Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes

The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella-containing vacuole (SCV) and tubular extensions from the SCV, Salmonella-induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N-terminal 225-amino-acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non-ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent-resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.     

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776–789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884–899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon γ secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination. Received: 10 April 2000 / Accepted: 12 July 2000     

Tissue-specific developmental changes in cell-wall ferulate and dehydrodiferulates in sugar beet

Sugar beet (Beta valgaris L.) seedlings were grown for 8-14 weeks, and then separated into leaf, petiole, inner and outer storage root and absorptive root fractions. Cell-wall ferulate and dehydrodiferulate esters were analysed by HPLC. In leaves, ferulate dimers were mostly 8-8 linked, while 8-O-4 and sometimes 8-5 linkages were most abundant in all other tissues. The total dimer content and percentage of dimerisation were much higher in the absorptive root than in other tissues. These results indicated varying patterns of ferulate and dehydrodiferulate ester content in different tissues, suggesting corresponding variations in the biosynthetic processes. When [14C]-cinnamate was applied to the leaves at 4 weeks, and [14C]-dimers measured in root cell walls at 8 and 14 weeks, a much higher proportion of 8-5 linkages was found in the [14C]-dimers than in total (non-radioactive) dimers in all parts of the root, especially at 14 weeks, indicating further complexity in the metabolism of cell-wall phenolics.     

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine β-lactoglobulin gene that confer locus commitment to heterogeneous tissues

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine -lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus     

Vegetation of tuscan ultramafic soils in relation to edaphic and physical factors

Vegetation and soil sampling were carried out in 80 plots located in five different ultramafic (serpentine) sites of Tuscany, central Italy. The physical and chemical features of each plot were determined and the species composition and cover recorded. The exchangeable fraction of soil metals was analysed because it gives a measure of their concentrations available to plants. The plots were classified by cluster analysis and ANOVA was used to compare the environmental variables of the groups of plots. Canonical correspondence analysis was used to detect the principal factors for gradients of species composition within the plant communities. A higher content of exchangeable metals was found under the more evolved and structured plant communities, suggesting that serpentine vegetation of Tuscany is not strongly limited by soil metals, such as chromium, cobalt, nickel and magnesium, typically associated with ultramafic soils. The low nutrient content of the soils and drought stress mainly due to topographical features, appear to have a more significant role in determining the typical scattered vegetation of the Tuscan ultramafics.     

Concurrent resistance and endurance training influence basal metabolic rate in nondieting individuals

Thirty physically active healthy men (20.1 ± 1.6 yr) wererandomly assigned to participate for 10 wk in one of the following training groups: endurance trained (ET; 3 days/wk joggingand/or running), resistance trained (RT; 3 days/wkresistance training), or combined endurance and resistance trained(CT). Before and after training, basal metabolic rate (BMR), percentbody fat (BF), maximal aerobic power, and one-repetition maximum forbench press and parallel squat were determined for each subject.Urinary urea nitrogen was determined pre-, mid-, and posttraining. BMRincreased significantly from pre- to posttraining for RT (7,613 ± 968 to 8,090 ± 951 kJ/day) and CT (7,455 ± 964 to 7,802 ± 981 kJ/day) but not for ET (7,231 ± 554 to 7,029 ± 666 kJ/day).BF for CT (12.2 ± 3.5 to 8.7 ± 1.7%) was significantly reducedcompared with RT (15.4 ± 2.7 to 14.0 ± 2.7%) and ET (11.8 ± 2.9 to 9.5 ± 1.7%). Maximal aerobic power increasedsignificantly for ET (13%) but not RT (0.2%) or CT (7%),whereas the improvements in one-repetition maximum bench press andparallel squat were greater in RT (24 and 23%, respectively) comparedwith CT (19 and 12%, respectively). Urinary urea nitrogen loss wasgreater in ET (14.6 ± 0.9 g/24 h) than in RT (11.7 ± 1.0 g/24h) and CT (11.5 ± 1.0 g/24 h) at the end of 10 wk oftraining. These data indicate that, although RT alone will increase BMRand muscular strength, and ET alone will increase aerobic power anddecrease BF, CT will provide all of these benefits but to a lessermagnitude than RT and ET after 10 wk of training.