Substance P induces localization of MIF/α1-inhibitor-3 complexes to umbrella cells via paracellular transit through the urothelium in the rat bladder

BACKGROUND: Macrophage migration inhibitory factor (MIF) is released into the intraluminal fluid during bladder inflammation in the rat complexed to α1-inhibitor-3 (A1-I3; a rodent proteinase inhibitor in the α-macroglobulin family). The location of A1-I3 in the bladder had not been investigated. Therefore, we examined the location of A1-I3 and MIF/A1-I3 complexes in the bladder and changes due to experimental inflammation. METHODS: Anesthetized male rats had bladders removed with no treatment (intact) or were injected with Substance P (SP; s.c.; saline vehicle). After one hour intraluminal fluid was removed, bladder was excised and MIF and A1-I3 levels were determined using ELISA and/or western-blotting. MIF co-immunoprecipitation determined MIF/A1-I3 complexes in the bladder. Bladder sections were immunostained for A1-I3 and MIF/A1-I3. RESULTS: A1-I3 immunostaining was observed in interstitial spaces throughout the bladder (including submucosa) but not urothelium in intact and saline-treated rats. RT-PCR showed that the bladder does not synthesize A1-I3, therefore, A1-I3 in the interstitial space of the bladder must be plasma derived. In SP-treated rats, A1-I3 in the bladder increased and A1-I3 was observed traversing through the urothelium. Umbrella cells that do not show MIF and/or A1-I3 immunostaining in intact or saline-treated rats, showed co-localization of MIF and A1-I3 after SP-treatment. Western blotting demonstrated that in the bladder MIF formed non-covalent interactions and also binds covalently to A1-I3 to form high molecular weight MIF/A1-I3 complexes (170, 130 and 75-kDa, respectively, verified by co-immunoprecipitation). SP-induced inflammation selectively reduced 170-kDa MIF/A1-I3 in the bladder while increasing 170 and 130-kDa MIF/A1-I3 in the intraluminal fluid. CONCLUSION: A1-I3 and MIF/A1-I3 complexes are resident in bladder interstitium. During SP-induced inflammation, MIF/A1-I3 complexes are released from the bladder into the lumen. Binding of MIF/A1-I3 complexes to urothelial cells during inflammation suggests these complexes participate in the inflammatory reaction through activation of receptors for MIF and/or for A1-I3

Substance P Increases Cell-Surface Expression of CD74 (Receptor for Macrophage Migration Inhibitory Factor): In Vivo Biotinylation of Urothelial Cell-Surface Proteins

Macrophage migration inhibitory factor (MIF), an inflammatory cytokine, and its receptor CD74 are upregulated by bladder inflammation. MIF-mediated signal transduction involves binding to cell-surface CD74, this study documents, in vivo, MIF-CD74 interactions at the urothelial cell surface. N-hydroxysulfosuccinimide biotin ester-labeled surface urothelial proteins in rats treated either with saline or substance P (SP, 40 μg/kg). The bladder was examined by histology and confocal microscopy. Biotinylated proteins were purified by avidin agarose, immunoprecipitated with anti-MIF or anti-CD74 antibodies, and detected with strepavidin-HRP. Only superficial urothelial cells were biotinylated. These cells contained a biotinylated MIF/CD74 cell-surface complex that was increased in SP-treated animals. SP treatment increased MIF and CD74 mRNA in urothelial cells. Our data indicate that intraluminal MIF, released from urothelial cells as a consequence of SP treatment, interacts with urothelial cell-surface CD74. These results document that our previously described MIF-CD74 interaction occurs at the urothelial cell surface

Anatomical location of Macrophage Migration Inhibitory Factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat

BACKGROUND: Previous work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat. RESULTS: In the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons. CONCLUSIONS: Therefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIF's expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes

(S)-6-Methyl-∊-caprolactone

The chiral title compound, C7H12O2, a lactone derivative, features a seven-membered ring that adopts a chair conformation. The crystal structure is stabilized by weak C—H⋯O inter­actions occurring in the (100) plane. The absolute configuration was assigned on the basis of the enantioselective synthesis

