<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of human osteopontin

Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin.     

Sepsis correlated with increased erythrocyte Na<Superscript>+</Superscript> content and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> pump activity

The aims of the present study were twofold: (1) simultaneous determinations of Na(+) transport parameters of erythrocytes from 40 healthy donors and 28 septic patients as assessed by a score of severity of sepsis (SSS), and (2) examination of the correlation between the SSS and specific Na(+) transport abnormalities. Erythrocytes were obtained and loaded with different ionic compositions and cellular Na(+) contents before determination of the near-maximal Na(+) pump rate (Vmax), the physiological extrusion rate of Na(+) (v) and the number of ouabain-binding sites (Bmax). In erythrocytes from septic patients, the cellular Na(+) content was 28% higher (p < 0.001), with no differences in water content compared to erythrocytes from healthy donors. This elevated Na(+) content was accompanied by significantly higher values for Vmax (43%), v (24%) and Bmax (48%) of the Na(+) pump in septic erythrocytes. Moreover, significant positive correlations existed between Vmax and SSS (p = 0.028) and between cellular Na(+) content and SSS (p = 0.005). These data suggest that during sepsis, membrane alterations occur and result in an increased cellular Na(+) content. Active Na(+) transport (Vmax and v) was significantly stimulated, possibly as a consequence of a secondary response to the elevated Na(+) of cells. Both cellular Na(+) and Vmax correlated well with the severity of sepsis, suggesting that these altered transport parameters may reflect the progress of sepsis.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

<Superscript>137</Superscript>Cs, <Superscript>40</Superscript>K, <Superscript>238</Superscript>Pu, <Superscript>239+240</Superscript>Pu and <Superscript>90</Superscript>Sr in biological samples from King George Island (Southern Shetlands) in Antarctica

There are few data reported on radionuclide contamination in Antarctica. The aim of this paper is to report ¹³⁷Cs, ⁹⁰Sr and ^238,239+240Pu and ⁴⁰K activity concentrations measured in biological samples collected from King George Island (Southern Shetlands, Antarctica), mostly during 2001–2002. The samples included: bones, eggshells and feathers of penguin Pygoscelis papua, bones and feathers of petrel Daption capense, bones and fur of seal Mirounga leonina, algae Himantothallus grandifolius, Desmarestia anceps and Cystosphaera jacquinotii, fish Notothenia corriceps, sea invertebrates Amphipoda, shells of limpet Nacella concina, lichen Usnea aurantiaco-atra, vascular plants Deschampsia antarctica and Colobanthus quitensis, fungi Omphalina pyxidata, moss Sanionia uncinata and soil. The results show a large variation in some activity concentrations. Samples from the marine environment had lower contamination levels than those from terrestrial ecosystems. The highest activity concentrations for all radionuclides were found in lichen and, to a lesser extent, in mosses, probably because lichens take up atmospheric pollutants and retain them. The only significant correlation (except for that expected between ²³⁸Pu and ^239+240Pu) was noted for moss and lichen samples between plutonium and ⁹⁰Sr. A tendency to a slow decrease with time seems to be occurring. Analyses of the activity ratios show varying fractionation between various radionuclides in different organisms. Algae were relatively more highly contaminated with plutonium and radiostrontium, and depleted with radiocesium. Feathers had the lowest plutonium concentrations. Radiostrontium and, to a lesser extent, Pu accumulated in bones. The present low intensity of fallout in Antarctic has a lower ²³⁸Pu/^239+240Pu activity ratio than that expected for global fallout.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Affordable uniform isotope labeling with <Superscript>2</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N in insect cells

For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for ¹⁵N and ¹³C with yields comparable to expression in full media. For ²H,¹⁵N and ²H,¹³C,¹⁵N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of sialyl Lewis<Superscript>a</Superscript> (sLe<Superscript>a</Superscript>, CA19-9) and construction of an immunogenic sLe<Superscript>a</Superscript> vaccine

Sialyl Lewis^a (sLe^a), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe^a is known to be the ligand for endothelial cell selectins suggesting a role for sLe^a in cancer metastases and adhesion. For these reasons, sLe^a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe^a is structurally similar to sLe^x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe^a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe^a hexasaccharide and its conjugation to KLH to construct a sLe^a-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe^a-HSA by ELISA and against sLe^a positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe^a plus GPI-0100 or unconjugated sLe^a mixed with KLH plus GPI-0100 failed to produce antibodies against sLe^a. However, mice immunized with sLe^a-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe^a-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe^a by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe^a positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le^y, Le^x, and sLe^x by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe^a positive cancer in clinical settings. Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA 92121. The sLe^a vaccine is licensed to MabVax.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.