Relationship between selenium, immunity and resistance against infection

1. Food selenium content, selenium supply and selenium needs are presented, along with methods of evaluation of selenium status. Glutathione peroxidase, a selenium-containing enzyme, is ubiquitous in the organism. 2. Some experimental studies on animal models reported a positive relationship between selenium status and resistance against infections. 3. Only one study in humans concerned the mechanisms of immune functions in selenium deficiency. Several experimental works suggest that severe selenium deficiency compromises T-cell dependent immune functions such as the blastogenic response to mitogens, but selenium deficiency was concomitant with vitamin E deficiency in most of them. Delayed hypersensitivity response is controversial in selenium-supplemented rats and guinea-pigs. 4. Selenium deficiency in animals decreases the antibody response, especially if associated with vitamin E deficiency. Low dietary selenium supplementation of healthy animals has a positive effect upon humoral responses. 5. Despite some controversies, most experimental studies on selenium-deficient animals report normal phagocytosis and an altered bactericidal capacity of neutrophils. The decrease in glutathione peroxidase activity of polymorphonuclear cells following selenium deficiency could explain some of these alterations. 6. Splenic Natural Killer cells activity is enhanced in selenium-supplemented, healthy animals.     

Synthesis and structural study of 5-nitro-6-(penta-O-acetylpentitol-1-yl)norbornenes

Uncatalyzed reaction between cyclopentadiene and (E)-3,4,5,6,7-pentaacetoxy-1-nitrohept-1-enes having the -manno, -galacto, and -gluco configurations at C-3—C-7 led, in each case, to the four stereoisomeric 5-nitro-6-(1,2,3,4,5-penta-O-acetylpentitol-1-yl)bicyclo[2.2.1]hept-2-enes. Face selectivity is discussed in terms of the sugar-chain configuration. The structures assigned the adducts are based on their n.m.r. spectra, and, in the case of the -manno compounds, on X-ray data. Also described are the ¹³C-n.m.r. spectra of the starting nitroalkenes. The crystal structures of (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-exo-nitrobicyclo[2.2.1]hept-2-en-6-endo-yl- -manno-pentitol (3a) and (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-endo-nitrobicyclo[2.2.1]hept-2-en-6-exo-yl- -manno-pentitol (5a) were determined from three-dimensional, X-ray data. Crystals of 3a are monoclinic, space group P2₁, with two molecules in a cell of dimensions a = 9.054(3), b = 15.580(11), c = 10.138(4) Å, β = 116.27(3)°. The structure was refined to an R-factor of 0.050 on the basis of 1485 observations. Crystals of 5a are triclinic, space group P1, with one molecule in a cell of dimensions a = 8.680(4), b = 9.760(4), c = 8.695(7) Å, = 98.69(5), β = 103.13(5), γ = 112.09(3)°. The structure was refined to an R-factor of 0.074 based on 970 observations.     

Multiple Mating,Paternity and Complex Fertilisation Patterns in the Chokka Squid Loligo reynaudii

Polyandry is widespread and influences patterns of sexual selection, with implications for sexual conflict over mating. Assessing sperm precedence patterns is a first step towards understanding sperm competition within a female and elucidating the roles of male- and female-controlled factors. In this study behavioural field data and genetic data were combined to investigate polyandry in the chokka squid Loligo reynaudii. Microsatellite DNA-based paternity analysis revealed multiple paternity to be the norm, with 79% of broods sired by at least two males. Genetic data also determined that the male who was guarding the female at the moment of sampling was a sire in 81% of the families tested, highlighting mate guarding as a successful male tactic with postcopulatory benefits linked to sperm deposition site giving privileged access to extruded egg strings. As females lay multiple eggs in capsules (egg strings) wherein their position is not altered during maturation it is possible to describe the spatial / temporal sequence of fertilisation / sperm precedence There were four different patterns of fertilisation found among the tested egg strings: 1) unique sire; 2) dominant sire, with one or more rare sires; 3) randomly mixed paternity (two or more sires); and 4) a distinct switch in paternity occurring along the egg string. The latter pattern cannot be explained by a random use of stored sperm, and suggests postcopulatory female sperm choice. Collectively the data indicate multiple levels of male- and female-controlled influences on sperm precedence, and highlights squid as interesting models to study the interplay between sexual and natural selection.     

Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco’s modified Eagle’s medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.     

Substrate specificity of strictosidine synthase

Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and the enzymatically generated compounds are processed by the second enzyme in this biosynthetic pathway.     

Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ∼5 m^-1 s^-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of arsenate (As(V))⁴ (1). Arsenate reductases (ArsCs) are unusual among well studied enzyme classes, because there is not a single family of evolutionarily related sequences. The structural folds and mechanisms that they are using are fundamentally different and arose independently during evolution (2). Arsenate reductases are small cytoplasmic redox enzymes that reduce arsenate to arsenite (As(III)) by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. As such, arsenate reductases from different organisms often work together with the thiol/disulfide mechanism in the cell.The major and most ubiquitous system for protection against oxidative stress and to maintain the intracellular thiol homeostasis is the thioredoxin system that is composed of Trx (thioredoxin) and TrxR (thioredoxin reductase) (3). In addition to the thioredoxin system, most living organisms contain low molecular weight thiol compounds that serve as a buffer to avert disulfide stress. In eukaryotes and Gram-negative bacteria, the redox level is maintained by redox cycling of glutathione (GSH) with Grx (glutaredoxin) and glutathione reductase (4). Gram-positive bacteria, like Staphylococcus aureus, produce no glutathione, but millimolar quantities of reduced coenzyme A is the predominant thiol, which is kept reduced with a NADPH-dependent flavoenzyme, coenzyme A disulfide reductase (5). Also actinobacteria, like Corynebacterium glutamicum, produce no GSH, but instead they contain millimolar concentrations of MSH (mycothiol; chemically 1D-myo-inosityl-2-[N-acetyl-l-cysteinyl] amido-2-deoxy-α-d-glucopyranoside), a pseudodisaccharide containing a cysteine moiety as a reactive thiol (6). Its oxidized form is mycothione (MSSM). In actinobacteria, MTR (mycothione reductase) is the NADPH-dependent flavoenzyme that reduces MSSM in order to maintain the intracellular redox homeostasis to allow the proper functioning of a variety of biological functions (7).Arsenate reductases are part of a defense mechanism of the cell against toxic arsenate. Their genes are most of the time found in an operon together with arsenite sensing and efflux genes (8). Based on the mechanism used to reduce arsenate to arsenite, two distinct ArsC classes can be defined. The first one is the thioredoxin-coupled ArsC class represented by S. aureus pI258 ArsC and Bacillus subtilis ArsC (9–11). Both enzymes use the structural fold of low molecular weight tyrosine phosphatase and need Trx to start a second catalytic cycle (12–14). The second class is the GSH/glutaredoxin-coupled class represented by Escherichia coli plasmid R773 ArsC (15, 16), the eukaryotic Acr2p reductase from Saccharomyces cerevisiae (17), and ArsC from Leishmania major (18). In this second class, two different structural folds are found; E. coli R773 ArsC partially resembles glutaredoxin (19), whereas the eukaryotic ArsCs have a rhodanese fold like the Cdc25a cell cycle control phosphatase (20). Notably, all arsenate reductases have a thiolate nucleophile at the N-terminal end of an α-helix. The active site of the ArsCs with a phosphatase-like scaffold is conserved (root mean square deviation of 0.54 Å) with a catalytically important Arg on position Cys⁺⁶.In C. glutamicum, there are four arsC genes located on different places in the chromosome (21): one orphan arsC gene (arsC4) and three arsC genes (arsC1-arsC1′ and arsC2) present in two ars operons. We show here that two of the encoded proteins, Cg_ArsC1 and Cg_ArsC2 (with 66% sequence identity) are members of a new third class, the mycothiol- and mycoredoxin-dependent arsenate reductases. Both the genes of arsC1 and arsC2, together with the genes for the enzymes of the mycothiol biosynthesis pathway are involved in arsenate resistance in C. glutamicum. We have reconstituted in vitro a novel electron transfer network containing, next to Cg_ArsC1 or Cg_ArsC2, mycothiol, mycoredoxin, and mycothione reductase. As such, the mechanism for the reduction of arsenate by C. glutamicum could be unraveled.     

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria.     

Effects of iron deficiency upon the antibody response to influenza virus in rats

The effects of severe and moderate iron deficiency upon the antibody response to influenza virus were investigated in rats. Three groups of weanling male Wistar rats were fed one of two iron-deficient diets (5 mg and 15 mg iron/kg diet) or a normal iron-containing diet (35 mg iron/kg diet). A group of individually pair-fed rats was introduced with the low iron-consuming rats. The effects of the diets upon various iron status parameters were followed during the 4th, 5th, 6th, and 7th week of diet. After 4 weeks of feeding different diets, an intraperitoneal injection of inactivated influenza virus A/New Jersey/76 was performed and a recall injection was done at 5 weeks. Primary and secondary antibody responses were assayed. Rats were sacrificed at 7 weeks of diet. After 4 weeks of feeding different diets, the rats fed the 5 mg iron/kg diet were severely anemic and rats fed 15 mg iron/kg diet were moderately iron-deficient, as shown by their iron status parameters. Growth was delayed in anemic and matched pair-fed rats. A primary antibody response was almost nonexistent in all groups. Secondary antibody titers were significantly weaker in anemic rats than in ad libitum controls, but were not different from those of pair-fed rats. This response was similar in moderately iron-deficient, ad libitum, and pair-fed rats. These results show that antibody synthesis in response to the influenza virus vaccine is preserved in moderate iron deficiency but is reduced in severe anemia. The reduction in energy consumption associated with severe iron deficiency in the rat could play a part in the altered humoral response.     

Aeropalynological and phenological study in two different Mediterranean olive areas: Cordoba (Spain) and Perugia (Italy)

ABSTRACT

An aerobiological and phenological investigation on the olive tree was carried out during three years in two areas: Cordoba (Spain) and Perugia (Italy). In these countries, this species is economically important and those areas were chosen because of the long series of aeropalynological data (1982–1998) available, obtained by means of identical volumetric pollen traps. The aim of this study was to use phenological observations to prove the real contribution to the pollen curves in different cultivated areas. Results show that in Cordoba province (302.152 ha) the pollen curve is characterised by different peaks because of the pollination of different cultivated crops. In some cases, these crops are located far from the pollen trap (50 km) but pollen is transported thanks to favourable winds during the flowering period. In Perugia (750 ha) the pollen curve is characterised by only one peak; it is very concentrated because of the proximity of the investigated crops. The objective of this research was to obtain information on this species in order to elaborate statistical models aimed at forecasting the potential fruit production based on the amount of pollen released into the atmosphere.