Does the chloroplast envelope contain carotenoids and quinones in vivo?

Leaves, cotyledons, isolated chloroplasts and subplastid fractions (thylakoids and envelopes) of radish ( Raphanus sativus L. cv. Saxa) and spinach ( Spinacia oleracea L. cv. Matador) were assayed for their pigment and quinone content and composition. Evidence is presented suggesting that the chloroplast envelope does not contain carotenoids and quinones in vivo. Envelopes prepared by the method described contained very low amounts of chlorophyll a and b , violaxanthin and neoxanthin, but no β-carotene, lutein, zeaxanthin and antheraxanthin. Among the quinones, trace amounts of plastoquinone and α-tocopherol but no plastohydroquinone, α-tocoquinone and phylloquinone were detected. The data presented suggest that, contrary to previous findings, carotenoids and quinones are not located in the chloroplast envelope.     

Is a ferroxidase involved in the high-affinity iron uptake in Chlamydomonas reinhardtii?

In phosphorus deficient soils and under smallscale farming systems, the development of efficient management strategies for P fertilizers is crucial to sustain food production. A field experiment was conducted on a P-fixing Acrisol in western Kenya to study possibilities of replenishing soil P with seasonal additions of small rates of P fertilizers. Triple superphosphate was applied at 0, 10, 25, 50 and 150 kg P ha^–1 for 5 consecutive maize growing seasons followed by 4 seasons of residual crops. Maize yields and soil P fractions were determined. Although maize responded to additions of 10 kg P ha^–1 with a cumulative grain yield of 16.8 Mg ha^–1, at the end of the experiment, compared to 8.8 Mg ha^–1 in the non-P fertilized plots, soil labile P did not increase correspondingly. Seasonal additions of 150 kg P ha^–1 increased maize yields to a cumulative value of 39 Mg ha^–1 at the end of the experiment, and increased all soil inorganic P fractions. At the third season of residual phase, treatment with a cumulative addition of 750 kg P ha^–1 gave the highest yields compared to treatments in the same residual stage, but these yields were considered less than the maximum yield of the season. This indicates that the large build up of soil P was not available for crop uptake. The inorganic P fraction extracted by NaHCO₃ was the most affected by changes in management, increasing during the input phase and decreasing after interruption of P addition, for all P rates. The decrease in this pool during the residual phase could be explained by the maize uptake. This study showed that seasonal additions of 25 kg P ha^–1 can increase maize yield with gradual replenishment of soil P.     

Transferrin, is a mixed chelate-protein ternary complex involved in the mechanism of iron uptake by serum-transferrin in vitro?

Iron uptake by transferrin from triacetohydroxamatoFe(III) (Fe(AHA)3) in the presence of bicarbonate has been investigated between pH 7 and 8.2. The protein transits from the opened apo- to the closed holoform by several steps with the accumulation of at least three kinetic intermediates. All these steps are accompanied by proton losses, probably occurring from the protein ligands and the side-chains involved in the interdomain H-bonding nets. The minor bihydroxamatoFe(III) species Fe(AHA)2 exchanges its iron with the C-site of apotransferrin in interaction with bicarbonate without detectable formation of any intermediate protein-iron-ligand mixed complex; direct second-order rate constant k1=4.15(+/-0.05)x10(7) M(-1) s(-1). The kinetic product loses a single proton and undergoes a modification in its conformation followed by the loss of two or three protons; first-order rate constant k2=3.25(+/-0.15) s(-1). This induces a new modification in the conformation; first-order rate constant k3=5.90(+/-0.30)x10(-2) s(-1). This new modification in conformation rate controls iron uptake by the N-site of the protein and is followed by a single proton loss; K3a=6.80 nM. Finally, the holoprotein or the monoferric transferrin in its thermodynamic equilibrated state is produced by a last modification in the conformation occurring in about 4000 seconds. But for the Fe(AHA)3 dissociation and the involvement of Fe(AHA)2 in the first step of iron uptake, this mechanism is identical to that reported for iron uptake from FeNAc3. This implies that the exchange of iron between a chelate and serum-transferrin occurs by a single general mechanism. The nature of the iron-providing chelate is only important for the first kinetic step of the exchange, which can be slowed to such an extent that it rate limits the exchange of iron.     

Does the peroxide compound of cytochrome oxidase contain a ferryl iron?

