Mode of in vitro interaction of mercuric mercury with selenite to form high-molecular weight substance in rabbit blood

Mode of interaction of mercuric mercury and selenite in rabbit blood was investigated in vitro. After the incubation of rabbit blood with 10^?5 M each of ²⁰³HgCl₂ and Na₂⁷⁵SeO₃, the amounts of both ²⁰³Hg and ⁷⁵Se incorporated into erythrocytes were markedly larger than the case where the blood was treated separately with one of these compounds. Most of ²⁰³Hg and ⁷⁵Se distributed into plasma and erythrocytes were found in high-molecular weight substance(s) (HMWS) fractionated by gel filtration at a molar ratio of 1:1. The ²⁰³Hg and ⁷⁵Se in HMWS found in plasma and erythrocytes were hardly diffusable through the erythrocytes membrane. The formation of the HMWS containing mercury and selenium was observed in stroma-free hemolysate incubated with mercuric chloride and selenite, but not in plasma. Addition of reduced glutathione (GSH) to the plasma, however, gave the HMWS as reaction products containing equimolar amounts of mercury and selenium. Further the binding properties of selenium to proteins were studied in the plasma incubated with selenodiglutathione (GSSeSG) or with selenite in the presence of GSH. The results indicated that GSH, a cellular component, is essential for the formation of an active selenium compound from selenite and that the interaction of mercuric mercury and selenite in plasma in the presence of GSH may occur through the other mechanism than the formation of GSSeSG.     

Effect of selenite and selenate on rat liver nuclear 3,5,3′-triiodothyronine (T3) receptor

The present study was undertaken to investigate the effects of selenite (Se^IV) or selenate (Se^VI) on nuclear T₃ receptors of rat liver. Selenite at 0.1 μM (p<0.01) inhibited the T₃ specific binding to rat liver nuclear receptors. The specific binding of the T₃ receptor was fully restored when even 1.0 μM selenite was separated from the T₃ receptor by gel filtration. No inhibitory effect of selenite (up to 100 μM) on the T₃ binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T₃-nuclear receptor complex was effectively increased by 0.1 μM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T₃ receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T₃ binding on the receptor molecule.     

Abnormalities of blood selenium and glutathione peroxidase activity in patients with acquired immunodeficiency syndrome and aids-related complex

Severe protein-calorie malnutrition is common in patients with AIDS and could contribute to the progressive deterioration characteristic of that disease. Selenium deficiency could also have a negative impact on immune function and other organ functions vital for recovery from infectious diseases. Therefore, to assess any role for selenium in AIDS, we determined plasma and erythrocyte selenium levels and glutathione peroxidase activity in 13 patients with AIDS compared to 8 patients with AIDS-related complex (ARC) and 14 healthy controls. Plasma selenium levels were significantly reduced in AIDS patients compared to controls (p<.0001) and to ARC (p<.02). Erythrocyte selenium levels in both AIDS and ARC were also reduced compared to controls (p<.02), but not to each other. Glutathione peroxidase activity in AIDS was 28.9±1.4 U/g Hb vs 38.4±6.9 in ARC (p=NS) and 52.3±1.7 in controls (p<.0001 vs AIDS;p<.02 vs ARC). When all groups were combined, there were significant correlations between total lymphocyte count and both plasma selenium (r=.53;p<.002) and erythrocyte glutathione peroxidase activity (r=.65;p<.0001). In addition, strong correlations were noted between plasma selenium and serum albumin (r=.68;p<.0001), plasma selenium and glutathione peroxidase (r=.77;p<.0001), and glutathione peroxidase and hematocrit (r=.66;p<.0001). In AIDS or ARC, no correlations between selenium with disease duration or weight loss were present. We conclude that, in comparison to normals, patients manifesting infection with human immunodeficiency virus have evidence of selenium deficiency as determined by diminished plasma and erythrocyte levels and glutathione peroxidase activity. These abnormalities are most marked in patients with AIDS, but are also present in patients with AIDS-related complex. Selenium deficiency has important implications for the progression and pathogenesis of clinical disease in AIDS.     

