USDA CSREES Research Articles

Efficacy of an Extract from Garlic, Allium sativum , Against Infection with the Furunculosis Bacterium, Aeromonas salmonicida , in Rainbow Trout, Oncorhynchus mykiss

Journal of the World Aquaculture SocietyVolume 46, Issue 5 p. 569-569 CorrigendumFree Access Efficacy of an Extract from Garlic, Allium sativum, Against Infection with the Furunculosis Bacterium, Aeromonas salmonicida, in Rainbow Trout, Oncorhynchus mykiss This article corrects the following: Efficacy of an Extract from Garlic, Allium sativum, Against Infection with the Furunculosis Bacterium, Aeromonas salmonicida, in Rainbow Trout, Oncorhynchus mykiss Kate E. Breyer, Rodman G. Getchell, Emily R. Cornwell, Gregory A. Wooster, H. George Ketola, Paul R. Bowser, Volume 46Issue 3Journal of the World Aquaculture Society pages: 273-282 First Published online: June 1, 2015 First published: 03 October 2015 https://doi.org/10.1111/jwas.12218Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In Breyer et al. (2015), an error was published in the Acknowledgment section. The contribution number for the USGS Great Lakes Science Center should have been published as 1878 instead of 1868.The corrected Acknowledgement section is shown below. Acknowledgments This work was funded in part by the Northeast Region of the National Research Support Project No. 7 (NRSP7), The Minor Use Animal Drug Program through USDA CSREES grant no. 2010-34143-21171. K. E. Breyer, DVM, was supported on a Residency Program in Laboratory Animal Medicine stipend from the Cornell University College of Veterinary Medicine and the Center for Animal Resources and Education, Ithaca, New York. We thank the USGS Tunison Laboratory of Aquatic Science (Cortland, NY) for their collaboration and donation of fish and feed for this study, K. J. Maurer, DVM, DACLAM, PhD, for his assistance with statistical analysis and data interpretation, as well as L. L. Coffee, DVM, DACVP, for her gross pathology expertise, and H. Priest, DVM, DACVP, for her clinical pathology expertise. This article is contribution 1878 of the USGS Great Lakes Science Center. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. The authors apologize for this error. Literature Cited Breyer, K. E., R. G. Getchell, E. R. Cornwell, and G. A. Wooster. 2015. Efficacy of an Extract from Garlic, Allium sativum, Against Infection with the Furunculosis Bacterium, Aeromonas salmonicida, in Rainbow Trout, Oncorhynchus mykis. Journal of the World Aquaculture Society 46: 273– 282. Citing Literature Volume46, Issue5October 2015Pages 569-569 ReferencesRelatedInformation

The satiety response to a standard lunch in habitual breakfast eaters and skippers

BackgroundInsulin, Leptin, GLP‐1, and Glucagon are known regulators of food intake and glucose metabolism. Past studies showed a benefit of eating breakfast on satiety but were possibly confounded by disruption of habitual meal patterns. Thus, we conducted a cross‐sectional study of 30 women who were habitual breakfast eaters (Br‐E) or habitual skippers (Br‐S) to assess the hormones Insulin, Leptin, GLP‐1, and Glucagon following a standard lunch meal.MethodsBr‐E ate their typical breakfast meal between waking and 10am, whereas Br‐S followed their normal Br‐S routine. Both Br‐E and Br‐S did not consume any food or caloric beverage after 10am until the standard lunch was served. Blood was drawn 5 min pre‐lunch and 5, 20, 35, 50, 110 min post‐lunch. A multiplexed sandwich immunoassay (MSD) was used to measure hormones.ResultsRepeated measures ANOVA revealed group differences that were time dependent for Insulin (p=0.02), GLP‐1 (p=0.02), and Gluc (p=0.02). GLP‐1 was higher in Br‐E at 5 (p=0.02) and 110 (p=0.03) min.DiscussionClear differences in circulating hormones between Br‐E and Br‐S suggest that the previous breakfast meal influenced the postprandial response, even though all subjects were post absorptive at the start of the protocol. Our data further support the idea that regularly eating breakfast promotes satiety. Funding: USDA CSREES 2009–35215‐05364; USDA Intramural project 5306–51530‐019–00D.Grant Funding Source: USDA CSREES 2009–35215‐05364; USDA Intramural project 5306–51530‐019–00D

