Triplicate Assays Research Articles

Endothelin-1 Promotes Osteosarcoma Cell Invasion and Survival against Cisplatin-induced Apoptosis

Endothelin-1 (ET-1) participates in a wide range of cancer-relevant processes including cell proliferation, inhibition of apoptosis, matrix remodeling, bone deposition, and metastases. Although ET-1 reportedly promotes osteosarcoma (OS) cell invasion, suggesting an important role of ET-1 in OS metastasis, the role of ET-1 in OS remains unclear. We asked whether (1) ET-1 expression is associated with the malignancy of OS, (2) ET-1 enhances the cell invasion ability of OS, and (3) ET-1 promotes OS cell survival against apoptotic stress. We cultured primary OS specimens from 22 patients with Stages II (OS-II) and III (OS-III) in real-time quantitative RT-PCR and ELISA to compare ET-1 expression. We used Transwell(®) cell invasion assays (in triplicate) to assess the invasion ability of cells in the presence or absence of exogenous ET-1 and/or ET receptor antagonists. We compared cell apoptosis rate among the cells treated with cisplatin in the presence or absence of exogenous ET-1 and/or ET receptor antagonists. We used OS cell line MG-63 in all experiments as a reference. Real-time quantitative RT-PCR and ELISA showed OS-III cells had greater ET-1 expression than OS-II cells at the mRNA and the secreted protein levels. Transwell(®) cell invasion assays showed OS-III cells had a greater migrated cell number than OS-II cells, which could be abrogated by ET(A) receptor antagonist BQ123 (100pmol/L), but not ET(B) receptor antagonist BQ788 (1 μmol/L); exogenous ET-1 dose-dependently promoted OS cell migration, which could be inhibited by BQ123 (100pmol/L). Cisplatin (10nmol/L) induced less apoptosis in OS-III cells than in OS-II cells; exogenous ET-1 dose-dependently promoted OS cell survival against cisplatin-induced apoptosis; both effects were reversed by BQ123 (1 μmol/L), but not BQ788 (1 μmol/L). Increased ET-1 expression appears to be associated with increased malignancy of OS. ET-1 promotes OS cell invasion and survival against cisplatin-induced apoptosis through the ET(A) receptor. The ET-1/ET(A) pathway may represent an important target for treating OS, because blocking the ET(A) receptor with a selective antagonist can inhibit OS cell invasion and potentiate a chemotherapeutic agent's effect on OS.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Effect of Ammonia Soaking Pretreatment and Enzyme Addition on Biochemical Methane Potential of Switchgrass

This article presents the biochemical methane potential (BMP) results from the anaerobic digestion (AD) of switchgrass. Triplicate BMP assays were performed on: untreated switchgrass, aqueous ammonia soaking (AAS) pretreated switchgrass (soaked in 29.5% reagent-grade aqueous ammonia at 5 L kg-1 switchgrass for 5 d), and AAS-pretreated switchgrass plus cellulytic enzymes at 12.5, 25, 62.5, and 125 filter paper units (FPU) enzyme g-1 volatile solids (VS). Biogas production and biogas methane content were measured daily in all treatments for 21 d. Both biogas and corrected methane production varied significantly among treatments, especially during the first 7 d of the BMP period. Total methane production at 21 d was corrected for enzyme degradation, and methane yields ranged from 0.15 to 0.36 m3 CH4 kg-1 VS. We compared the corrected energy yield of biogas from switchgrass to prior reports of the energy yield of ethanol from switchgrass via simultaneous saccharification and fermentation (SSF). The AD of AAS-pretreated switchgrass at the highest enzyme loading rates resulted in a 120% increase in energy extracted as compared to AAS-pretreated switchgrass converted to ethanol via SSF. Overall, the addition of enzymes to AAS-pretreated switchgrass greatly accelerated the rate of methane production over the untreated switchgrass and AAS-pretreated switchgrass without enzymes. However, the process economics are not clear, and additional work is needed to determine whether pretreating switchgrass with aqueous ammonia and/or enzymes before AD is economically advantageous.

