Evidence of Antineutrinos from Distant Reactors Using Pure Water at SNO.

The SNO+ Collaboration reports the first evidence of reactor antineutrinos in a Cherenkov detector. The nearest nuclear reactors are located 240 km away in Ontario, Canada. This analysis uses events with energies lower than in any previous analysis with a large water Cherenkov detector. Two analytical methods are used to distinguish reactor antineutrinos from background events in 190 days of data and yield consistent evidence for antineutrinos with a combined significance of 3.5σ.

A paediatric ECMO case of plasma leakage through a polymethylpentene oxygenator.

Polymethylpentene (PMP) oxygenators, utilised for ECMO, are commonly believed to be resistant to plasma leakage. Whilst uncommon, plasma leakage has been previously reported with PMP fibres, both in vivo and in vitro. We describe a paediatric ECMO case during which plasma leakage occurred and oxygenator function gradually deteriorated, ultimately necessitating device replacement. To our knowledge, this is the first case of plasma leakage described using a PMP device during paediatric ECMO. Subsequent investigation is described, demonstrating that a protein coating reduces the free passage of solution across the PMP membrane.

Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods.

Animal-bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host-associated bacteria might establish tissue-specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue-specific Wolbachia-microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain-specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co-evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia-infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare populations.

Structural and oxidation state changes of the photosystem II manganese complex in four transitions of the water oxidation cycle (S0 --> S1, S1 --> S2, S2 --> S3, and S3,4 --> S0) characterized by X-ray absorption spectroscopy at 20 K and room temperature.

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.

Importance of ets1 proto-oncogene for breast cancer progression.

Ets proteins are transcription factors, which share a unique DNA binding domain, the Ets domain. Some members of the Ets family are implicated in tumorigenesis. Ets1, the founder of the Ets family, is predominantly expressed in invasive tumors and able to activate certain genes encoding ECM-degrading proteases. We used RNA-interference in combination with DNA chip analysis to identify Ets1-regulated genes in MDA-MB-231 breast cancer cells. Of the Ets1-responsive proteases, matrix metalloproteases MMP1 and MMP9, but not MMP3 or uPA, showed reduced RNA levels when endogenous Ets1 expression was suppressed. These data suggest that Ets1 regulates only a certain subset of ECM-degrading proteases. How Ets1 is regulated in invasive breast cancer cells is unknown. The observations that protein kinase C inhibitors abrogated Ets1 expression and that protein kinase C was able to increase Ets1-dependent transcription imply that protein kinase C is a potential regulator of Ets1 activity in breast cancer cells.

Transforming growth factor beta regulates parathyroid hormone-related protein expression in MDA-MB-231 breast cancer cells through a novel Smad/Ets synergism.

The majority of breast cancers metastasizing to bone secrete parathyroid hormone-related protein (PTHrP). PTHrP induces local osteolysis that leads to activation of bone matrix-borne transforming growth factor beta (TGF beta). In turn, TGF beta stimulates PTHrP expression and, thereby, accelerates bone destruction. We studied the mechanism by which TGF beta activates PTHrP in invasive MDA-MB-231 breast cancer cells. We demonstrate that TGF beta 1 up-regulates specifically the level of PTHrP P3 promoter-derived RNA in an actinomycin D-sensitive fashion. Transient transfection studies revealed that TGF beta 1 and its effector Smad3 are able to activate the P3 promoter. This effect depended upon an AGAC box and a previously described Ets binding site. Addition of Ets1 greatly enhanced the Smad3/TGF beta-mediated activation. Ets2 had also some effect, whereas other Ets proteins, Elf-1, Ese-1, and Erf-1, failed to cooperate with Smad3. In comparison, Ets1 did not increase Smad3/TGF beta-induced stimulation of the TGF beta-responsive plasminogen activator inhibitor 1 (PAI-1) promoter. Smad3 and Smad4 were able to specifically interact with the PTHrP P3-AGAC box and to bind to the P3 promoter together with Ets1. Inhibition of endogenous Ets1 expression by calphostin C abrogated TGF beta-induced up-regulation of the P3 transcript, whereas it did not affect the TGF beta effect on PAI expression. In TGF beta receptor II- and Ets1-deficient, noninvasive MCF-7 breast cancer cells, TGF beta 1 neither influenced endogenous PTHrP expression nor stimulated the PTHrP P3 promoter. These data suggest that TGF beta activates PTHrP expression by specifically up-regulating transcription from the PTHrP P3 promoter through a novel Smad3/Ets1 synergism.

The tetra-manganese complex of photosystem II during its redox cycle - X-ray absorption results and mechanistic implications.