The reaction of peroxide with cytochrome oxidase generates a peroxide compound having a Soret maximum at 428 nm. X-ray absorption spectroscopy analysis of the local structure of the active site iron shows marked similarity to that of the cytochrome c peroxidase intermediate Compound ES, which contains a short iron to proximal nitrogen distance compared to globins. Reductive titration of the 580 nm band of this compound indicates that the iron is one oxidizing equivalent above the resting oxidized form. These results support the presence of a ferryl iron (Fe(IV) = O) in the peroxide compound similar to that found for the peroxidases.     

Does a quorum sensing mechanism direct the behavior of immune cells?

Quorum sensing is a decision-making process used by decentralized groups such as colonies of bacteria to trigger a coordinated behavior. The existence of decentralized coordinated behavior has also been suggested in the immune system. In this paper, we explore the possibility for quorum sensing mechanisms in the immune response. Cytokines are good candidates as inducer of quorum sensing effects on migration, proliferation and differentiation of immune cells. The existence of a quorum sensing mechanism should be explored experimentally. It may provide new perspectives into immune responses and could lead to new therapeutic strategies.     

Does a redox cycle provide a mechanism for setting the capacity of neuroglobin to protect cells from apoptosis?

We hypothesize that the various, previously reported, reactivities of neuroglobin with redox partners and oxygen provide for the establishment of a redox cycle within cells, such as neurons and retinal rod cells. Using native cell lysates, from cultured human cells of neuronal origin, we have estimated the rate of reduction of the oxidized form of neuroglobin in vivo. Furthermore we provide evidence that the cytosol of these cells contains factors (presumably enzymes) capable of employing either glutathione or NADH as re-reductants of ferric neuroglobin. Taken in conjunction with previous rate data, for the various redox reactions of neuroglobin, this information allows us to set up a computer model to estimate the steady state cellular level of the antiapoptotic ferrous form of neuroglobin. This model indicates that the steady state level of antiapoptotic neuroglobin is very sensitive to the cellular oxygen tension and moderately sensitive to the redox status of the cell. Further analysis indicates that such a system would be capable of significant modification, on the seconds time scale, following hypoxic transition, as is likely in stroke. We hypothesize that this mechanism might provide a moderately rapid mechanism for adjusting the antiapoptotic status of a cell, whilst the reaction of neuroglobin with mitochondrial cytochrome c provides a very rapid, but limited, capacity to intervene in the apoptotic pathway.     

Are lysosomes directly involved in the iron uptake by reticulocytes?

• 1.1. The mechanism by which iron is transferred from the plasma protein transferrin into erythroid precursors for incorporation in heme is not completely understood.
• 2.2. To show a direct functional role of lysosomes in the process of iron uptake we tried to isolate lysosomes from reticulocytes, which have been incubated with ¹²⁵ITf⁵⁹Fe.
• 3.3. However, with various cell fractionating techniques described for liver cells no pure lysosomes from reticulocytes could be obtained.
• 4.4. Fluorescence and electron microscopy showed that reticulocytes hardly contain well-defined lysosomes.
• 5.5. There are several indications that in reticulocytes acid vacuoles instead of lysosomes are involved in the removal of iron from endocytosed transferrin.
• 6.6. The presence of apoTf, monoferric TfFe(A), monoferric TfFe(B) in the medium after incubation of reticulocytes with diferric transferrin, together with the fact that both iron binding sites of transferrin release their iron at pH present in acid vacuoles, suggests a second mechanism of iron uptake by reticulocytes, in which acid vacuoles are not involved.

     

Could root-excreted iron ligands contribute to cadmium and zinc uptake by the hyperaccumulator Noccaea caerulescens?

Plant and Soil - Evaluation of various mechanisms of Cd2+ and Zn2+ root uptake through modelling, including interactions with Fe uptake. Four increasingly complex models were developed and tested...     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Does sphingomyelin inhibit the erythrocyte anion transport system?

The anion transport protein of the human erythrocyte membrane, band 3, was incorporated into unilamellar sphingomyelin vesicles. The vesicles showed a rapid sulfate efflux which could be inhibited by specific inhibitors of the erythrocyte anion transport system. All band 3 molecules contributing to the inhibitor-sensitive flux component were arranged 'right-side-out'. The turnover number of the transport protein for sulfate transport was virtually identical to that in phosphatidylcholine bilayers and around 6 times larger than in human erythrocyte membranes. Thus, in contrast to other claims, sphingomyelin does not inhibit the erythrocyte anion transport system.     

Intra-Golgi transport: a way to a new paradigm?

The morpho-functional principles of intra-Golgi transport are, surprisingly, still not clear, which is in marked contrast to our advanced knowledge of the underlying molecular machineries. Recently, the conceptual and technological hindrances that had delayed progress in this area have been disappearing, and a cluster of powerful morphological techniques has been revealing new glimpses of the organization of traffic in intact cells. Here, we discuss the new concepts around the present models of intra-Golgi transport.     