Evaluation of AZD8931, an Equipotent Inhibitor of erbB1, erbB2, and erbB3 Receptor Signaling, on Ligand Stimulated Breast Cancer Cell Lines with Differing Levels of erbB2 Expression.

Abstract Background: Further treatment options for patients whose breast cancers do not overexpress erbB2 are required. AZD8931, an equipotent, reversible inhibitor of erbB1 (HER1, EGFR), erbB2 (HER2), and erbB3 (HER3) receptor signaling may be useful in this setting and may lead to inhibition of tumor cell proliferation, invasion, metastasis, angiogenesis and tumor cell survival.Objectives: To compare the activity of AZD8931 with other erbB inhibitors (gefitinib [G] and lapatinib [L]) in breast cancer cell lines stimulated with erbB ligands.Methods: A panel of 9 breast cancer cell lines with differing erbB2 expression levels were used: erbB2+/ER+ (BT474c; MDA-MB-361); erbB2+/ER- (MDA-MB-453; SKBR-3); erbB2-/ER+ (MCF7; T47D; ZR75-1); or erbB2-/ER- (MDA-MB-231; MDA-MB-468). Following overnight serum starvation, cells were incubated with AZD8931, G or L (0-10 µM) for 90 min and then stimulated with erbB ligands (50 ng/ml; EGF, TGFα, amphiregulin, epiregulin, betacellulin, neuregulin1, or HBEGF) for 5 min before lysis. Levels of phosphorylated erbB1, erbB2, and erbB3 were analyzed by ELISA. For IC50 determination, mean basal phosphorylation was subtracted. Geometric mean IC50s were calculated from triplicate assays and 2-sided unequal variance t-tests compared logIC50s.Results: AZD8931 demonstrated potent inhibitory activity (IC50s ≤10610 nM) when phosphorylation of erbB1, erbB2 or erbB3 receptors was driven by any erbB ligand. G demonstrated potent inhibitory activity (IC50s ≤20 nM) when the phosphorylation of erbB1 and erbB2 was driven by any erbB ligand. L more strongly inhibited the phosphorylation of erbB2 (IC50s ≤10 nM) than erbB1 (IC50s <400 nM) and showed a ligand and cell-dependant range of activities against erbB3 phosphorylation. AZD8931 was particularly differentiated from G and L in the inhibition of neuregulin1-driven erbB3 phosphorylation: IC50s were lower for AZD8931 (1-5 nM) than for G (1-120 nM) or L (20-80 nM) in the majority of the cell lines tested. Inhibition of erbB1 phosphorylation driven by EGF (IC50s ≤ vs >20 nM), TGFα (≤5 vs >20 nM), HB-EGF (≤ vs >25 nM), or betacellulin (≤6 vs 10-118 nM) also indicated more potent inhibitory activity for AZD8931 over L in cell lines that respond to ligand stimulation. No phosphorylation response to amphiregulin was seen in any of the cell lines.Conclusion: This study demonstrates that in a range of breast cell lines with varying levels of erbB2 expression, AZD8931 is a potent and balanced inhibitor of erbB1, erbB2, and erbB3 signaling. The pharmacological profile of AZD8931 is thus distinct from G and L and suggests that AZD8931 offers an agent to test the hypothesis that combined, balanced inhibition of erbB signaling could provide clinical benefit. AZD8931 may be particularly useful in the treatment of solid tumors that do not overexpress erbB2 including trastuzumab-ineligible breast cancer, an area of unmet medical need. AZD8931 is being evaluated in a Phase I clinical trial. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5059.

An In Vivo Mouse Model for Testing the Effect On Hematopoietic Stem Cell Mobilization of Bortezomib Alone and in Combination with G-CSF.

