Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

The functional role of the tumor suppressor MMAC /PTEN in glioblastoma multiforme and prostate cancer

Investigations into the molecular basis of glioblastoma multiforme led to the identification of a putative tumor suppressor gene, MMAC/ PTEN. Initial studies implicated MMAC/PTEN in many different tumor types, and identified a protein phosphatase motif in its sequence. This project aimed to identify the biological and biochemical functions of MMAC/PTEN by transiently expressing the gene in cancer cells that lack a functional gene product. ^ Expression of MMAC/PTEN mildly suppressed the growth of U251 human glioma cells and abrogated the growth advantage mediated by overexpression of the epidermal growth factor receptor (EGFR). Immunoblotting demonstrated that MMAC/PTEN expression did not affect the phosphorylation of the EGFR itself, or the intermediates of several downstream signaling pathways. However, MMAC/PTEN expression significantly reduced the phosphorylation and catalytic activity of the proto-oncogene Akt/PKB. While Akt/PKB regulates the survival of many cell types, expression of MMAC/PTEN did not induce apoptosis in adherent U251 cells. Instead, MMAC/PTEN expression sensitized the cells to apoptosis when maintained in suspension (anoikis). As the survival of suspended cells is one of the hallmarks leading to metastasis, MMAC/PTEN expression was examined in a system in which metastasis is more clinically relevant, prostate cancer. ^ Expression of MMAC/PTEN in both LNCaP and PC3-P human prostate cancer cells specifically inhibited Akt/PKB phosphorylation. MMAC/PTEN expression in LNCaP cells resulted in a profound inhibition of growth that was significantly greater than that achieved with expression of p53. Expression of MMAC/PTEN in PC3-P cells resulted in greater growth inhibition than was observed in U251 glioma cells, but less than was observed in LNCaP cells, or upon p53 expression. To determine if MMAC/PTEN could function as a tumor suppressor in vivo, the effects of MMAC/PTEN expression on PC3-P cells implanted orthotopically in nude mice were examined. The ex-vivo expression of MMAC/PTEN did not decrease tumor incidence, but it did significantly decrease tumor size and metastasis. In-vivo expression of MMAC/PTEN in pre-established PC3-P tumors did not significantly inhibit tumor incidence or size, but did inhibit metastasis formation. ^ These studies demonstrate that MMAC/PTEN is a novel and important tumor suppressor gene, which functions to downregulate an important cell survival signaling pathway. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

Major element concentrations in spinel, olivine and pyroxene from peridotites of ODP Hole 210-1277 of the Newfoundland margin

Serpentinized spinel peridotites of the Newfoundland margin drilled during ODP Leg 210 at Site 1277 have preserved, relic mineral compositions similar to the most depleted abyssal peridotites worldwide and different from those of the conjugate Iberian margin. The samples are derived from mass flows containing clasts of peridotite and gabbro and from in-situ basement, and are mostly mylonitic cpx-poor spinel harzburgites with Cr-rich spinels (Cr#0.35-0.66). Melting of the Newfoundland mantle occurred in the spinel peridotite field and probably exceeded the cpx-out phase boundary for some samples. Using proposed spinel peridotite melting models and experimentally derived phase diagrams, the Newfoundland harzburgites can be modeled as a residue after extraction of 14 to 20-25% melting. Basalts that are interleaved with mass flow deposits on top of the peridotite basement resemble normal to transitional mid-ocean ridge basalt. This, together with the unusually high Cr# of some spinel harzburgites suggest that the formation of basalts and partial melting of the underlying peridotite are not cogenetic. Among mantle samples some of the Newfoundland harzburgites approach mineral compositions of the Bay of island ophiolite and ophiolites from Japan that represent peridotites formed in an arc-setting. Thus, the peridotites drilled at Site 1277 may represent inherited (Caledonian or older) subarc mantle that was exhumed close to the ocean floor during the rifting evolution of the Atlantic. Compared to the spinel harzburgites from Newfoundland, the peridotites from the conjugate Iberian margin are, on average, less depleted and provide evidence for local equilibration in the plagioclase stability field. This can either be explained by an inherited, primary, Ca-richer composition of the Iberia peridotite, or, alternatively, by local melt impregnation and stagnation during continental rifting, and thus refertilizing previously depleted (arc-related) peridotite.

