Search

Quantitative trait loci (QTL) analysis genetically dissects complex traits, discerns their genetic control and genotype‐by‐environment interactions, and ultimately helps marker development for assisted breeding selection. A mapping population of 124 F5:7recombinant inbred lines (RILs) derived from the cross of ‘TAM 112’ × ‘TAM 111’ was grown under seven diverse environments and evaluated for end‐use quality traits including kernel, flour, and dough‐mixing characteristics. The objective of this study was to detect QTL associated with end‐use quality traits in these two cultivars. Through 5,948 single nucleotide polymorphisms (SNPs), 39 QTL regions were consistently identified in two or more environments or QTL analyses. Thirteen QTL regions associated to two or more traits were also identified. Among them, 11 QTL regions were in common on chromosomes 1A, 1B, 1D, 2D, and 4D associated to kernel hardness (HARD), kernel diameter (DIAM), single kernel weight (SKW), flour yield (FYLD), midline peak time (MLPT) with logarithm of odds (LOD) up to 42.0, and explained up to 49.3% of the phenotypic variation. Notably, Glu‐D1loci at 414.3 Mb on chromosome 1D strongly influenced dough rheology and explained up to 54.6% of the trait variation for MLPT, with the favorable allele derived from TAM 112. Novel QTL for HARD, FYLD, and ASH were also identified on the chromosome 1A and 2A, 6B, and 7D, respectively. This study confirmed previously identified loci at major genes, some newly identified QTL on chromosomes, and the favorable alleles carried forward for improved end‐use quality.

A novel class of 5-substituted 5H-imidazo[5,1-a]isoindoles are described as potent inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). A structure-based drug design approach was used to elaborate the 5H-imidazo[5,1-a]isoindole core and to improve potency and pharmacological properties. Suitably placed hydrophobic and polar functional groups in the lead molecule allowed improvement of IDO1 inhibitory activity while minimizing off-target liabilities. Structure–activity relationship studies focused on optimizing IDO1 inhibition potency and a pharmacokinetic profile amenable to oral dosing while controlling CYP450 and hERG inhibitory properties.

Stripe rust, caused by Puccinia striiformisWestend. f. sp. triticiErikss. (Pst), is an important disease of wheat (Triticum aestivumL.) in the United States and many other areas of the world. To identify the genetic basis of resistance in the winter wheat cultivar ‘TAM 111’, a mapping population of 124 F6recombinant inbred lines (RILs) developed from the cross TAM 112/TAM 111 was evaluated against Pstpopulations over eight field environments in the United States and against race PST‐100 in the greenhouse. A high‐density genetic map was constructed using the wheat 90K iSelect array and genotyping‐by‐sequencing (GBS) markers. A set of 6343 markers covering 11.8 Gb of all 21 chromosomes, including 16 simple sequence repeat (SSR) and sequence‐tagged site (STS), 3335 GBS, and 2992 single nucleotide polymorphism (SNP) markers from the 90K array were used for quantitative trait locus (QTL) analyses. Eight QTL on chromosomes 1A, 2A, 2B, 4A, 4B, 6B, and 7D were identified, and two of them were novel. Six tightly linked SNP markers were converted to Kompetitive allele specific polymerase chain reaction (KASP) markers for high‐throughput screening of the largest and most consistent QTL at 154.3 Mb of chromosome 2B. This QTL, QYr.tamu‐2B, was involved with significant epistatic interactions on both disease severity and infection type, and epistasis × environment interactions with QYr.tamu‐2A1on disease severity only. These QTL can be combined with effective major genes to enhance the stripe rust resistance, and corresponding diagnostic markers can be applied through marker‐assisted breeding.

The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1and Cyp1b1mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Pergene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1and CYP1B1gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor–induced P450gene expression and BaP metabolism was similar to BaP-induced Cyp1A1and Cyp1B1and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.

Many mitochondrial mRNAs in trypanosomes are subject to extensive RNA editing involving both insertion and deletion of blocks of uridine residues. This paper examines the RNA constituents of distinct protein complexes known as mitochondrial RNA-binding complexes or MRBs. The evidence supports the existence of multiple subcomplexes of MRBs containing different RNA/protein composites.

