Study of the phagocytic process in neutrophils from elite sportswomen

All the stages of the phagocytic process of blood neutrophils were compared in sedentary young women (no formal exercise during the previous 24 months) and elite sportswomen (basketball players from the Siglo XXI Spain Selection, in the middle of their competitive season) at rest. The sportswomen had performed no exercise since the day before taking the blood samples. Adherence of neutrophils to nylon fibre, which is similar to endothelium adherence, was not different between the two groups [62 (SD 14) and 58 (SD 18) in control and sport groups respectively]. Chemotaxis (studied in a Boyden chamber using a filter with 3 m pore diameter) was found to be stimulated (P<0.001) in the sportswomen [105 (SD 30)] with respect to the controls [39 (SD 9)]. Attachment, ingestion and killing by neutrophils was measured by incubation of the neutrophils with serum and a suspension ofCandida albicans. While no statistical differences were found in attachment ofC. albicans after 15 min incubation [71 (SD 8) in the control group, and 74 (SD 20) in the sport group], the sportswomen showed a higher (P<0.001) ingestion capacity forC. albicans at both 15 min [53 (SD 13) and 111 (SD 32) in control and sportswomen respectively] and 60 min [control 90 (SD 10), and sport group 224 (SD 21)] incubation. The greater phagocytic capacity in sportswomen was correlated with a higher plasma cortisol concentration (P <0.05) and a lower plasma ACTH concentration (P <0.001) in this group. It is concluded that elite women basketball players have a greater phagocytic capacity than sedentary women, possibly mediated by the higher plasma cortisol concentration in the sportswomen.     

Recent developments in the molecular genetics and biology of self-incompatibility

A summary of recent work on molecular aspects of self-incompatibility in Nicotiana alata is presented. The amino acid sequences of style proteins corresponding to different S-alleles of N. alata have a high level of homology in some regions and are variable in other regions. The regions of homology include N-terminal sequences as well as most of the glycosylation sites and cysteine residues. The glycosyl substituents may consist of a number of glycoforms. The isolated style S-glycoproteins inhibit in vitro growth of pollen tubes. The S-glycoproteins tested inhibited the growth of pollen of several S-genotypes, and there was some specificity in the interaction. Heat treatment of the isolated S-glycoproteins dramatically increased their activity as inhibitors of pollen tube growth, although the specificity in the interaction was lost. The nature of the S-allele products in pollen is not yet established.     

Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

Summary Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.Abbreviations WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt - MTT 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt - MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymemoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt - TTFA thenoyltrifluoroacetone - pCMBS p-chloromercuriphenylsul-fonic acid - SOD Superoxide dismutase - PMOR plasma membrane - NADH oxidoreductase - PMS phenazine methosulfate - PMA phorbol myristate acetate     

Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows

Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P < 0.01) when fresh serum was included in the medium (1226 cells/mm² in serum vs. 1110 cells/mm² in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm²) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P < 0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P < 0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm²) was also decreased (P < 0.01) in the presence of caffeine (1090 cells/mm²). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P < 0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs.     

Recent progress in the biology, chemistry and structural biology of DNA glycosylases

Since the discovery in 1974 of uracil DNA glycosylase (UDG), the first member of the family of enzymes involved in base excision repair (BER), considerable progress has been made in the understanding of DNA glycosylases, the polypeptides that remove damaged or mispaired DNA bases from DNA. We also know the enzymes that act downstream of the glycosylases, in the processing of abasic sites, in gap filling and in DNA ligation. This article covers the most recent developments in our understanding of BER, with particular emphasis on the mechanistic aspects of this process, which have been made possible by the elucidation of the crystal structures of several glycosylases in complex with their respective substrates, substrate analogues and products. The biological importance of individual BER pathways is also being appreciated through the inactivation of key BER genes in knockout mouse models.     

Seasonal changes in phagocytic capacity and superoxide anion production of blood phagocytes from tench (Tinca tinca,L.)

Seasonal variations in the ex vivo phagocytic function of blood cells from tench, including ingestion capacity of inert particles and its destruction (microbicide capacity) assessed by measurement of superoxide anion production, were studied. Tench were maintained under natural conditions throughout the year, and the different assays of samples taken during each season were initially performed in vitro at 22°C and the results compared. Subsequently, assays were performed at the same temperature as that of the water ponds in which the fish were kept (“seasonal temperature”: 12°C in winter, 22°C in spring and autumn and 30°C in summer) and the results compared seasonally. The results at 22°C showed that phagocytic capacity was greatest in spring and summer and lowest in winter. However, when phagocytic capacity was measured at seasonal temperature, highest values appeared in winter and lowest in summer and autumn. Nitroblue tetrazolium reduction by tench phagocytes after phagocytosing latex beads demonstrated a similar seasonal behaviour at both 22°C in each season and at seasonal temperature. The highest values appeared in summer, which suggests a better microbicide capacity in this season. The results obtained in this study suggest that for a correct interpretation of ex vivo phagocytic capacity of fish through the year it is necessary to use the same assay temperature as that of the water in which the fish is kept.     