Interactions between selenium and tin,selenium and lead,and their effects on alad activity in blood

Interactions of tin and selenium, as well as of lead and selenium, were investigated in male ICR mice. The toxic effects of selenium on mortality and body weight loss were reduced by simultaneous injection with tin or lead; among mice that were injected ip with selenium at 100 μmol/kg, the 24 h survival rate was 20%, whereas among mice that were administered selenium and tin or selenium and lead at the dose of 100 μmol/kg each, the rates were 100% and 90%, respectively. As for δ-aminolevulinic acid dehydratase (ALAD, EC 4.2.1.24), lead and tin were strong inhibitors, which is well known; selenium showed no effect. When more than an equimolar dose of sodium selenite was injected ip simultaneously with stannous chloride, the ALAD activity was completely retained. On the other hand, in the simultaneous injection with sodium selenite and lead acetate of differing ratios (Se/Pb), 1, 2.5, 5, and 7.5, selenium did not exhibit an obvious protective effect against the inhibition of ALAD activity caused by lead. It is suggested that selenium protects essential thiol groups in ALAD that are otherwise blocked by invading tin; in contrast, selenium, under similar conditions, does not prevent interactions of lead with enzyme thiol groups.     

Uptake,distribution, and turnover rates of selenium in barley

The present communication elucidates initially the topographic distribution of selenium in barley grains. Then by the fluorimetric method the uptake of selenium (selenite) in 8–16 d old germinating barley was estimated. Finally by means of⁷⁵Se the anabolic and catabolic rates (turnover) of⁷⁵Se (selenite) was compared. The distribution of selenium in barley was evaluated after microdissection of barley grains. In dried grains the highest concentration was found in husk and pericarp with about 0.6 ppm Se. Then followed Scutellum with 0.4 and 0.3 ppm in embryon. The aleurone layer, embryonic leaves, and initial root did only have 0.2 ppm Se. In order to know more about the uptake and distribution of selenium in 8-d-old barley, the plants were cultivated for a further 8 d in the culture medium with variation in selenite concentration. In roots and leaves, the uptake did not arrive at saturation during the period studied since the dose-response curve increased up to 0.34 mM selenite in the medium, whereas the selenium levels were about 200 ppm in roots and 30 ppm in leaves. However, the uptake was linear, with concentration during 8 d of cultivation up to 0.84 μM selenite for grain and stem. At higher concentrations the dose-response curve diminished its slope. At 0.34 mM selenite the concentration in grain increased to 6.87 ppm and in the stem to 8.13 ppm. The uptake, distribution, and catabolic rate of selenium components in germinating barley were further evaluated by exposing the plants to 0.0492 μCi⁷⁵Se (12.6 μM selenite) for up to 4 d. Then the plants were moved to a selenium deficient medium for further 4 d. Then finally the medium was supplemented with high doses of cold selenite (0.126 mM selenite) for further 4 d. The first third period made it possible to estimate the rate of uptake. It was highest in roots (313 fmol/h/mg dw), i.e., about 10 times those of grains, stems, and leaves. The intermediate period where the barley was transferred to a selenium deficient medium made it possible to estimate the kinetics and eventual sparing mechanisms. The selenium losses were highest for leaves (39%), then followed by roots and stems (22 and 25%, respectively). The losses were lowest in grain with 9% Se losses. The losses were three times more pronounced during the first day than in the following 3 d. These data may argue that the selenium is distributed into different pools and that sparing mechanisms may be in function. The last period, i.e., the chase experiment, revealed the rate of elimination of selenium under conditions with surplus selenium. The catabolic rate was about 10 times faster in roots (169 fmol/h/mg dw) than in grains and about 8 times faster than in leaves.     

