Diverse phenotypes resulting from polyphosphate kinase gene (ppk1) inactivation in different strains of Helicobacter pylori

Connections among biochemical pathways should help buffer organisms against environmental stress and affect the pace and trajectory of genome evolution. To explore these ideas, we studied consequences of inactivating the gene for polyphosphate kinase 1 (ppk1) in strains of Helicobacter pylori, a genetically diverse gastric pathogen. The PPK1 enzyme catalyzes synthesis of inorganic polyphosphate (poly P), a reservoir of high-energy phosphate bonds with multiple roles. Prior analyses in less-fastidious microbes had implicated poly P in stress resistance, motility, and virulence. In our studies, ppk1 inactivation caused the expected near-complete absence of poly P (>250-fold decrease) but had phenotypic effects that differed markedly among unrelated strains: (i) poor initial growth on standard brain heart infusion agar (five of six strains tested); (ii) weakened colonization of mice (4 of 5 strains); (iii) reduced growth on Ham's F-12 agar, a nutritionally limiting medium (8 of 11 strains); (iv) heightened susceptibility to metronidazole (6 of 17 strains); and (v) decreased motility in soft agar (1 of 13 strains). Complementation tests confirmed that the lack of growth of one Deltappk1 strain on F-12 agar and the inability to colonize mice of another were each due to ppk1 inactivation. Thus, the importance of ppk1 to H. pylori differed among strains and the phenotypes monitored. We suggest that quantitative interactions, as seen here, are common among genes that affect metabolic pathways and that H. pylori's high genetic diversity makes it well suited for studies of such interactions, their underlying mechanisms, and their evolutionary consequences.     

Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)beta/delta occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARbeta/delta expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-beta1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARbeta/delta accelerate wound closure. The results reveal a dual role of TGF-beta1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARbeta/delta expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-beta1 at wound sites.     

RING finger ubiquitin-protein isopeptide ligase Nrdp1/FLRF regulates parkin stability and activity

Parkin is a ubiquitin-protein isopeptide ligase. It has been suggested that loss of function in parkin causes accumulation and aggregation of its substrates, leading to death of dopaminergic neurons in Parkinson disease. Using the yeast two-hybrid screen, we isolated a RING finger protein that interacted with the N terminus of parkin in a Drosophila cDNA library. Interaction between human parkin and the mammalian RING finger protein homologue Nrdp1/FLRF, a ubiquitin-protein isopeptide ligase that ubiquitinates ErbB3 and ErbB4, was validated by in vitro binding assay, co-immunoprecipitation, and immunofluorescence co-localization. Significantly, pulse-chase experiments showed that cotransfection of Nrdp1 and parkin reduced the half-life of parkin from 5 to 2.5 h. Consistent with these findings, we further observed that degradation of CDCrel-1, a parkin substrate, was facilitated by overexpression of parkin protein. However, co-transfection of Nrdp1 with parkin reversed the effects of parkin on CDCrel-1 degradation. We conclude that Nrdp1 is a parkin modifier that accelerates degradation of parkin, resulting in a reduction of parkin activity.     

Receptor-type protein-tyrosine phosphatase-kappa regulates epidermal growth factor receptor function

Epidermal growth factor receptor (EGFR), the prototypic receptor protein tyrosine kinase, is a major regulator of growth and survival for many epithelial cell types. We report here that receptor-type protein-tyrosine phosphatase-kappa (RPTP-kappa) dephosphorylates EGFR and thereby regulates its function in human keratinocytes. Protein-tyrosine phosphatase (PTP) inhibitors induced EGFR tyrosine phosphorylation in intact primary human keratinocytes and cell-free membrane preparations. Five highly expressed RPTPs (RPTP-beta, delta, kappa, mu, and xi) were functionally analyzed in a Chinese hamster ovary (CHO) cell-based expression system. Full-length human EGFR expressed in CHO cells, which lack endogenous EGFR, displayed high basal (i.e. in the absence of ligand) tyrosine phosphorylation. Co-expression of RPTP-kappa, but not other RPTPs, specifically reduced basal EGFR tyrosine phosphorylation. RPTP-kappa also reduced epidermal growth factor-dependent EGFR tyrosine phosphorylation in CHO cells. Purified RPTP-kappa preferentially dephosphorylated EGFR tyrosines 1068 and 1173 in vitro. Overexpression of wild-type or catalytically inactive RPTP-kappa reduced or enhanced, respectively, basal and EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Furthermore, siRNA-mediated knockdown of RPTP-kappa increased basal and EGF-stimulated EGFR tyrosine phosphorylation and augmented downstream Erk activation in human keratinocytes. RPTP-kappa levels increased in keratinocytes as cells reached confluency, and overexpression of RPTP-kappa in subconfluent keratinocytes reduced keratinocyte proliferation. Taken together, the above data indicate that RPTP-kappa is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes.     

The crystal structure of the plexin-semaphorin-integrin domain/hybrid domain/I-EGF1 segment from the human integrin beta2 subunit at 1.8-A resolution

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

Estimating haplotype relative risks on human survival in population-based association studies

Association-based linkage disequilibrium (LD) mapping is an increasingly important tool for localizing genes that show potential influence on human aging and longevity. As haplotypes contain more LD information than single markers, a haplotype-based LD approach can have increased power in detecting associations as well as increased robustness in statistical testing. In this paper, we develop a new statistical model to estimate haplotype relative risks (HRRs) on human survival using unphased multilocus genotype data from unrelated individuals in cross-sectional studies. Based on the proportional hazard assumption, the model can estimate haplotype risk and frequency parameters, incorporate observed covariates, assess interactions between haplotypes and the covariates, and investigate the modes of gene function. By introducing population survival information available from population statistics, we are able to develop a procedure that carries out the parameter estimation using a nonparametric baseline hazard function and estimates sex-specific HRRs to infer gene-sex interaction. We also evaluate the haplotype effects on human survival while taking into account individual heterogeneity in the unobserved genetic and nongenetic factors or frailty by introducing the gamma-distributed frailty into the survival function. After model validation by computer simulation, we apply our method to an empirical data set to measure haplotype effects on human survival and to estimate haplotype frequencies at birth and over the observed ages. Results from both simulation and model application indicate that our survival analysis model is an efficient method for inferring haplotype effects on human survival in population-based association studies.     

Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.     

Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.