Knocking out peroxisome proliferator-activated receptor (PPAR) alpha inhibits radiation-induced apoptosis in the mouse kidney through activation of NF-kappaB and increased expression of IAPs

Peroxisome proliferator-activated receptor (PPAR) alpha, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at testing the hypothesis that PPARalpha knockout mice would exhibit decreased radiation-induced apoptosis due to exacerbated activation of NF-kappaB (NFKB) and expression of pro-survival factors. Thirty wild-type mice (29S1/SvImJ) and 30 PPARalpha knockout mice were irradiated with a single total-body dose 10 Gy of (137)Cs gamma rays; controls were sham-irradiated. Tissue samples were collected at 3, 6, 12, 24 and 48 h postirradiation. Apoptosis was quantified using immunohistochemical staining for apoptotic bodies and cleaved caspase 3. Radiation-induced apoptosis was observed in both mouse strains in a time-dependent manner. However, the level of apoptosis was significantly suppressed in PPARalpha knockout mice compared with wild-type mice at 6 h postirradiation (P < 0.05). This inhibition of radiation-induced apoptosis was associated with time-dependent increases in NF-kappaB DNA-binding activity, IkappaBalpha phosphorylation, and expression of other antiapoptosis factors in the PPARalpha knockout mouse kidneys but not in wild-type animals. These data support the hypothesis that the loss of PPARalpha expression leads to the suppression of radiation-induced apoptosis in the mouse kidney, mediated through activation of NF-kappaB and up-regulation of anti-apoptosis factors.     

Prevention of human PC-346C prostate cancer growth in mice by a xenogeneic tissue vaccine

Vaccination, as an approach to prostate cancer, has largely focused on immunotherapy utilizing specific molecules or allogeneic cells. Such methods are limited by the focused antigenic menu presented to the immune system and by immunotolerance to antigens recognized as “self”. To examine if a xenogeneic tissue vaccine could stimulate protective immunity in a human prostate cancer cell line, a vaccine was produced by glutaraldehyde fixation of harvested PAIII prostate cancer cells tumors (GFT cell vaccine) from Lobund-Wistar rats. Immunocompetent Ncr-Foxn1<nu> mice were vaccinated with the GFT cell vaccine four times, 7 days apart. The control animals were either not vaccinated or vaccinated with media or glutaraldehyde-fixed PC346C human prostate cancer cells and adjuvant. About 8 days after the final boost, serum and spleens were harvested. The splenocytes were co-incubated with PC346C cells and then transplanted orthotopically into sygneneic immunodeficient nude mice. About 10 weeks later, the prostates were weighed and sampled for histolologic examination. The spleens were harvested from additional mice, and the splenocytes were cultured, either with or without pulsing by GFT cells, and the supernatants harvested 72 h later for cytokine analysis. Results showed that vaccination with GFT cells resulted in increased serum antibody to a PAIII cell lysate; reduced weight of the prostate/seminal vesicle complex and reduced incidence of prostate cancer in nude mice; increased splenocyte supernatant levels of TNF-α, IL-2, IFN-γ and IL-12, cytokines associated with Th1 immunity; and increased splenocyte supernatant levels of IL-4 and IL-10, cytokines associated with Th2 immunity. In summary, the results suggest that use of a xenogeneic tissue vaccine can stimulate protective immunity against human prostate cancer cells.     

Structure-based mutation analysis shows the importance of LRP5 beta-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling

A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Wnts with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf 1 (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5-G171V) induces beta-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity. HBM variant suppresses Dkk1 function and this results in reduced inhibition of TCF activity as compared to that with LRP5. Structural analysis of LRP5 revealed that the HBM mutation lies in the 4th blade of the first beta-propeller domain. To elucidate the functional significance and consequence of the LRP5-G171V mutation in vitro, we took a structure-based approach to design 15 specific LRP5 point mutations. These included (a) substitutions at the G171 in blade 4, (b) mutations in blades 2-6 of beta-propeller 1, and (c) mutations in beta-propellers 2, 3 and 4. Here we show that substitutions of glycine at 171 to K, F, I and Q also resulted in HBM-like activity in the presence of Wnt1 and Dkk1. This indicates the importance of the G171 site rather than the effect of specific amino acid modification to LRP5 receptor function. Interestingly, G171 equivalent residue mutations in other blades of beta-propeller 1 (A65V, S127V, L200V, A214V and M282V) resulted in LRP5-G171V-like block of Dkk1 function. However G171V type mutations in other beta-propellers of LRP5 did not result in resistance to Dkk1 function. These results indicate the importance of LRP5 beta-propeller 1 for Dkk1 function and Wnt signaling. These data and additional comparative structural analysis of the LRP5 family member LDLR suggest a potential functional role of the first beta-propeller domain through intramolecular interaction with other domains of LRP5 wherein Dkk1 can bind. Such studies may also lead to a better understanding of the mechanisms underlying the reduced function of Dkk1-like inhibitory ligands of LRP5 with HBM-like mutations and its relationship to increased bone density phenotypes.     

Arthromyces and Blastosporella, two new genera of conidia-producing lyophylloid agarics (Agaricales, Basidiomycota) from the neotropics

Two new genera encompassing three new species of lyophylloid agarics that produce conidia on the basidiomata are described. Arthromyces is a genus comprised of two very different arthrospore-producing mushroom species found in the Greater Antilles and Central America. Blastosporella is a monotypic genus with spherical balls of blastospores covering the pileus surface with age and is known from Hispaniola and Colombia. A key to the species of Arthromyces is included.     

Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling

The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.     

Roles for Drosophila melanogaster myosin IB in maintenance of enterocyte brush-border structure and resistance to the bacterial pathogen Pseudomonas entomophila

Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both singed (fascin) and Dmoesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection.     

Hormonal regulation of hair follicles exhibits a biological paradox

Hair's importance for insulation and camouflage or human communication means that hairs need to change with season, age or sexual development. Regular, regenerating hair follicle growth cycles produce new hairs which may differ in colour and/or size, e.g., beard development. Hormones of the pineal-hypothalamus-pituitary axis coordinate seasonal changes, while androgens regulate most sexual aspects with paradoxically different effects depending on body site; compare beard growth and balding! Hormones affect follicular mesenchymal-epithelial interactions altering growing time, dermal papilla size and dermal papilla cell, keratinocyte and melanocyte activity. Greater understanding of these mechanisms should improve treatments for poorly controlled hair disorders, alopecia and hirsutism.