Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Structure, regulation and function of ghrelin

Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein-coupled receptor. Ghrelin is a 28-amino acid peptide existing in two major forms: n-octanoyl-modified ghrelin, which possesses an n-octanoyl modification on serine-3 and des-acyl ghrelin. Fatty acid modification of ghrelin is essential for ghrelin-induced growth hormone release from the pituitary and appetite stimulation. This acyl-modification of ghrelin is catalysed by ghrelin-O-acyl transferase recently identified. Despite the number of innovative advancements in this field of research, there are still many aspects of ghrelin function and biosynthesis process that remain to be clarified. Here, we review the current understanding of the structure, regulation and function of ghrelin; this review is intended for researchers who will be involved in this field in the future.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

Regioselective phosphorylation of branched cyclodextrins with cyclo-mono-mu-imidotriphosphate

The phosphorylation of the branched cyclodextrins, mono-6-O-(alpha-D-glucopyranosyl)cyclomaltohexaose, mono-6-O-(alpha-D-maltosyl)cyclomaltohexaose, mono-6-O-(alpha-D-glucopyranosyl)cyclomaltoheptaose, and mono-6-O-(alpha-D-maltosyl)cyclomaltoheptaose, in aqueous solution by sodium cyclo-mono-mu-imidotriphosphate (cMITP) was examined. In these reactions, only the 2-OH group of a single alpha-D-glucopyranosyl residue of the cyclodextrin ring was phosphorylated, in a maximum yield of 67%. A possible mechanism for the phosphorylation is discussed.     

Expression of starvation-induced genes in zygotes ofDictyostelium discoideum

Two cDNA fragments induced in developing zygotes ofDictyostelium discoideum were isolated by mRNA differential display. the relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms inD. discoideum.     

Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3��-Hydroxysterol ��24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.     

Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.     

A novel screening method for microorganisms that efficiently remove heavy metals from aqueous solution

A novel bioassay system for estimating concentrations of several heavy metal ions was carried out with yeast mutants which are highly sensitive to heavy metal ions. The method does not need an atomic adsorption spectrometer or other special equipment. It is suitable for screening of microorganisms that efficiently remove heavy metal ions from aqueous solution.     

Molecular breeding of yeast with higher metal-adsorption capacity by expression of histidine-repeat insertion in the protein anchored to the cell wall

A fusion protein of hexa-histidine repeat (His) and glycosylphosphatidylinositol (GPI)-anchor region of Saccharomyces cerevisiae Cwp1 with Aspergillus oryzae Taka-amylase A (TAA) was expressed on the yeast cell surface. The expressed fusion protein (TAA-His-Cwp1) was localized on the cell wall and demonstrated amylolytic activity. In comparison with the TAA-Cwp1 expressing strain, these cells exhibited 1.6- to 2.8-fold higher adsorbing capacity for Cu(2+), Ni(2+), and Zn(2+).     

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells

Background

Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.

Methodology/Principal Findings

Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.

Conclusions/Significance

RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.