The immune modulatory drug chloroquine (CQ) has been demonstrated to enhance survival following radiotherapy in patients with high-grade glioma in a clinical trial, but the efficacy in patients with brain metastases is unknown. We hypothesized that short-course CQ during whole brain radiotherapy (WBRT) would improve response to local therapy in patients with brain metastases.A prospective, single-cohort study was performed combining WBRT with concurrent CQ to assess both the feasibility of and intracranial response to combined therapy in patients with brain metastases. Safety, tolerability, and overall survival of this combination were also examined, along with allelic status of IDO2(indoleamine 2,3-dioxygenase 2), an immune modulatory enzyme inhibited by chloroquine that may affect survival outcomes. CQ therapy (250 mg by mouth daily) was initiated 1 week before WBRT (37.5 in 2.5 Gy daily fractions) in patients with newly diagnosed brain metastases from biopsy-proven, primary lung, breast, or ovarian solid tumors (n= 20). The primary endpoint was radiologic response 3 months after combined CQ and WBRT therapy. Secondary endpoints included toxicity and overall survival. Patients were stratified by IDO2allelic status.After a median clinical follow-up of 5 months (range, 0.5–31), 16 patients were evaluable for radiologic response which was complete response in two patients, partial response in 13 patients, and stable disease in one patient. There were no treatment-related grade ≥ 3 toxicities or treatment interruption due to toxicity. Median and mean overall survival was 5.7 and 8.9 months, respectively (range, 0.8–31). A trend toward increased overall survival was observed in patients with wild-type IDO2compared to patients with heterozygous or homozygous configurations that ablate IDO2enzyme activity (10.4 vs. 4.1 months; p= 0.07).WBRT with concurrent, short-course CQ is well-tolerated in patients with brain metastases. The high intracranial disease control rate warrants additional study.

Tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) alters inflammation and favors T-cell tolerance in cancer, but the underlying molecular mechanisms remain poorly understood. The integrated stress response kinase GCN2, a sensor of uncharged tRNA that is activated by amino acid deprivation, is recognized as an important effector of the IDO pathway. However, in a mouse model of inflammatory carcinogenesis, ablation of Gcn2did not promote resistance against tumor development like the absence of IDO does, implying the existence of additional cancer-relevant pathways that operate downstream of IDO. Addressing this gap in knowledge, we report that the IDO-mediated catabolism of tryptophan also inhibits the immunoregulatory kinases mTOR and PKC-Θ, along with the induction of autophagy. These effects were relieved specifically by tryptophan but also by the experimental agent 1-methyl-D-tryptophan (D-1MT, also known as NLG8189), the latter of which reversed the inhibitory signals generated by IDO with higher potency. Taken together, our results implicate mTOR and PKC-Θ in IDO-mediated immunosuppressive signaling, and they provide timely insights into the unique mechanism of action of D-1MT as compared with traditional biochemical inhibitors of IDO. These findings are important translationally, because they suggest broader clinical uses for D-1MT against cancers that overexpress any tryptophan catabolic enzyme (IDO, IDO2 or TDO). Moreover, they define mTOR and PKC-Θ as candidate pharmacodynamic markers for D-1MT responses in patients recruited to ongoing phase IB/II cancer trials, addressing a current clinical need.

Aging entails the accumulation of neoantigens comprised of aggregated, oxidized, mutated and misfolded biomolecules, including advanced-glycation end projects (AGEs). There is evidence that the immune system can recognize and clear cells fouled by these molecular debris, which contribute to the emergence of cancer and other major age-associated diseases such as atherogenic and neurodegenerative disorders. However, this process may become increasingly inefficient with aging, perhaps in part because of an insufficiency of adjuvant signals normally associated with infection that can program productive inflammatory states and properly orient the immune system toward regenerative healing. Here we propose conceptual foundations for exploring a small set of infection-associated molecules as potential immune adjuvants to reprogram non-productive inflammatory states in aging tissues, and to improve the clearance of cellular pathologies that engender age-associated disease. The proposed adjuvant classes include a subset of D-amino acids used by bacteria to disrupt biofilms; nucleoside derivatives of N6-methyladenine, which functions at the core of bacterial dam restriction systems; and derivatives of the galactosyl trisaccharide α-Gal, which invokes the hyperacute response in primates. These foreign amino acids, nucleosides and sugar molecules are generally rare or absent in humans, except in association with infections by bacteria, protists or nematodes. A rationale for exploration of these candidate adjuvant principles and their chemical derivatives is discussed in terms of their use in generalized strategies to improve the prevention or treatment of cancer and other age-associated diseases, as negative modifiers of aging.

Chronic inflammation drives the development of many cancers, but a genetic definition of what constitutes ‘cancer-associated’ inflammation has not been determined. Recently, a mouse genetic study revealed a critical role for the immune escape mediator indoleamine 2,3-dioxygenase (IDO) in supporting inflammatory skin carcinogenesis. IDO is generally regarded as being immunosuppressive; however, there was no discernable difference in generalized inflammatory processes in IDO-null mice under conditions where tumor development was significantly suppressed, implicating IDO as key to establishing the pathogenic state of ‘cancer-associated’ inflammation. Here we review recent findings and their potential implications to understanding the relationship between immune escape and inflammation in cancer. Briefly, we propose that genetic pathways of immune escape in cancer are synonymous with pathways that define ‘cancer-associated’ inflammation and that these processes may be identical rather than distinct, as generally presumed, in terms of their genetic definition.