Effects of selenium oxyanions on the white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The ability of Phanerochaete chrysosporium to reduce the oxidized forms of selenium, selenate and selenite, and their effects on the growth, substrate consumption rate, and pellet morphology of the fungus were assessed. The effect of different operational parameters (pH, glucose, and selenium concentration) on the response of P. chrysosporium to selenium oxyanions was explored as well. This fungal species showed a high sensitivity to selenium, particularly selenite, which inhibited the fungal growth and substrate consumption when supplied at 10 mg L^?1 in the growth medium, whereas selenate did not have such a strong influence on the fungus. Biological removal of selenite was achieved under semi-acidic conditions (pH 4.5) with about 40 % removal efficiency, whereas less than 10 % selenium removal was achieved for incubations with selenate. P. chrysosporium was found to be a selenium-reducing organism, capable of synthesizing elemental selenium from selenite but not from selenate. Analysis with transmission electron microscopy, electron energy loss spectroscopy, and a 3D reconstruction showed that elemental selenium was produced intracellularly as nanoparticles in the range of 30–400 nm. Furthermore, selenite influenced the pellet morphology of P. chrysosporium by reducing the size of the fungal pellets and inducing their compaction and smoothness.     

Analysis of human erythrocyte glucose 6-phosphate dehydrogenase isozymes by isoelectric focusing in polyacrylamide gel

The method of isoelectric focusing in polyacrylamide gel was used to separate G6PD isozymes in crude hemolysates of human, rabbit, and rat erythrocytes. G6PD (B) from erythrocytes of a normal human male donor revealed six bands of activity. Their mean isoelectric points, using pH 3–10 and 5–8 range empholytes, were pI 7.04 for band I, pI 6.60 for band II, pI 6.37 for band III, pI 6.11 for band IV, pI 5.94 for band V, pI 5.79 for band VI. G6PD from rabbit and rat erythrocytes revealed completely different multiple band patterns. The method of isoelectric focusing in polyacrylamide gel is presented as a new way of detecting G6PD isozyme patterns.     

The separation and comparison of albumin complexes of seed proteins in three cultivars ofPhaseolus vulgaris

Complexes of water soluble proteins (albumins) were investigated in three cultivarsof Phaseolus vulgaris, viz: Yeltruská Saxa, Vainica Saavegra B, and Krupnaya sakharnaya. The first two cultivars exhibit haemagglutinating activity against rabbit erythrocytes, but have different elution profiles on Sephadex G-100. Their individual peaks have a different subunit composition, as revealed by SDS gel electrophoresis, as well as a different immunoelectrophoretic pattern, although proteins I and II of the specificity Veltruská Saxa are present in both cultivars. The cultivar Krupnaya sakharnaya expressively differs from the preceding lectin cultivars; it has no erythroagglutinating activity, its albumin complex has a high-molecular component, absent in the preceding ones, and has no lectin peak in the region of molecular mass of 100 000 to 200 000. Immunoelectrophoresis gave no evidence of protein I and II of the specificity Veltruská Saxa.     

Haemagglutinin inTriticale

Saline extracts of several varieties ofTriticale had haemagglutinin activity against rabbit, rat and fowl erythrocytes. In contrast to the wheat germ lectin theTriticale lectin was inactive against human B, 0 blood group type erythrocytes and rather high concentrations of the lectin are needed to agglutinate human A blood group type erythrocytes. TheTriticale lectin was purified about 20-fold with a 10% recovery of activity from one of the varieties (DTS 138) by (NH₄)₂SO₄ fractionation followed sequentially by chromatography on DEAE-cellulose and sulphopropyl-Sephadex. Approximately 4 μg of the purified lectin caused visible agglutination with trypsinised rabbit erythrocytes. Among a variety of sugars tested D-glucose, D-mannose and N-acetyl-D-glucosamine (2·5-7·5mM) caused inhibition of agglutination.     

Comparison of whole blood selenium values and erythrocyte glutathione peroxidase activities of normal individuals on supplementation with selenate,selenite,l-selenomethionine,and high selenium yeast