Design and characterization of Thermotoga maritima glycerol dehydrogenase to reduce inactivation by Dihydroxyacetone

Enzyme catalysis for production of commercially useful compounds is a competitive alternative to traditional chemical synthesis. Thermotoga maritima glycerol dehydrogenase (TmGlyDH) is a potential industrial enzyme for the production of dihydroxyacetone (DHA), the active ingredient of sunless tanning products, from glycerol. Our studies showed that TmGlyDH loses up to 40% of activity when incubated with 200 mM DHA at 50°C for 15 min, with inactivation roughly fitting an exponential decay model. Inactivation likely occurs by the Maillard reaction, where DHA reacts with the amino acids lysine and arginine forming a Schiff base then undergoing rearrangements. Intact and inactivated proteins were digested with trypsin and chymotrypsin and fifteen modified residues were identified by electrospray ionization mass spectrometry. The targeted Lys and Arg residues were substituted with Gln by site‐directed mutagenesis of the TmGlyDH gene. Mutants were expressed in E. coli BL21(DE3), and purified. We will report on the mutant enzymes’ activity and their resistance to inactivation by DHA.This project was supported by an MSU Honors College Professorial Assistantship and MSU Lyman Briggs College Undergraduate Research Support to PGG, by a US Department of Education GAANN fellowship to JB, and by NSF grant CBET 0756703 and USDA CSREES National Research Initiative grant 2008–35504‐04611 to CV.

A putative Fe+3/phytosiderophore (PS) transporter isolated from oats (Avena sativa)

The objectives of this project were: 1) to isolate and characterize a Fe+3/phytosiderophore (PS) transporter from Oats (Avena sativa), and, 2) develop a method for the synthesis of the oat phytosiderophore avenic acid. The putative transporter's functionality will be evaluated by complementation assays in yeast (Saccharomyces cerevisiae) using synthetic avenic acid (Stocksdale).Total RNA was isolated from the roots of hydroponically grown, iron‐starved oats (var. Rodeo). Full‐length clones were obtained using 5′RACE and RT/PCR. Sequence analysis of the clone, AvsYS1, (Genbank Accession# FJ477297) shows>;85% similarity at both the nucleotide and amino acid level to HvYS1, a functionally confirmed Fe+3/ (PS) transporter from barley (Hordeum vulgare). AvsYS1 was subcloned into pYES2 (Invitrogen), a galactose‐inducible shuttle vector.DDY4 ( a gift from Dr. David Eide) is a mutant S. cervisiae lacking a Fe+3 uptake ability. DDY4 will be transformed with pYES2/BS4YAT, pYES2 containing the AvsYS1 insert. To evaluate functionality, transformants will be grown in minimal media (plates and liquid) supplemented with FeCl3, synthetic avenic acid or complexed FeCl3/avenic acid. If AvsYS1 encodes a functional transporter we will be able to rescue DDY4 in a the medium is supplemented with FeCl3/avenic acid.This research was supported by a USDA CSREES Grant, #2007–35318

Within subject variation of satiety hormone responses to a standard lunch

BackgroundInsulin (Ins), leptin (Lep), GLP‐1, and glucagon (Glg) are known regulators of glucose metabolism and food intake, but reproducibility in response to a meal challenge is not well characterized. We assessed within‐subject variation of these hormones in 14 young adult women.MethodsSubjects were studied on 2 days during the follicular phase of the menstrual cycle. On test days blood was drawn at 20, 35, 50, 65 & 125 min after a standard lunch meal. Meso Scale Discovery assay system was used to measure the hormones in a multiplexed sandwich immunoassay.ResultsAnalysis of variance revealed that there were no differences between test days for all 4 hormones. Correlations between values for test days 1 & 2 were significant for Ins (r = 0.68; p < 0.0001), Lep (r = 0.86; p < 0.0001) and GLP‐1 (r = 0.97; p < 0.0001) but not for Glg (r = 0.18; p = 0.143). Mean within‐subject variation (% of mean value at each time point) was 42, 37, 48, and 87 for Ins, Lep, GLP‐1 and Glg, respectively. Averaging values for 5 time points, to represent postprandial response, reduced within‐subject variation to 30, 37, 23, and 35% of mean value, respectively.DiscussionDespite controlled test meals, we observed large within‐subject variation that was partly reduced by using summary measures. Other contributors to variation may include pre‐test diet intake, environmental influences, or analytical error. Funding: USDA CSREES 2009‐35215‐05364.

