Measurement of Protein in Nearshore Marine Sediments

Proteinaceous material in marine sediments which is available to proteolytic hydrolysis has been measured using a new method. This technique utilizes Coomassie Blue dye binding, which has the advantage of being sensitive only to larger polypeptides. Substantial interferences from other sedmentary organic substances are overcome by using a standard additions approach in conjunction with enzymatic digestion of the protein. Although tedious, the technique provides acceptable precision and accuracy. Measurements of protein in surficial nearshore sediments of the Gulf of Maine and St. Croix yield values ranging from 0.1 to 2.2 mg g-1, which account for a minor fraction of total nitrogen or acid-hydrolyzable amino acids. Protein decreases downcore at a faster rate than either of these 2 indicators of nitrogenous material, indicating the greater lability of the truly proteinaceous material. Biomass comprises a minor portion of the measured protein.

Part of the global DOC versus AOU (dissolved organic carbon/apparent oxygen utilization) data compilation, Ross Sea

Dissolved and particulate organic matter was measured during six cruises to the southern Ross Sea. The cruises were conducted during late austral winter to autumn from 1994 to 1997 and included coverage of various stages of the seasonal phytoplankton bloom. The data from the various years are compiled into a representative seasonal cycle in order to assess general patterns of dissolved organic matter (DOM) and particulate organic matter (POM) dynamics in the southern Ross Sea. Dissolved organic carbon (DOC) and particulate organic carbon (POC) were at background concentrations of approximately 42 and 3 µM C, respectively, during the late winter conditions in October. As the spring phytoplankton bloom progressed, organic matter increased, and by January DOC and POC reached as high as 30 and 107 µM C, respectively, in excess of initial wintertime conditions. Stocks and concentrations of DOC and POC returned to near background values by autumn (April). Approximately 90% of the accumulated organic matter was partitioned into POM, with modest net accumulation of DOM stocks despite large net organic matter production and the dominance of Phaeocystis antarctica. Changes in NO3 concentration from wintertime values were used to calculate the equivalent biological drawdown of dissolved inorganic carbon (DICequiv). The fraction of DICequiv drawdown resulting in net DOC production was relatively constant (ca. 11%), despite large temporal and spatial variability in DICequiv drawdown. The C : N (molar ratio) of the seasonally produced DOM had a geometric mean of 6.2 and was nitrogen-rich compared to background DOM. The DOM stocks that accumulate in excess of deep refractory background stocks are often referred to as "semi-labile" DOM. The "semi-labile" pool in the Ross Sea turns over on timescales of about 6 months. As a result of the modest net DOM production and its lability, the role DOM plays in export to the deep sea is small in this region.

Concentrations of total organic carbon on vertical profiles in waters of the Weddell Sea measured on water bottle sample during POLARSTERN cruise ANT-X/6

Concentrations of dissolved organic carbon (DOC) and nitrogen (DON) were measured during early austral Spring 1992 at a number of stations along the 6°W meridian between 47° and 60°S. This included the Polar Front in the north, the zone of melting sea-ice in the south, and waters of the Antarctic Circumpolar Current in between. Concentrations of DOC were low in deep water (34-38 ?M) with generally similar or slightly higher values in the surface mixed layer (38-55 ?M). DOC:DON ratios are wider in surface water than in deep water, i.e. surface accumulations contain relatively C-rich dissolved organic matter. The highly variable distribution of the surface DOC was not related to hydrographic or biotic features (fronts, plankton development) indicating the lability and transient occurrence of this material. Growth rates of bacteria were determined in subsamples from 51 0.8-?m-filtered batches of seawater incubated in the dark at in-situ temperature. Thymidine and leucine uptake and bacterial biomass change as well as changes in dissolved organic carbon in the batches, and oxygen consumption in parallel incubations correlated linearly over 2 weeks of incubation which allowed extrapolation to in-situ conditions. Bacterial growth in these experiments depended strongly on the amount of initial DOC. Growth in water from greater depth (1000 m) containing 38 ?M DOC was minimal, as were DOC-decrease and oxygen consumption. Higher rates were observed in surface water slightly enriched with DOC, and highest rates in surface water amended with DOC-rich melted sea ice. Bacterial growth efficiencies (biomass C-increase vs DOC consumed) were about 30%. The experiments showed that at least 40-60% of the DOC in excess of deep water concentrations was available to bacteria.

