Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease

Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs)in vitro.Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21.Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH.Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21.Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system

Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae

The CD1d pathway present lipid antigens resulting in the activation of iNKT cells but the complete pathway remains to be fully elucidated. Here, Chandra et al. use an siRNA screen and identify Mrp1 as crucial for CD1d lipid presentation and activation of iNKT in the context of Streptococcus pneumoniae infection

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp <it>Chelonus inanitus </it>(Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.</p> <p>Results</p> <p>About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein.</p> <p>An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the <it>Chelonus </it>lineage. Venom components specific to <it>C. inanitus </it>included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins.</p> <p>Conclusions</p> <p>The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of <it>C. inanitus </it>appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.</p

Toward osteogenic differentiation of marrow stromal cells and in vitro production of mineralized extracellular matrix onto natural scaffolds

Uncorrected proofTissue engineering has emerged as a new interdisciplinary field for the repair of various tissues, restoring their functions by using scaffolds, cells, and/or bioactive factors. A temporary scaffold acts as an extracellular matrix analog to culture cells and guide the development of new tissue. In this chapter, we discuss the preparation of naturally derived scaffolds of polysaccharide origin, the osteogenic differentiation of mesenchymal stem cells cultured on biomimetic calcium phosphate coatings, and the delivery of biomolecules associated with extracellular matrix mineralization

Intraspecific competition between adult females of the hyperparasitoid Trichomalopsis apanteloctena (Hymenoptera: Chelonidae), for domination of Cotesia kariyai (Hymenoptera: Braconidae) cocoons

The development of parasitoid wasps is dependent on the finite resources contained in a single item of resource (=host) that is frequently not much larger than the adult parasitoid. When the costs of egg production are high, and host distribution is highly aggregated, parasitoid females may spend prolonged periods guarding their eggs and host resources as an adaptive strategy to optimize their inclusive fitness. Here, we examine aggressive interactions between the females of the secondary hyperparasitoid Trichomalopsis apanteloctena (Crawford) (Hymenoptera: Chelonidae), for control of cocoon clusters of their primary parasitoid host Cotesia kariyai (Watanabe) (Hymenoptera: Braconidae). Generally, larger female hyperparasitoids were more successful at defending cocoon clusters than smaller female hyperparasitoids. However, when first encountering host cocoons, larger females behaved more aggressively toward conspecific wasps than smaller females. After occupation of a host co! coon cluster, females of similar size rarely engaged in physical combat, but both females primarily exhibited threatening behavior toward each other. However, larger females usually displaced smaller females which had initially occupied cocoon clusters. Some small females chewed through the outer cocoon silk layer to avoid being displaced by larger females and these wasps were able to continue parasitizing cocoons of C. kariyai. Extended bouts of aggression tended to reduce the number of eggs laid by the guarding female because of disruption of oviposition behavior. The relationship between the size of host cocoons and body mass in T. apanteloctena was also examined. The size of hyperparasitoid progeny was strongly correlated with host size. However, the relationship between maternal size, the number of matured eggs in her ovarioles and body mass in her offspring was not significant.

Cotesia kariyai larvae need an anchor to emerge from the host Pseudaletia separata

Mature larvae of the gregarious endoparasitoid Cotesia kariyai construct cocoons for pupation approximately 10 days after parasitization and emerge from their host Pseudaletia separata under a long day photo-regime (16L8D) at 25 ± 1°C. The parasitoid larvae make capsules in the host hemocoel just prior to their emergence. These capsules function as anchors, which enable them to press against the host integument from inside the host. It was predicted that this anchor might be composed of silk proteins secreted from the parasitoid larvae, because a previous study showed that the anchor was made up of a glycoprotein and that the silk gland of parasitoid larvae developed from 2nd larval stage. Fibroin-like proteins in C. kariyai larva mainly consist of two proteins with molecular masses of the 300.6 and 46.7 kDa estimated by SDS-PAGE. The fibroin-like proteins with the same molecular mass were detected from the anchor proteins just prior to parasitoid emergence. These results indicate that the anchor was assembled with fibrion-like proteins and was formed just before parasitoid emergence while in the host body cavity. Injection of bovine pancreatic trypsin inhibited the emergence of parasitoid larvae from the host because the anchor was decomposed by trypsin. Trypsin activity in the parasitized host hemolymph increased only after parasitoid emergence.

Influence of nutrient deficiency caused by host developmental arrest on the growth and development of a koinobiont parasitoid

Koinobiont parasitoids utilize nutrients obtained from hosts that contine to feed and grow after parasitization. However, if the ecdysis of early host instars is prevented, parasitized larvae will fail to grow large enough to support the development of the parasitoid brood and both organisms will perish. When L5 instar larvae (the penultimate stage) of Pseudaletia separata were parasitized by Cotesia kariyai and injected with Euplectrus separatae venom (5PV), the development of these hosts was arrested before molting to the next stage and the caterpillars thus failed to gain weight. These hosts remained at approximately 300 mg until parasitoid emergence. In contrast, hosts parasitized as L5 but without the injection of venom (5P) exhibited an increase in weight after molting to the next stage and ultimately grew to approximately 700 mg. The inhibition of ecdysis reduced the amount of food resource (e.g. fat body) for the parasitoid larvae. On the other hand, when final (=L6) host instars were parasitized and injected with E. separatae venom (6PV), the maximum weight attained by these larvae was about 710 mg, although weight gain was depressed compared to hosts parasitized without the injection of E. separatae venom (6P). The adult weight of C. kariyai that emerged from 5PV hosts was less than conspecifics that emerged from 5P, 6P, and 6PV respectively, although the egg-pupal period of the parasitoid from 5PV hosts was extended. The offspring sex ratio (percentage males) of adult wasps did not vary significantly with treatment. Female parasitoids that eclosed from 5PV hosts laid almost the same number of eggs in day 0–6th host instars as those emerging from 5P, 6P, 6PV hosts. Their egg-pupal period was extended a