early steps of infection. The slight viral inhibition shown by the citrate complexes of the different metals could indicate that the antiviral effect was not significantly influenced by Fe , Mn or Zn ions delivered by BLf into the cells. © Rapid Science 1998     

Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes

Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449–2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.     

Manganese and iron interactions on their uptake and distribution in soybean (Glycine max (L.) Merr.)

Summary The uptake and distribution of iron and manganese were studied in a manganese-sensitive soybean cultivar (‘Bragg’) grown over a range of supply levels of these nutrients in solution culture. At high (90 and 275 μM) manganese levels, increasing the iron concentration in solution from 2 to 100 μM partially overcame the effects of manganese toxicity. Interactions between manganese and iron occurred for dry matter yields, rate of Mn absorption by the roots, and the proportions of manganese and iron transported to the tops. No interaction was observed for the rate of root absorption of iron. The percentage distribution of manganese in the plant top increased with increasing iron, despite a reduced rate of Mn uptake. On the other hand, iron uptake was independent of solution Mn concentration and increased with increasing solution Fe. Also more iron was retained in the roots at high Mn and/or Fe levels in solution. Concentrations of manganese and iron in roots, stems and individual leaves were affected independently by the manganese and iron supplyi.e. without any interaction occurring between the two elements. In general, the concentration in a plant part was related directly to the solution concentration. Symptoms resembling iron deficiency correlated poorly with leaf Fe concentrations whereas high levels of manganese were found in leaves displaying Mn toxicity symptoms.