Discovery of a 66 mas Ultracool Binary with Laser Guide Star Adaptive Optics

We present the discovery of 2MASS J21321145+1341584AB as a closely separated (0.066") very low-mass field dwarf binary resolved in the near-infrared by the Keck II Telescope using laser guide star adaptive optics. Physical association is deduced from the angular proximity of the components and constraints on their common proper motion. We have obtained a near-infrared spectrum of the binary and find that it is best described by an L5+/-0.5 primary and an L7.5+/-0.5 secondary. Model-dependent masses predict that the two components straddle the hydrogen burning limit threshold with the primary likely stellar and the secondary likely substellar. The properties of this sytem - close projected separation (1.8+/-0.3 AU) and near unity mass ratio - are consistent with previous results for very low-mass field binaries. The relatively short estimated orbital period of this system (~7-12 yr) makes it a good target for dynamical mass measurements. Interestingly, the system's angular separation is the tightest yet for any very low-mass binary published from a ground-based telescope and is the tightest binary discovered with laser guide star adaptive optics to date.Comment: 10 pages, 3 figures; accepted for publication to A

Protoplanetary and Transitional Disks in the Open Stellar Cluster IC 2395

We present new deep UBVRI images and high-resolution multi-object optical spectroscopy of the young (~ 6 - 10 Myr old), relatively nearby (800 pc) open cluster IC 2395. We identify nearly 300 cluster members and use the photometry to estimate their spectral types, which extend from early B to middle M. We also present an infrared imaging survey of the central region using the IRAC and MIPS instruments on board the Spitzer Space Telescope, covering the wavelength range from 3.6 to 24 microns. Our infrared observations allow us to detect dust in circumstellar disks originating over a typical range of radii ~ 0.1 to ~ 10AU from the central star. We identify 18 Class II, 8 transitional disk, and 23 debris disk candidates, respectively 6.5%, 2.9%, and 8.3% of the cluster members with appropriate data. We apply the same criteria for transitional disk identification to 19 other stellar clusters and associations spanning ages from ~ 1 to ~ 18 Myr. We find that the number of disks in the transitional phase as a fraction of the total with strong 24 micron excesses ([8] - [24] > 1.5) increases from 8.4 +\- 1.3% at ~ 3 Myr to 46 +\- 5% at ~ 10 Myr. Alternative definitions of transitional disks will yield different percentages but should show the same trend.Comment: accepted by the Astrophysical Journa

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

Hypoxia is a characteristic feature of solid tumors leading to the over expression of hypoxia-inducible factor (HIF)-1α protein and therefore to a specific cellular behavior. However, even though the oxygen tension in tumors is low (<5 %), most of the cell lines used in cancer studies are grown under 21 % oxygen tension. This work focuses on the impact of oxygen conditions during in vitro cell culture on glucose metabolism using 1-13C-glucose. Growing U87-MG glioma cells under hypoxic conditions leads to a two- to threefold reduction of labeled glutamine and an accumulation of fructose. However, under both hypoxic and normoxic conditions, glucose is used for de novo synthesis of pyrimidine since the 13C label is found both in the uracil and ribose moieties. Labeling of the ribose ring demonstrates that U87-MG glioma cells use the reversible branch of the non-oxidative pentose phosphate pathway. Interestingly, stereotactic implantation of U87-MG cells grown under normoxia or mild hypoxia within the striatum of nude mice led to differential growth; the cells grown under hypoxia retaining an imprint of the oxygen adaptation as their development is then slowed down

Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum) levels. Recent studies from another laboratory failed to document these results. This study's aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate. METHODS: MIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis. RESULTS: Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87 ± 3.91 ng/ml; ± interquartile range; n = 115) compared with patients with no documented diagnosis of prostate cancer (2.19 ± 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected in culture medium from prostate cancer cell lines (LNCaP and PC-3). CONCLUSION: Increased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer

(4aS,5R,7R,8S,8aR)-8-(1,3-Dioxolan-2-yl)-7,8-dimethyl-5-(1-methyl­ethen­yl)perhydro­naphthalen-2-one

In the chiral title compound, C18H28O3, the two six-membered rings of the perhydronaphthalenone adopt a rigid chair–chair conformation and the five-membered dioxolanyl ring adopts an envelope conformation. The crystal structure is stabilized only by weak inter­actions