Caffeine, dibutyryl cyclic-AMP and heparin affect the chemotactic and phagocytotic activities of neutrophils for boar sperm in vitro

The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P < 0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm² vs serum, 1226.3 cells/mm²), regardless of the presence of sperm (control, 1121.1 cells/mm² vs serum, 1245.8 cells/mm²), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm² and with sperm, 1132.6 cells/mm²). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P < 0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P < 0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm²) was also reduced (P < 0.05) with 2 mM caffeine (942.5 cells/mm²). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P < 0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P < 0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm² to 1008.8-1026.3 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities.     

Domestication of plants in the Americas: insights from Mendelian and molecular genetics

BACKGROUND: Plant domestication occurred independently in four different regions of the Americas. In general, different species were domesticated in each area, though a few species were domesticated independently in more than one area. The changes resulting from human selection conform to the familiar domestication syndrome, though different traits making up this syndrome, for example loss of dispersal, are achieved by different routes in crops belonging to different families. GENETIC AND MOLECULAR ANALYSES OF DOMESTICATION: Understanding of the genetic control of elements of the domestication syndrome is improving as a result of the development of saturated linkage maps for major crops, identification and mapping of quantitative trait loci, cloning and sequencing of genes or parts of genes, and discoveries of widespread orthologies in genes and linkage groups within and between families. As the modes of action of the genes involved in domestication and the metabolic pathways leading to particular phenotypes become better understood, it should be possible to determine whether similar phenotypes have similar underlying genetic controls, or whether human selection in genetically related but independently domesticated taxa has fixed different mutants with similar phenotypic effects. CONCLUSIONS: Such studies will permit more critical analysis of possible examples of multiple domestications and of the origin(s) and spread of distinctive variants within crops. They also offer the possibility of improving existing crops, not only major food staples but also minor crops that are potential export crops for developing countries or alternative crops for marginal areas.     

HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes. Evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 × g particulate fraction of the postnuclear supernatant obtained from guinea-pig phagocytosing polymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O^?₂ generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH.2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1 : 1.3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O^?₂ generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation.4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O^?₂ formed during the oxidation of NADPH.5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH.6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH.7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible.8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase.9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O₂; an enzymatic, apparently non-univalent, O₂ reduction and a non-enzymatic chain reaction.10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O₂ is optimal at pH 7.0 with 1 mM NADPH.     

Disciplinary baptisms: a comparison of the naming stories of genetics, molecular biology, genomics, and systems biology

Understanding how scientific activities use naming stories to achieve disciplinary status is important not only for insight into the past, but for evaluating current claims that new disciplines are emerging. In order to gain a historical understanding of how new disciplines develop in relation to these baptismal narratives, we compare two recently formed disciplines, systems biology and genomics, with two earlier related life sciences, genetics and molecular biology. These four disciplines span the twentieth century, a period in which the processes of disciplinary demarcation fundamentally changed from those characteristic of the nineteenth century. We outline how the establishment of each discipline relies upon an interplay of factors that include paradigmatic achievements, technological innovation, and social formations. Our focus, however, is the baptism stories that give the new discipline a founding narrative and articulate core problems, general approaches and constitutive methods. The highly plastic process of achieving disciplinary identity is further marked by the openness of disciplinary definition, tension between technological possibilities and the ways in which scientific issues are conceived and approached, synthesis of reductive and integrative strategies, and complex social interactions. The importance--albeit highly variable--of naming stories in these four cases indicates the scope for future studies that focus on failed disciplines or competing names. Further attention to disciplinary histories could, we suggest, give us richer insight into scientific development.     

Origin and early evolution of the vertebrates: new insights from advances in molecular biology, anatomy, and palaeontology

Recent advances in molecular biology and microanatomy have supported homologies of body parts between vertebrates and extant invertebrate chordates, thus providing insights into the body plan of the proximate ancestor of the vertebrates. For example, this ancestor probably had a relatively complex brain and a precursor of definitive neural crest. Additional insights into early vertebrate evolution have come from recent discoveries of Lower Cambrian soft body fossils of Haikouichthys and Myllokunmingia (almost certainly vertebrates, possibly related to modern lampreys) and Yunnanozoon and Haikouella (evidently stem-group vertebrates). The earliest vertebrates had an unequivocally marine origin, probably evolved mineralised pharyngeal denticles before the dermal skeleton, and evidently utilised elastic recoil of the visceral arch skeleton for suction feeding. Moreover, the new data emphasise that the advent of definitive neural crest was supremely important for the evolutionary origin of the vertebrates.     

Ultrastructural,cytochemical and functional studies on the eosinophilic granulocytes of larval lampreys

Summary Blood from larval lampreys (ammocoetes) contains a small number of eosinophilic granulocytes which are formed in the protospleen and kidney. Both immature and mature forms of this cell type are present in the blood and these are easily identified from other cell types due to the prominent eosinophilic granules that fill the cytoplasm. Ultrastructurally, these granules are electron-dense, largely unstructured, Golgi-derived and contain acid phosphatase but not peroxidase. Eosinophilic granulocytes ingest bacteria but fail to internalise colloidal carbon. The functional and phylogenetic significance of these cells is discussed.     

Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage

Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.     

Recent developments in the cell and molecular biology of root hairs

Recent results in root hair research show that these tip-growing cells are useful models in plant cell biology research. The review covers a range of topics, but there is particular emphasis on the use of mutants in molecular (genetic) analysis.