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (P_M) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the P_M across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the P_M of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~ 10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.     

Does a targeting ligand influence nanoparticle tumor localization or uptake?

Inclusion of a tumor-targeting molecule in nanosized delivery systems increases their in vivo efficacy. However, the biodistribution and pharmacokinetics of the uptake of such particles have not yet been well addressed. Several recent papers have suggested that tumor-targeting ligands function primarily to increase intracellular uptake of the nanocomplex and do not influence tumor localization. However, other reports indicate that they do play a role in the accumulation in the tumor. One difference might be the presence or absence of poly-[ethylene glycol] (PEG) in the complex and its impact on the enhanced permeability and retention (EPR) effect. Further studies are clearly needed to more fully elucidate the influence of composition on tumor-targeted, systemic delivery of nanoparticles.     

Does high bicarbonate supply to roots change availability of iron in the leaf apoplast?

The role of the leaf apoplast in iron (Fe) uptake into the leaf symplast is insufficiently understood, particularly in relation to the supposed inactivation of Fe in leaves caused by elevated bicarbonate in calcareous soils. It has been supposed that high bicarbonate supply to roots increases the pH of the leaf apoplast which decreases the physiological availability of Fe in leaf tissues. The study reported here has been carried out with sunflower plants grown in nutrient solution and with grapevine plants grown on calcareous soil under field conditions. The data obtained clearly show that the pH of the leaf apoplastic fluid was not affected by high bicarbonate supply in the root medium (nutrient solution and field experiments). The concentrations of total, symplastic and apoplastic Fe were decreased in chlorotic leaves of both sunflower (nutrient solution experiment) and grapevine plants in which leaf expansion was slightly inhibited (field experiment). However, in grapevine showing severe inhibition of leaf growth, total Fe concentration in chlorotic leaves was the same or even higher than in green ones, indicative to the so-called `chlorosis paradox'. The findings do not support the hypothesis of Fe inactivation in the leaf apoplast as the cause of Fe deficiency chlorosis since no increase was found in the relative amount of apoplastic Fe (% of total leaf Fe) either in the leaves of sunflower or grapevine plants. It is concluded that high bicarbonate concentration in the soil solution does not decrease Fe availability in the leaf apoplast.     

Investigation of iron pools in cucumber roots by Mössbauer spectroscopy: direct evidence for the Strategy I iron uptake mechanism

Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without ⁵⁷Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM ⁵⁷Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe^III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe^II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM ⁵⁷Fe^III-citrate for 30 min contained significant amount of Fe^II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe^II–Fe^III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants.     

Diesel emissions revisited: is the carcinogenicity due to a genotoxic mechanism?

The induction of recA, umuC and sfiA genes by quercetin was studied in the presence and in the absence of S9 mix. The inducing activity of quercetin is higher for sfiA than for recA and umuC genes in the absence of S9 mix. The putative genotoxic metabolites of quercetin produced by S9 mix display different inducing activities of the three SOS genes as compared to quercetin. The induction of sfiA gene is decreased by the presence of S9 mix, whereas an opposite effect was observed concerning umuC and recA. These data suggest that the error-prone repair pathway participates in mutagenesis by quercetin and its metabolites. Moreover, the type of DNA damage exerted by quercetin seems to be determined by its metabolic fate. The importance of testing for the induction of other SOS genes, together with sfiA, in the study of SOS functions as a genotoxic index is emphasized.     

Mutations introduced along the HTLV-I envelope gene result in a non-functional protein: a basis for envelope conservation?

The envelope protein of the human T-cell leukemia virus type I (HTLV-I) is highly conserved among the isolates sequenced so far, as opposed to what is observed for the human immunodeficiency virus (HIV) envelope. By linker insertion scanning, we have produced 33 random mutations along the HTLV-I envelope gene, cloned into a eukaryotic expression vector. The resulting envelope products were analysed by immunoprecipitation and syncytia formation after transfection into COS-1 cells. We show here that 25 out of 33 mutations result in a non-functional envelope product as assessed by the lack of ability to form syncytia. In the majority of these mutants, the processing of the envelope gp61 precursor into the mature gp45 and gp20 proteins was affected. We propose that conformational constraints for processing and fusion abilities tend to limit the variability of the HTLV-I envelope. In three mutants, processing was observed but no syncytia were formed. These mutations might affect regions important for HTLV-I envelope functions, such as the receptor binding region.     

Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.