Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.     

HybF, a zinc-containing protein involved in NiFe hydrogenase maturation

HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a K(D) of 1.87 microM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.     

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.     

Sulfur stable isotopes separate producers in marine food-web analysis

Ecological applications of stable isotope analysis rely on different producers having distinct isotopic ratios to trace energy and nutrient transfer to consumers. Carbon (C) and nitrogen (N) are the usual elements analysed. We tested the hypothesis that producers unable to be separated using C and N would be separated by sulphur (S), by reviewing estuarine and marine food web studies using all three elements (total of 836 pairwise comparisons between producers). S had a wider range of values across all producers than C and N (S: 34.4, C: 23.3, N: 18.7), and a higher mean difference among producers (S: 9.3, C: 6.5, N: 3.3). We varied from 1 to 10 the distance producers must be apart to be considered separate. For each of these gap distances, S-separated producers tied on C and N in 40% or more of cases. Comparing the three elements individually, S had fewer tied pairs of producers for any gap distance than C or N. However, S also has higher within-producer variability. Statistical tests on simulated data showed that this higher variability caused S to be less effective than C for analysing differences among mean producer values, yet mixing models showed that S had the smallest confidence intervals around mean estimates of source contributions to consumers. We also examined the spatial and temporal scales over which S isotope signatures of the saltmarsh plant Spartina alterniflora varied. Differences between samples taken within tens of metres were smallest, but between samples hundreds of metres apart were as different as samples thousands of kilometres apart. The time between samples being taken did not influence S signatures. Overall, the use of S is recommended because it has a high probability of distinguishing the contribution of different producers to aquatic food webs. When two elements are employed, the combination of S and C separates more producers than any other combination.     

Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Orbitrap mass analyzer--overview and applications in proteomics

The orbitrap mass analyzer is proving itself as a useful addition to a proteomics tool box. The key attributes of this analyzer are accurate mass and high resolution similar to those achievable with FT ICR instrumentation. The basic principles underlying these capabilities, and how they translate into benefits in real-life proteomics experiments are discussed. The focus is on reviewing examples of protein identification with bottom-up and top-down approaches, and detection of post-translational modifications.     

Suberoylanilide hydroxamic acid (SAHA) at subtoxic concentrations increases the adhesivity of human leukemic cells to fibronectin

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL‐MK1, K562, CML‐T1, Karpas‐299, HL‐60, and ML‐2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose‐dependent and cell type‐dependent cell death which displayed apoptotic features (caspase‐3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5–1 µM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma‐derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin β1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA‐induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate). J. Cell. Biochem. 109: 184–195, 2010. © 2009 Wiley‐Liss, Inc.     

The Archaeal Lsm Protein Binds to Small RNAs

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.