Inhibition of epithelial ductal branching in the prostate by sonic hedgehog is indirectly mediated by stromal cells

Sonic hedgehog (Shh), a vertebrate homologue of the Drosophila segment-polarity gene hedgehog, has been reported to play an important role during normal development of various tissues. Abnormal activities of Shh signaling pathway have been implicated in tumorigenesis such as basal cell carcinomas and medulloblastomas. Here we show that Shh signaling negatively regulates prostatic epithelial ductal morphogenesis. In organotypic cultures of developing rat prostates, Shh inhibited cell proliferation and promoted differentiation of luminal epithelial cells. The expression pattern of Shh and its receptors suggests a paracrine mechanism of action. The Shh receptors Ptc1 (Patched1) and Ptc2 were found to be expressed in prostatic stromal cells adjacent to the epithelium, where Shh itself was produced. This paracrine model was confirmed by co-culturing the developing prostate in the presence of stromal cells transfected with a vector expressing a constitutively active form of Smoothened, the real effector of the Shh signaling pathway. Furthermore, expression of activin A and TGF-beta1 that were shown previously to inhibit prostatic epithelial branching was up-regulated following Shh treatment in the organotypic cultures. Taken together, these results suggest that Shh negatively regulates prostatic ductal branching indirectly by acting on the surrounding stromal cells, at least partly via up-regulating expression of activin A and TGF-beta1.     

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.     

Native Australian Species are Effective in Extracting Multiple Heavy Metals from Biosolids

Selecting native plant species with characteristics suitable for extraction of heavy metals may have multiple advantages over non-native plants. Six Australian perennial woody plant species and one willow were grown in a pot trial in heavy metal-contaminated biosolids and a potting mix. The plants were harvested after fourteen months and above-ground parts were analysed for heavy metal concentrations and total metal contents. All native species were capable of growing in biosolids and extracted heavy metals to varying degrees. No single species was able to accumulate heavy metals at particularly high levels and metal extraction depended upon the bioavailability of the metal in the substrate. Metal extraction efficiency was driven by biomass accumulation, with the species extracting the most metals also having the greatest biomass yield. The study demonstrated that Grevillea robusta, Acacia mearnsii, Eucalyptus polybractea, and E. cladocalyx have the greatest potential as phytoextractor species in the remediation of heavy metal-contaminated biosolids. Species survival and growth were the main determinants of metal extraction efficiency and these traits will be important for future screening of native species.     

Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1)

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.     

The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI(+)], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI(+)]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI(+)] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI(+)] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI(+)] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI(+)].