Relationship between prostate-specific antigen levels and ambient temperature

We examined the association between prostate-specific antigen (PSA) and daily mean ambient temperature on the day of the test in healthy men who had three annual checkups. We investigated 9,694 men who visited a hospital for routine health checkups in 2007, 2008, and 2009. Although the means and medians of ambient temperature for the three years were similar, the mode in 2008 (15.8 °C) was very different from those in 2007 and 2009 (22.4 °C and 23.2 °C). After controlling for age, body mass index, and hematocrit, a multiple regression analysis revealed a U-shaped relationship between ambient temperature and PSA in 2007 and 2009 (P?<?0.001 and P?=?0.004, respectively), but not in 2008 (P?=?0.779). In 2007, PSA was 13.5 % higher at 5 °C and 10.0 % higher at 30 °C than that at 18.4 °C (nadir). In 2009, PSA was 7.3 % higher at 5 °C and 6.8 % at 30 °C compared with the level at 17.7 °C (nadir). In logistic regression analysis, a U-shaped relationship was found for the prevalence of a higher PSA (> 2.5 ng/mL) by ambient temperature, with the lowest likelihood of having a high PSA at 17.8 °C in 2007 (P?=?0.038) and 15.5 °C in 2009 (P?=?0.033). When tested at 30 °C, there was a 57 % excess risk of having a high PSA in 2007 and a 61 % higher risk in 2009 compared with those at each nadir temperature. We found a U-shaped relationship between PSA and ambient temperature with the lowest level of PSA at 15–20 °C.     

Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxonil, and heat shock in Neurospora crassa

Two-component signal transduction comprising of OS-1 (histidine kinase), OS-4 (MAPKK kinase), OS-5 (MAPK kinase), and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. To identify the genes regulated downstream of OS-2 MAP kinase, quantitative real-time RT-PCR analysis was conducted in selected genes based on Hog1 MAP kinase regulated genes in yeast. In response to osmotic stress and fludioxonil, expression of six genes that for glycerol synthesis (gcy-1, gcy-3, and dak-1), gluconeogenesis (fbp-1 and pck-1), and catalase (ctt-1) was activated in the wild-type strain, but not in the os-2 mutant. A heat shock treatment also induced their expression in the same way. Consisting with the gene expression, the enzyme activity of glycerol dehydrogenase, but not glycerol-3-phosphate dehydrogenase, was increased in response to osmotic stress and fludioxonil in the wild-type strain. OS-2 was phosphorylated by the OS-1 cascade in response to relatively low osmotic stress and fludioxonil. However, OS-2 phosphorylation by heat shock and a higher osmotic stress was found in the os-1 mutant normally but not in the os-4 and os-5 mutants. These results suggested that non-OS-1 signaling activates OS-2 in an OS-4-dependent manner in such conditions.     

Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis

The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs.     

Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

Background

Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition.

Methodology

We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB.

Results and Conclusion

Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.     

Solution Structure of Human Pex5��Pex14��PTS1 Protein Complexes Obtained by Small Angle X-ray Scattering