The selenium levels and the glutathione peroxidase activity GSH-PX of whole blood and of erythrocytes, respectively, were determined in 139 normal Danes and related to sex and smoking habits. No differences were found in relation to sex apart from a higher GSH-PX activity of females when assayed with tertiary butyl hydroperoxide. Smokers showed significantly lower selenium values than non-smokers (p<0.05), but the two groups had identical GSH-PX activities. Individuals from the above-mentioned group were divided into four groups, receiving daily oral doses of 200 μg of selenium in the form of selenite, selenate, L-selenomethionine, and selenium as contained in yeast. Whole blood selenium values and the erythrocyte glutathione peroxidase activities were determined during three months of supplementation followed by a withdrawal period of four months. Both the inorganic selenium compounds and the organic derivatives gave rise to steady state levels of GSH-PX after one month of supplementation. However, the selenium levels in the groups receiving organic selenium showed a steady rise during the whole period, whereas those supplemented with inorganic selenium leveled off after a period of one to three months. The data for smokers and non-smokers revealed identical results when organic selenium was supplemented. However, selenite gave rise to significantly higher selenium levels and GSH-PX activities in smokers than in non-smokers. Less significant (p<0.08) elevations of both parameters were also observed among the smokers in the selenate group. By taking both the selenium level and the GSH-PX activity into consideration, organic selenium (i.e.,l-(+) selenomethionine) was judged to be more bioavailable than selenite and selenate.     

Ion transport in broad bean leaf mesophyll under saline conditions

Main conclusion

Salt stress reduces the ability of mesophyll tissue to respond to light. Potassium outward rectifying channels are responsible for 84 % of Na ⁺ induced potassium efflux from mesophyll cells. Modulation in ion transport of broad bean (Vicia faba L.) mesophyll to light under increased apoplastic salinity stress was investigated using vibrating ion-selective microelectrodes (the MIFE technique). Increased apoplastic Na⁺ significantly affected mesophyll cells ability to respond to light by modulating ion transport across their membranes. Elevated apoplastic Na⁺ also induced a significant K⁺ efflux from mesophyll tissue. This efflux was mediated predominately by potassium outward rectifying channels (84 %) and the remainder of the efflux was through non-selective cation channels. NaCl treatment resulted in a reduction in photosystem II efficiency in a dose- and time-dependent manner. In particular, reductions in Fv′/Fm′ were linked to K⁺ homeostasis in the mesophyll tissue. Increased apoplastic Na⁺ concentrations induced vanadate-sensitive net H⁺ efflux, presumably mediated by the plasma membrane H⁺-ATPase. It is concluded that the observed pump’s activation is essential for the maintenance of membrane potential and ion homeostasis in the cytoplasm of mesophyll under salt stress.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Selenium uptake in Zea mays supplied with selenate or selenite under hydroponic conditions

Background and aims

Selenium is an essential micro-nutrient for animals, humans and microorganisms; it mainly enters food chains through plants. This study proposes to explore effect of inorganic Se forms on its uptake and accumulation in Zea mays.

Methods

Zea mays was grown in a controlled-atmosphere chamber for 2 weeks in a hydroponic solution of low-concentration selenium (10 μg/L (i.e.0.12 μM) or 50 μg/L (i.e. 0.63 μM) of Se). For each concentration, four treatments were defined: control (without selenium), selenite alone, selenate alone and selenite and selenate mixed.

Results

At low concentrations, selenium did not affect the biomass production of Zea mays. However, for both concentrations, Se accumulation following a selenite-only treatment was always higher than with selenate-only. Moreover, in the selenate-only treatment, Se mainly accumulated in shoots whereas in the selenite-only treatment, Se was stocked more in the roots. Interactions between selenate and selenite were observed only at the higher concentration (0.63 μM of selenium in the nutrient solution).

Conclusions

Se form and concentration in the nutrient solution strongly influenced the absorption, allocation and metabolism of Se in Zea mays. Selenate seems to inhibit selenite absorption by the roots.     

