Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent kcat of 0.1 min–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ∼5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (Kd ∼ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (Kd ∼ 23.4 nM) and the affinity for its final β-elimination product was much lower (Kd ∼ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

Top-down morphogenesis of colorectal tumors

One of the fundamental tenets of oncology is that tumors arise from stem cells. In the colon, stem cells are thought to reside at the base of crypts. In the early stages of tumorigenesis, however, dysplastic cells are routinely found at the luminal surface of the crypts whereas the cells at the bases of these same crypts appear morphologically normal. To understand this discrepancy, we evaluated the molecular characteristics of cells isolated from the bases and orifices of the same crypts in small colorectal adenomas. We found that the dysplastic cells at the tops of the crypts often exhibited genetic alterations of adenomatous polyposis coli (APC) and neoplasia-associated patterns of gene expression. In contrast, cells located at the base of these same crypts did not contain such alterations and were not clonally related to the contiguous transformed cells above them. These results imply that development of adenomatous polyps proceeds through a top-down mechanism. Genetically altered cells in the superficial portions of the mucosae spread laterally and downward to form new crypts that first connect to preexisting normal crypts and eventually replace them.

Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer.

The predisposition to colon cancer is multigenetically controlled in animals and probably also in humans. We have analyzed the multigenic control of susceptibility to 1,2-dimethylhydrazine-induced colon tumors in mice by using a set of 20 homozygous CcS/Dem recombinant congenic strains, each of which contains a different random subset of approximately 12.5% of genes from the susceptible strain STS/A and 87.5% of genes from the relatively resistant strain BALB/cHeA. Some CcS/Dem strains received the alleles from the susceptible strain STS/A at one or more of the multiple colon tumor susceptibility loci and are susceptible, whereas others are resistant. Linkage analysis shows that these susceptibility genes are different from the mouse homologs of the genes known to be somatically mutated in human colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2, and probably also MLH1). Different subsets of genes control tumor numbers and size. Two colon cancer susceptibility genes, Scc1 and Scc2, map to mouse chromosome 2. The Scc1 locus has been mapped to a narrow region of 2.4 centimorgans (90% confidence interval).

Truncation of the class II beta-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes.

Previous studies have established that antigen presenting cells (APC) expressing major histocompatibility complex class II beta chains with truncated cytoplasmic domains are impaired in their capacity to activate T cells. While it had been widely accepted that this impairment is due to a defect in class II cytoplasmic domain-dependent signal transduction, we recently generated transgenic mice expressing only truncated class II beta chains, and functional analyses of APC from these mice revealed signaling-independent defects in antigen presentation. Here, we demonstrate that T cells primed on such transgenic APC respond better to stimulation by APC expressing truncated beta chains than by wild-type APC. This finding suggests that APC expressing truncated class II beta chains are not inherently defective in their antigen presenting capacity but, rather, may differ from wild-type APC in the peptide antigens that they present. Indeed, analysis of the peptides bound to class II molecules isolated from normal and transgenic spleen cells revealed clear differences. Most notably, the level of class II-associated invariant chain-derived peptides (CLIP) is significantly reduced in cells expressing only truncated beta chains. Prior studies have established that CLIP and antigenic peptides compete for binding to class II molecules. Thus, our results suggest that the cytoplasmic domain of the class II beta chain affects antigen presentation by influencing the level of CLIP/class II complexes.

Trends in mortality due to motor vehicle traffic accident injuries between 1987 and 2011 in a Spanish region (Comunitat Valenciana)

Objective: To analyse the time evolution of the rates of mortality due to motor vehicle traffic accidents (MVTA) injuries that occurred among the general population of Comunitat Valenciana between 1987 and 2011, as well as to identify trend changes by sex and age group. Methods: An observational study of annual mortality trends between 1987 and 2011. We studied all deaths due to MVTA injuries that occurred during this period of time among the non-institutionalised population residing in Comunitat Valenciana (a Spanish Mediterranean region that had a population of 5,117,190 inhabitants in 2011). The rates of mortality due to MVTA injuries were calculated for each sex and year studied. These rates were standardised by age for the total population and for specific age groups using the direct method (age-standardised rate – ASR). Joinpoint regression models were used in order to detect significant trend changes. Additionally, the annual percentage change (APC) of the ASRs was calculated for each trend segment, which is reflected in statistically significant joinpoints. Results: For all ages, ASRs decrease greatly in both men and women (70% decrease between 1990 and 2011). In 1990 and 2011, men have rates of 36.5 and 5.2 per 100,000 men/year, respectively. In the same years, women have rates of 8.0 and 0.9 per 100,000 women/year, respectively. This decrease reaches up to 90% in the age group 15–34 years in both men and women. ASR ratios for men and women increased over time for all ages: this ratio was 3.9 in 1987; 4.6 in 1990; and 5.8 in 2011. For both men and women, there is a first significant segment (p < 0.05) with an increasing trend between 1987 and 1989–1990. After 1990, there are 3 segments with a significant decreasing APC (1990–1993, 1993–2005 and 2005–2011, in the case of men; and 1989–1996, 1999–2007 and 2007–2011, in the case of women). Conclusion: The risk of death due to motor vehicle traffic accidents injuries has decreased significantly, especially in the case of women, for the last 25 years in Comunitat Valenciana, mainly as of 2006. This may be a consequence of the road-safety measures that have been implemented in Spain and in Comunitat Valenciana since 2004. The economic crisis that this country has undergone since 2008 may have also been a contributing factor to this decrease. Despite the decrease, ASR ratios for men and women increased over time and it is still a high-risk cause of death among young men. It is thus important that the measures that helped decrease the risk of death are maintained and improved over time.

