Sensitivity and response time of three common Antarctic marine copepods to metal exposure.

Understanding the sensitivity of Antarctic marine organisms to metals is essential in order to manage environmental contamination risks. To date toxicity studies conducted on Antarctic marine species are limited. This study is the first to examine the acute effects of copper and cadmium on three common coastal Antarctic copepods: the calanoids Paralabidocera antarctica and Stephos longipes, and the cyclopoid Oncaea curvata. These copepods responded slowly to metal exposure (4-7d) emphasising that the exposure period of 48-96 h commonly used in toxicity tests with temperate and tropical species is not appropriate for polar organisms. We found that a longer 7 d exposure period was the minimum duration appropriate for Antarctic copepods. Although sensitivity to metal exposure varied between species, copper was more toxic than cadmium in all three species. P.antarctica was the most sensitive with 7d LC50 values for copper and cadmium of 20 µg L(-1) and 237 µg L(-1) respectively. Sensitivities to copper were similar for both O. curvata (LC50=64 µg L(-1)) and S. longipes (LC50=56 µg L(-1)), while O. curvata was more sensitive to cadmium (LC50=901 µg L(-1)) than S. longipes (LC50=1250 µg L(-1)). In comparison to copepods from lower latitudes, Antarctic copepods were more sensitive to copper and of similar sensitivity or less sensitive to cadmium. This study highlights the need for longer exposure periods in toxicity tests with slow responding Antarctic biota in order to generate relevant sensitivity data for inclusion in site-specific environmental quality guidelines for Antarctica.

A TIR domain protein from E. faecalis attenuates MyD88-mediated signaling and NF-κB activation.

Toll-like receptor signaling, mediated by functional Toll/interleukin-1 receptor (TIR) domains, plays a critical role in activating the innate immune response responsible for controlling and clearing infection. Bacterial protein mimics of components of this signaling pathway have been identified and function through inhibition of interactions between Toll-like receptors (TLRs) and their adaptor proteins, mediated by TIR domains. A previously uncharacterized gene, which we have named tcpF (for TIR domain-containing protein in E. faecalis) was identified in the genome of Enterococcus faecalis V583, and predicted to encode a protein resembling mammalian and bacterial TIR proteins. We overexpressed and purified TcpF from E. coli and found that the recombinant protein could bind to phosphatidylinositol phosphates in vitro, suggesting a mechanism by which TcpF may be anchored to the plasma membrane in close proximity to TIR domains of TLRs and adaptor proteins. Purified TcpF was also found to interact specifically with the TIR adaptor protein MyD88, and this interaction was dependent on the BB loop domain in the Box 2 region of TcpF. Despite no evidence of TcpF being a secreted protein, recombinant TcpF was effectively able to enter RAW264.7 cells in vitro although the mechanism by which this occurs remains to be determined. Overexpression of TcpF in mammalian cells suppressed the NF-κB activation induced by bacterial lipoteichoic acid. A mutant lacking the tcpF gene was attenuated for survival in macrophages, with increased ability to activate NF-κB compared to the wild type strain. Complementation in trans restored growth, and inhibition of NF-κB, to that of wild type levels. No appreciable difference in bacterial persistence, dissemination or pathogenesis was observed between the wild type and mutant in a mouse peritonitis model however, which suggested either a subtle role for TcpF or functional overlap with other redundant factor(s) in this virulence model.

Temporal changes in the sensitivity of coastal Antarctic zooplankton communities to diesel fuel: a comparison between single- and multi-species toxicity tests.

Despite increasing human activity and risk of fuel spills in Antarctica, little is known about the impact of fuel on Antarctic marine fauna. The authors performed both single- and multi-species (whole community) acute toxicity tests to assess the sensitivity of an Antarctic coastal zooplankton community to the water-accommodated fraction of Special Antarctic Blend diesel. Single-species tests using abundant copepods Oncaea curvata, Oithona similis, and Stephos longipes allowed comparisons of sensitivity of key taxa and of sensitivity estimates obtained from traditional single-species and more novel multi-species tests. Special Antarctic Blend diesel caused significant mortality and species compositional change in the zooplankton community within 4 d to 7 d. The sensitivity of the community also increased across the summer sampling period, with decreasing 7-d median lethal concentration (LC50) values for total petroleum hydrocarbons (TPH): 1091 µg TPH/L in early January 2011, 353 µg TPH/L in mid January 2011, and 186 µg TPH/L in early February 2011. Copepods showed similar sensitivities to Special Antarctic Blend diesel in single-species tests (7-d LC50s: O. curvata, 158 µg TPH/L; O. similis, 176 µg TPH/L; S. longipes, 188 µg TPH/L). The combined use of single- and multi-species toxicity tests is a holistic approach to assessing the sensitivity of key species and the interactions and interdependence between species, enabling a broader understanding of the effects of fuel exposure on the whole zooplankton community.

Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of αvß6 integrin in DCIS identifies high-risk patients and predicts recurrence.