The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by a subset of murine plasmacytoid DCs (pDCs) in tumor-draining lymph nodes (TDLNs), where it can potently activate Foxp3+regulatory T cells (Tregs). We now show that IDO functions as a molecular switch in TDLNs, maintaining Tregs in their normal suppressive phenotype when IDO was active, but allowing inflammation-induced conversion of Tregs to a polyfunctional T-helper phenotype similar to proinflammatory T-helper-17 (TH17) cells when IDO was blocked. In vitro, conversion of Tregs to the TH17-like phenotype was driven by antigen-activated effector T cells and required interleukin-6 (IL-6) produced by activated pDCs. IDO regulated this conversion by dominantly suppressing production of IL-6 in pDCs, in a GCN2-kinase dependent fashion. In vivo, using a model of established B16 melanoma, the combination of an IDO-inhibitor drug plus antitumor vaccine caused up-regulation of IL-6 in pDCs and in situ conversion of a majority of Tregs to the TH17 phenotype, with marked enhancement of CD8+T-cell activation and antitumor efficacy. Thus, Tregs in TDLNs can be actively reprogrammed in situ into T-helper cells, without the need for physical depletion, and IDO serves as a key regulator of this critical conversion.

The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by a subset of murine plasmacytoid DCs (pDCs) in tumor-draining lymph nodes (TDLNs), where it can potently activate Foxp3+ regulatory T cells (Tregs). We now show that IDO functions as a molecular switch in TDLNs, maintaining Tregs in their normal suppressive phenotype when IDO was active, but allowing inflammation-induced conversion of Tregs to a polyfunctional T-helper phenotype similar to proinflammatory T-helper-17 (TH17) cells when IDO was blocked. In vitro, conversion of Tregs to the TH17-like phenotype was driven by antigen-activated effector T cells and required interleukin-6 (IL-6) produced by activated pDCs. IDO regulated this conversion by dominantly suppressing production of IL-6 in pDCs, in a GCN2-kinase dependent fashion. In vivo, using a model of established B16 melanoma, the combination of an IDO-inhibitor drug plus antitumor vaccine caused up-regulation of IL-6 in pDCs and in situ conversion of a majority of Tregs to the TH17 phenotype, with marked enhancement of CD8+ T-cell activation and antitumor efficacy. Thus, Tregs in TDLNs can be actively reprogrammed in situ into T-helper cells, without the need for physical depletion, and IDO serves as a key regulator of this critical conversion.

The mammary gland microenvironment during postlactational involution shares similarities with inflammation, including high matrix metalloproteinase activity, fibrillar collagen deposition, and release of bioactive fragments of fibronectin and laminin. Because inflammation can promote tumorigenesis, we evaluated whether the tissue microenvironment of the involuting gland is also promotional. Extracellular matrix was isolated from mammary glands of nulliparous rats or rats with mammary glands undergoing weaning-induced involution. Using these matrices as substratum, nulliparous matrix was found to promote ductal organization of normal mammary epithelial MCF-12A cells in three-dimensional culture and to suppress invasion of mammary tumor MDA-MB-231 cells in transwell filter assays. Conversely, involution matrix failed to support ductal development in normal cells and promoted invasiveness in tumor cells. To evaluate the effects of these matrices on metastasis in vivo, MDA-MB-231 cells, premixed with Matrigel, nulliparous matrix, or involution matrix, were injected into mammary fat pads of nude mice. Metastases to lung, liver, and kidney were increased in the involution matrix group, and correlated with a twofold increase in tumor vascular endothelial growth factor expression and increased angiogenesis. These data suggest that the mammary gland microenvironment becomes promotional for tumor cell dissemination during involution, thus providing a plausible mechanism to explain the high rate of metastases that occur with pregnancy-associated breast cancer.

Mouse mammary epithelial cells (HC‐11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC‐11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation‐dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC‐11 cells and mammary glands, elevated Per2 expression was positively correlated with c‐Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with β‐casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation. Developmental Dynamics 235:263–271, 2006. © 2005 Wiley‐Liss, Inc.

The authors outline the development of a form of nontraditional high-end, or luxury, healthcare known as concierge care—also often called boutique medicine or retainer practice. Physicians choosing to work within this new patient care model have provoked important medical, ethical, and legal questions in the medical community. Although some regulatory changes have taken place on the state and federal levels to address concerns about this trend, the authors argue for some of the perhaps lesser-known benefits of this model of care.

