A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70

Heat shock protein 70 (HSP70) is a member of a highly conserved superfamily of intracellular chaperones called stress proteins that can activate innate and adaptive immune responses. We evaluated the effect of a fusion DNA vaccine that encoded mycobacterial HSP70 and MPT51, a major secreted protein of Mycobacterium tuberculosis. Spleen cells from mice immunized with fusion DNA of full-length HSP70 and MPT51 produced a higher amount of interferon-γ (IFN-γ) in response to the CD4+, but not the CD8+ T-cell epitope peptide on MPT51 than those from mice immunized with MPT51 DNA. Furthermore, because HSP70 comprises the N-terminal ATPase domain and the C-terminal peptide-binding domain, we attempted to identify the domain responsible for its enhancing effect. The fusion DNA vaccine that encoded the C-terminal domain of HSP70 and MPT51 induced a higher MPT51-specific IFN-γ production by CD4+ T cells than the vaccine that encoded MPT51 alone, whereas that with the N-terminal domain did not. Similar results were obtained by immunization with the fusion proteins. These results suggest that the DNA vaccine that encodes a chimeric antigen molecule fused with mycobacterial HSP70, especially with its C-terminal domain, can induce a stronger antigen-specific T-helper cell type 1 response than antigen DNA alone.     

Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium

The uptake of labeled inorganic phosphate into primary rabbit kidney proximal tubule cells has been examined. Phosphate was accumulated into the primary proximal tubule cells against a concentration gradient. This accumulation was sensitive to inhibition by metabolic inhibitors. The dependence of phosphate uptake on the extracellular phosphate concentration was examined. Similarities were observed between primary proximal tubule cells and the LLC-PK1 cell line in these regards. These phosphate uptake data were then plotted on a Lineweaver-Burke plot. A nonlinear plot was obtained, which suggested that phosphate uptake occurs by means of a Na+ dependent, carrier mediated process, as well as by another Na+ independent mechanism. The pH dependence of phosphate uptake was also examined. Unlike previous observations with LLC-PK1 cells, optimal phosphate uptake occurred at pH 6.5. However, this difference between the two cell culture systems may possibly be explained by differences in uptake conditions. The dependence of phosphate uptake on the extracellular NaCl concentration was examined at three different pH values. The rate of phosphate uptake at pH 7.0 was observed to saturate at a lower NaCl concentration than at either pH 6.0 or pH 6.5. Furthermore, the optimal rate of phosphate uptake at pH 7.0 was observed to be higher than at the other two pH values studied when the NaCl concentration was below 120 mM. However, when the NaCl concentration was raised to 150 mM, optimal phosphate was observed to occur at pH 6.5 rather than at pH 7.0. These observations may be explained if the pH affects not only the rate of phosphate uptake but also the affinity of the phosphate uptake system for sodium. Phosphate uptake was also observed to be sensitive to several agents, Na2 X SO4 and NaSCN, which affect the membrane potential. As observed with phosphate uptake by LLC-PK1 (and renal brush border membrane vesicles), phosphate uptake was highly sensitive to inhibition by the phosphate analogue arsenate. Novel observations were that the phosphate analogue vanadate and its cellular metabolite vanadyl stimulated the initial rate of phosphate uptake.     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Biosynthesis of GA73 Methyl Ester in Lygodium Ferns

Biosynthesis of GA73 methyl ester (GA73-Me), the principal antheridiogen in Lygodium ferns, was investigated. From the methanol extract of prothallia of Lygodium circinnatum, GA25, GA73, GA73-Me, GA88-Me, and a few unknown GA73 derivatives were detected by GC-MS. Because the presence of GA25 suggests that GA24, a direct precursor of GA25, could also be present in L. circinnatum prothallia, we used feeding experiments to investigate the possibility that GA24 is a precursor of GA73-Me. In L. circinnatum prothallia, [2H2]GA24 was converted into [2H2]GA73-Me and a trace amount of [2H2]GA9-Me, whereas [2H3]GA9 was converted into [2H3]GA9-Me and [2H3]monohydroxy-GA9-Me. Because GA73-Me, GA9-Me, and their monohydroxy derivatives had been identified by GC-MS from the culture medium of L. circinnatum prothallia, our results suggest that GA73-Me is biosynthesized from GA24 via GA73, and that neither GA9 nor GA9-Me is a precursor of GA73-Me. Though the possibility had been suggested that GA73-Me is biosynthesized from 9,15-cyclo-GA9 (GA103), [2H2]GA103 was not converted into [2H2]GA73-Me.     

Subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli induces stress granule formation

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)‐negative strains of Shiga‐toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase‐dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB‐induced SG formation is regulated by activation of double‐stranded RNA‐activated protein kinase (PKR)‐like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH₄Cl and chloroquine, suppressed SubAB‐induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death‐associated protein 1 (DAP1) knockdown increased basal phospho‐PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA‐treated cells. Our findings show that SubAB‐induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin‐induced SG formation.     

Mycorrhizas influence functional traits of two tallgrass prairie species

Over the past decade, functional traits that influence plant performance and thus, population, community, and ecosystem biology have garnered increasing attention. Generally lacking, however, has been consideration of how ubiquitous arbuscular mycorrhizas influence plant allometric and stoichiometric functional traits. We assessed how plant dependence on and responsiveness to mycorrhizas influence plant functional traits of a warm‐season, C4 grass, Andropogon gerardii Vitman, and the contrasting, cool‐season, C3 grass, Elymus canadensis L. We grew both host species with and without inoculation with mycorrhizal fungi, across a broad gradient of soil phosphorus availabilities. Both host species were facultatively mycotrophic, able to grow without mycorrhizas at high soil phosphorus availability. A. gerardii was most dependent upon mycorrhizas and E. canadensis was weakly dependent, but highly responsive to mycorrhizas. The high dependence of A. gerardii on mycorrhizas resulted in higher tissue P and N concentrations of inoculated than noninoculated plants. When not inoculated, E. canadensis was able to take up both P and N in similar amounts to inoculated plants because of its weak dependence on mycorrhizas for nutrient uptake and its pronounced ability to change root‐to‐shoot ratios. Unlike other highly dependent species, A. gerardii had a high root‐to‐shoot ratio and was able to suppress colonization by mycorrhizal fungi at high soil fertilities. E. canadensis, however, was unable to suppress colonization and had a lower root‐to shoot ratio than A. gerardii. The mycorrhiza‐related functional traits of both host species likely influence their performance in nature: both species attained the maximum responsiveness from mycorrhizas at soil phosphorus availabilities similar to those of tallgrass prairies. Dependence upon mycorrhizas affects performance in the absence of mycorrhizas. Responsiveness to mycorrhizal fungi is also a function of the environment and can be influenced by both mycorrhizal fungus species and soil fertility.