Transcriptional regulation of the chicken MLC3f gene

The expression of the chicken fast skeletal myosin alkali light chain (MLC) 3f is subject to complex patterns of control by developmental and physiologic signals. Regulation over MLC3f gene expression is thought to be exerted primarily at the transcriptional level. The purpose of this dissertation was to identify cis-acting elements on the 5$\sp\prime$ flanking region of chicken MLC3f gene that are important for transcriptional regulation. The results show that the 5$\sp\prime$ flanking region of MLC3f gene contains multiple cis-acting elements. The nucleotide sequence of these elements demonstrates a high degree of conservation between different species and are also found in the 5$\sp\prime$ flanking regions of many muscle protein genes. The first regulatory region is located between $-$185 and $-$150 bp from the transcription start site and contains an AT-rich element. Linker scanner analyses have revealed that this element has a positive effect on transcription of the MLC3f promoter. Furthermore, when linked to a heterologous viral promoter, it can enhance reporter gene expression in a muscle-specific manner, independent of distance or orientation.^ The second regulatory region is located between $-$96 and $-$64 from the transcription start site. Sequences downstream of $-$96 have the capacity to drive muscle-specific reporter gene expression, although the region between $-$96 and $-$64 has no intrinsic enhancer-like activity. Linker scanner analyses have identified a GC-rich motif that required efficient transcription of the MLC3f promoter. Mutations to this region of DNA results in diminished capacity to drive reporter gene expression and is correlated with disruption of the ability to bind sequence-specific transcription factors. These sequence-specific DNA-binding proteins were detected in both muscle and non-muscle extracts. The results suggest that the mere presence or absence of transcription factors cannot be solely responsible for regulation of MLC3f expression and that tissue-specific expression may arise from complex interactions with muscle-specific, as well as more ubiquitous transcription factors with multiple regulatory elements on the gene. ^

Provenance versus weathering control on the composition of tropical river mud (southern Africa)

This study presents an integrated mineralogical-geochemical data base on fine-grained sediments transported by all major rivers of southern Africa, including the Zambezi, Okavango, Limpopo, Olifants, Orange and Kunene. Clay mineralogy, bulk geochemistry, Sr and Nd isotopic signatures of river mud, considered as proxy of suspended load, are used to investigate the influence of source-rock lithology and weathering intensity on the composition of clay and silt produced in subequatorial to subtropical latitudes. Depletion in mobile alkali and alkaline-earth metals, minor in arid Namibia, is strong in the Okavango, Kwando and Upper Zambezi catchments, where recycling is also extensive. Element removal is most significant for Na, and to a lesser extent for Sr. Depletion in K, Ca and other elements, negligible in Namibia, is moderate elsewhere. The most widespread clay minerals are smectite, dominant in muds derived from Karoo or Etendeka flood basalts, or illite and chlorite, dominant in muds derived from metasedimentary rocks of the Damara Orogen or Zimbabwe Craton. Kaolinite represents 30-40% of clay minerals only in Okavango and Upper Zambezi sediments sourced in humid subequatorial Angola and Zambia. After subtracting the effects of recycling and of local accumulation of authigenic carbonates in soils, the regional distribution of clay minerals and chemical indices consistently reflect weathering intensity primarily controlled by climate. Bulk geochemistry identifies most clearly volcaniclastic sediments and mafic sources in general, but cannot discriminate the other sources of detritus in detail. Instead, Sr and Nd isotopic fingerprints are insensitive to weathering, and thus mirror faithfully the tectonic structure of the southern African continent. Isotopic tools thus represent a much firmer basis than bulk geochemistry or clay mineralogy in the provenance study of mudrocks.

