Synthesis and characterization of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine: crystal structures of 4,6-O-butylidene-alpha-D-glucopyranose, 4,6-O-butylidene-beta-D-glucopyranosylamine and 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine

4,6-O-Butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. 1H and 13C NMR studies showed the presence of the beta-anomer, which has also been confirmed by the crystal structure. The molecular structure of this compound showed the presence of the tridentate ONO ligation-core. Both precursors, 4,6-O-butylidene-alpha-D-glucopyranose and 4,6-O-butylidene-beta-D-glucopyranosylamine were characterized using single crystal X-ray diffraction. The alpha-anomeric nature of the former and beta-anomeric nature of the latter were proposed based on 1H NMR studies and were confirmed by determining the crystal structures. In addition, the crystal structure of 4,6-O-butylidene-beta-D-glucopyranosylamine revealed the C-1-N-glycosylation. In all the three molecules, the saccharide unit exhibits a 4C(1) chair conformation. In the lattice, the molecules are connected by hydrogen-bond interactions. The conformation of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine is stabilized via an O-H...N intramolecular interaction, and each molecule in the lattice interacts with three neighboring molecules through hydrogen bonds of the type O-H...O and C-H...O.     

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Suppressive subtractive hybridization detects extensive genomic diversity in Thermotoga maritima

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.     

Distribution of spontaneous mutants and inferences about the replication mode of the RNA bacteriophage phi6

When a parent virus replicates inside its host, it must first use its own genome as the template for replication. However, once progeny genomes are produced, the progeny can in turn act as templates. Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly. While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus. We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage phi6 and find that the distribution closely matches a Poisson distribution. The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication. However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate. By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10(-4). We discuss whether different replication modes may confer an adaptive advantage to viruses.     

The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers

Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.     

Structure of the human argininosuccinate synthetase gene and an improved system for molecular diagnostics in patients with classical and mild citrullinemia

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia, an autosomal recessive inherited defect of the urea cycle. Most patients described so far have presented with the classical form of the disease. There are also patients with a mild form of citrullinemia in whom the exact molecular basis and clinical relevance are uncertain. Mutations in the human ASS gene have not yet been described in mildly affected or asymptomatic patients with citrullinemia. The genomic sequence of the human ASS gene is not precisely known making mutation analysis difficult. Here, the entire genomic DNA sequence and mutations in the ASS gene of patients with the classical and mild form of the disease are described. The mutations c.1168G-->A (G390R) and IVS13+5 G-->A and the novel mutation c.323G-->T (R108L) have been found to be associated with classical citrullinemia, whereas the novel mutations c.535T-->G (W179R), and c.1085G-->T (G362V) have been detected on alleles of the mildly affected patients. Thus, mutations found in the human ASS gene of asymptomatic children with biochemical abnormalities and in some cases enzymatically proven citrullinemia have allowed us to classify these cases as ASS-deficient patients. The elucidation of the structure of the human ASS gene has made possible the use of intronic primers for molecular analysis of patients with mild disease and the classical form, and provides another option for prenatal diagnostics in affected families with the severe type.     

Focal adhesion kinase activated by beta(4) integrin ligation to mCLCA1 mediates early metastatic growth

Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by beta(4) integrin adhesion to the mouse lung endothelial Ca(2+)-activated chloride channel protein mCLCA1. In a manner independent of Met, beta(4) integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, beta(4) integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth.     

Location of N-unsubstituted glucosamine residues in heparan sulfate

Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH(2)) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either N-sulfated or N-unsubstituted glucosamine units followed by reduction with NaB(3)H(4). The labeled products were characterized following complementary deamination steps. The proportion of GlcNH(2) units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH(2) units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20-30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharide-protein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH(2)6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH(2) residues are formed through regulated, incomplete action of an N-deacetylase/N-sulfotransferase enzyme.