Abstract 2156Poster Board II-133 Introduction:Hematopoietic stem cell (HSC) collection for autologous transplantation is a standard of care for lymphoma and myeloma patients due to old age and extensive prior chemotherapy. However, about 25% of patients fail to collect adequate amount of HSC sufficient for a transplant. Hence, new strategies are being sought to increase HSC collection in these patients. Cell adhesion and chemotaxis are involved in retaining HSC in the marrow. The CXCR4 receptor and its specific ligand, SDF-1 are involved in retaining HSC in the marrow, hence, blocking of CXCR4 results in egress of HSC from the marrow to the peripheral blood. We and others have previously shown that the small bicyclam molecule AMD-3100, by itself can induces >a 5-fold increase in HSC mobilization to the peripheral blood and a 2-3 fold increase in blood HSC over G-CSF alone in myeloma and lymphoma patients in phase 2 and 3 studies. Recently, this drug was approved by the FDA as a mobilizing agent in combination with G-CSF under the name Mozobil. Bortezomib, a proteasome inhibitor is widely used for the treatment of MM and mantle cell lymphoma. However, in addition to blocking of the proteasome, bortezomib has also many other ensuing activities such as immune modulation, blocking of the CXCR4/SDF-1 axis and cell adhesion. Therefore, we hypothesized that Bortezomib will have similar effect as AMD3100 as a mobilizer of HSC. Mice and methods:Phase 1 was designed for optimization of HSC mobilization by Bortezomib. Mice (C57/Bl; x10/group) were injected with saline, or Bortezomib (IV,0.1 to 0.8 mg/kg). Mice were sacrificed after 1 hour,2,4,8, 16 and 24 hours and blood was collected by cardiac puncture. Red cell were lysed and white blood cells (WBS) were counted and cells used for CFU-G, CFU-M and CFU-GM colonies in a standard 14-day, methyl cellulose colony assay (Stem Cell Technologies). Cells were seeded at 200,000/plate in triplicate assay. In phase2 –Bortezomib was used in combination with G-CSF for HSC mobilization. Mice (C57/Bl; x10/group) were injected with saline, or Bortezomib (IV,0.8 mg/kg). Mice were sacrificed after 16 hours and blood was collected and analyzed as above. Two other groups of mice were injected SC with r-hG-CSF (2ug/mouse/dayx4days), or with G-CSF (x4days) followed by 16 hours of Bortezomib (IV,0.8 mg/kg). Mice from these 2 groups were sacrificed and blood was collected by cardiac puncture and processed for WBC and colony assays as above. Cells from each mouse were seeded at 100,000 cells/plate (x5 mice) and 40,000 cells/plate in triplicate assay. Results of phase1:A peak increase in total WBC (p<0.0001, vs. saline) was observed at 8 and 24 hours after Bortezomib. Similarly, significant increase of ∼3 fold and ∼4 fold was observed in the number of blood CFU-GM and BFU-E colonies/plate, respectively after 8 and 24 hours of mobilization with Bortezomib. In a similar study with fixed amount of Bortezomib (0.8mg/kg) administered for 8, 16 and 24 hours, a peak increase in peripheral blood WBC, CFU-GM and BFU-E of 3.5 fold, 4 fold and 8 fold, respectively was observed at 16 hour of Bortezomib (saline vs. Bortezomib, p<0.0001). Results of phase2:An increase of 2 fold, 4 fold and 5 fold in the mean WBC/μl, over saline was observed in the peripheral blood of mice treated with 0.8mg/kg of Bortezomib, G-CSF, or Bortezomib+ G-CSF, respectively. Similarly, an increase of 3 fold, 4.8 fold and 12.9 fold was observed in the number of blood CFU-GM colonies/plate in mice treated with Bortezomib, G-CSF, or Bortezomib+ G-CSF, respectively. Similar to CFU-GM colonies, we observed a 5.7 fold, 7.8 fold and 15 fold increase in the number of BFU-E progenitors in mice treated with Bortezomib, G-CSF, or G-CSF+ Bortezomib, respectively. One way ANOVA revealed a p value <0.0001 between the 4 groups for all comparisons. Conclusions:Bortezomib is a potent mobilizer of HSC and augment G-CSF mobilization of HSC in an additive fashion. Based on these results a pilot study has been initiated to study HSC mobilization in lymphoma and myeloma patients with the combination of bortezomib and G-CSF. Disclosures:Gazitt:millennium: Research Funding.