NADW Photodegradation FTICR-MS

Duplicate, filtered samples of North Atlantic Deep Water (NADW) were irradiated for 28 days in a solar simulator. Duplicate dark controls were placed alongside the irradiated samples. After 28 days, samples were extensively photo-degraded based upon colored dissolved organic matter (CDOM) photo-bleaching (> 95%). Samples were solid phase extracted using PPL resin to isolate, concentrate and desalt the dissolved organic matter (DOM) in the samples. Ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) enabled 3024 molecular formulas to be identified in the dark control. Photo-degradation decreased molecular diversity, with 2402 formulas assigned post-irradiation. Molecular formulas were classified based upon their photo-lability as 1) photo-resistant; 2) photo-labile; and, 3) photo-produced. Photo-resistant DOM made up 73% of all formulas and was dominated by highly unsaturated molecular signatures consistent with carboxylic-rich alicyclic molecules, suggesting that these apparently bio-refractory compounds may also survive multiple passages through sunlit surface waters and thus accumulate for timeframes exceeding ocean ventilation. Photo-labile DOM was enriched in low molecular weight formulas, aromatic molecular formulas, and sulfur and phosphorous containing formulas. Compounds containing both sulfur and nitrogen were particularly photo-labile and may represent an underappreciated component of the photo-reactive CDOM pool. Photo-produced DOM was enriched in higher molecular weight formulas, as well as aliphatic and peptide formulas. Molecular formulas are indexed by their photo-lability classification in the supplementary information.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

Hypoxic Regulation of Vascular Endothelial Growth Factor mRNA Stability Requires the Cooperation of Multiple RNA Elements

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3′ untranslated region (3′UTR), but also contains destabilizing elements in the 5′UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5′UTR, coding region, and 3′UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

Contaminant bioavailability in soils, sediments, and aquatic environments

The aqueous concentrations of heavy metals in soils, sediments, and aquatic environments frequently are controlled by the dissolution and precipitation of discrete mineral phases. Contaminant uptake by organisms as well as contaminant transport in natural systems typically occurs through the solution phase. Thus, the thermodynamic solubility of contaminant-containing minerals in these environments can directly influence the chemical reactivity, transport, and ecotoxicity of their constituent ions. In many cases, Pb-contaminated soils and sediments contain the minerals anglesite (PbSO4), cerussite (PbCO3), and various lead oxides (e.g., litharge, PbO) as well as Pb2+ adsorbed to Fe and Mn (hydr)oxides. Whereas adsorbed Pb can be comparatively inert, the lead oxides, sulfates, and carbonates are all highly soluble in acidic to circumneutral environments, and soil Pb in these forms can pose a significant environmental risk. In contrast, the lead phosphates [e.g., pyromorphite, Pb5(PO4)3Cl] are much less soluble and geochemically stable over a wide pH range. Application of soluble or solid-phase phosphates (i.e., apatites) to contaminated soils and sediments induces the dissolution of the “native” Pb minerals, the desorption of Pb adsorbed by hydrous metal oxides, and the subsequent formation of pyromorphites in situ. This process results in decreases in the chemical lability and bioavailability of the Pb without its removal from the contaminated media. This and analogous approaches may be useful strategies for remediating contaminated soils and sediments.

d-Ribulose-5-Phosphate 3-Epimerase: Cloning and Heterologous Expression of the Spinach Gene, and Purification and Characterization of the Recombinant Enzyme1

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding d-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by dl-α-glycerophosphate or ethanol and destabilized by d-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

Relationship Maintenance in Couples with a Partner Who Has Attention Deficit Hyperactivity Disorder

