Interferon-gamma plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus

Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into the rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N(G)-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUNEL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS. Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus.     

Comparative mapping and rapid karyotypic evolution in the genus helianthus

Comparative genetic linkage maps provide a powerful tool for the study of karyotypic evolution. We constructed a joint SSR/RAPD genetic linkage map of the Helianthus petiolaris genome and used it, along with an integrated SSR genetic linkage map derived from four independent H. annuus mapping populations, to examine the evolution of genome structure between these two annual sunflower species. The results of this work indicate the presence of 27 colinear segments resulting from a minimum of eight translocations and three inversions. These 11 rearrangements are more than previously suspected on the basis of either cytological or genetic map-based analyses. Taken together, these rearrangements required a minimum of 20 chromosomal breakages/fusions. On the basis of estimates of the time since divergence of these two species (750,000-1,000,000 years), this translates into an estimated rate of 5.5-7.3 chromosomal rearrangements per million years of evolution, the highest rate reported for any taxonomic group to date.     

Interchangeability and distinct properties of bacterial Fe-S cluster assembly systems: functional replacement of the isc and suf operons in Escherichia coli with the nifSU-like operon from Helicobacter pylori

The assembly of iron-sulfur (Fe-S) clusters, a key step in the post-translational maturation of Fe-S proteins, is mediated by a complex apparatus. In E. coli, this process involves two independent systems called ISC and SUF encoded by the iscSUA-hscBA-fdx gene cluster and sufABCDSE operon, respectively. Another system, termed NIF (nifSU), is required for the maturation of nitrogenase in nitrogen-fixing bacteria. We have developed a novel genetic system to gain further insight into these multi-component systems, and to determine how ISC, SUF and NIF might differ in their roles in Fe-S assembly. We have constructed an E. coli mutant lacking both the isc and suf operons, and this strain can only survive in the presence of a complementing plasmid. Using the plasmid replacement technique, we examined the isc and suf operons, and identified the genes essential for the function. Additionally, we have found that nifSU-like genes cloned from Helicobacter pylori are functionally exchangeable with the isc and suf operons. Thus, the NIF-like system participates in the maturation of a wide variety of Fe-S proteins. An increased ability of NIF to complement isc and suf loss was seen under anaerobic conditions. This may explain why the NIF system is only found in a limited number of bacterial species, and most other organisms prefer the ISC and/or SUF systems. While the differences between ISC and SUF were small with respect to the complementing activity, the SUF system appears to be more advantageous for bacterial growth in the presence of hydrogen peroxide.     

Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system

A protein-synthesizing system based on a minimal set of purified components was used to investigate the roles molecular chaperones play in the folding of newly synthesized polypeptides. After we ascertained that this system lacks intrinsic chaperones, the effect of adding chaperones in a co-translational or post-translational manner was directly evaluated. An aggregation-prone single-chain antibody was used as the model nascent chain. The participation of the trigger factor or the DnaK system during translation efficiently increased the level of functional protein that was generated. In addition, both systems also acted as chaperones after translation had been stopped. In contrast, the GroEL/ES system showed little or no co- or post-translational assistance in folding.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

AP-1-independent sensitization to oxidative stress-induced apoptosis by proteasome inhibitors

Hydrogen peroxide (H(2)O(2)) induces apoptosis of mesangial cells via c-Jun N-terminal kinase (JNK)-activator protein-1 (AP-1) and extracellular signal-regulated kinase (ERK)-AP-1 pathways. We recently found that subtoxic doses of proteasome inhibitors, MG132 and lactacystin, dramatically enhanced H(2)O(2)-induced apoptosis in mesangial cells. In this report, we examined molecular mechanisms involved in this phenomenon, especially focusing on AP-1 pathways. Reporter assays showed that MG132 induced activation of AP-1. However, pharmacological inhibitors of AP-1, retinoic acid, and curcumin, did not suppress the proapoptotic effect of MG132. Suppression of JNK-AP-1 by transfection with either a dominant-negative mutant of JNK or a dominant-negative mutant of c-Jun did not attenuate the apoptosis enhancement by MG132. Similarly, suppression of ERK-AP-1 by PD98059 or dominant-negative mutants of ERK did not affect the apoptosis-promoting effect of MG132. Interestingly, pretreatment with MG132 did not enhance activation of AP-1 by H(2)O(2). These data suggested a novel, AP-1-independent promotion of apoptosis by proteasome inhibitors.