Phosphoglucose Isomerase/Autocrine Motility Factor Promotes Melanoma Cell Migration through ERK Activation Dependent on Autocrine Production of Interleukin-8

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Effectiveness of Peripheral Blood (PB) Molecular Monitoring by Quantitative RT-PCR (Q-PCR) in Phase II Acute Promyelocytic Leukemia (APL) Trial J0422.

Bone marrow (BM) is the accepted source for monitoring post-therapy minimal residual disease (MRD) in APL. PB is easier and less painful to obtain but variable, sometimes contradictory evidence has been presented for its efficacy in MRD monitoring. In this study, PB vs BM monitoring, as well as conventional, qualitative RT-PCR (C-PCR) vs Q-PCR were directly and prospectively compared for their effectiveness as part of an intensive MRD monitoring schedule applied as a safety measure to an investigative Phase II trial (J0422) designed to test the efficacy of minimizing chemotherapy exposure and treatment duration. This trial consisted of one cycle of induction with all-trans retinoic acid (ATRA) and daunorubicin, followed by consolidation with single-agent arsenic trioxide (ATO), followed by a maintenance phase of intermittent ATRA alone or with 6-mercaptopurine and methotrexate for patients (pts) with a presenting white blood cell count (WBC) of >10,000 WBC/uL. The MRD monitoring schedule was as follows: BM and PB after the induction and consolidation treatment modules (modules 1 & 2), then, PB every month and BM every 3 months during 2 years of maintenance therapy. C-PCR and Q-PCR were performed according to published procedures for monitoring the APL-specific fusion gene PML-RARα by the BIOMED-1 Concerted Action and the North American Cooperative Oncology Groups, respectively. Criteria for positive assays were: C-PCR, confirmed visualization of an appropriate-sized gel band after conventional, double-nested PCR amplification; Q-PCR, demonstration of a CT value <40 cycles by real-time PCR amplification in ≥2 of triplicate assays. Positive dilution controls to document detection with approximately 1 in 104 sensitivity were included in all experiments. Test samples were available from 26 patients with 3 to 38 months (mo) post-module-2 follow-up (median, 16 mo). After module-1 induction, 4 pts were positive by C-PCR (3 BM-only; 1 PB-only) and 9 pts were positive by Q-PCR (7 BM & PB, 2 PB-only). All 4 C-PCR and 4 Q-PCR positives occurred in pts with WBC <1,000 (6 pts), while 0 C-PCR and 2 Q-PCR positives occurred in pts with WBC >10,000 (7 pts), suggesting an association of pretreatment WBC with a difference in the initial dynamics of treatment response. After module-2 consolidation, 0 pts were positive by C-PCR and 3 by Q-PCR (1 BM & PB, 1 BM-only, 1 PB-only). During maintenance at the 3 mo shared BM/PB checkpoints, 0 C-PCR and 5/114 Q-PCR assays were positive (2 BM & PB, 1 BM-only, 2 PB-only), distributed among 3 pts. At monthly PB-only checkpoints, an additional 12/252 assays were positive by Q-PCR, none in a progressive pattern suggestive of impending molecular relapse. Overall, 1 – 3 Q-PCR assays were positive in 9 patients during the maintenance and follow-up periods. Among 8 pts who completed 24 mo maintenance, only 1/6 positive Q-PCR assays occurred at >12 mo, suggesting continued reduction of MRD during first 12 mo of maintenance therapy. No C-PCR assays were positive beyond module-1. In 1 exceptional pt, excluded from the above maintenance analysis, the Q-PCR assays became recurrently positive in BM and/or PB after 6 mo maintenance at a level below the criterion for molecular relapse (normalized quotient relative to the housekeeping gene GAPDH, NQGAPDH, ≥10−5). After 18 mo, the C-PCR became repeatedly positive in PB but not BM with Q-PCRs positive (2 BM & PB, 1 PB-only at shared checkpoints) in the NQGAPDH 4×10−7 to 4×10−6 range, which was associated with relapse in the central nervous system but not the BM. These results indicate that molecular monitoring of PB or BM was equally effective in detecting MRD and that Q-PCR was a more critical measure of MRD than C-PCR on protocol J0422 after single-cycle ATO-based consolidation therapy. The results further suggest that PB monitoring may be more effective in detecting extramedullary relapse, a relatively increasing cause of disease relapse with improved overall therapy for APL.

MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27Kip1

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHT(R))) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHT(R) cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27(Kip1), a known target of miR-221/222, was reduced by 50% in OHT(R) cells and by 28-50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27(Kip1) in the resistant OHT(R) cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.

Interaction of daptomycin with two recombinant thromboplastin reagents leads to falsely prolonged patient prothrombin time/International Normalized Ratio results

A cluster of patients experiencing elevations of International Normalized Ratio without clinical bleeding in temporal association with daptomycin therapy was identified during postmarketing safety surveillance. A common element was the thromboplastin reagent used for the laboratory assay. The present study evaluated the effect of daptomycin on measured prothrombin time using commercially available thromboplastin reagent kits commonly used in the United States. Thirty reagent kits were obtained. Daptomycin was added to pooled normal human plasma samples to achieve final concentrations of 0-200 microg/ml. Quality control ranges were established for each reagent kit using normal and abnormal control plasmas. Triplicate assays of the prothrombin time were performed on the daptomycin-spiked plasma samples using each of the 30 kits. The activated partial thromboplastin time and thrombin time were also assessed. Statistical comparisons of interest were performed using analysis of variance with the Bonferroni t-test for multiple comparisons; alpha = 0.05 was used. Addition of daptomycin to human plasma samples dose-dependently prolonged measured prothrombin times when two recombinant thromboplastin reagents were utilized. The findings were statistically and clinically significant. No clinically meaningful effect was observed with the other reagents. The activated partial thromboplastin time and thrombin time were not affected. Prolonged International Normalized Ratio patient values were an artifact caused by the interaction of daptomycin with the in-vitro prothrombin time test reagent; an in-vivo anticoagulant effect was not observed. Healthcare providers should consider a possible drug-laboratory test interaction if prolonged prothrombin time or elevated International Normalized Ratio values are observed in patients receiving daptomycin.

Digestive enzyme activity of the red porgy (Pagrus pagrus, L.) during larval development under culture conditions

The ontogenic development of the main digestive enzymes (proteases, amylase and lipase) in the red porgy, Pagrus pagrus, larvae was assayed during the larval development. The green water technique was carried out for larval rearing and whole-body homogenates were used for enzymatic assays in triplicate. Significant alterations in specific activities of all digestive enzymes measured during the period of this study were mostly related to metamorphosis and weaning. Trypsin- and chymotrypsin-specific activities were first detected on day 3, together with opening of the mouth, and slightly increased until 25 days after hatching (DAH). After this period, the specific activities of these enzymes slightly decreased. Pepsin was first detected on day 28, concurrent with stomach formation, and a sharp increase was observed until 30 DAH. A slight decrease was measured from this date until the end of the experiment. Both amylase and lipase were measured for the first time on days 2 and 4 respectively, and the specific activities of these enzymes showed similar patterns during the first week of the study. Then, slight variations were observed until 30 DAH and while lipase-specific activity declined, an increase in the specific activity of amylase was found until the end of the experiment. It is concluded that the variations observed in the specific activity of digestive enzymes were related to either metamorphosis, such as the formation of the stomach (28 DAH), or to changes in food composition. The profile of the developmental pattern of the main digestive enzymes detected in P. pagrus is similar to that described for other Sparid species.