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (⁷⁸Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.In recent years selenium (Se)¹ has received considerable attention as an essential element for human health. Severe Se deficiency is linked to oxidative stress and aging (1), elevated mortality with HIV (2), and irreversible brain injury (seizures, Parkinson''s disease) (3). Se occurs in nature principally in four inorganic chemical forms: the highly toxic selenide (Se²⁻⁻) (4), the moderately toxic selenate (SeO₄²⁻⁻) and selenite (SeO₃²⁻⁻), and elemental selenium (Se⁰) which is essentially nontoxic and can be stored by several bacterial species as nanoparticles on the cell surface (5, 6, 7). Inorganic selenium can be converted by biological systems (microorganisms, plants, and mammals) into seleno-amino acids, which are then incorporated into proteins. The two most common seleno-amino acids are selenomethionine (SeMet) and selenocysteine (SeCys). The former is synthesized via a route similar to the sulfur metabolic pathway in which selenium substitutes sulfur with no alteration of the protein structure (8, 9). The insertion of SeCys is genetically encoded by the UGA (TGA) codon and requires a SECIS element downstream of such a codon, a specific tRNA^[Ser]Sec and accessory proteins (10).Selenoproteins containing genetically encoded SeCys are known to be synthesized by several bacteria. Among Gram-negative ones, E. coli produces three forms of selenated formate dehydrogenase (FdhN, FdhO, FdhH) (11). Among Gram-positive bacteria, all the selenoproteins experimentally known were found exclusively in anaerobic bacteria belonging to the clostridial clade. Examples include glycine reductase from Clostridium sticklandii (12) and Eubacterium acidaminophilum (13), proline reductase in C. sticklandii (14), xanthine dehydrogenase in C. acidiurici (15), and several antioxidant defense proteins (16). Enterococcus faecalis is the only member of the Firmicutes/Lactobacillales subdivision containing a SeCys-decoding trait (SelD) (17).Lactobacillus reuteri species has been widely described as a probiotic: it produces antimicrobial compounds, such as reuterin, with a broad spectrum of action (18), it is effective against diarrhea in children (19) and possesses immunomodulatory (potent TNF-inhibitory activity) effects in humans (20). Because L. reuteri species are native inhabitants of human microbiota, the association of the probiotic feature L. reuteri Lb2 BM-DSM 16143 with its ability to fix selenium into proteins, offers an innovative approach to combat human selenium deficiency.The objectives of this study were to investigate the ability of Firmicutes/Lactobacillales subdivision, Lactobacillus reuteri Lb2 BM-DSM 16143to incorporate selenium into proteins, and to investigate, for the first time, its speciation in order to identify the pathway(s) of this process (SeMet or SeCys). For this purpose an analytical approach based on laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) of Se-containing proteins in 2D gel electrophoresis, followed by their identification by capillary HPLC - electrospray Orbitrap MS/MS assisted by the quantitative control of selenium elution by ICP MS, was developed.     

An evolving picture of the interactions between malaria parasites and their host erythrocytes

