The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins –major histocompatibility complex II (MHCII), CD86 and CD97 –from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Enhancement of keratinocyte growth factor potential in inducing adipose‐derived stem cells differentiation into keratinocytes by collagen‐targeting

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials.     

Vasoactive intestinal peptide receptor activity in human fetal enterocytes

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind ¹²⁵I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of ¹²⁵I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 × 10⁻¹⁰ M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10⁻⁷M and above) in the following order of potency: VIP> PHI> GRF> secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9–12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.     

Field evaluation of two biorational compounds in the control of pear psylla,Cacopsylla pyricola (Förster), on pear trees

Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) is an important pest of commercial pear in all pear-growing regions of Iran. In the scope of an integrated pest management, a research was carried out on the impact of treatment with biorational compounds in comparison with conventional chemical insecticides for controlling the pear psyllid. The experiments were done with five treatments consisted of diflubenzuron and lufenuron as biorational insecticides and thiacloprid and diazinon as conventional chemical insecticides and untreated check. The trials were set up in a randomised complete block design. The treatments were replicated four times. Samplings were carried out one day before spraying and 3, 7, 15, 30 and 45 days after spraying through clipping 15 leaves in each replicate and counting the number of pear psyllid live nymphs. Mortality percentage was calculated using Henderson–Tilton formula. The data were subjected to analysis of variance (ANOVA) and the means comparison was performed using Duncan’s multiple range test. The results indicated that the highest mortality in diflubenzuron and lufenuron treatments occurred after 15 days, with 82.09% and 71.01% mortality, respectively. In comparison with conventional chemical insecticides, the efficacy of biorational compounds was higher or not significantly different. The results of the trials are discussed in terms of improving management of the populations of pear psylla.     

Interaction Between Opioidergic and Dopaminergic Systems on Food Intake in Neonatal Layer Type Chicken

Central regulatory mechanisms for food intake regulation vary among animals. Evidence from animal studies suggests central opioids and dopamine have prominent role on appetite regulation but their interaction(s) have not been studied in layer-type chicken. Thus, in this study six experiments designed to investigate intracerebroventricular (ICV) administration of SCH23390 (D₁ like receptors antagonist), Sulpride (D₂ like receptors antagonist), DAMGO (μ-opioid receptors agonist), DPDPE (δ-opioid receptors agonist), U-50488H (κ-opioid receptors agonist) on feeding behavior in 3 h food deprived neonatal layer-type chickens. In experiment 1, chicks ICV injected with control solution, SCH23390 (2.5 nmol), DAMGO (125 pmol) and their combination (SCH23390 + DAMGO). In experiment 2: control solution, SCH23390 (2.5 nmol), DPDPE (δ-opioid receptors agonist, 40 pmol) and SCH23390 + DPDPE were applied to the birds. In experiment 3, injections were control solution, SCH23390 (2.5 nmol), U-50488H (30 nmol) and SCH23390 + U-50488H. In experiments 4–6 were similar to experiments 1–3 except Sulpride (2.5 nmol) applied instead of SCH23390. Then, cumulative food intake was recorded until 120 min after injection. According to the results, ICV injection of DAMGO (125 pmol) significantly decreased food intake but co-injection of DAMGO + SCH23390 diminished DAMGO-induced hypophagia (P < 0.05). Also, SCH23390 was not able to decrease the DPDPE- and U-50488H-induced hyperphagia (P > 0.05). Furthermore, Sulpride had no role on DAMGO, DPDPE and U-50488H-induced food intake (P > 0.05). These results suggest there is an interaction between opioidergic and dopaminergic systems via μ and D₁ receptors in appetite regulation in chicken.