Vellozone,a tetracyclic triterpene from Vellozia stipitata

The isolation of (20R)-20-hydroxy-24-methylenedammar-3-one from Vellozia stipitata is described.     

Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.     

Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum

Abscisic acid-treated callus of the resurrection plant Craterostigma plantagineum tolerates extreme desiccation. Nuclear proteins from tolerant callus bind specific sequence elements in the promoter region of the ABA and desiccation-inducible CDeT27–45 gene. One specific region of the promoter, which is protected from DNAse I treatment by DNA-binding activities, is different from previously reported ABA response elements. Four complexes of nuclear proteins and this DNA region are detected by electrophoretic mobility shift assay: two of these complexes (I and II) are readily detectable in untreated samples and are increased by ABA treatment while two other complexes (III and IV) accumulate only following ABA treatment and are prevented from accumulating by protein synthesis inhibitors. When a fragment containing the novel binding site is deleted from the wild-type promoter the ABA responsiveness of the promoter is removed; however, gain of function experiments using synthetic promoters in a protoplast transient assay suggest that besides the binding site other promoter elements are required. A second region of the promoter, containing the sequence element ACGT which is found in abscisic acid response elements, is also bound by nuclear proteins. The level of this second binding activity is similar in both untreated and ABA-treated cells and promoter/reporter gene constructs which contain only the four ACGT elements of the CDeT27–45 promoter are not ABA responsive in a C. plantagineum transient assay system     

PARP1 ADP-ribosylates lysine residues of the core histone tails

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.     

Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1

The rfb_kpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfb_kpO1cluster encode Rrfb_kpO1and RfbB_KpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbB_KpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbA_KpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbA_KpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbA_KpO1 and RfbB_KpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfb_KpO1 gene cluster deleted in rfbA_KpO1 and rfbB_KpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbA_KpO1 and RfbB_KpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan l-substituted lipopolysaccharide is produced. RfbA_KpO1 and RfbB_KpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbA_KpO1 represents the membrane-spanning translocator and RfbB_KpO1 couples the energy of ATP hydrolysis to the transport process.