Serum 25(OH)D response to soy isoflavone supplementation in postmenopausal women

Soy isoflavones (ISO) are believed to have anti-osteoporotic properties possibly mediated through vitamin D. The impact of ISO supplementation on serum 25(OH)D was assessed in a multisite, randomized, double-blind, 2-yr bone study in 403 early postmenopausal women (aged 54.0±4.0 yr) with 1/3 on placebo (PL), 1/3 on 80 mg/d ISO and 1/3 on 120 mg/d ISO. All subjects received 1,000 mg/d CaCO3 and 400 IU/d vitamin D3. Serum 25(OH)D was measured at baseline and annually using radioimmunoassay (Diasorin Inc.). Repeated measures ANCOVA adjusting for baseline 25(OH)D was employed in the statistical analysis. Baseline characteristics were similar in all groups, including mean 25(OH)D values (78.9, 74.9 and 82.4 nmol/L for the PL, 80 mg and 120 mg groups, respectively). ISO supplementation did not alter 25(OH)D concentrations, yet there was a significant trend such that 25(OH)D increased over time (p<0.05). The percentage of participants with serum 25(OH)D <50 nmol/L fell from 11.0% at baseline to 5.8% at yr 1 and 4.1% at yr 2. Soy ISO supplementation did not alter circulating 25(OH)D over 2 yr, but 400 IU/d vitamin D was effective at improving serum 25(OH)D in healthy postmenopausal women with high baseline values. Supported by Initiative for Future Agriculture and Food Systems Grant no. 2001-52102-11255 from the USDA CSREES.

Histone biotinylation is a naturally occurring phenomenon

Modifications of histones such as methylation and acetylation are involved in gene regulation. We recently identified a diet‐dependent histone modification, biotinylation of lysines, mediated by holocarboxylase synthetase (HCS). HCS is a chromatin protein. Knockdown of HCS decreases histone biotinylation in human cells. We identified ten biotinylation sites in histones using histone‐based synthetic peptides and biotinylation site‐specific antibodies. A recent study (Healy et al., 2009) has questioned the occurrence of histone biotinylation in vivo. Here, we provide unambiguous evidence in support of histone biotinylation in a number of primary and transformed human cells. Our biotinylation site‐specific antibodies, unlike the commercial antibodies used by Healy, do not cross react with methylated and acetylated histones. We detected biotinylated histones in various cell lines using anti‐biotin, streptavidin, and target‐specific antibodies. Cells of the lymphoid lineage produced the strongest biotinylation signal, which might explain the failure to detect histone biotinylation in HeLa cells in Healy's studies. Current studies focus on detecting biotin in native histones using [3H]biotin, customized [14C]carbonyl‐labeled biotin, and mass spectrometry in Jurkat cells. (UNL ARD Hatch grant, NIH DK063945, DK077816, DK082476 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPS 0701892.)

Identification of a potential role of K9‐biotinylated histone H3 in honeybee (Apis mellifera) development

Diet plays a unique role in the development of honeybees. Larvae fed honey develop into worker bees, and larvae fed royal jelly develop into queens. We hypothesized that the binding of biotin to histones is an important epigenetic mechanism contributing towards bee development. First, we demonstrated that the biotin content in royal jelly (45 ± 9.3 nM) is significantly greater than in honey (1.7 ± 0.6 nM). Second we screened various tissues from bees with site‐specific antibodies to various histone biotinylation marks. Lysine‐9 methylated histone H3 (H3K9bio) was easily detectable; highest concentrations were observed in bee brain. The abundance of H3K9bio was about three times greater in brain from queens compared with workers. Currently, we are conducting biotin intervention studies to determine whether dietary biotin can cause shifts in the bee populations that develop into queens and larvae, and chromatin immunoprecipitation assays to identify genomic loci where H3K9bio is enriched in bee brains. Collectively, this study is consistent with the hypothesis that biotinylation of histones occurs in bees; that H3K9bio is the predominant biotinylation mark in bees; and that biotin plays a role in bee development. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816, DK082476 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Characterization of the H4K16bio Mark in Human Lymphoid Cells

Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to lysine (K) residues in histones. Histone biotinylation is biologically important for gene regulation in humans. For example, biotinylation of the K‐12 in histone H4 (H4K12bio) represses the transcription of genes coding for transposable elements, interleukin‐2, and the sodium‐dependent multivitamin transporter. Mass spectrometry studies provided preliminary evidence for the existence of a novel biotinylation site, H4K16bio, in addition to the 11 known biotinylation sites in histones H2A, H3 and H4. In this study we developed a new antibody to H4K16bio and conducted an extensive series of tests to confirm specificity. Bulk extracts of human histones tested positive for H4K16bio, using the novel antibody as a probe. Currently, we are using ChIP/qRT‐PCR to obtain first insights into potential biological functions of H4K16bio. (Supported by ARD, NIH DK063945, DK077816, DK082476 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPS 070189).