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitroDNA repair assay and an Escherichia colireadout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.

UDP-glucuronosyltransferase UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene metabolites and other bulky aromatic compounds. Both UGT1A7 mRNA and an associated enzyme activity (benzo(a)pyrene7,8-dihydrodioltransferase activity) are markedly increased in livers of rats treated with β-naphthoflavone or 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz). Nuclear runoff assays show that the effects of both inducers are primarily due to transcriptional activation. A 27-kilobase region that included the UGT1A7/UGT1A6promoter regions was cloned. Primer extension and RNase protection studies indicated ≥30 transcription start sites in five clusters between bases −85 and −40 respective to the translation start codon. There was no recognizable TATA box, but the promoter region is TA-rich. Sequence analysis revealed potential binding sites for CCAAT enhancer-binding protein, activator protein 1, and hepatic nuclear factors 1, 3, and 4, but no xenobiotic response elements or antioxidant response elements, implicated in the regulation of other genes by β-naphthoflavone or oltipraz, were found. A UGT1A7gene reporter plasmid directed strong constitutive expression in transient transfection assays using primary rat hepatocytes. Treatment with 3-methylcholanthrene or oltipraz had no effect compared with similarly treated pGL3-Basic-transfected cells. These results suggest that the regulatory elements controlling xenobiotic inducibility of UGT1A7transcription are located either 5′ or 3′ of bases −1600 to +54. One possibility is that the polycyclic aromatic-mediated regulation of UGT1A7occurs via the xenobiotic response element flanking the UGT1A6locus 7 kilobase pairs downstream.

Previous work has shown that polycyclic aromatic hydrocarbons and oltipraz both induce an unidentified rat liver UDP-glucuronosyltransferase with activity toward benzo(a)pyrene-7,8-diol, the proximate carcinogenic form of benzo(a)pyrene. Here we report the isolation of a benzo(a)pyrene-7,8-diol transferase-encoding cDNA, LC14, from an adult rat hepatocyte-derived cell line (RALA255-10G LCS-3). The predicted amino acid sequence of LC14 is nearly identical (5 differences out of 531 residues) to that deduced from UGT1A7, recently cloned at the genomic DNA level (Emi, Y., Ikushiro, S., and Kyanagi, T. (1995) J. Biochem.(Tokyo) 117, 392-399). Northern analysis of RNA from female F344 rat liver and LCS-3 cells revealed over a 40-fold and 4.4-fold enhancement by oltipraz treatment, respectively. Benzo(a)pyrene-7,8-diol glucuronidating activity was detected (0.4 nmol/106cells/16 h) in AHH-1 cells transfected with the LC14 expression vector, pMF6-LC14-3. The LC14-encoded transferase exhibited even higher activity toward certain benzo(a)pyrene phenols, including the major 3- and 9-phenol metabolites (4.1 and 2.8 nmol/106cells/16 h, respectively). The Kmof the enzyme for (−)-transbenzo(a)pyrene-7,8-diol and 3-OH-BP was 15.5 and 12.3 μM, respectively. Northern analyses of total RNA revealed expression of LC14 or LC14-like RNA in all extrahepatic tissues tested. Marked inducibility by oltipraz was observed only in liver and (to a lesser extent) intestine. The results suggest that induction of UGT1A7 may explain the increased glucuronidating activities toward benzo(a)pyrene-7,8-diol and other metabolites that occur following treatment with polycyclic aromatic hydrocarbon-type inducing agents and oltipraz. UGT1A7 appears to represent an important cellular chemoprotective enzyme which mediates conjugation and elimination of toxic benzo(a)pyrene metabolites.

Background: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function. Results: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. Conclusions: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

Summary Morbidly obese patients are significantly more susceptible to clotting phenomena, including recurrent deep venous thrombosis, pulmonary emboli, inferior vena caval thrombosis, and renal vein thrombosis. The patient described in this report developed the sudden onset of massive ascites two years following jejunoileal bypass for morbid obesity. Occlusion of the portal vein and its tributaries was demonstrated. Our experience and one previous report suggest that portal vein thrombosis may be a rare complication of this surgical procedure.

Metz: New Link Genetics Corporation: Employment.

Abstract 1047

Metz: New Link Genetics Corporation: Employment. Prendergast:New Link Genetics Corporation: Employment.

Abstract 3231

Abstract 4298

Metz: NewLink Genetics: Employment. Prendergast:New Link Genetics Corp: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17–18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-β. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-α, β or γ are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4–8 hrs and decreases after 16 hrs.