Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.     

Early disruption of pregnancy as a manifestation of seasonal infertility in pigs

All gilts and sows in production from which the detailed production information was available in a 160-sow unit were included to the study. In winter-spring, there were complete data available from 47 animals and in summer-autumn from 64 animals. The farm had a consistent history of the seasonally reduced farrowing rate in summer-autumn. Success of inseminations was monitored during a 4-month breeding period in winter-spring and in summer-autumn. Each animal was bled twice a week for 6 weeks starting a day before insemination and the blood samples were assayed to determine serum progesterone concentration. The blood samples were also assayed for cortisol to detect any acute infectious response. Starting on day 18, animals were pregnancy tested by transcutaneous real time ultrasound twice a week. In winter-spring, the farrowing rate was 72% (58 inseminations, 1.2 inseminations/sow) and in summer-autumn 63% (81 inseminations, 1.3 inseminations/sow). In winter-spring, there was only one detected case of early disruption of pregnancy (EDP), whereas nine such cases were recognised in summer-autumn. Five out of those nine animals returned to oestrus with a mean insemination to oestrus interval of 25.8+/-1.6 days. One sow returned to oestrus 35 days after insemination and three sows did not return to oestrus within 45 days. However, two of these sows had progesterone profiles that indicated an undetected oestrus around day 25. In those nine animals, no acute phase infectious response as indicated by a rise in serum cortisol was evident. Serum progesterone concentrations in the animals eventually loosing the pregnancy tended to be lower on day 13 (no significant difference) and were significantly lower on day 20 when compared with animals remaining pregnant. There was no difference in serum progesterone levels of pregnant animals between winter-spring and summer-autumn. Litter size was not affected by the season. The weaning to oestrus interval tended to be longer in summer-autumn. This study showed that the seasonally decreased farrowing rate is partly caused by EDP. The lowered progesterone concentrations in summer-autumn were demonstrable only in "problem animals".     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

Predictive modelling of Fe(III) precipitation in iron removal process for bioleaching circuits

In this study, the applicability of three modelling approaches was determined in an effort to describe complex relationships between process parameters and to predict the performance of an integrated process, which consisted of a fluidized bed bioreactor for Fe³⁺ regeneration and a gravity settler for precipitative iron removal. Self-organizing maps were used to visually evaluate the associations between variables prior to the comparison of two different modelling methods, the multiple regression modelling and artificial neural network (ANN) modelling, for predicting Fe(III) precipitation. With the ANN model, an excellent match between the predicted and measured data was obtained (R ² = 0.97). The best-fitting regression model also gave a good fit (R ² = 0.87). This study demonstrates that ANNs and regression models are robust tools for predicting iron precipitation in the integrated process and can thus be used in the management of such systems.     

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.     

ADAM12 produced by tumor cells rather than stromal cells accelerates breast tumor progression

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression.     

RNA reveals a succession of active fungi during the decay of Norway spruce logs

Current knowledge of the succession of fungi in decaying wood is mostly based on fruit bodies and in vitro culture. Here, we investigated the changing community of metabolically active fungi during the decomposition of fallen Picea abies logs by directly extracting and barcode sequencing precursor rRNA. We also compared rRNA-derived amplicons of the 18S and ITS regions in 21 isolates and discuss the use of RNA as a marker of metabolically active fungi. The richness of active fungi, revealed as separated bands in DGGE, peaked in logs at an advanced stage of decay. Soft-rot fungi were common in the early stages but white- and brown-rot fungi became dominant as decay progressed. Ectomycorrhizal fungi were detected at an early stage, and they became the most abundant group in the late stages of succession. A comparison of rRNA-derived amplicons revealed that although ITS was detected in the form of precursor rRNA, introns within 18S rDNA were already spliced. As such, rRNA- and rDNA-derived amplicons would yield different profiles of active and total communities if profiling method is affected by amplicon length.     

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.     

Changes in soil carbon with stand age – an evaluation of a modelling method with empirical data

Forest soils store a substantial amount of carbon, often more than the forest vegetation does. Estimates of the amount of soil carbon, and in particular estimates of changes in these amounts are still inaccurate. Measuring soil carbon is laborious, and measurements taken at a few statistically unrepresentative sites are difficult to scale to larger areas. We combined a simple dynamic model of soil carbon with litter production estimated on the basis of stand parameters, models of tree allometry and biomass turnover rates of different biomass components. This integrated method was used to simulate soil carbon as forest stands develop. The results were compared with measurements of soil carbon from 64 forest sites in southern Finland. Measured carbon stocks in the organic soil layer increased by an average of 4.7±1.4 g m^?2 a^?1 with increasing stand age and no significant changes were measured in the amount of carbon in mineral soil. Our integrated method indicated that soil carbon stocks declined to a minimum 20 years after clear‐cutting and the subsequent increase in the soil carbon stock (F/H ? 1 m) was 5.8±1.0 g m^?2 a^?1 averaged over the period to next harvesting (～125 years). Simulated soil carbon accumulation slowed down considerably in stands older than 50 years. The carbon stock measured (F/H ? 1 m) for the study area averaged 6.8±2.5 kg m^?2. The simulated carbon stock in soil was 7.0±0.6 kg m^?2 on average. These tests of the validity of the integrated model suggest that this method is suitable for estimating the amount of carbon in soil and its changes on regional scales.     

Developments in craniomaxillofacial surgery: use of self-reinforced bioabsorbable osteofixation devices.

Because of the problems associated with the conventional osteofixation devices used in craniomaxillofacial surgery, absorbable devices present an appealing alternative. Devices made of the polymers polylactide, polyglycolide, and their copolymers (PLGA and P[L/DL]LA) are currently the most commonly used. Ultrahigh-strength implants can be manufactured from these polymers with the self-reinforcing technique. Over the authors' almost two decades of study, both in experimental and clinical settings, self-reinforced devices have proved to be biocompatible, easy to handle, and mechanically strong, even for the fixation of femoral neck fractures. In craniomaxillofacial surgery, the authors have used self-reinforced devices for over 8 years without complications. Because of the more favored degradation characteristics, currently the copolymeric self-reinforced devices (P[L/DL]LA, Biosorb FX and PLGA, Biosorb PDX; Elite Performance Technologies, Solana Beach, Calif.) represent the advancing front in the application of absorbable devices in craniomaxillofacial surgery. The authors' share their experience and their studies of self-reinforced devices, which possess the highest strength and ductility of all bioabsorbable products.