K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle

KCl causes smooth muscle contraction by elevating intracellular free Ca2+, whereas receptor stimulation activates an additional mechanism, termed Ca2+ sensitization, that can involve activation of RhoA-associated kinase (ROK) and PKC. However, recent studies support the hypothesis that KCl may also increase Ca2+ sensitivity. Our data showed that the PKC inhibitor GF-109203X did not, whereas the ROK inhibitor Y-27632 did, inhibit KCl-induced tonic (5 min) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited BAY K 8644- and ionomycin-induced MLC phosphorylation and force but did not inhibit KCl-induced Ca2+ entry or peak ( approximately 15 s) force. Moreover, KCl and BAY K 8644 nearly doubled the amount of ROK colocalized to caveolae at 30 s, a time that preceded inhibition of force by Y-27632. Colocalization was not inhibited by Y-27632 but was abolished by nifedipine and the calmodulin blocker trifluoperazine. These data support the hypothesis that KCl caused Ca2+ sensitization via ROK activation. We discuss a novel model for ROK activation involving translocation to caveolae that is dependent on Ca2+ entry and involves Ca2+-calmodulin activation.     

Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton-gated cation channels

Acid-sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton-sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three-dimensional structure in solution was determined by means of (1)H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the beta-sheet and the beta-turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.     

What is the nature of the replicative niche of a stealthy bug named Brucella?

Brucella spp. are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts. The so-called phagosome to which they have adapted is their natural living niche. Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria. This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen. We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients. Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

Induction of antigen-specific CTL responses using antigens conjugated to short peptide vectors

Linear peptides (SynB vectors) with specific sequence motifs have been identified that are capable of enhancing the transport of a wide range of molecules into cells. These peptide vectors have been used to deliver exogenous peptides and protein Ags across the cell membrane and into the cytoplasm of cells. Specifically, in vitro analysis indicated that these SynB peptides enhanced the uptake of two 9-mer peptide Ags, NP(147-155) and Mtb(250-258) (T cell epitopes of influenza nucleoprotein and Mycobacterium tuberculosis, respectively) and the M. tuberculosis Ag Mtb8.4 protein, into K562 cells when covalently linked to the respective Ags. Furthermore, selected SynB vectors, when conjugated to these same Ags and used as immunogens, resulted in considerably enhanced Ag-specific CTL responses. Several SynB vectors were tested and resulted in varying levels of cellular uptake. The efficiency of uptake correlated with the ability of the SynB construct to deliver each epitope in vivo and induce specific CTL responses in mice. These data suggest that peptide vectors, such as SynB that transport target Ags across the cell membrane in a highly efficient manner, have significant potential for vaccine delivery.     

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.     

The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae

iota-Carrageenans are sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of red algae, which auto-associate into crystalline fibers made of aggregates of double-stranded helices. iota-Carrageenases, which constitute family 82 of glycoside hydrolases, fold into a right-handed beta-helix. Here, the structure of Alteromonas fortis iota-carrageenase bound to iota-carrageenan fragments was solved at 2.0A resolution (PDB 1KTW). The enzyme holds a iota-carrageenan tetrasaccharide (subsites +1 to +4) and a disaccharide (subsites -3, -4), thus providing the first direct determination of a 3D structure of iota-carrageenan. Electrostatic interactions between basic protein residues and the sulfate substituents of the polysaccharide chain dominate iota-carrageenan recognition. Glu245 and Asp247 are the proton donor and the base catalyst, respectively. C-terminal domain A, which was highly flexible in the native enzyme structure, adopts a alpha/beta-fold, also found in DNA/RNA-binding domains. In the substrate-enzyme complex, this polyanion-binding module shifts toward the beta-helix groove, forming a tunnel. Thus, from an open conformation which allows for the initial endo-attack of iota-carrageenan chains, the enzyme switches to a closed-tunnel form, consistent with its highly processive character, as seen from the electron-microscopy analysis of the degradation of iota-carrageenan fibers.     

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.     

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.