Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5

Assessment of a Siloxane Poly(Urethane‐Urea) Elastomer Designed for Implantable Heart Valve Leaflets

Synthetic polymer leaflets in prosthetic cardiac valves hold the potential to reduce calcification and thrombus, while improving blood flow, durability, and device economics. A recently developed siloxane poly(urethane‐urea) (LifePolymer™, LP) exhibits properties essential for heart valve leaflets, including low dynamic modulus, high tensile strength, minimal creep, and excellent biostability. LP properties result from carefully designed “linked co‐macrodiol” chemistry that maximizes silicone content and virtual crosslinks between soft and hard phases. Characterization of multiple commercial batches demonstrates a robust synthesis process with minimal variation. Extensive ISO 10993‐based biocompatibility testing resulted in no observable toxicity or other adverse reactions. An ex vivo AV shunt thrombogenicity investigation revealed nearly undetectable levels of platelet attachment and thrombus formation on LP surfaces. Chronic ovine implantation of prototype heart valves with LP leaflets showed no differences in thrombogenicity or systemic tissue response when compared to a clinically standard tissue‐based valve. Toxicological risk assessment, based on extractables and leachables analysis of LP‐based heart valves, confirmed minimal toxicological risk. Lastly, 24‐week, strain‐accelerated in vivo LP biostability testing confirmed previous favorable in vitro biostability findings. These studies demonstrate that this newly developed elastomer exhibits ideal biomaterial properties for the flexible leaflets of a totally synthetic heart valve replacement

UCS protein function is partially restored in the Saccharomyces cerevisiae she4 mutant with expression of the human UNC45-GC, but not UNC45-SM

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90β isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90β. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90β in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved-yeast to man-in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking-events also facilitated by the myosins in yeast

Mechanisms of resistance to Hsp90 inhibitor drugs: a complex mosaic emerges

The molecular chaperone Hsp90 holds great promise as a cancer drug target, despite some of the initial clinical trials of Hsp90 inhibitor drugs having not lived up to expectation. Effective use of these drugs will benefit greatly from a much more detailed understanding of the factors that contribute to resistance, whether intrinsic or acquired. We review how cell culture studies have revealed a number of different mechanisms whereby cells can be rendered less susceptible to the effects of Hsp90 inhibitor treatment. A major influence is Hsp90 inhibition causing strong induction of the heat shock response, a stress response that increases cellular levels of prosurvival chaperones such as Hsp27 and Hsp70. Another problem seems to be that these inhibitors do not always access the Hsp90 proteins of the mitochondrion, forms of Hsp90 that-in cancer cells-are operating to suppress apoptosis. It should be possible to overcome these drawbacks through the appropriate drug redesign or with the combinatorial use of an Hsp90 inhibitor with a drug that targets either heat shock factor or the chaperone Hsp70. Still though, cells will often differ in the key antiapoptotic versus proapoptotic activities that are dependent on Hsp90, in the key steps in their apoptotic pathways responsive to Hsp90 inhibition or Hsp70 level, as well as the extents to which their survival is dependent on oncogenic tyrosine kinases that are clients of Hsp90. A systems approach will therefore often be required in order to establish the most prominent effects of Hsp90 inhibition in each type of cancer cell. © 2011 by the authors; licensee MDPI, Basel, Switzerland

Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer

BACKGROUND: The clinical benefit of determining the status of HER-2/neu amplification in breast cancer patients is well accepted. Although immunohistochemistry (IHC) is the most frequently used method to assess the over-expression of HER-2 protein, fluorescent in-situ hybridization (FISH) is recognized as the "gold standard" for the determining of HER-2/neu status. The greatest discordance between the two methods occurs among breast tumors that receive an indeterminate IHC score of 2+. More recently, a real-time polymerase chain reaction (PCR) assay using the LightCycler(® )has been developed for quantifying HER-2/neu gene amplification. In this study, we evaluated the sensitivity and specificity of a commercially available LightCycler assay as it compares to FISH. To determine whether this assay provides an accurate alternative for the determination of HER-2/neu status, we focused primarily on tumors that were deemed indeterminate or borderline status by IHC. METHODS: Thirty-nine breast tumors receiving an IHC score of 2+ were evaluated by both FISH and LightCycler(® )technologies in order to determine whether quantitative real-time PCR provides an accurate alternative for the determination of HER-2/neu status. RESULTS: We found a high concordance (92%) between FISH and real-time PCR results. We also observed that 10% of these tumors were positive for gene amplification by both FISH and real-time PCR. CONCLUSION: The data show that the results obtained for the gene amplification of HER-2/neu by real-time PCR on the LightCycler(® )instrument is comparable to results obtained by FISH. These results therefore suggest that real-time PCR analysis, using the LightCycler(®), is a viable alternative to FISH for reassessing breast tumors which receive an IHC score of 2+, and that a combined IHC and real-time PCR approach for the determination of HER-2 status in breast cancer patients may be an effective and efficient strategy