Mesozooplankton biomass data from the Kapisigdlit an inner fjord branch of the Godthaabsfjord system, West Greenland, 2010

Sampling was conducted from March 24 to August 5 2010, in the fjord branch Kapisigdlit located in the inner part of the Godthåbsfjord system, West Greenland. The vessel "Lille Masik" was used during all cruises except on June 17-18 where sampling was done from RV Dana (National Institute for Aquatic Resources, Denmark). A total of 15 cruises (of 1-2 days duration) 7-10 days apart was carried out along a transect composed of 6 stations (St.), spanning the length of the 26 km long fjord branch. St. 1 was located at the mouth of the fjord branch and St. 6 was located at the end of the fjord branch, in the middle of a shallower inner creek . St. 1-4 was covering deeper parts of the fjord, and St. 5 was located on the slope leading up to the shallow inner creek. Mesozooplankton was sampled by vertical net tows using a Hydrobios Multinet (type Mini) equipped with a flow meter and 50 µm mesh nets or a WP-2 net 50 µm mesh size equipped with a non-filtering cod-end. Sampling was conducted at various times of day at the different stations. The nets were hauled with a speed of 0.2-0.3 m s**-1 from 100, 75 and 50 m depth to the surface at St. 2 + 4, 5 and 6, respectively. The content was immediately preserved in buffered formalin (4% final concentration). All samples were analyzed in the Plankton sorting and identification center in Szczecin (www.nmfri.gdynia.pl). Samples containing high numbers of zooplankton were split into subsamples. All copepods and other zooplankton were identified down to lowest possible taxonomic level (approx. 400 per sample), length measured and counted. Copepods were sorted into development stages (nauplii stage 1 - copepodite stage 6) using morphological features and sizes, and up to 10 individuals of each stage was length measured.

Radiogenic isotope trace composition of the Labrador Sea

Geological reconstructions and general circulation models suggest that the onset of both Northern Hemisphere glaciation, 2.7 Myr ago, and convection of Labrador Sea Water (LSW) were caused by the closure of the Panama Gateway ~4.5 Myr ago. Time series data that have been obtained from studies of ferromanganese crusts from the northwestern Atlantic suggest that radiogenic isotopes of intermediate ocean residence time (Pb and Nd) can serve as suitable tracers to reconstruct these events. However, it has been unclear until now as to whether the changes that have been observed in isotope composition at this time are the result of increased thermohaline circulation or due to the effects of increased glacial weathering. In this paper we adopt a box model approach to demonstrate that the shifts in radiogenic isotope compositions are unlikely to be due to changes in convection in LSW but can be explained in terms of increases of erosion levels due to the glaciation of Greenland and Canada. Furthermore, we provide experimental evidence for the incongruent release of a labile fraction of strongly radiogenic Pb and nonradiogenic Nd from continental detritus eroding into the Labrador Sea. This can be attributed to the glacial weathering of old continents and accounts for the paradox that one of the areas of the world most deficient in radiogenic Pb should provide such a rich supply of radiogenic Pb to the oceans. An important general conclusion is that the compositions of radiogenic isotopes in seawater are not always a reflection of their continental sources. Perhaps more importantly, the transition from chemical weathering to mechanical erosion is likely to result in significant variations in radiogenic tracers in seawater.