Geochemical variability in distal and proximal glass from the Youngest Toba Tuff eruption

The Youngest Toba Tuff (YTT, erupted ca. 74 ka ago) is a distinctive and widespread tephra marker across south and southeast Asia. The climatic, human and environmental consequences of the YTT eruption are widely debated. Although a considerable body of geochemical data is available for this unit, there has not been a systematic study of the variability of the ash geochemistry. Intrinsic (magmatic) and extrinsic (post-depositional) chemical variations bring fundamental information regarding the petrogenesis of the magma, the distribution of the tephra and the interaction between the ash and the receiving environment. Considering the importance of the geochemistry of the YTT for stratigraphic correlations and eruptive models, it is central to the YTT debate to quantify and interpret such variations. Here we collate all published geochemical data on the YTT glass, including analyses from 68 sites described in the literature and three new samples. Two principal sources of chemical variation are investigated: (i) compositional zonation of the magma reservoir, and (ii) post-depositional alteration. Post-depositional leaching is responsible for up to ca. 11% differences in Na2O/K2O and ca. 1% differences in SiO2/Al2O3 ratios in YTT glass from marine sites. Continental tephra are 2% higher in Na2O/K2O and 3% higher in SiO2/Al2O3 respect to the marine tephra. We interpret such post-depositional glass alteration as related to seawater induced alkali migration in marine environments, or to site-specific water pH. Crystal fractionation and consequential magmatic differentiation, which produced order-of-magnitude variations in trace element concentrations reported in the literature, also produced major element differences in the YTT glass. FeO/Al2O3 ratios vary by about 50 %, which is analytically significant. These variations represent magmatic fractionation involving Fe-bearing phases. We also compared major element concentrations in YTT and Oldest Toba Tuff (OTT) ash samples, to identify potential compositional differences that could constrain the stratigraphic identity of the Morgaon ash (Western India); no differences between the OTT and YTT samples were observed.

Melting of metasomatized peridotite at 4–6 GPa and up to 1200 °C: an experimental approach

The phase assemblages and compositions in a K-bearing lherzolite + H2O system are determined between 4 and 6 GPa and 850–1200 °C, and the melting reactions occurring at subarc depth in subduction zones are constrained. Experiments were performed on a rocking multi-anvil apparatus. The experiments had around 16 wt% water content, and hydrous melt or aqueous fluid was segregated and trapped in a diamond aggregate layer. The compositions of the aqueous fluid and hydrous melt phases were measured using the cryogenic LA-ICP-MS technique. The residual lherzolite consists of olivine, orthopyroxene, clinopyroxene, and garnet, while diamond (C) is assumed to be inert. Hydrous and alkali-rich minerals were absent from the run products due to preferred dissolution of K2O (and Na2O) to the aqueous fluid/hydrous melt phases. The role of phlogopite in melting relations is, thus, controlled by the water content in the system: at the water content of around 16 wt% used here, phlogopite is unstable and thus does not participate in melting reactions. The water-saturated solidus, i.e., the first appearance of hydrous melt in the K–lherzolite composition, is located between 900 and 1000 °C at 4 GPa and between 1000 and 1100 °C at 5 and 6 GPa. Compositional jumps between hydrous melt and aqueous fluid at the solidus include a significant increase in the total dissolved solids load. All melts/fluids are peralkaline and calcium-rich. The melting reactions at the solidus are peritectic, as olivine, clinopyroxene, garnet, and H2O are consumed to generate hydrous melt plus orthopyroxene. Our fluid/melt compositional data demonstrate that the water-saturated hybrid peridotite solidus lies above 1000 °C at depths greater than 150 km and that the second critical endpoint is not reached at 6 GPa for a K2O–Na2O–CaO–FeO–MgO–Al2O3–SiO2–H2O–Cr2O3(–TiO2) peridotite composition.

Syntheses, characterization and crystal structures of a new functionalised TTF derivative and its Ni(II) complex

The new ligand 4,5-bis (2-pyridylmethylsulfanyl)-4',5'-bis(cyanoethylthio)tetrathiafulvalene (BPM-BCET-TTF) and its nickel(II) complex have been prepared and crystallographically characterized. The Ni(II) complex shows octahedral geometry around the metal ion with the coordination site occupied by the pyridyl nitrogen atoms, the thioether sulfur atoms of the ligand and cis coordination of the halide ions.

