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.     

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.     

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.     

Insulin‐ and Exercise‐Stimulated Skeletal Muscle Blood Flow and Glucose Uptake in Obese Men

Objective: Insulin resistance in obese subjects results in the impaired use of glucose by insulin‐sensitive tissues, e.g., skeletal muscle. In the present study, we determined whether insulin resistance in obesity is associated with an impaired ability of exercise to stimulate muscle blood flow, oxygen delivery, or glucose uptake. Research Methods and Procedures: Nine obese (body mass index = 36 ± 2 kg/m²) and 11 age‐matched nonobese men (body mass index = 22 ± 1 kg/m²) performed one‐legged isometric exercise during hyperinsulinemia. Rates of femoral muscle blood flow, oxygen consumption, and glucose uptake were measured simultaneously in both legs using [¹⁵O]H₂O, [¹⁵O]O₂, [¹⁸F]fluoro‐deoxy‐glucose, and positron emission tomography. Results: The obese subjects exhibited resistance to insulin stimulation of glucose uptake in resting muscle, regardless of whether glucose uptake was expressed per kilogram of femoral muscle mass (p = 0.001) or per the total mass of quadriceps femoris muscle. At similar workloads, oxygen consumption, blood flow, and glucose uptake were lower in the obese than the nonobese subjects when expressed per kilogram of muscle, but similar when expressed per quadriceps femoris muscle mass. Discussion: We conclude that obesity is characterized by insulin resistance of glucose uptake in resting skeletal muscle regardless of how glucose uptake is expressed. When compared with nonobese individuals at similar absolute workloads and under identical hyperinsulinemic conditions, the ability of exercise to increase muscle oxygen uptake, blood flow, and glucose uptake per muscle mass is blunted in obese insulin‐resistant subjects. However, these defects are compensated for by an increase in muscle mass.     

Changes in carbohydrates and freezing tolerance during cold acclimation of red raspberry cultivars grown in vitro and in vivo

Changes in LT50 and carbohydrate levels in response to cold acclimation were monitored in vitro and in vivo in red raspberry ( Rubus idaeus L.) cultivars with different levels of cold hardiness. Entire micropropagated plantlets or shoot tips from 3 cultivars were harvested before, during and after cold acclimation. Cane samples from container-grown plants of 4 cultivars were harvested before and during cold acclimation and deacclimation. Samples were evaluated for cold hardiness (LT50) by controlled freezing, then analyzed for carbohydrates, including starch, sucrose, glucose, fructose and raffinose. Hardiness of cold-acclimated 'Muskoka' and 'Festival' was superior to that of 'Titan' or 'Willamette'. In vitro plantlets had higher levels of soluble carbohydrates on a dry weight basis and higher ratios of sucrose:(glucose+fructose) than the container-grown plants. Total soluble carbohydrates, primarily sucrose, accumulated during cold acclimation in both plantlets (33–56% relative increase) and plants (143–191% relative increase). Sucrose increased 124–165% in plantlets and 253–582% in container-grown plants during acclimation and declined rapidly to the level of control plants during deacclimation. Glucose and fructose also accumulated, but to a lesser extent than sucrose. Raffinose concentrations were very low, but increased significantly during cold acclimation. In vitro, genotype hardiness was related to the high concentrations of total soluble carbohydrates, sucrose and raffinose. In vivo, hardier genotypes had lower concentrations of starch than the less hardy genotypes. These results demonstrated the importance of soluble carbohydrates, especially sucrose, in cold hardening of red raspberry and that the in vitro conditions or controlled acclimation conditions do not necessarily reflect the phenomena observed in vivo.     

DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method

During pregnancy, mothers are exposed to complex chemical mixtures, such as air pollution and smoke from incomplete combustion. In this study DNA adducts were measured in human placentas from 29 mothers. Environmental exposure and several possible biomarkers in relation to levels of DNA adducts were measured. Placental aromatic and bulky DNA adducts were measured with the ³²     

Healthy human salivary glands contain a DHEA-sulphate processing intracrine machinery, which is deranged in primary Sjögren's syndrome

Sjögren's syndrome (SS) patients have low salivary dehydroepiandrosterone (DHEA) and androgen biomarker levels, but high salivary oestrogen levels. The hypothesis was that the healthy glands contain DHEA‐sulphate processing intracrine machinery; the local androgen/oestrogen imbalance suggests that this is disarranged in SS. Indirect immunofluorescence and quantitative real‐time PCR (qRT‐PCR) of steroid sulphatase, sulfotransferase, 3β‐ and 17β‐hydroxysteroid dehydrogenases (3β‐ and 17β‐HSD), 5α‐reductase and aromatase were performed for labial salivary glands of healthy controls and persons with SS. In control acini steroid sulphatase and sulfotransferase immunoreactivities were located in the basolateral cell parts. 3β‐ and 17β‐HSD formed strong, interrupted bands along the basal cell parts. 5α‐reductase was mainly located in acinar cell nuclei and aromatase in the apical cell membrane. All enzymes were more widespread in ducts. In SS, steroid sulphatase was weak and deranged, 3β‐ and 17β‐HSD had lost their strict basal acinar cell localization and 5α‐reductase was mainly found in the cytoplasm of the acinar cells, whereas aromatase showed similar staining in SS and controls. qRT‐PCR of labial salivary glands disclosed all corresponding enzyme mRNAs with the levels of 3β‐HSD in SS being the lowest. Healthy tubuloacinar epithelial cells contain complete intracrine machineries for DHEA(‐sulphate) pro‐hormone processing. These enzymes have in healthy acini an organized architecture, which corresponds with DHEA uptake from the circulation, nuclear site of production of the active dihydrotestosterone (DHT) end product and production of oestrogens into saliva for export to ductal and oral epithelial cells. SS is characterized by low 3β‐HSD levels, which together with impaired subcellular compartmentalization of HSDs and 5α‐reductase may explain the low local DHT and androgen biomarker levels in SS.