(Figure 3d) Export production reconstruction based on planktonic foraminiferal sampled in surface sediments

To reconstruct Export Productivity (Pexp), 27 taxonomic categories of the planktonic foraminifera census data were used with the modern analog technique SIMMAX 28 (Pflaumann et al., 1996, doi:10.1029/95PA01743; 2003, doi:10.1029/2002PA000774). To the 26 taxonomic groups widely used and listed in Kucera et al. (2005, doi:10.1016/j.quascirev.2004.07.014), Turborotalita humilis was added in our calibration as it is associated with the PCC source region (Meggers et al., 2002, doi:10.1016/S0967-0645(02)00103-0). The modern analog file is based on the Iberian margin database (Salgueiro et al., 2008, doi:10.1016/j.marmicro.2007.09.003) combined with the North Atlantic surface samples used by the MARGO project (Kucera et al., 2005). This results in a total of 999 analogs for Pexp. Modern oceanic primary productivity (PP) is obtained for each site by averaging 12 monthly primary productivity values for a 8-year period (1978-1986) that were estimated from satellite color data (CZCS) and gridded at 0.5° latitude - longitude fields (Antoine et al., 1996, doi:10.1029/95GB02832). Export Productivity (Pexp) was calculated from the PP values following the empirical relationship Pexp = PP**2/400 for primary production below 200 gC/m**2/yr, and Pexp = PP/2 for primary production above 200 gC/m2/yr (Eppley and Peterson, 1979, doi:10.1038/282677a0; Sarnthein et al., 1988, doi:10.1029/PA003i003p00361). The residuals gives the differences between satellite based Pexp and foraminiferal Pexp.

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80–84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445–448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlidea and thereby may regulate the steady-state level of light-harvesting POR-Pchlide comple

CO2and Rn degassing from the natural analog of Campo de Calatrava(Spain): Implications for monitoring of CO2storage sites

Natural analogs offer a valuable opportunity to investigate the long-term impacts associated with thepotential leakage in geological storage of CO2.Degassing of CO2and radon isotopes (222Rn?220Rn) from soil, gas vents and thermal water dischargeswas investigated in the natural analog of Campo de Calatrava Volcanic Field (CCVF; Central Spain) todetermine the CO2?Rn relationships and to assess the role of CO2as carrier gas for radon. Furthermore,radon measurements to discriminate between shallow and deep gas sources were evaluated under theperspective of their applicability in monitoring programs of carbon storage projects.CO2flux as high as 5000 g m?2d?1and222Rn activities up to 430 kBq m?3were measured;220Rn activi-ties were one order of magnitude lower than those of222Rn. The222Rn/220Rn ratios were used to constrainthe source of the Campo de Calatrava soil gases since a positive correlation between radon isotopic ratiosand CO2fluxes was observed. Thus, in agreement with previous studies, our results indicate a deepmantle-related origin of CO2for both free and soil gases, suggesting that carbon dioxide is an efficientcarrier for Rn. Furthermore, it was ascertained that the increase of222Rn in the soil gases was likely pro-duced by two main processes: (i) direct transport by a carrier gas, i.e., CO2and (ii) generation at shallowlevel due to the presence of relatively high concentrations of dissolved U and Ra in the thermal aquiferof Campo de Calatrava.The diffuse CO2soil flux and radon isotopic surveys carried out in the Campo de Calatrava VolcanicFields can also be applicable to geochemical monitoring programs in CCS (Carbon Capture and Storage)areas as these parameters are useful to: (i) constrain CO2leakages once detected and (ii) monitor both theevolution of the leakages and the effectiveness of subsequent remediation activities. These measurementscan also conveniently be used to detect diffuse leakages.

Structural requirements for 5-HT2A and 5-HT1A serotonin receptor potentiation by the biologically active lipid oleamide

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT1A receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT2A and 5-HT1A response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT2A receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the Δ9-cis double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT1A potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT2A, but not the 5-HT1A, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.

Calculated 11B–13C NMR chemical shift relationship in hypercoordinate methonium and boronium ions

The boronium-carbonium continuum was extended to include hypercoordinated protonated methanes and their boron analogs. The 11B NMR chemical shifts of the hypercoordinated hydriodo boron compounds and the 13C NMR chemical shifts of the corresponding isoelectronic and isostructural carbocations were calculated by using the GIAO-MP2 method. The data show good linear correlation between 11B and 13C NMR chemical shifts, which indicates that the same factors that determine the chemical shifts of the boron nuclei also govern the chemical shifts of carbon nuclei of these hypercoordinated hydriodo compounds.

The role of the ClpA chaperone in proteolysis by ClpAP

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA–ClpP–substrate complexes assemble either by ClpA–substrate complexes interacting with ClpP or by ClpA–ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA–ClpP–substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09–0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-{N-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to ≈40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

Catalytic antibodies have shown great promise for catalyzing a tremendously diverse set of natural and unnatural chemical transformations. However, few catalytic antibodies have efficiencies that approach those of natural enzymes. In principle, random mutagenesis procedures such as phage display could be used to improve the catalytic activities of existing antibodies; however, these studies have been hampered by difficulties in the recombinant expression of antibodies. Here, we have grafted the antigen binding loops from a murine-derived catalytic antibody, 17E8, onto a human antibody framework in an effort to overcome difficulties associated with recombinant expression and phage display of this antibody. “Humanized” 17E8 retained similar catalytic and hapten binding properties as the murine antibody while levels of functional Fab displayed on phage were 200-fold higher than for a murine variable region/human constant region chimeric Fab. This construct was used to prepare combinatorial libraries. Affinity panning of these resulted in the selection of variants with 2- to 8-fold improvements in binding affinity for a phosphonate transition-state analog. Surprisingly, none of the affinity-matured variants was more catalytically active than the parent antibody and some were significantly less active. By contrast, a weaker binding variant was identified with 2-fold greater catalytic activity and incorporation of a single substitution (Tyr-100aH → Asn) from this variant into the parent antibody led to a 5-fold increase in catalytic efficiency. Thus, phage display methods can be readily used to optimize binding of catalytic antibodies to transition-state analogs, and when used in conjunction with limited screening for catalysis can identify variants with higher catalytic efficiencies.

Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin–myosin–ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1–SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1–SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.

Human deoxycytidine kinase is located in the cell nucleus

Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.

Topoisomerase II drives DNA transport by hydrolyzing one ATP

DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme’s mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme’s mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10−8 M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using 35S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [35S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent Kd values for [35S]PSK-α binding were found to be 1 × 10−9 M for the high-affinity type and 1 × 10−7 M for the low-affinity type, with maximal numbers of binding sites of 1 × 104 sites per cell and 1 × 105 sites per cell, respectively. Competition studies with [35S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.