Structural Studies of Transition Metal Complexes with 4,5-Bis(2-pyridylmethylsulfanyl)-4‘,5‘-ethylenedithiotetrathiafulvalene: Probing Their Potential for the Construction of Multifunctional Molecular Assemblies

Three divalent transition metal complexes of 4,5-bis(2-pyridylmethylsulfanyl)-4‘,5‘-ethylenedithiotetrathiafulvalene have been prepared and crystallographically characterized. The isostructural Co(II) and the Ni(II) complexes show octahedral geometries around the metal ions with the coordination sites occupied by the pyridyl nitrogen atoms and the thioether sulfur atoms of the ligand and cis coordination of the halide ions. Cyclic voltammetry reveals that the complexation leads to a small anodic shift in the first oxidation potential of the TTF system.

A Method for Preparing Oligodeoxynucleotides Containing an Apurinic Site

10.1002/hlca.19900730309.abs In three steps, 2-deoxy-D-ribose has been converted into a phosphoramidite building block bearing a (t-Bu)Me2Si protecting group at the OH function of the anomeric centre of the furanose ring. This building block was subsequently incorporated into DNA oligomers of various base sequences using the standard phosphoramidite protocol for automated DNA synthesis. The resulting silyl-oligomers have been purified by HPLC and selectively desilylated to the corresponding free apurinic DNA sequences. The hexamer d (A-A-A-A-X-A) (X representing the apurinic site) which was prepared in this way was characterized by 1H- and 31P-NMR spectroscopy. The other sequences as well as their fragments, which formed upon treatment with alkali base, were analyzed by polyacrylamide gel electrophoresis.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

OBJECTIVE Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. MATERIALS AND METHODS Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. RESULTS Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. CONCLUSION Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface.

Dynamics of Sodium‐22 and Cesium‐134 in Young Wheat Plants

The dynamics of the two alkali metals sodium and cesium in crop plants are relevant in an ecological context. Redistribution processes for these elements in young wheat plants were investigated in the work reported here. Two days old wheat plants (Triticum aestivum L. cv. Arina) were fed for 24 h with sodium‐22 (22Na) and cesium‐134 (134Cs) via the main root and incubated afterwards in a culture room. Cesium‐134 accumulated in newly formed parts of the main root and in the expanding leaves during the first 20 days after labeling, while 22Na accumulated transiently in these plant parts, reached a peak and declined after a few days. A high percentage of 22Na was released from the roots to the medium. Total Na in leaves also accumulated transiently, but its highest accumulation appeared later than the peak of 22Na. Therefore, the distribution and retranslocation processes differ considerably for sodium and cesium in wheat plants. Such differences must be considered for the evaluation of environmental effects (e.g., release of pollutants into the environment) on the quality of harvested cereal products.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers

Mutations in the vacuolar–type H+-ATPase B1 subunit gene ATP6V1B1 cause autosomal–recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G.A; p.E161K) that causes greatly diminished pump function in vitro. To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in theDallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild–type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low–Ca and low–Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; P,0.01). Kidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H+-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate– containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA.