PURPOSE: This study investigated the functional and clinical significance of integrin αvß6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS). EXPERIMENTAL DESIGN: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvß6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvß6 expression, were used to measure the effect of αvß6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFß signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography. RESULTS: Expression of αvß6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvß6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvß6-positive myoepithelial cells is dependent on TGFß-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway. CONCLUSION: These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvß6 on myoepithelial cells generates a tumor promoter function through TGFß upregulation of MMP-9. These data suggest that expression of αvß6 may be used to stratify patients with DCIS.

Laser phosphoroscope and applications to room-temperature phosphorescence.

A simple phosphoroscope with no moving parts is described. In one scan the total luminescence, the long-lived phosphorescence, and the short-lived fluorescence can be determined. A 50% duty cycle excitation from a diode laser is used to excite the sample, and from the digitized waveform the phosphorescence is extracted from the off period, the total emission from the full cycle, and the fluorescence from the on period corrected for the phosphorescence contribution. The performance of the system is demonstrated using room-temperature phosphorescence of organic dyes in boric acid glasses, a multi-emissive boron-polymer dye, and a europium chelate.

Outcomes in unresectable and locally advanced resected cholangiocarcinoma.

Patients with cholangiocarcinomas often present with unresectable disease, which is associated with a poor clinical outcome and survival. A number of palliative options are available to patients; the evaluated article presented experience from a single institution of treating cholangiocarcinoma, either unresectable or locally advanced, with conformal radiotherapy and concurrent chemotherapy. Patients who had received biliary radiation for cholangiocarcinoma were identified from the hospital database, and information on the patients sourced from notes and reports. In total, 20 patients with a diagnosis of biliary tract cancer were included and received radical conformal radiotherapy with concurrent cisplatin/5-fluorouracil and sequential gemcitabine. The median overall survival was 20.4 months and the relapse-free survival was 9.6 months. Treatment failure within the radiotherapy field was recorded in 45% of patients; adverse events were minimal. This study adds to the retrospective data available regarding the management of patients with biliary tract carcinomas, and we have found in our own cohort of 45 patients that gemcitabine/platinum was a more effective combination than monotherapy.

Influence of the tumor microenvironment on angiogenesis.

It is becoming increasingly recognized that the host microenvironment is essential for regulating tumor cell behavior. The cellular stromal compartment can modulate angiogenesis either directly through enhanced secretion of pro-angiogenic factors or reduced secretion of antiangiogenic factors, or indirectly by modulating the surrounding extracellular matrix. Control of angiogenesis represents a critical step in cancer progression and is a potential therapeutic target. This article focuses on the role of the tumor microenvironment in the control of angiogenesis and how dissection of the molecular interactions may enhance prognostic and predictive power and facilitate therapeutic targeting.

Luminescence oxygen sensor based on a ruthenium(II) star polymer complex.

A novel quenchometric oxygen sensor based on a low polydispersity (PDI) star polymer [Ru(bpyPS(2))(3)](PF(6))(2) (bpy = 2,2'-bipyridine, PS = polystyrene) is reported. The synthesis, characterization, photophysics, and oxygen sensing properties are examined. Combining the polystyrene support with the oxygen sensing ruthenium complex provides much higher doping levels without microcrystallization of the complex than traditional two-component sensors. The single molecule approach also avoids sensor leaching. While the polydispersity was 1.10, indicating a very tight distribution of molecular weights, sensor heterogeneity was not completely eliminated, as the luminescence decays were still multiexponentials. The likely source of this heterogeneity and possible methods for generating more homogeneous materials are discussed.

Abnormal behavior in Stern-Volmer luminescence quenching measurements via apparent lifetime methods.

Luminescence lifetimes are widely used as an analysis tool. Since decays in analytical systems are frequently complex decays rather than single exponentials, apparent lifetime methods based on the rapid lifetime determination (RLD) method or single frequency phase shift (SFPS) measurements are frequently used to reduce cost and simplify data analysis. It is demonstrated here that these methods can produce large errors under the right conditions. Both methods can give unexpected and uncharacteristic Stern-Volmer quenching plots (SVQPs) in two-component systems. Behaviors include bimodal quenching curves as well as "anti-quenching" curves. These phenomena are exacerbated by small fractions of long unquenched components.

Ruthenium tris(bipyridine) complexes with sulfur substituents: model studies for PEG coupling.

Ruthenium polypyridyl complexes are incorporated into polymers for sensing and light emitting materials applications. Coupling reactions between metal complexes and polymers are one route to polymeric metal complexes. In an effort to increase conjugation efficiency, tune materials properties, and introduce a responsive crosslink, ruthenium tris(bipyridine) derivatives with sulfur substituents were synthesized and compared to oxygen analogues. Difunctional thiols, thioesters, thioethers, and disulfides, as well as hexafunctional nonpolymeric model systems, were explored. Upon exposure to oxygen, the thiol derivative was readily oxidized. These studies guided Ru(bpy)3 PEG coupling reactions with disulfide and thioether linkages, which proceeded to approximately 80% and approximately 60% yield, respectively. The luminescence properties of the Ru PEG derivatives and model systems were investigated. The emission spectra and lifetimes for all complexes in CH3CN under an inert atmosphere are comparable to [Ru(bpy)3]Cl2. Lifetime data for nonpolymeric analogues fit to a single exponential decay indicating heterogeneity, suggesting sample homogeneity, whereas data for polymers fit to a multiexponential decay. In contrast to certain [Ru(bpy)3](2+)/thiol mixtures, no intramolecular quenching by the sulfide is observed for [Ru(bpy)2{bpy(CH2SH)2}](PF6)2. Emission spectra red shift and multiexponential decay are noted for the oxidized Ru thiol product. The rates of oxygen quenching are slower for Ru PEG derivatives than those for nonpolymeric analogues, which may be attributed to shielding effects of the polymer chain.

