Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

Hybrids of two closely related tropical sea urchins (genus Echinometra): evidence against postzygotic isolating mechanisms

A series of cross-fertilization experiments were conducted with two unnamed, sympatric species of sea urchins in the Echinometra mathaei species complex, Echinometra sp. A (Ea) and Echinometra sp. C (Ec). Heterogametic fertilization success was high when eggs of Ec and sperm of Ea were involved, and low with eggs of Ea and sperm of Ec. Hybrids produced from crosses in either direction developed normally to sexually mature adults; Ea x Ea were largest in test size, followed by Ec (ova) x Ea (sperm), Ea (ova) x Ec (sperm), and Ec x Ec, respectively. Color patterns of the hybrids were closer to the maternal coloration, whereas other characters such as relative test dimensions and spine lengths, morphology of tubefoot and gonad spicules, and gamete sizes were intermediate. Fertilization rates in F1 backcrosses were high, minimizing the possibility that hybrid infertility is a postzygotic mechanism of reproductive isolation. On the other hand, intensive surveys failed to find individuals with hybrid characteristics in the field, suggesting that natural hybridization between the two species is rare. Prezygotic isolating mechanisms, such as microhabitat separation and gamete incompatibility, at least between Ea eggs and Ec sperm, most likely maintain the genetic integrity of these two closely related species.     

Organ-specific pancreatic tumor growth properties and tumor immunity

We established a model of orthotopic injection of a syngeneic pancreatic tumor cell line in C57BL/6 mice and evaluated the effects of organ site on induction of immunity to a tumor-specific antigen, MUC1. Mice were challenged with a syngeneic pancreatic adenocarcinoma cell line that expressed MUC1 (Panc02-MUC1) by orthotopic injection into the pancreas, or by subcutaneous injection. Tumor cells injected into the pancreas grew much faster than those injected subcutaneously. Mice challenged subcutaneously with Panc02-MUC1 rejected tumors or developed slowly growing tumors that were negative for MUC1 expression. In contrast, mice challenged orthotopically into the pancreas developed progressive tumors that were positive for MUC1 expression. Sera from mice that rejected Panc02-MUC1 (tumor-immune mice) showed no detectable IgG1 and IgM titers against the MUC1 tandem-repeat peptide, whereas mice with progressive tumor growth had significant titers of IgG1 and IgM specific for MUC1. This suggests that the humoral immune response was ineffective in mediating tumor rejection. The results show that the growth properties and immunological rejection of pancreatic tumors is affected by the organ site at which the tumor grows. Received: 25 April 1998 / Accepted: 7 October 1998     

Phase 1 study of multiple biomarkers for metabolism and oxidative stress after one-week intake of broccoli sprouts

Little is known about the direct effect of broccoli sprouts on human health. So we investigated the effect of broccoli sprouts on the induction of various biochemical oxidative stress markers. Twelve healthy subjects (6 males and 6 females) consumed fresh broccoli sprouts (100 g/day) for 1 week for a phase 1 study. Before and after the treatment, biochemical examination was conducted and natural killer cell activity, plasma amino acids, plasma PCOOH (phosphatidylcholine hydroperoxide), the serum coenzyme Q(10), urinary 8-isoprostane, and urinary 8-OHdG (8-hydroxydeoxyguanosine) were measured. With treatment, total cholesterol and LDL cholesterol decreased, and HDL cholesterol increased significantly. Plasma cystine decreased significantly. All subjects showed reduced PCOOH, 8-isoprostane and 8-OHdG, and increased CoQ(10)H(2)/CoQ(10) ratio. Only one week intake of broccoli sprouts improved cholesterol metabolism and decreased oxidative stress markers.     

A double classification tree search algorithm for index SNP selection

Background  

In population-based studies, it is generally recognized that single nucleotide polymorphism (SNP) markers are not independent. Rather, they are carried by haplotypes, groups of SNPs that tend to be coinherited. It is thus possible to choose a much smaller number of SNPs to use as indices for identifying haplotypes or haplotype blocks in genetic association studies. We refer to these characteristic SNPs as index SNPs. In order to reduce costs and work, a minimum number of index SNPs that can distinguish all SNP and haplotype patterns should be chosen. Unfortunately, this is an NP-complete problem, requiring brute force algorithms that are not feasible for large data sets.     

Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants

Certain beta-lactam antibiotics induce the chromosomal ampC beta-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that beta-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an ftsZ mutant and other cell division mutants (ftsA, ftsQ, and ftsI) and a PBP2 mutant for induction of beta-lactamase. In all mutants, beta-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress beta-lactamase expression in vitro. Our results suggest that beta-lactamase is not induced because the fts mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.     

A role for CCR9 in T lymphocyte development and migration

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic alphabeta-T cell development, and thymocyte selection appeared unimpaired in adult CCR9-deficient (CCR9(-/-)) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9(-/-) mice was less efficient at repopulating the thymus of lethally irradiated Rag-1(-/-) mice than bone marrow from littermate CCR9(+/+) mice. CCR9(-/-) mice had increased numbers of peripheral gammadelta-T cells but reduced numbers of gammadeltaTCR(+) and CD8alphabeta(+)alphabetaTCR(+) intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating the development and/or migration of both alphabeta(-) and gammadelta(-) T lymphocytes.     

Molecular requirements for CD8-mediated rejection of a MUC1-expressing pancreatic carcinoma: implications for tumor vaccines

Previous studies have indicated that different effector cells are required to eliminate MUC1-expressing tumors derived from different organ sites and that different vaccine strategies may be necessary to generate these two different MUC1-specific immune responses. In this study, we characterized molecular components that are required to produce immune responses that eliminate Panc02.MUC1 tumors in vivo by utilizing mice genetically deficient in molecules related to immunity. A parallel study has been reported for a B16.MUC1 tumor model. We confirmed that a CD8(+) effector cell was required to eliminate MUC1-expressing Panc02 tumors, and demonstrated that T cells expressing TCR-alpha/beta and co-stimulation through CD28 and CD40:CD40L interactions played critical roles during the initiation of the anti-Panc02.MUC1 immune response. TCR-alpha/beta(+) cells were required to eliminate Panc02.MUC1 tumors, while TCR-gamma/delta(+) cells played a suppressive non-MUC1-specific role in anti-Panc02 tumor immunity. Type 1 cytokine interferon-gamma (IFN-gamma), but not interleukin-12 (IL-12), was essential for eliminating MUC1-expressing tumors, while neither IL-4 nor IL-10 (type 2 cytokines) were required for tumor rejection. In vitro studies demonstrated that IFN-gamma upregulated MHC class I, but not MHC class II, on Panc02.MUC1 tumor cells. Surprisingly, both perforin and FasL played unique roles during the effector phase of immunity to Panc02.MUC1, while lymphotoxin-alpha, but not TNFR-1, was required for immunity against Panc02.MUC1 tumors. The findings presented here and in parallel studies of B16.MUC1 immunity clearly demonstrate that different effector cells and cytolytic mechanisms are required to eliminate MUC1-expressing tumors derived from different organ sites, and provide insight into the immune components required to eliminate tumors expressing the same antigen but derived from different tissues.