Kinetics and Mechanism of Oxygen Delignification

Considerable research has been conducted into the kinetics and selectivity of the oxygen delignification process to overcome limitation in its use. However most studies were performed in a batch reactor whereby the hydroxide and dissolved oxygen concentrations are changing during the reaction time in an effort to simulate tower performance in pulp mills. This makes it difficult to determine the reaction order of the different reactants in the rate expressions. Also the lignin content and cellulose degradation of the pulp are only established at the end of the experiment when the sample is removed from the batch reactor. To overcome these deficiencies, we have adopted a differential reactor system used frequently for fluid-solid rate studies (so-called Berty reactor) for measurement of oxygen delignification kinetics. In this reactor, the dissolved oxygen concentration and the alkali concentration in the feed are kept constant, and the rate of lignin removal is determined from the dissolved lignin content in the outflow stream measured by UV absorption. The mass of lignin removed is verified by analyzing the pulp at several time intervals. Experiments were performed at different temperatures, oxygen pressures and caustic concentrations. The delignification rate was found to be first order in HexA-free residual lignin content. The delignification rate reaction order in caustic concentration and oxygen pressure were determined to be 0.42 and 0.44 respectively. The activation energy was found to be 53kJ/mol. The carbohydrate degradation during oxygen delignification can be described by two contributions: one due to radicals produced by phenolic delignification, and a much smaller contribution due to alkaline hydrolysis. From the first order of the reaction and the pKa of the active lignin site, a new oxygen delignification mechanism is proposed. The number 3 carbon atom in the aromatic ring with the attached methoxyl group forms the lignin active site for oxygen adsorption and subsequent electrophic reaction to form a hydroperoxide with a pKa value similar to that of the present delignification kinetics. The uniform presence of the aromatic methoxyl groups in residual lignin further support the first order in lignin kinetics.

DNA-DAMAGING REACTIONS OF NEOCARZINOSTATIN (ANTIBIOTICS, DNA REPAIR, CYTOTOXICITY)

Sensitive assays utilizing a cell-free and an intracellular system were employed to study the molecular bases of the DNA-damaging reactions of neocarzinostatin (NCS). In the cell-free DNA system, super-helical form I DNA from the bacteriophage PM2 was used as the substrate. The three forms of DNA present after treatment with NCS were separated by agarose gel electrophoresis. When NCS-damaged DNA was assayed under neutral conditions, there was a progressive decrease in the amount of surviving form I DNA and a corresponding increase in form II (nicked, relaxed circular) DNA, but very little increase in form III (linear duplex) DNA. This indicates that NCS introduces primarily single-strand breaks. However later studies showed that there were some site-specific double-strand breaks mediated by NCS on PM2 DNA. Seven such specific sites were mapped on the PM2 genome. When the damage was assayed under nondenaturing alkaline conditions or with the apurinic/apyrimidinic endonuclease IV, there was a slightly greater decrease in the amount of surviving form I DNA compared with neutral conditions indicating the presence of some alkali-labile sites.^ NCS-mediated DNA damage and repair were examined with cultured Chinese hamster ovary (CHO) cells using either alkaline elution for analysis of single-strand breaks or neutral elution for analysis of double-strand breaks. Most of the strand breaks introduced by NCS were capable of being rejoined. However, there was a small amount of residual DNA damage remaining unrejoined at 24-hr after removal of the drug. The amount of residual DNA damage was higher in a CHO mutant cell line (EM9) having a higher sensitivity to killing by NCS than its parental strain (AA8). Other lesions, DNA-protein complexes and alkali-labile sites, were detected after NCS treatment but they constituted only a small fraction of the DNA damage.^ Based on the above information, it can be postulated that NCS introduces some very lethal DNA damage. It is likely that the lethal lesions are a subset of the total DNA lesions representing the residual DNA damage. This DNA damage may be composed of site-specific, unrejoinable double-strand breaks and are thus the primary lesion leading to NCS-mediated lethality.^

Characterization of the MLC box: A conserved {\it cis\/}-acting DNA element present in the chicken MLC1s/1c gene