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S(1)-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S(3)-state; (f) XAS-based working model for the S(2)-S(3) transition; (g) XAS-based working model for the S(0)-S(1) transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S(3)-(S(4))-S(0) transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.

Intravascular circulation and distribution of human 51Cr-DBBF stroma-free hemoglobin, 51Cr-plasma, 51Cr-saline, 59FE-plasma, and 125I-albumin in the mouse.

Male B6C3HF1 mice were infused with human 51Cr-labeled DBBF (bis 3,5-dibromosalicyl fumarate) crosslinked stroma-free hemoglobin (SFH). In the first hour following SFH infusion, 11.2% of the infused radioactivity was found in the skin, 11.4% in muscle, 9.1% in the skeleton, and 5% in the liver. Twenty-four hours after infusion, 15.4% of the radioactivity was found in the skin, 10.3%, in the muscle, 16.6% in the skeleton, and 6.7% in the liver. The circulation and distribution of 51Cr-labeled DBBF-SFH were compared with levels of 51Cr labeled plasma, 51Cr in saline, 59Fe labeled plasma, and 125I albumin. The radioactivity in the blood was similar for 51Cr-DBBF-SFH, 51Cr-plasma, and 59Fe-plasma. During the 24-hour post-infusion period, extravascular distribution of the 51Cr-saline, 51Cr-plasma, and 125I albumin within the organs was similar to that of 51Cr-DBBF-SFH, with the highest levels being in skin, muscle, skeleton and liver, and no increase in the levels in the lung or spleen. The distribution of 59Fe compared to that of 51Cr-DBBF, 51Cr-plasma, 51Cr-saline, and 125I albumin can be explained by the fact that 59Fe is utilized in the production of new red blood cells.

Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum.

Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.

Regulation of the human interleukin-5 promoter by Ets transcription factors. Ets1 and Ets2, but not Elf-1, cooperate with GATA3 and HTLV-I Tax1.

Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.

Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes.

To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.

X-ray absorption spectroscopy on layered photosystem II membrane particles suggests manganese-centered oxidation of the oxygen-evolving complex for the S0-S1, S1-S2, and S2-S3 transitions of the water oxidation cycle.

By application of microsecond light flashes the oxygen-evolving complex (OEC) was driven through its functional cycle, the S-state cycle. The S-state population distribution obtained by the application of n flashes (n = 0. 6) was determined by analysis of EPR spectra; Mn K-edge X-ray absorption spectra were collected. Taking into consideration the likely statistical error in the data and the variability stemming from the use of three different approaches for the determination of edge positions, we obtained an upshift of the edge position by 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the S0-S1, S1-S2, and S2-S3 transitions, respectively, and a downshift by 2.3-3.1 eV for the S3-S0 transition. These results are highly suggestive of Mn oxidation state changes for all four S-state transitions. In the S0-state spectrum, a clearly resolved shoulder in the X-ray spectrum around 6555 eV points toward the presence of Mn(II). We propose that photosynthetic oxygen evolution involves cycling of the photosystem II manganese complex through four distinct oxidation states of this tetranuclear complex: Mn(II)-Mn(III)-Mn(IV)2 in the S0-state, Mn(III)2-Mn(IV)2 in the S1-state, Mn(III)1-Mn(IV)3 in the S2-state, and Mn(IV)4 in the S3-state.

Structure and orientation of the oxygen-evolving manganese complex of green algae and higher plants investigated by X-ray absorption linear dichroism spectroscopy on oriented photosystem II membrane particles.

X-ray absorption spectroscopy at the Mn K-edge has been performed on multilayers of photosystem II-enriched fragments of the native thylakoid membrane prepared from a higher plant (spinach) and a unicellular green alga (Scenedesmus obliquus). Spectra collected for various angles between the prevailing orientation of the thylakoid membrane normal and the X-ray electric field vector contain information on the atomic structure of the tetranuclear manganese complex of photosystem II (PS II) and its orientation with respect to the membrane normal. The previously used approach for evaluation of the dichroism of extended X-ray absorption fine structure (EXAFS) spectra [George, G. N., et al. (1989) Science 243, 789-791] is modified, and the following results are obtained for PS II in its dark-stable state (S1-state): (1) structure and orientation of the PS II manganese complexes of green algae and higher plants are highly similiar or fully identical; (2) two 2.7-A vectors, which, most likely, connect the Mn nuclei of a planar Mn2(mu-O2) structure, are at an average angle of 80 degrees +/- 10 degrees with respect to the thylakoid normal; (3) the plane of the Mn2(mu-O2) structures is rather in parallel with the thylakoid plane than perpendicular. Structural models for the oxygen-evolving manganese complex and its orientation in the thylakoid membrane are discussed within the context of the presented results.