Geochemistry of glass shards in the volcanic ash layers of the Izu-Bonin Arc

To examine the processes and histories of arc volcanism and of volcanism associated with backarc rifting. 130 samples containing igneous glass shards were taken from the Plioccne-Quatemai^ succession on the rift Hank (Site 788) and the Quaternary fill in the basin fill of the Sumisu Rift (Sites 790 and 791). These samples were subsequently analyzed at the University of Illinois at Chicago and Shizuoka University. The oxides determined by electron probe do not account for the total weight of the material; differences between summed oxides and 100% arise from the water contents, probably augmented by minor losses thai result from alkali vaporization during analysis. Weight losses in colorless glasses are up to 9%; those in brown glasses (dacitcs to basalts) arc no more than 4.5%; shards from the rift-flank (possibly caused by prolonged proximity to ihc scafloor) generally have higher values than those from the rift-basin fill How much of the lost water is magmatic, and how much is hydrated is uncertain; however, although the shards absorb potassium, calcium, and magnesium during hydration in the deep sea, they do so only to a minor extent that does not significantly alter their major element compositions. Therefore, the electron-probe results are useful in evaluating the magmatism recorded by the shards. Pre- and syn-rift Izu-Bonin volcanism were overwhelmingly dominated by rhyolile explosions, demonstrating that island arcs may experience significant silicic volcanism in addition to the extensive basaltic and basaltic andestic activity, documented in many arcs since the 1970s, that occurs in conjunction with the andesitic volcanism formerly thought to be dominant. Andesitic eruptions also occurred before rifting, but the andesitic component in our samples is minor. All the pre- and syn-rift rhyolites and andesites belong to the low-alkali island-arc tholeiitic suite, and contrast markedly with the alkali products of Holocene volcanism on the northernmost Mariana Arc that have been attributed to nascent rifting. The Quaternary dacites and andesites atop the rift flank and in the rift-basin fill are more potassic than those of Pliocene age, as a result of assimilation from the upper arc crust, or from variations in degrees of partial melting of the source magmas, or from metasomatic fluids. All the glass layers from the rift-flank samples belong to low-K arc-tholeiitic suites. Half of those in the Pliocene succession are exclusively rhyolitic: the others contain minor admixtures of dacite and andesite, or andesite and either basaltic andesite or basalt. In Contrast, the Quaternary (syn-rift) volcaniclastics atop the rift-flank lack basalt and basaltic andesite shards. These youngest sediments of the rift flank show close compositional affinities with five thick layers of coarse, rhyolitic pumice deposits in the basin fill, the two oldest more silicic than the younger ones. The coarse layers, and most thin ash layers that occur in hemipelagites below and intercalated between them, are low-K rhyolites and therefore probably came from sources in the arc. However, several thin rhyolitic ash beds in the hemipelagites are abnormally enriched in potassium and must have been provided by more distal sources, most likely to the west in Japan. Remarkably, the Pliocene-Pleistocene geochemistry of the volcanic front does not appear to have been influenced by the syn-rift basaltic volcanism only a few kilometers away. Rare, thin layers of basaltic ash near the bases of the rift-basin successions are not derived from the arc. They deviate strongly from trends that the arc-derived glasses display on oxide-oxide plots, and show close affinities to the basalts empted all over the Sumisu Rift during rifting. These basalts, and the basaltic ashes in the basal rift-basin fill, arc compositionally similar to those erupted from mature backarc basins elsewhere.

Review of the Performance of Residential PV systems in Belgium

The main objective of this paper is to review the state of the art of residential PV systems in Belgium by the analysis of the operational data of 993 installations. For that, three main questions are posed: how much energy do they produce? What level of performance is associated to their production? Which are the key parameters that most influence their quality? This work brings answers to these questions. A middling commercial PV system, optimally oriented, produces a mean annual energy of 892 kWh/kWp. As a whole, the orientation of PV generators causes energy productions to be some 6% inferior to optimally oriented PV systems. The mean performance ratio is 78% and the mean performance index is 85%. That is to say, the energy produced by a typical PV system in Belgium is 15% inferior to the energy produced by a very high quality PV system. Finally, on average, the real power of the PV modules falls 5% below its corresponding nominal power announced on the manufacturer's datasheet. Differences between real and nominal power of up to 16% have been detected.

Representation of sound localization cues in the auditory thalamus of the barn owl

Barn owls can localize a sound source using either the map of auditory space contained in the optic tectum or the auditory forebrain. The auditory thalamus, nucleus ovoidalis (N.Ov), is situated between these two auditory areas, and its inactivation precludes the use of the auditory forebrain for sound localization. We examined the sources of inputs to the N.Ov as well as their patterns of termination within the nucleus. We also examined the response of single neurons within the N.Ov to tonal stimuli and sound localization cues. Afferents to the N.Ov originated with a diffuse population of neurons located bilaterally within the lateral shell, core, and medial shell subdivisions of the central nucleus of the inferior colliculus. Additional afferent input originated from the ipsilateral ventral nucleus of the lateral lemniscus. No afferent input was provided to the N.Ov from the external nucleus of the inferior colliculus or the optic tectum. The N.Ov was tonotopically organized with high frequencies represented dorsally and low frequencies ventrally. Although neurons in the N.Ov responded to localization cues, there was no apparent topographic mapping of these cues within the nucleus, in contrast to the tectal pathway. However, nearly all possible types of binaural response to sound localization cues were represented. These findings suggest that in the thalamo-telencephalic auditory pathway, sound localization is subserved by a nontopographic representation of auditory space.

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.

The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB∷LUC reporter genes containing the promoter and 5′ untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB∷Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4–KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.