The slow/cardiac alkali myosin light chain (MLC1s/1c) is a member of a multigene family whose protein products are essential for activation of the myosin ATPase. In the adult, the MLC1s/1c isoform is expressed in both cardiac and slow-twitch skeletal muscles, while it is expressed by all skeletal muscles during development.^ To elucidate the molecular mechanisms that underlie the transcriptional regulation of MLC1s/1c gene expression, the immediate 5$\sp\prime$ flanking region of the gene was isolated and shown to be capable of directing reporter gene expression. Analysis of this region revealed a 110 bp muscle-specific enhancer that includes a myocyte-specific enhancer-binding factor 2 (MEF-2) site, E-boxes, which are potential binding sites for the basic-helix-loop-helix proteins such as MyoD, and a MLC box. The focus of the thesis was to identify the role of the MLC box in expression of the MLC1s/1c gene.^ The MLC box is a member of the family of CArG box containing cis-acting DNA elements. Mutagenesis showed that the MLC box is necessary, but not sufficient, for the expression of a reporter gene linked to the 5$\sp\prime$ flanking region of the MLC1s/1c gene. Linker scanner and site-directed mutagenesis identified a number of potential sites within the 110 bp muscle-specific enhancer that may cooperate with the MLC box. These are the MEF-2 site, the E-box site, and a 10 bp element located upstream of the MEF-2 site that does not have sequence similarity with any known cis-acting element. The MLC box is capable of binding to factors present in muscle nuclear extracts, as well as to human recombinant serum response factor (SRF). Binding of SRF to the MLC box was correlated with the ability of the 5$\sp\prime$ flanking region of the MLC1s/1c gene to drive reporter gene expression. Results suggest a model in which binding of SRF to the MLC box activates expression of the MLC1s/1c gene while binding of the factors present in the nuclear extracts suppresses the expression of the gene. (Abstract shortened with permission of author.) ^

Geochemistry of minerals in CRP sediment cores from the Ross Sea, Antarctica

Sixty-four volcanic chists, sandstones and tephras between 5.95 and 618.19 meters below sea floor (mbsf) in the Cape Roberts Project cores 2 and 2A cores (CRP-2/2A) were examined for Cenozoic and Mesozoic volcanic components, using optical and Scanning Electron Microscopy. Minerals and glass shards in a selection of samples were analysed by electron microprobe fined with an EDAX detector. Laser-Ablation ICP-Mass-Spectrometry (ICP-MS) was used to determine rare earth elements and 14 additional trace elements in glass shards, pyroxenes and feldspars in order to pin-point the onset of McMurdo Volcanic Group (MVG) activity in the stratigraphic column. Pumices in tephra layers of peralkaline phonolite composition in Unit 7.2 -between 108 and 114 mbsf - were also analysed for trace elements by ICP-MS. This tephra unit is not reworked and its isotopic age (21.44 ± 0.05 Ma) is the age of deposition. The height of the eruptive column responsible for the deposition of the tephra was probably less than 8 km; the source was local, probably within 30 km from the drill site. Phonolite of unit 7.2 of CRP-2/2A has no direct petrogenetic relation with the peralkaline trachyte in the tephra-enriched layer of CRP-1 at 116.55 mbsf. Volcanic clasts and sand grains (glass shards, aegirine-augite, anorthoclase) related to Cenozoic activity of MVG were observed only starting from Unit 9.8, where they are dated at 24.22 ± 0.06 Ma at c. 280 mbsf. In this unit the lowest- occurring basaltic glass shard is found at 297.54 mbsf. Sampled McMurdo volcanics are generally vesicular and vary in composition from alkali basalt to trachyte and peralkaline phonolite. By contrast, below 320 mbsf, aphyric or slightly-porphyritic volcanic clasts become more abundant but they are all non-vesiculated, pigeconite and ilmenite-bearing basalts and dolerite of tholeiitic affinity. These rocks are considered to be related to lava flows and associated intrusions of Jurassic age (Kirkpatrick basalts and Ferrar dolerite). As in CRP-1, McMurdo volcanics appear to derive from a variety of lithologics. Besides glaciers, a dominant role of wind transportation from exposed volcanic rocks may be inferred from the contemporary occurrence of glass shards of different compositions at depths above 297.54 mbsf. These data confirm that the onset of magmatic activity in southern Victoria Land is considerably delayed (by about 24 Ma) with respect to northern Victoria Land.