Rock-magnetic data of ODP Site 105-645 (Appendix)

We present the rock-magnetic stratigraphy for Ocean Drilling Program (ODP) Leg 105, Site 645 (Baffin Bay). Variations in magnetic mineral concentration (X) and particle size (XAKM/X) are used to correlate advanced piston corer (APC) cores from several holes at Site 645. Downsite variations in rock-magnetic parameters and carbonate content are placed within the framework of inferred changes in sedimentation. These results suggest an initiation of Northern Hemisphere ice rafting in Baffin Bay in the early-late Pliocene (approximately 3.4 Ma).

Polarity chron boundaries and interval sedimentation rates of ODP Leg 162 sites

Magnetic polarity stratigraphies for Sites 980-984 are based on shipboard measurements from the pass-through magnetometer after alternating field (AF) demagnetization at a peak field of 25 mT and shore-based stepwise AF demagnetization of discrete samples. The characteristic magnetization component was determined after AF demagnetization removed the steep downward drill-string-related magnetic overprint. Peak AF fields in the 20-30 mT range were required to resolve the component, carried by magnetite, that was used to produce unambiguous Pliocene-Pleistocene magnetic stratigraphies at all five sites. At Sites 980 and 983, magnetic stratigraphies were resolved to the base of the recovered advanced hydraulic piston corer (APC) section, which lies in the Matuyama Chron (1r.2r) and Olduvai Subchron (2n), respectively. At Sites 981 and 982, magnetization intensities decrease sharply in the normal polarity zone corresponding to the Gauss Chron (2An), and magnetic stratigraphies below this level could not be resolved. At Site 984, the resolution of magnetic stratigraphy was curtailed at ~250 meters below seafloor (Olduvai Subchron) by core deformation at the base of the APC section and in the underlying extended core barrel section. As the magnetic stratigraphies at all four sites are unequivocal, polarity chron interpretations can be made without aid from the biostratigraphy. Mean sedimentation rates within polarity chrons have been calculated and Pliocene-Pleistocene biomagnetostratigraphic correlations tested.

Illinois reasonable further progress report for 1985 : ozone and carbon monoxide.

"September, 1986."

Illinois reasonable further progress report for 1986 : ozone and carbon monoxide.

"September, 1987."

Project plan for the sampling and analysis of polychlorinated biphenyls in the Lake Calumet area of southeast Chicago.

Cover title: Southeast Chicago air quality: a plan for the evaluation of PCB's.

Granulocyte colony-stimulating factor modulates alpha-galactosylceramide-responsive human V alpha 24(+) V beta 11(+) NKT cells

Despite more than a 10-fold increase in T cell numbers in G-CSF-mobilized peripheral blood stem cell (PBSC) grafts, incidence and severity of acute graft-vs-host disease (GVHD) are comparable to bone marrow transplantation. As CD1d-restricted, Valpha24(+)Vbeta11(+) NKT cells have pivotal immune regulatory functions and may influence GVHD, we aimed to determine whether G-CSF has any effects on human NKT cells. In this study, we examined the frequency and absolute numbers of peripheral blood NKT cells in healthy stem cell donors (n = 8) before and following G-CSF (filgrastim) treatment. Effects of in vivo and in vitro G-CSF on NKT cell cytokine expression profiles and on responsiveness of NKT cell subpopulations to specific stimulation by alpha-galactosylceramide (alpha-GalCer) were assessed. Contrary to the effects on conventional T cells, the absolute number of peripheral blood NKT cells was unaffected by G-CSF administration. Furthermore, responsiveness of NKT cells to alpha-GalCer stimulation was significantly decreased (p < 0.05) following exposure to G-CSF in vivo. This hyporesponsiveness was predominantly due to a direct effect on NKT cells, with a lesser contribution from G-CSF-mediated changes in APC. G-CSF administration resulted in polarization of NKT cells toward a Th2, IL-4-secreting phenotype following alpha-GalCer stimulation and preferential expansion of the CD4(+) NKT cell subset. We conclude that G-CSF has previously unrecognized differential effects in vivo on NKT cells and conventional MHC-restricted T cells, and effects on NKT cells may contribute to the lower than expected incidence of GVHD following allogeneic peripheral blood stem cell transplantation.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

Failure of a medulloblastoma-derived mutant of SUFU to suppress WNT signaling

Germline mutations of APC in patients with Turcot syndrome (colon cancer and medulloblastoma), was well as somatic mutations of APC, beta-catenin, and Axin in sporadic medulloblastomas (MBs) have shown the importance of WNT signaling in the pathogenesis of MB. A subset of children with MB have germline mutations of SUFU, a known inhibitor of Hedgehog signal transduction. A recent report suggested that murine Sufu can bind beta-catenin, export it from the nucleus, and thereby repress beta-catenin/T-cell factor (Tcf)-mediated transcription. We show that an MB-derived mutant of SUFU has lost the ability to decrease nuclear levels of beta-catenin, and cannot inhibit beta-catenin/Tcf-mediated transcription as compared to wild type SUFU. Our results suggest that loss of function of SUFU results in overactivity of both the Sonic Hedgehog, and the WNT signaling pathways, leading to excessive proliferation and failure to differentiate resulting in MB.