Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10(5) Hz in view of the suppressed peaks from [3-(13)C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal alpha-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.     

Roles of Ser130 and Thr126 in chloride binding and photocycle of pharaonis halorhodopsin

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl(-)binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl(-) ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.     

Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon,Thermoplasma acidophilum

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.     

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10(-11) mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.     

Characterization of entry mechanisms of human herpesvirus 8 by using an Rta-dependent reporter cell line

To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.     

Induction of protective immune responses against R5 human immunodeficiency virus type 1 (HIV-1) infection in hu-PBL-SCID mice by intrasplenic immunization with HIV-1-pulsed dendritic cells: possible involvement of a novel factor of human CD4(+) T-cell origin

The potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1(JR-CSF). Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4(+) T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1alpha, MIP-1beta, alpha interferon (IFN-alpha), IFN-beta, IFN-gamma, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-alpha), or TNF-beta failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4(+) T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.     

Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting

The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl(3), which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.