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) for gene delivery.

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) (Ru PEI) was synthesized via acid hydrolysis of Ru tris(bipyridine)-centered poly(2-ethyl-2-oxazoline) (Ru PEOX), and the luminescence, DNA entrapment, and transfection efficiencies were evaluated. Emission maxima for Ru PEI samples are red-shifted compared to Ru PEOX precursors, and the luminescence lifetimes are shorter in both methanol and aqueous solutions. Slower oxygen quenching of Ru PEOX and Ru PEI luminescence versus [Ru(bpy)3]Cl2 (bpy = bipyridine) is attributed to polymer shielding effects. Ru PEI luminescence is similar in the presence and absence of DNA. Ru PEI (7900 Da) and linear PEI (L-PEI; 22,000 Da) fully entrapped DNA (5.4 kb; pcDNA) at an N/P ratio of 2. LNCaP prostate cancer cells were transfected with a plasmid encoding for green fluorescent protein using Ru PEI and L-PEI vectors for comparison. For N/P = 48, the transfection efficiency for Ru PEI was approximately 50% relative to that of L-PEI.

Bradykinin receptors as a therapeutic target.

Biologically-active kinins, including bradykinin (BK) and Lys(0)-BK (kallidin), are short-lived peptide mediators predominantly generated by the enzymatic action of kallikreins on kininogen precursors. A diverse spectrum of physiological and pathological actions attributed to local kinin production is a consequence of the activation of G-protein-coupled receptors (GPCRs). Currently, two major subtypes of kinin receptor, designated B(1) and B(2), are recognised, although there is much evidence for pharmacological heterogeneity, particularly within the B(2) receptors. Considering these facts and the widespread distribution of kinin receptors in many human tissues, it is no surprise that the therapeutic potential of kinins and kinin receptor antagonists remains the focus of numerous investigations. Studies in animals and animal tissues, instrumental in elucidating the biological roles of kinins, are well-documented in numerous excellent reviews. Unfortunately, and despite the enormous potential illustrated by animal studies, attempts to develop kinin analogues as therapeutic agents to combat human disease have largely proven disappointing. Consequently, this review selectively focuses upon studies that are directly relevant to the targeting of human BK receptors as a therapeutic intervention. In addition to providing a succinct review of well-documented pathological conditions to which kinin receptors contribute, the authors have also included more recent data that illustrate new avenues for the therapeutic application of kinin analogues.

Cold storage of rabbit thoracic aorta in University of Wisconsin solution attenuates P2Y(2) purine receptors.

Post-transplantation thrombosis may occur in donor segments of iliac arteries and livers following surgical removal and storage in University of Wisconsin (UW) solution for transplantation. We have previously suggested that purine receptors are vulnerable to denaturation after UW storage. The aims of the present study were to determine what particular subtypes of purine P2Y receptors in rabbit thoracic aorta deteriorate after 8 days of UW storage by studying vascular reactivity to acetylcholine, ATP, 2MeSATP and UTP. Ring segments of aortae from male New Zealand White rabbits were mounted upon fine-wire myographs and vasodilatation to the above agents tested on fresh tissue, and after 8 days of UW storage. Vasodilatation to ATP was attenuated by 100 microM L-NAME in fresh tissue suggesting that the relaxant response was, in part, due to nitric oxide (NO). P2Y-mediated relaxation to ATP was significantly attenuated by UW storage and cholinergic responses were not. This attenuated relaxation to ATP was not further attenuated by L-NAME, suggesting a loss of the NO-dependent mechanism. De-endothelialisation indicated that UTP-mediated vasorelaxation, via P2Y(2) receptors, was endothelium-dependent. Any residual endothelium-independent relaxation to UTP was abolished by UW storage and endothelium-dependent UTP relaxation was reduced to the same level as that seen in fresh, de-endothelialised tissue. In contrast responses to 2MeSATP, via P2Y(1) receptors, were predominantly endothelium-independent and were only partially attenuated by UW storage. Responses to pyridoxalphosphate-6-azophenyl-2('),4(')-disulphonic acid (PPADS) and L-NAME suggested that vasorelaxation to 2MeSATP and UTP was mediated by P2Y(1) and P2Y(2) receptors, respectively. It is therefore concluded that UW storage predominantly decreases P2Y(2) receptor-mediated vascular reactivity.