In patients with malaria, Plasmodium falciparum parasites multiply to enormous numbers in the bloodstream, initiating processes of erythrocyte destruction, endothelial activation and microvascular inflammation that cause devastating pathological effects on host tissues and organs. Recent research casts new light on a mechanism by which hemoglobin mutations may protect against these effects, and on a critical receptor-ligand interaction that provides fresh opportunities for the development of vaccines against blood-stage infection.The symptoms of malaria occur in the period of the Plasmodium life cycle when erythrocytes are infested by parasites. This period commences when parasites emerge from the liver after replicating from the sporozoites introduced by a mosquito bite. The new blood-stage parasites multiply quickly as haploid, asexual forms in one-, two- or three-day cycles (depending on the Plasmodium species) and increase their numbers enormously, often infecting 1% or more of the trillions of erythrocytes in the bloodstream. These populations of asexual forms are required for the production of sexual gametocyte forms that enter feeding mosquitoes, in which the parasites mate and produce new sporozoites for transmission to other human hosts. While large numbers of erythrocytes support the propagation and survival of malaria parasites, the inflammatory and erythrocyte-destroying effects of the parasite biomass cause devastating pathological effects on host tissues and organs.These pathological effects and the deaths that they cause have exerted powerful selection pressure on the human genome over thousands of generations. Outcomes of this pressure include the sickle cell hemoglobin (HbS) mutation, which protects young children against the life-threatening complications of Plasmodium falciparum malaria, and various blood group antigens, which affect the ligand-receptor interactions utilized by different Plasmodium parasites for erythrocyte invasion. Two recent papers in Science and Nature offer fresh and interesting discoveries from research in these areas: (i) a molecular mechanism by which mutation of hemoglobin may protect against malaria ¹, and (ii) a newly discovered ligand-receptor interaction that may be critical for P. falciparum invasion of human erythrocytes ².HbS represents a classic example of a balanced polymorphism: the heterozygous sickle cell trait (HbAS, from the inheritance of one normal HbA and one sickle HbS β-globin-coding gene) protects against severe life-threatening malaria, while the homozygous HbAA condition offers no protection from malaria and the homozygous HbSS condition produces frequent fatalities from sickle cell anemia ³. Although early studies implicated poor growth of P. falciparum in HbAS erythrocytes as a mechanism of protection that keeps parasitemias low, other work found that laboratory-adapted parasite clones grew normally in HbAS erythrocytes even under reduced oxygen conditions ^{4, 5}. Further, it was clear that substantial parasitemias and frequent episodes of uncomplicated malaria occurred in HbAS as well as HbAA children, despite marked differences in the incidence of severe malaria between these groups ⁶. Differential protection against severe malaria is also provided by hemoglobin C (HbC) in West Africa ⁷. These field observations suggest a mechanism of protection that, instead of merely reducing the numbers of parasitized erythrocytes in the circulation, works to ameliorate the inflammation that arises at the host-parasite interface from the interactions of infected erythrocytes with the endothelium and other blood elements. Indeed, parasitized HbAC and HbAS erythrocytes show significant impairment of cytoadherence in association with perturbed display of the P. falciparum major cytoadherence protein (PfEMP1) on abnormal knob protrusions ^{5, 8}.Using cryo-electron tomography, Cryklaff et al. ¹ show that P. falciparum hijacks and remodels erythrocyte actin into a network that may support the trafficking of PfEMP1 and other virulence proteins to the host cell membrane, where these proteins form the knobs involved in sequestration-related events and inflammation (Figure 1). Branching patterns of actin were found in association with Maurer''s clefts, membranous compartments that have an important role in exporting proteins from the parasite to the periphery of the host cell cytoplasm ⁹. Further, Cryklaff et al. ¹ observed significantly reduced actin remodeling and aberrant Maurer''s clefts in HbCC and HbSC erythrocytes, suggesting that these mutant hemoglobin states may interfere with the installation of actin scaffolds that help to tether Maurer''s clefts and support vesicle and protein trafficking to the erythrocyte membrane.Open in a separate windowFigure 1Junctures of pathogenesis in P. falciparum malaria: cytoadherence of parasitized erythrocytes to microvascular endothelium and parasite invasion of erythrocytes. P. falciparum parasites display knobs at the surface of their host erythrocytes as they mature from ring to trophozoite and schizont forms. PfEMP1 cytoadherence proteins are concentrated on knobs, where they bind receptors (e.g., CD36 and ICAM-1), activate endothelial cells, and recruit blood elements including platelets and white blood cells. By adhering in microvessels, the mature parasites avoid being carried by the bloodstream to the spleen where they are destroyed. Sequestration-related events lead to upregulation of tissue factor, resulting in thrombin and complement activation, platelet activation, cytokine production, endothelial dysfunction and inflammation ¹⁴. To support the transport of PfEMP1 and other proteins including the knob-associated histidine-rich protein (KAHRP) to the erythrocyte membrane, P. falciparum parasites tether membranous Maurer''s clefts beneath the cytoskeleton ⁹. In knobs, PfEMP1 associates with KAHRP anchored to spectrin-actin-protein 4.1 complexes, to spectrin-actin junctions, and to the band 3-binding domain of ankyrin ^{15, 16}. Cryklaff et al. ¹ report that P. falciparum remodels host actin into a network of filaments associated with Maurer''s clefts and the erythrocyte membrane; this network may support protein and vesicle trafficking to the knobs. Hemoglobin variants HbS and HbC interfere with proper knob formation and PfEMP1 display, weaken the binding of parasitized erythrocytes to endothelium and may thereby reduce sequestration-related pathology ^{5, 8}. Aberrant Maurer''s clefts and compromised remodeling of the actin network occur in HbSC and HbCC erythrocytes ¹; these abnormalities remain to be demonstrated in HbAS and HbAC erythrocytes. Inset (left) is adapted from ref ¹⁷. Merozoites released from mature schizonts