The Pex5p receptor recognizes newly synthesized peroxisomal matrix proteins which have a C-terminal peroxisomal targeting signal to the peroxisome. After docking to protein complexes on the membrane, these proteins are translocated across the membrane. The docking mechanism remains unclear, as no structural data on the multicomponent docking complex are available. As the interaction of the cargo-loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step, we have investigated the solution structure of these complexes by small angle x-ray scattering and static light scattering. Titration studies yielded a 1:6 stoichiometry for the Pex5p·Pex14p complex, and low resolution structural models were reconstructed from the x-ray scattering data. The free full-length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. The model of the complex reveals that the N terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with the Pex5p moiety. These results suggest that the extended structure of Pex5p may play a role in interactions with other substrates such as lipids and membrane proteins during the formation of functional multiprotein complexes.Peroxisomes are ubiquitous organelles in eukaryotes which are involved in different metabolic pathways (1). Peroxisomal matrix proteins, which contain a peroxisomal targeting signal (PTS),⁴ are imported into the peroxisome by recognition of two different import receptors, Pex5p or Pex7p. These receptors recognize specific signal sequences, PTS1 and PTS2, respectively (1). At the molecular level the C-terminal PTS1 signal is bound in a central cavity of the ring-like structure of the seven tetrapeptide repeat (TPR) domains of the C-terminal part of Pex5p (Pex5p(C)) (2–5). It was recently proposed that some of the structural principles of the Pex5p/cargo interaction may also apply to the PTS2 cargo recognition of the Pex7p receptor (5).The next step of PTS-protein import, docking of the cargo loaded receptor to the translocon, involves the peroxisomal protein Pex14p (6). Multiple Pex14p binding sites with di-aromatic pentapeptide motifs (WXXX(F/Y)) were shown to be present in the N terminus of Pex5p (7–9). The number of these motifs, however, varies among species. The human Pex5p receptor, which has been investigated in this contribution, has a total of seven motifs. A recent NMR structure of the N-terminal domain of Pex14p and the first WXXX(F/Y) motif of Pex5p reveals an α-helical conformation of the motif (10). Interactions between Pex5p and other proteins and by their association with the peroxisomal membrane possibly lead to dissociation of the PTS-protein from Pex5p (11–13). The exact sequence of events in the import mechanism remains, however, unknown. It is in particular unclear how, in contrast with other organelles, peroxisomes can import folded oligomeric, functional proteins (14).Previous biophysical work indicated that the N terminus half of Pex5p is unfolded in vitro (15, 16). Recent protease sensitivity assays showed that the proteolytic profiles of the full-length receptor Pex5p(F) change in the presence of PTS1 peptide and the Pex13p Src homology 3 domain, which is another docking factor (16, 17), indicating conformational changes of Pex5p upon binding these receptor ligands. Furthermore, it was found that Pex5p may even traverse the peroxisomal membrane, leaving only a small N-terminal fragment in the cytosol while exposing the C-terminal TPR domain to the luminal side of the membrane (11).Although recognition of many PTS cargos seems to be confined to the C-terminal TPR domains of Pex5p, it has become clear that the N-terminal part of Pex5p is primarily involved in docking of the receptor onto the peroxisomal membrane and other docking factors. Because only poorly diffracting crystals have been purified to date, we investigated its solution structure by small angle x-ray scattering (SAXS) and static light scattering (SLS). Complexes with the PTS1 cargo sterol carrier protein 2 (SCP2), which functions as lipid transfer protein, were also studied as the crystal structure of Pex5p(C)/SCP2 is already known (4). Our results indicate that human Pex5p(F) is a monomer with an extended N terminus. The stoichiometry of Pex5p(F)·Pex14p(N)·PTS1 complex has been assessed by titration with SAXS, SLS, and gel filtration, and a low resolution structural model of the complex has been reconstructed in which Pex5p(F) remains extended upon Pex14p(N) binding.     

Disseminated prostate cancer cells can instruct hematopoietic stem and progenitor cells to regulate bone phenotype

Prostate cancer metastases and hematopoietic stem cells (HSC) frequently home to the bone marrow, where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMP)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. As it is not known, we examined whether metastatic prostate cancer cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPC). HSC/HPCs isolated from mice bearing nonmetastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line, which produces mixed osteoblastic and osteolytic lesions in bone, HPCs, but not HSCs, were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase-positive osteoclasts through an interleukin-6-mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of prostate cancer.     

c-Fos/activator protein-1 transactivates wee1 kinase at G(1)/S to inhibit premature mitosis in antigen-specific Th1 cells

M-phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. We isolated the wee1 gene promoter and found that it contains one AP-1 binding motif and is directly activated by the immediate early gene product c-Fos at cellular G(1)/S phase. In antigen-specific Th1 cells stimulated by antigen, transactivation of the c-fos and wee1 kinase genes occurred sequentially at G(1)/S, and the substrate of wee1 kinase, cdc2-Tyr15, was subsequently phosphorylated at late G(1)/S. Under prolonged expression of the c-fos gene, however, the amount of wee1 kinase was increased and its target cdc2 molecule was constitutively phosphorylated on its tyrosine residue, where Th1 cells went into aberrant mitosis. Thus, an immediate early gene product, c-Fos/AP-1, directly transactivates the wee1 kinase gene at G(1)/S. The transient increase in c-fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G(1)/S phase of the cell cycle.