Coarse-sand ice-rafted debris components and mass accumulation rates of ODP Site 152-918

To understand the late Cenozoic glacial history of the Northern Hemisphere, continuous long-term proxy records from climatically sensitive regions must be examined. Ice-rafted debris (IRD) from Ocean Drilling Program (ODP) Site 918, located in the Irminger Basin, is one such record. IRD in marine sediments is a direct indicator of the presence of glacial ice extending to sea level on adjacent landmasses, and, therefore, is an important paleoclimatic signal from the mid- to high latitudes. The IRD record at Site 918 is the first long-term ice-rafting record available for southeast Greenland, a region that may have been a key nucleation area for widespread glaciation during the late Cenozoic (Larsen et al, 1994, doi:10.2973/odp.proc.ir.152.1994). This data report presents the results of coarse sand-size IRD mass accumulation rate (MAR) analyses for Site 918 from the late Miocene through the Pleistocene. In addition, a preliminary analysis of IRD compositions is included. Detailed discussions of the local, regional, and global paleoclimatic implications of this data, and of the companion Site 919 Pleistocene IRD MAR data (Krissek, 1999, doi:10.2973/odp.proc.sr.163.118.1999), are in preparation. Such future work will include comparisons of these IRD MAR data sets to the Site 919 oxygen isotope stratigraphy developed by Flower (1998, doi:10.2973/odp.proc.sr.152.219.1998).

Epigeal spider occurrence, abundance and diversity in moist acidic and dry heath tundra at Toolik Lake, Alaska

For the 2004-2006 growing seasons, we trapped a total of 6980 spiders (5066 adults, 1914 immatures) using pitfall traps at the Arctic Long Term Experimental Research (LTER) site in Toolik Lake, Alaska. We found 10 families and 51 putative species, with 45 completely identified, in two distinct habitats: Moist Acidic Tundra (MAT) and Dry Heath (DH) Tundra. We captured spiders belonging to the following families (number of species captured): Araneidae (1), Clubionidae (1), Dictynidae (1), Gnaphosidae (4), Linyphiidae (26), Lycosidae (11), Philodromidae (2), Salticidae (1), Theridiidae (1), and Thomisidae (3). Statistical comparisons of families captured at MAT and DH Tundra indicate that the habitats have significantly different spider communities (Chi Square Test: p < 0.0001, and Fisher's Exact Test: p = 0.0018). This finding is further supported by differences in similarity, diversity, evenness, and species richness between the two habitats. In this report, we present eight new state records and five extensions of previously described ranges for spider species. The following species are new state records for Alaska: Emblyna borealis (O.P.-Cambridge 1877), Horcotes strandi (Sytschevskaja 1935), Mecynargus monticola (Holm 1943), Mecynargus tungusicus (Eskov 1981), Metopobactrus prominulus (O.P. -Cambridge 1872), Poeciloneta theridiformis Emerton 1911, and Poeciloneta vakkhanka (Tanasevitch 1989). The following five species have been reported previously in Alaska, but not near Toolik Lake: Hypsosinga groenlandica Simon 1889, Gnaphosa borea Kulczyn'ski 1908, Gnaphosa microps Holm 1939, Haplodrassus hiemalis (Emerton 1909), and Islandiana cristata Eskov 1987. Pairwise similarity indices were calculated across 13 other arctic and subarctic spider communities and statistical tests show that all sites are dissimilar (p = 0.25). These results fit the general pattern of both the patchiness and habitat specificity of arctic spider fauna.

Depth tie points and ages of IODP Site 306-U1314

Integrated Ocean Drilling Program (IODP) Site U1314 of the North Atlantic is a critical sedimentary archive record of subpolar deep water from the southern Gardar Drift for which we derived an age model of orbital resolution for the last 1.8 Ma. This chronology combined with high-resolution (cm scale) X-ray fluorescence core scanning measurements of major elements allows tracking changes in terrigenous provenance during the last 1.1 Ma. Low Potassium to Titanium (K/Ti) ratios reflect enhanced transport of basalt-derived titanomagnetites during warm climate intervals, while high K/Ti ratios indicate a dominance of acidic sediment sources typical for glacial and stadial events. Changes in K/Ti and magnetic concentration at Site 1314 are coeval with fluctuations in smectite content and grain size data from nearby piston cores, suggesting that the provenance changes are mainly controlled by variable flow of the Iceland-Scotland Overflow Water, an important branch of North Atlantic Deep Water. Furthermore, K/Ti variations on orbital time scales show a striking similarity to the deep sea d13C record from ODP Site 607. Pervasive features of the K/Ti time series during and after the Mid-Pleistocene Transition are suborbital changes similar to Dansgaard/Oeschger and Bond oscillations that appear to be strongly amplified during ice growth phases when global benthic d18O was within the range of ~4.1-4.6 per mil. The strong increase in variability of sediment provenance and subsequently deep hydrography at benthic d18O values below ~4.1 suggests that the extent of glaciations and, therefore, sea level corresponding to this value constitutes an important physical threshold that was persistent at least for the last 1.1 Ma.

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.

Characterization of Mutants with Alterations of the Phosphorylation Site in the D2 Photosystem II Polypeptide of Chlamydomonas reinhardtii1

We have changed the potential phosphorylation site, a threonine residue at position 2 of the D2 polypeptide of the photosystem II complex of Chlamydomonas reinhardtii, to alanine, valine, aspartate, proline, glycine, or glutamate. Mutants with neutral amino acid changes did not display any phenotype with regard to photoautotrophic growth, light sensitivity, fluorescence transients, or photoinhibition. Pulse labeling of these mutants with 32P indicated that a phosphorylated protein of the same size as D2 is absent in these mutants, suggesting that threonine-2 is indeed the unique phosphorylation site of D2. In contrast, mutants in which threonine-2 has been replaced with acidic residues are deficient in photosystem II. Use of chimeric genes containing the psbD 5′-untranslated region revealed that the initiation of translation was not affected in these mutants, but the mutations interfered with a later step of D2 synthesis and accumulation.

Negative pH, efflorescent mineralogy, and consequences for environmental restoration at the Iron Mountain Superfund site, California

The Richmond Mine of the Iron Mountain copper deposit contains some of the most acid mine waters ever reported. Values of pH have been measured as low as −3.6, combined metal concentrations as high as 200 g/liter, and sulfate concentrations as high as 760 g/liter. Copious quantities of soluble metal sulfate salts such as melanterite, chalcanthite, coquimbite, rhomboclase, voltaite, copiapite, and halotrichite have been identified, and some of these are forming from negative-pH mine waters. Geochemical calculations show that, under a mine-plugging remediation scenario, these salts would dissolve and the resultant 600,000-m3 mine pool would have a pH of 1 or less and contain several grams of dissolved metals per liter, much like the current portal effluent water. In the absence of plugging or other at-source control, current weathering rates indicate that the portal effluent will continue for approximately 3,000 years. Other remedial actions have greatly reduced metal loads into downstream drainages and the Sacramento River, primarily by capturing the major acidic discharges and routing them to a lime neutralization plant. Incorporation of geochemical modeling and mineralogical expertise into the decision-making process for remediation can save time, save money, and reduce the likelihood of deleterious consequences.

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.