invade erythrocytes by steps of initial contact, reorientation with attachment, junction formation, and entry and membrane resealing. Most members of the PfEBL and PfRH protein families have overlapping and individually dispensable functions that support a diversity of invasion pathways by attachment to different blood group antigens. These events are followed by binding of P. falciparum AMA1 and RON proteins at the attachment interface, triggering junction formation (not shown) ¹⁸. The interaction of the PfRH5-PfRipr complex ¹² with basigin ² appears to be essential for invasion and may have a critical function beyond the roles of binding and attachment that characterize other members of the PfEBL and PfRH families. Inset (right) is adapted from ref ¹⁸.Are there also significant differences between the actin networks and/or Maurer''s clefts of HbAS, HbAC and HbAA erythrocytes? HbAS and HbAC are, after all, the prevalent malaria-protective states of HbS and HbC. On this question the report of Cryklaff et al. ¹ is unfortunately silent; actin remodeling and Maurer''s clefts in HbAS and HbAC erythrocytes still need to be investigated, as do the effects of HbAS and HbAC erythrocyte extracts on actin polymerization in vitro. Mechanisms apart from disturbed actin remodeling may still account for abnormal PfEMP1 display and knob formation on parasitized HbAC and HbAS erythrocytes. For example, hemichromes generated more readily from oxidation of HbC or HbS may physically hinder the docking of virulence proteins and thus interrupt the formation of regular knob arrays in the erythrocyte cytoskeleton (where hemichromes are known to bind). Another possibility is that HbC and HbS elevate the levels of oxidative stress in parasitized erythrocytes, damaging membranes and biochemically hampering the functions of Maurer''s clefts and their associated vesicles in the transport of knob-forming proteins. The challenge for the research field in the coming years will be to sort out these possible mechanisms and their contributions to malaria protection by the heterozygous HbAS and HbAC conditions.P. falciparum invades erythrocytes by various pathways involving different ligand-receptor interactions ¹⁰. Partner ligands in these interactions include members of two molecular families known as the EBA (erythrocyte binding antigen) and RBL (reticulocyte binding-like) proteins. In P. falciparum, members of these families are termed PfEBL (P. falciparum erythrocyte binding-like) and PfRH (P. falciparum reticulocyte binding-like homolog) proteins. Evidence suggests that the roles of many of these proteins are overlapping: P. falciparum lines can often invade erythrocytes that lack receptors for specific PfEBL or PfRH proteins (because of inherent mutations or enzyme treatment) and, conversely, knockout parasites that do not express individual PfEBL or PfRH proteins have been found to efficiently invade erythrocytes. It thus came as a surprise that focused efforts to knock out the gene encoding an atypical, foreshortened member of the PfRH family, PfRH5, did not succeed in two laboratories ^{10, 11}. While polymorphisms in PfRH5 could be linked to receptor preferences and an ability of a P. falciparum line to also invade Aotus monkey erythrocytes ¹¹, the function of PfRH5 as a parasite ligand and the erythrocyte receptor(s) that it uses for invasion remained unknown.Crosnier et al. ² have now identified the PfRH5 receptor by applying an avidity-based extracellular interaction screen (AVEXIS) to the expressed proteins of an erythrocyte ectodomain library. Results show that PfRH5 binds an isoform of basigin on erythrocytes (BSG-S, the Ok blood group antigen, CD147) and that P. falciparum appears to generally require this interaction to efficiently invade human erythrocytes. In contrast to the effects of glycan removal (by neuraminidase) from Aotus erythrocytes on PfRH5 binding and parasite invasion by particular P. falciparum lines ¹¹, removal of all glycans from human basigin does not alter PfRH5 binding ². Further, in experiments with multiple P. falciparum clones, Crosnier et al. show that the invasion of human erythrocytes is potently inhibited by a soluble pentamerized form of basigin, by anti-basigin monoclonal antibodies, and by reduction of basigin levels on the erythrocyte surface ².Two of five single amino acid polymorphisms (E92K, L90P) in basigin affected PfRH5 binding to or invasion of human erythrocytes. While one of these polymorphisms (E92K) is associated with a relatively rare Ok^a- blood group in Japan, there are no data to suggest that L90P (or perhaps another undiscovered basigin polymorphism) was naturally selected to high prevalence in malaria-endemic areas. Although the native function of basigin on human erythrocytes is unknown, its high level of conservation suggests that P. falciparum may have evolved to depend on basigin to a much greater extent than on other, more polymorphic receptors. Interestingly, PfRH5 is also unlike other members of the PfEBL and PfRH families in that it lacks a transmembrane domain; recent evidence indicates that it forms a complex with a processed EGF-like PfRH5-interacting protein (PfRipr) and that this complex, in turn, associates tightly with another partner on the merozoite membrane ¹² (Figure 1). Attempts to disrupt the PfRipr gene also have been unsuccessful ¹².These findings suggest a critical role for the PfRH5-PfRipr complex that goes beyond an additional contribution of mechanical attachment within the binding repertoire of PfEBL and PfRH proteins. Possibilities for essential function might include PfRH5-PfRipr participation in a signaling pathway required for invasion, or an essential partnership in the junction during merozoite entry (Figure 1). Recent results also show promise for the use of PfRH5 and PfRipr as targets for intervention: rabbit IgG antibodies against virally-vectored, full-length PfRH5 outperformed antibodies that were generated by the same strategy against nine other erythrocytic-stage vaccine candidates, including four other PfEBL and PfRH proteins ¹³; rabbit antibodies against PfRipr likewise inhibited merozoite attachment and parasite growth in culture ¹². Although variations in the susceptibility of different P. falciparum lines were observed in these studies (likely due to different utilization patterns of ligand-receptor interactions), striking neutralization was nevertheless achieved in all cases. These results support PfRH5 and other components of its binding complex as new targets for therapeutic intervention and give an important boost to vaccine efforts against the asexual blood stages of P. falciparum.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