NOD2 Transgenic Mice Exhibit Enhanced MDP-Mediated Down-Regulation of TLR2 Responses and Resistance to Colitis Induction

Mutations in the CARD15 gene encoding NOD2 are susceptibility factors in Crohn's disease. We explored the mechanism of this susceptibility using mice that over express NOD2. Cellular and molecular responses of mice bearing an NOD2 transgene or administered plasmids that express wild-type and mutated NOD2 constructs were examined. In initial studies, we showed that splenocytes from NOD2 transgenic mice as compared with littermate controls exhibit decreased interleukin (IL)-12p70 responses to peptidoglycan (PGN), a TLR2 ligand that contains muramyl dipeptide, but not other TLR ligands; in contrast, IL-12 responses to PAM(3)CSK(4), a TLR2 ligand that does not contain muramyl dipeptide, were normal. Similarly, transgenic mice as compared with controls exhibited greatly decreased IL-12p40 responses to intraperitoneal administration of PGN but not to lipopolysaccharide. In further studies, we showed using electrophoretic mobility shift assay that PGN-stimulated cells from transgenic mice exhibited decreased activation of nuclear factor kappaB. Finally, in a series of studies on the effect of the NOD2 on susceptibility to induced colitis, we found that (1) transgenic mice were highly resistant to induction of PGN colitis and partially resistant to induction of trinitrobenzene sulfonic acid (TNBS) colitis and (2) mice administered a plasmid expressing a wild-type NOD2 gene were completely resistant to TNBS colitis whereas mice administered a plasmid expressing an NOD2 gene with the Crohn's disease frameshift mutation were only slightly resistant to TNBS colitis. These data offer new evidence that NOD2 mutations contribute to inflammatory bowel disease by causing excessive TLR2 cytokine responses.

MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.

Post‐pneumonectomy (PNX) lung expansion enhances expression of erythropoietin receptor (EPO‐R) through an Akt‐associated pathway

To examine if the elevated EPO-R protein level post-PNX (AJP 287:, 2004) is related to lung expansion, we replaced the right lung in litter-matched adult foxhounds (22.2±4.0kg) by a custom-shaped silicone prosthesis inflated with SF6/air to prevent mediastinal shift and minimize expansion of the remaining lung. Normal (pre-PNX) lung tissue was sampled from the removed right upper lobe. Three wk post-PNX, the prosthesis was either acutely deflated (DEF, n=3) to impose lung expansion separate from other post-PNX stimuli, or kept inflated (INF, n=3). Three days later, lung tissue was sampled from the remaining left upper lobe and analyzed by real-time PCR (EPO-R mRNA) and immunoblots (EPO-R, total and phospho-Akt) normalized to cyclophilin and β-actin, respectively. Triplicate assays used separate tissue samples. Results are shown as ratios to pre-PNX (±SD). ∗p<0.05 vs. 1.0, paired t-test. In the INF group, no significant post-PNX change was seen. In the DEF group, EPO-R mRNA and protein were 183% and 71% higher and p-Akt/total-Akt 124% higher than pre-PNX. Results suggest that post-PNX lung expansion enhances EPO-R expression through an Akt-associated pathway. Supported by NHLBI RO1 HL40070, 62873, 45716, 54060 and R01-DK48482, DK-20543 and the Simmons Family Foundation.

Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

Ontogenic development of the digestive enzymes in common pandora, Pagellus erythrinus, L. larvae

Ontogenic development of some digestive enzymes (proteases, amylase and lipase) in common pandora Pagellus erythrinus larva was assayed during larval development. The green-water technique was employed for larval rearing, and whole-body homogenates were used for enzymatic assays in triplicate. Important alterations in specific activities of all digestive enzymes measured during the period of this study were mostly related to metamorphosis and weaning. Mouth opening was observed on day 3 at 2.23±0.01 mm total length synchronously with the first determination of trypsin and chymotrypsin activities. After this date, the specific activities of these slightly increased until 25 days after hatching (DAH), respectively, and then slightly decreased and changed. The pattern determined for pepsin was strongly related to stomach formation on day 25 at 9.72±2.3 mm total length and a sharp increase was found until 30 DAH and then a slight decrease was measured from this date until the end of the experiment. Both amylase and lipase were measured for the first time on days 2 and 4, respectively, and also the specific activities of these showed similar patterns during the first week of the study. Then, slight variations were observed until 30 DAH and while lipase-specific activity had declined, an increase in the specific activity of amylase was found until the end of the experiment. Finally, it is thought that the variations observed in the specific activity in the profile of digestive enzymes were related to either metamorphosis such as formation of stomach (25 DAH) or to changes in characteristics of food (30 DAH). The pattern of development of the main digestive enzymes found in P. erythrinus is similar to that described in other Sparid species.

Cost efficiency in the detection of chytridiomycosis using PCR assay

Chytridiomycosis is an emerging infectious disease of amphibians associated with mass mortalities and population declines worldwide. Recent technological advances have resulted in a highly sensitive, non-invasive technique for diagnosing the disease based on a quantitative (real-time) polymerase chain reaction (qPCR) assay. The qPCR assay yields the most accurate and informative data of any available detection technique. However, due to the relatively high costs involved, it has yet to attain widespread use by chytridiomycosis researchers. Using the results of a disease survey of 467 wild frogs from eastern Queensland, Australia, we examine the necessity of triplicate assays in qPCR detection of chytridiomycosis. We describe a singlicate qPCR assay that can be used to substantially decrease costs, with no significant decrease in sensitivity. We also demonstrate that detection of chytridiomycosis by use of the conventional PCR assay may lead to appreciable underestimations in disease prevalence. We recommend that amphibian disease researchers adopt the singlicate qPCR assay as the primary means of chytridiomycosis detection.

Probing Protein Ligand Interactions by Automated Hydrogen/Deuterium Exchange Mass Spectrometry

Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARgamma induced upon binding a full agonist and a partial agonist. PPARgamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARgamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARgamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARgamma/RXRalpha LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.

Tumor Suppressor in Lung Cancer (TSLC)1 Suppresses Epithelial Cell Scattering and Tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.

Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor

The analysis of carbaryl in natural water samples was accomplished using a portable immunosensor based on surface plasmon resonance (SPR) technology. The assay was based on a binding inhibition immunoassay format with the analyte derivative covalently immobilized on the sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed onto the gold-coated sensor surface to allow the reusability of the same sensing surface during 220 regeneration cycles. Reproducibility was evaluated by performing three independent assays in triplicate on 3 different days. The batch-assay variability was also calculated using three different gold-coated sensor surfaces. The intra- and inter-day relative standard deviation were 8.6 and 15.3%, respectively, whilst a variation of 7.4% in assay sensitivity was obtained by employing different sensor chips. The lowest detection limit, calculated as the concentration providing a 10% decrease of the blank signal, was of 1.38 microg L(-1). Matrix effects were also evaluated in different water types, showing I50 values (carbaryl concentrations that produced a 50% decrease of the blank signal) within the range of carbaryl standard curves in distilled water (2.78-3.55 microg L(-1)). The carbaryl immunoassay performance was validated with respect to conventional high-performance liquid chromatography-mass spectrometry (HPLC-MS). The correlation between methods was in good agreement (r2 = 0.998, 0.999 and 0.999) for the three types of natural water samples tested. A complete assay cycle, including regeneration, is accomplished in 20 min. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company SENSIA, SL (Spain). The small size and low-time of response of the beta-SPR platform would allow its utilization in real contaminated locations.