The body of research on the relationship functioning of adults with attention deficit hyperactivity disorder (ADHD) is relatively small; the aim of the present study is to advance our understanding of this topic. It has been estimated that three to ten percent of children and one to five percent of adults have impairing symptoms of ADHD, which is a total of 4 million children and 4-5 million adults in the U.S. (Wender, 2000). A recent prevalence study found that approximately 4.4% of adults in the U.S. meet the criteria for a diagnosis of ADHD (Kessler et al., 2006). Children with ADHD show innate temperamental characteristics, usually inattentiveness, distractibility, impulsivity, restlessness, demandingness, hyperreactivity, low tolerance for frustration, temperoutbursts, bossiness and stubbornness, and mood lability, along with an innate proclivity for academic underachievement. It has been estimated that one- to two-thirds of children with ADHD have symptoms that continue into adulthood, and for 40-50% of these adults, these symptoms are serious enough to cause impairment in functioning (Everett & Everett, 1999; Wender, 2000). Research suggests that the majority of cases are transmitted genetically, but some may be due to exposure to environmental toxins such as lead. Consumption of excess sugar or allergies to food may exacerbate or mimic ADHD symptoms in some children, but they are not a cause of ADHD (Wender, 2000). One hypothesized cause of the symptoms associated with ADHD is a deficit in the brain's executive functioning (Barkley & Gordon, 2002). Executive functioning can be conceptualized as the ability to inhibit, organize, and plan behaviors. Barkley and Gordon (2002) define it as the abilityto self-direct and regulate behaviors toward future goals, including social behaviors and goals. Other research suggests that executive functioning consists of inhibition, control of interference, verbal and nonverbal working memory, emotional regulation, attention, verbal fluency, visual scanning, and processing speed. Studies have shown impairments in these areas among adults with ADHD (Barkley & Gordon, 2002; Barkley, Murphy & Kwasnik, 1996; Goldstein, 2002).

Organic carbon and carbohydrates in waters of the Amazon River, Atlantic Ocean, and Tropical Indian Ocean

A method is presented to study carbohydrate composition of marine objects involved into sedimento- and diagenesis (plankton, particulate matter, benthos, and bottom sediments). Analysis of the carbohydrates is based on consecutive separation of their fractions with different solvents (water, alkali, and acid). Ratios of carbohydrate fractions allows to evaluate lability of carbohydrate complexes. They are also usable as an indicators of biogeochemical processes in the ocean, as well of genesis and degree of transformation of organic matter in bottom sediments and nodules. Similarity in monosaccharide composition is shown for dissolved organic matter and aqueous and alkaline fractions of seston and particulate matter.

Organic compounds in surface sediments from the Pakistan continental margin

In order to assess whether the oxygen-minimum zone (OMZ) in the Arabian sea has an effect on the preservation and composition of organic matter in surface sediments we investigated samples from three different transects on the Pakistan continental margin across the OMZ. In addition to determining the total amount of organic carbon (TOC), we analyzed the extractable lipids by gas chromatography, combined gas chromatography/mass spectrometry, and compound-specific stable carbon isotope measurements. The extractable lipids are dominated by marine organic matter as indicated by the abundance of lipids typical of marine biota and by the bulk and molecular isotopic composition. Sediments from within the OMZ are enriched in organic carbon and in several extractable lipids (i.e. phytol, n-alcohols, total sterols, n-C35 alkane) relative to stations above and below this zone. Other lipid concentrations, such as those of total n-fatty acids and total n-alkanes fail to show any relation to the OMZ. Only a weak correlation of TOC with mineral surface area was found in sediments deposited within the OMZ. In contrast, sediments from outside the OMZ do not show any relationship between TOC and surface area. Among the extractable lipids, only the n-alkane concentration is highly correlated with surface area in sediments from the Hab and Makran transects. In sediments from outside the OMZ, the phytol and sterol concentrations are also weakly correlated with mineral surface area. The depositional environment of the Indus Fan offers the best conditions for an enhanced preservation of organic matter. The OMZ, together with the undisturbed sedimentation at moderate rates, seems to be mainly responsible for the high TOC values in this area. Overall, the type of organic matter and its lability toward oxic degradation, the mineral surface area, the mineral composition, and possibly the secondary productivity by (sedimentary) bacteria also appear to have an influence on organic matter accumulation and composition.

Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T-166, that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.