The Development of Monoclonal Antibodies to the secA Protein of Cape St. Paul Wilt Disease Phytoplasma and Their Evaluation as a Diagnostic Tool

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.     

The identification of six novel proteins with fibronectin or collagen type I binding activity from Streptococcus suis serotype 2

Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type I. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type I binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type I binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type I.     

Different effects of selenium on cyclic AMP metabolism in hepatoma cells and normal liver cells

Sodium selenite administered to normal and hepatoma (HA)-bearing mice sc (2 mg/kg) or ip (1 mg/kg) led to a significant increase in cyclic AMP (cAMP) level and a decrease in cAMP phosphodiesterase (PDE) activity in HA cells. In contrast, in tumor host liver and normal liver the cAMP level was reduced, and the PDE activity was slightly elevated, whereas the cAMP adenylate cyclase (AC) was little affected by Na₂SeO₃, if at all. These results imply that selenite-induced changes in PDE activity play a decisive role in regulating intracellular cAMP level. Kinetic studies revealed that there were different forms of PDE. TheK _m value of PDE isozymes in normal liver and host liver were identical, but differed from those of HA. It seems likely that the selective responsiveness of PDE to selenite may be related to the difference in PDE isozyme patterns. Addition of DBcAMP to the culture medium resulted in an inhibition of³H-thymidine incorporation into hepatoma cells, indicating that the inhibition of HA cell proliferation was cAMP-mediated. Thus, selenium has been shown to exert a selective effect on cAMP metabolism of hepatoma cells, which may account for its inhibitory effects on cancer cells.     

The localization of auxin transporters PIN3 and LAX3 during lateral root development in Arabidopsis thaliana

Fluorophore tagged proteins are used in Arabidopsis thaliana to understand their functional role in plant development. This requires the analysis of their spatial localization in planta. However, the localization analysis is often perturbed by a significant overlap of the fluorophores used to label proteins of interest and the optical filtering methods available on the confocal microscope. This problem can be addressed by the use of spectral imaging with linear unmixing the image data. We applied this method to help us identify double transgenic A. thaliana lines which expressed two fluorescently tagged auxin transporter proteins: the auxin efflux protein PIN-FORMED-3 (PIN3), tagged with green fluorescent protein (GFP), and the auxin influx protein LIKE-AUX1-3 (LAX3), tagged with yellow fluorescent protein (YFP). This method allows the reliable separation of overlapping GFP and YFP fluorescence signals and subsequent localization analysis highlighting the potential benefit of this methodology in studies of lateral root development.