Inhibition of p53 transactivation function by the human T-cell lymphotropic virus type 1 Tax protein.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. HTLV-1 transforms lymphocytes, and there is increasing evidence that the virus-encoded protein, Tax, plays a primary role in viral transformation. We have shown that wild-type p53 in HTLV-1-transformed cells is stabilized. This study was initiated to directly analyze whether the p53 in HTLV-1-transformed cell lines was transcriptionally active and to identify the viral gene product responsible for stabilization and inactivation. Transfection experiments using a p53-responsive reporter plasmid and gamma-irradiation studies demonstrate that the wild-type p53 in HTLV-1-transformed cell lines is not fully active. Further, we demonstrate that the HTLV-1-transforming protein, Tax, stabilizes and inactivates p53 function. Cotransfection of Tax with p53 results in a greater than 10-fold reduction in p53 transcription activity. Using Ga14-p53 fusion proteins, we demonstrate that Tax inhibition of p53 transactivation function is independent of sequence-specific DNA binding. Moreover, Tax inhibits p53 function by interfering with the activity of the N-terminal activation domain (amino acids 1 to 52). We conclude that Tax is involved in the inactivation of p53 function and stabilization of p53 in HTLV-1-infected cells. The functional interference of p53 function by Tax may be important for transformation and leukemogenesis.

Physical and functional interaction between the human T-cell lymphotropic virus type 1 Tax1 protein and the CCAAT binding protein NF-Y.

Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis.

Interaction of human T-cell lymphotropic virus type I Tax, Ets1, and Sp1 in transactivation of the PTHrP P2 promoter.

We have previously shown that the parathyroid hormone-related protein (PTHrP) promoter contains binding sites for transcription factors Ets1 and Sp1 and that human T-cell lymphotropic virus type I (HTLV-I) Tax cooperates with Ets1 to transactivate the PTHrP P2 promoter. Using the yeast two-hybrid interaction system, we now provide evidence that Tax interacts with Ets1. Moreover, a double mutation (D22A,C23S) in the Tax protein that abrogated the Tax/Ets1 interaction also inhibited the Tax/Ets1 cooperative effect, suggesting that the interaction between Tax and Ets1 is important for transactivation of the PTHrP promoter. In coimmunoprecipitation assays, we find that Tax facilitates the interaction between Ets1 and Sp1, forming a ternary complex. When the Sp1 site in the PTHrP promoter was mutated, the Tax/Ets1 cooperative effect was dramatically decreased. This suggests that Sp1 plays an important role in the Ets1-dependent Tax transactivation of the PTHrP P2 promoter. Finally, we demonstrate that Gal4-Tax is a strong activator of the Gal PTHrP promoter, implying that Tax contributes directly to the transcriptional activation of the promoter. We propose a model in which the Tax/Ets1 cooperative effect on the PTHrP P2 promoter is based on the ability of Tax, Ets1, and Sp1 to form a ternary complex on the template DNA. Tax facilitates the interaction of Ets1/Sp1 and participates directly in the transcription initiation process.

Human T-cell leukemia virus type I Tax protein transactivates RNA polymerase III promoter in vitro and in vivo.

Tax protein of the human T-cell lymphotropic virus type 1 (HTLV-I) is critical for viral replication and is a potent transcriptional activator of viral and cellular polymerase II (pol II) genes. We report here that Tax is able to transactivate a classical pol III promoter, VA-I. In cotransfection experiments, Tax is shown to increase transcription of the VA-I promoter approximately 25-fold. Moreover, Tax is able to activate VA-I transcription when added exogenously to an in vitro transcription reaction. Using Tax affinity column chromatography, we demonstrate that Tax is able to deplete a HeLa cell extract for components required for transcription of VA-I. The transcriptional activity of the Tax-depleted extract can be restored by the 0.6 phosphocellulose fraction. Interestingly, a consensus binding site for cAMP-responsive element binding protein (CREB) is located upstream of the VA-I promoter, and deletion of this element results in the loss of Tax responsiveness. When this CREB binding site is replaced by a Gal-4 binding site, the VA-I promoter can be transactivated by a Gal4-Tax fusion protein. Taken together, these results suggest that Tax may activate pol III and pol II promoter through a similar mechanism involving the CREB activation pathway. It is also possible that Tax affects pol III transcription by direct interaction with a component of the pol III transcriptional machinery.

Isolation of a cDNA clone, TRX encoding a human T-cell lymphotrophic virus type-I Tax1 binding protein.

Tax1 is essential for human T-cell lymphotropic virus type I (HTLV-I) virus replication and transformation. We have identified and characterized a Tax1 binding protein, TRX, by cDNA screening of a Jurkat T-cell cDNA library. TRX mRNA is ubiquitously expressed in human tissues tested and cell lines analyzed.