Alpha 2-antiplasmin Enschede is not an inhibitor, but a substrate, of plasmin.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.     

Evolution of uni- and bifactorial sexual compatibility systems in fungi

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.     

Efficiency of intrathymic tolerance induction in various inbred rat strains: relationship with TH1/TH2 status of the recipient?

The simultaneous transplantation and intrathymic tolerance induction (STITTI) protocol induces a longlasting state of functional tolerance in over 90% of AO (RT1^u) recipients transplanted with a fully MHC-incompatible PVG (RT1^c) cardiac allograft. Similar results are obtained when using LEWIS (RT1¹) rats as recipients of either PVG or DA (RT1^avl) grafts. However, when STITTI is performed on PVG and BN (RT1ⁿ) as recipient animals receiving spleen cells intrathymically and a cardiac allograft from respectively AO and PVG rats, this procedure results in significantly shorter graft survival (MST PVG → BN 25 ± 9 days; AO → PVG 31 ± 8 days) as compared to the combinations using AO (MST PVG → AO > 236 ± 28 days) and LEWIS (MST PVG → LEW > 366 ± 51 days; DA → LEW > 123 ± 33 days) rats as recipients. Since both PVG and BN rats are relatively deficient in their ability to produce IFNγ and intrathymic IFNγ responses are very dominant upon intrathymic injection of alloantigens, it is argued that the inability to effectively induce a longlasting state of functional tolerance in BN and PVG rats using the STITTI protocol may be related to their decreased IFNγ-production potential.     

Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.     

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non‐coding regions in the gene promoter and 5′UTR regions of genes. Poly‐A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non‐crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non‐homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.     

Role of NKT cells in the digestive system. III. Role of NKT cells in intestinal immunity

Natural killer T (NKT) cells are a small subset of unconventional T cells that recognize lipid antigens presented by the nonclassical major histocompatibility complex (MHC) class I molecule CD1d. NKT cells are involved in the host response to a variety of microbial pathogens and likely commensals. In the intestine, invariant and noninvariant NKT cells can be found among intraepithelial lymphocytes and in the lamina propria. Activation of intestinal NKT cells by CD1d-expressing intestinal epithelial cells and professional antigen-presenting cells may contribute to induction of oral tolerance and protection from mucosal infections. On the other hand, sustained and uncontrolled activation of NKT cells may play a pivotal role in the pathogenesis of inflammatory bowel disease. Here we review the current literature on intestinal NKT cells and their function in the intestine in health and disease.     

Secretory leukoprotease inhibitor in mucosal lymph node dendritic cells regulates the threshold for mucosal tolerance

The notion that the mucosal immune system maintains a tolerogenic response to harmless Ags while continually being challenged with microbial products seems an enigma. The aim of this study was to unravel mechanisms that are involved in regulating the development of tolerance under constant microbial pressure. The tolerogenic response to Ags administered via the nasal mucosa is dependent on the organized lymphoid tissue of the cervical lymph nodes (LN). We show that cervical LN differentially express secretory leukoprotease inhibitor (SLPI) compared with peripheral LN. SLPI was expressed by dendritic cells (DCs) and because SLPI is known to suppress LPS responsiveness, it was hypothesized that its expression in mucosal DCs may be required to regulate cellular activation to microbial products. Indeed, compared with wild-type controls, bone marrow-derived DCs from SLPI(-/-) mice released more inflammatory cytokines and enhanced T cell proliferation after stimulation with low dose LPS. This increased sensitivity to LPS was accompanied by increased NF-kappaB p65 activation in SLPI(-/-) DCs. In vivo, nasal application of OVA with LPS to SLPI(-/-) mice resulted in enhanced DC activation in the cervical LN reflected by increased costimulatory molecule expression and release of inflammatory cytokines. This led to failure to maintain tolerance to nasal OVA application in the presence of low doses of LPS. We propose that expression of SLPI functions as a rheostat by controlling the level of bacterial stimuli that induce mucosal DC activation. As such, it regulates the quality of the ensuing Ag-specific immune response in the mucosa draining LN.     

Effect of ethidium bromide and DEAE-dextran on divalent cation accumulation in yeast. Evidence for an ion-selective extrusion pump for divalent cations

The larger accumulation of Mn2+ than of Sr2+ in Saccharomyces cerevisiae is ascribed to the operation of a specific extrusion pump, presumably a Ca2+ pump, which has a higher affinity for Sr2+ than for Mn2+. The differences in accumulation levels of Mn2+ and Sr2+ attained after prolonged incubation are completely abolished in cells of which the plasmamembrane has been permeabilized with the polybase DEAE-dextran under isotonic conditions. In the permeabilized cells Sr2+ and Mn2+ accumulation levels are attained as for Mn2+ in intact cells. It is suggested that the accumulation of divalent cations into the permeabilized cells mainly represents their accumulation into the vacuoles. Also the cationic dye ethidium abolishes the differences in Mn2+ and Sr2+ accumulation. The dye increases the accumulation of Sr2+ but decreases that of Mn2+ somewhat. It cannot be distinguished yet whether its action is due to an impairment of the efflux pump or to an increase in the permeability of the plasmamembrane facilitating the divalent cations to be accumulated into the vacuoles. Ethidium does not affect the initial rates of divalent cation uptake into the vacuoles, but it effectively reduces the ultimate accumulation of the divalent cations in the DEAE-dextran permeabilized cells, possibly by competing with the divalent cations for intravacuolar binding sites. Similar results are obtained for the accumulation of Ca2+. It is concluded that the efflux pump enables the yeast cell to regulate accumulation levels of the various divalent cations to different extents.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.