PAR1 cleavage and signaling in response to activated protein C and thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.     

Selective ligand purification using high-performance affinity beads

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.     

Cytochrome P450 CYP307A1/Spook: a regulator for ecdysone synthesis in insects

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450 gene designated Cyp307a1. Its expression level positively correlates with a change in the hemolymph ecdysteroid titer. In addition, Drosophila Cyp307a1 is encoded in the spook locus, one of the Halloween mutant family members showing a low ecdysone titer in vivo, suggesting that Cyp307a1 is involved in ecdysone synthesis. While Drosophila Cyp307a1 is expressed in the early embryos and adult ovaries, the expression is not observed in the PGs of embryos or third instar larvae. These results suggest a difference in the ecdysone synthesis pathways during larval development in these insects.     

Heat shock induces phosphorylation of histone H2AX in mammalian cells

Heat shock induces a variety of biological events including gene activation, cell cycle arrest, and apoptosis. Heat shock has recently been shown to be potentially useful when combined with radiation in cancer therapy, probably because, in mammalian cells, heat inhibits the repair of double-strand breaks (DSBs) induced by ionizing radiation. It remains unclear, however, whether heat shock by itself induces DSBs. In this communication, we present the first evidence that heat shock induces the phosphorylated form of histone H2AX, which is thought to be generated at the chromatin proximal to DSB sites. These results suggest that heat shock induces DSBs in mammalian cells and may provide direct evidence to explain previous reports on DSB-related events occurring after heat shock treatment.     

Can the low-resolution structures of photointermediates of bacteriorhodopsin explain their crystal structures?

To understand the molecular mechanism of light-driven proton pumps, the structures of the photointermediates of bacteriorhodopsin have been intensively investigated. Low-resolution diffraction techniques have demonstrated substantial conformational changes at the helix level in the M and N intermediates, between which there are noticeable differences. The intermediate structures at atomic resolution have also been solved by x-ray crystallography. Although the crystal structures have demonstrated local structural changes, such as hydrogen bond network rearrangements including water molecules, the large conformational changes at the helix level are not necessarily observed. Furthermore, the two reported crystal structures of an intermediate accumulated using a common method were distinct. To reconcile these apparent discrepancies, low-resolution projection maps were calculated from the crystal structures and compared to the low-resolution intermediate structures obtained using native membranes. The crystal structures can be categorized into three groups, which qualitatively correspond to the low-resolution structures of the M1-type, M2-type, and N-type determined in the native membrane. Based on these results, we conclude that at least three types of intermediate structures play a role during the photocycle.     

A novel adjuvant for mucosal immunity to HIV-1 gp120 in nonhuman primates

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4(+)) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.     

Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/JxDBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.     

Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.     

Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases

We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization. The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs). Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively. The point mutation of the Cys ligand of each type 1 Cu with Ala gives two mutants, C114A and C260A, possessing one type 1 Cu and one type 2 Cu. The spectroscopic data of C114A reveal that the C-terminal NIR-like domain has the green type 1 Cu (type 1 Cu(C)), showing two intense absorption peaks at 455 (epsilon = 2600 M(-1) cm(-1)) and 600 nm (epsilon = 2800 M(-1) cm(-1)) and a rhombic EPR signal like those of the green type 1 Cu of Achromobacter cycloclastes NIR (AcNlR). The spectroscopic data of C260A elucidate that the N-terminal Pc-like domain in HdNIR contains the blue type 1 Cu (type 1 Cu(N)), exhibiting an intense absorption band at 605 nm (epsilon = 2900 M(-1) cm(-1)) and an axial EPR signal like those of the blue type 1 Cu of Alcaligenes xylosoxidans NIR (AxNIR). The sum of the visible absorption or EPR spectra of C114A and C260A is almost equal to the corresponding spectrum of wild-type HdNIR. The spectroscopic characterization of the type 1 Cu indicates that the geometries of the type 1 Cu(N) and Cu(C) sites are slightly distorted tetrahedral (or axially elongated bipyramidal) and flattened tetrahedral, respectively. In the cyclic voltammograms, the midpoint potentials (E(1/2)), probably because of the type 1 Cu ions of C114A and C260A, are observed at +321 and +336 mV versus normal hydrogen electrode (NHE) at pH 7.0, respectively. These values, which are close to each other, are more positive than those ( approximately +0.24-0.28 V at pH 7.0) of the type 1 Cu sites of AcNIR and AxNIR. The electron-accepting capability of C114A from cytochrome c(550) is almost similar to that of wild-type HdNIR, whereas that of C260A is very low. This suggests that the type 1 Cu(C) in the C-terminal domain is essential for the enzyme functions of HdNIR.