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Palaeomagnetism of the Mahatta Humaid Group (Cambrian – Early Ordovician, Oman), including a re-interpretation of previous Neoproterozoic palaeomagnetic data

We carried out a palaeomagnetic study in the Al-Huqf region (Sultanate of Oman) on rocks that belong to different units of the Cambrian – Ordovician Mahatta Humaid Group. Thirty-nine samples were systematically collected from a succession ca. 520–495 Ma old. Seventeen samples showed characteristic remanent magnetization components with two antipodal polarities carried by hematite. Evidence suggests that these components have a primary origin. A detailed petrographic analysis revealed syntaxial overgrowths parallel to the easy plane of magnetization of the magnetic carriers that has probably enhanced and reinforced the primary magnetization. A palaeopole computed with the mean direction of the 17 characteristic remanent magnetization components was considered alongside previously published Neoproterozoic – early Palaeozoic palaeomagnetic data, which we placed in an updated chronostratigraphic framework for the Neoproterozoic – Cambrian Huqf Supergroup. Two interpretations were considered: (1) Oman was detached from the Arabian-Nubian craton until ca. 660 Ma, and it became attached (or was nearby) to it by ca. 630 Ma. In this interpretation, an apparent polar wander (APW) path of Arabia is proposed between ca. 630 and 500 Ma. The palaeomagnetic directions of Mirbat obtained by Killner et al. (2005) in rocks 720–660 Ma old are therefore assumed as primary, and taking into account that Oman was an independent block of the Arabian-Nubian craton, the corresponding palaeopole is not considered in the proposed segment of the Arabian APW path. (2) The Neoproterozoic data belong to two different tectonic blocks within the Arabian-Nubian craton and were involved in left-lateral, strikeslip movements along NW-trending faults. One block included the localities of Al Jabal al-Akhdar and Al-Huqf and may have rotated counter-clockwise ca. 45° about a vertical axis between ca. 600 and 500 Ma. The other block included the locality of Mirbat and rotated counter-clockwise ca. 25° about a vertical axis between ca. 600 and 550 Ma. These suggested block rotations may have played a role in generating the underlying fabrics for some of the sedimentary basins in Oman. In the second model, the rocks sampled by Killner et al. (2005) in Mirbat were re-magnetized during the intrusion of dike swarms at ca. 615–600 Ma.Fil: Vizan, Haroldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; ArgentinaFil: Turner, Peter. No especifica;Fil: Millson, John. No especifica;Fil: Ixer, Rob. No especifica

A simple yeast-based system for analyzing inhibitor resistance in the human cancer drug targets Hsp90alpha/beta.

Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, is one of the most promising targets for cancer drug development. Whether any resistance to these Hsp90 inhibitor drugs could arise by Hsp90 mutation is still unknown. Yeast is readily engineered so that its essential Hsp90 function is provided by either isoform of the human cytosolic Hsp90, Hsp90alpha or Hsp90beta. However, its high intrinsic resistance to most drugs poses a major obstacle to the use of such Hsp90alpha- or Hsp90beta-expressing yeast cells as a model system to analyse whether drug resistance might arise by Hsp90 mutation. In order to overcome this problem, we have generated a strain that is both hypersensitive to Hsp90 inhibitors as it lacks multiple drug resistance genes, and in which different heterologous and mutant Hsp90s can be expressed by plasmid exchange. It is not rendered appreciably stress sensitive when made to express Hsp90alpha or Hsp90beta as its sole form of Hsp90. Should there be any development of resistance to the Hsp90 drugs now in cancer clinic trials, this system can provide a rapid initial test of whether any single nucleotide polymorphism appearing within the coding regions of Hsp90alpha or Hsp90beta could be a contributory factor in this resistance. We have used this strain to demonstrate that significant levels of resistance to the Hsp90 inhibitors radicicol and 17-allylamino-demethoxygeldanamycin (17-AAG) are generated as a result of the same single point mutation within the native Hsp90 of yeast (A107N), the human Hsp90alpha (A121N) and the human Hsp90beta (A116N)