USDA CSREES NRI Merit Award Fellows

USDA CSREES NRI Merit Award Fellows

Linking Information to Real-Life Problems: An Interdisciplinary Collaboration of Librarians, Departments, and Food Businesses

Through a United States Department of Agriculture Cooperative State Research, Education, and Extension Service (USDA CSREES) Higher Education Challenge Grant, a team of teaching faculty and librarians at Rutgers University developed an interdisciplinary course, “Food and Nutrition Business Information and Communication.” The course employed an active learning approach that involved a unique collaboration between various academic disciplines; the Rutgers University Libraries; the School of Communication, Information, and Library Studies (SCILS); and the Rutgers Food Manufacturing Technology Facility (FMT). In the course, teams of students from the departments involved worked collaboratively to help solve an actual research problem from industry partners working at FMT. Three examples of student problem solving are presented and assessed using the rubric developed for the course. The course actively involves librarians and demonstrates the importance of information literacy skills in today's information economy.

Repression of the Intrinsic Pathway to Apoptosis in the Two-Cell Bovine Embryo Involves Histone De-Acetylation and DNA Methylation.

Apoptosis in the bovine embryo cannot be induced by activators of the intrinsic or extrinsic apoptosis pathways until the 8-16 cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in two-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by hyperacetylation of histones and methylation of DNA. To test this hypothesis, we evaluated whether reducing DNA methylation or histone deacetylation would make two-cell embryos susceptible to DNA fragmentation caused by CCCP. Bovine oocytes were maturated and fertilized in vitro for 18 h. Putative zygotes were then treated for 12 h with vehicle, 100 μM 5-aza 2′ deoxycytidine (AZA), to cause DNA demethylation, or 100 nM Trichostatin A (TSA), to inhibit histone deacethylase. Embryos were then treated + 100 μM CCCP. Fragmentation of DNA was determined at 24 h after CCCP using the TUNEL reaction. The experiment was replicated 6 times using a total of 458 embryos. The percent of blastomeres positive for TUNEL (% TUNEL positive nuclei) was affected by a treatment x CCCP interaction (P<0.0001). CCCP did not cause a large increase in % TUNEL positive nuclei in control embryos (2.0 +3.4% vs 7.7 + 5.5%) but caused a large increase in % TUNEL positive nuclei for embryos treated with AZA (5.4 + 2.9% vs 42.3 + 3.2%). Results for TSA indicated an increase in %TUNEL positive nuclei in embryos without CCCP and only a slight increase with CCCP treatment (17.1 + 2.8% vs 24.9 + 4.2%). Further analysis of TSA treated embryos focused on the subset of embryos that were < 8 cells (i.e., those that are ordinarily not susceptible to apoptosis). In this subset, which included 68% of the TSA-treated embryos, there was a clear increase in % TUNEL positive nuclei after CCCP treatment (10.0 + 4.2% vs 24 + 4.5%, P<0.025). In conclusion, disruption of DNA methylation and histone deacetylation can remove the block to apoptosis in bovine two-cell embryos. Moreover, inhibition of histone acetylation leads to some spontaneous apoptosis in the absence of a proapoptotic signal. It is likely that interactions between methylated DNA and histones masks DNA at early stages of development and prevents access of caspase-activated DNAses to DNA. Support: USDA CSREES Grant # 2007-35203-18070. (poster)

SGN Database: From QTLs to Genomes

AbstractQuantitative trait loci (QTL) analysis is used to dissect the genetic basis underlying polygenic traits. Several public databases have been storing and making QTL data available to research communities. To our knowledge, current QTL databases rely on manual curation where curators read literature and extract relevant QTL information to store in databases. Evidently, this approach is expensive in terms of expert manpower and time use and limits the type of data that can be curated. At the Solanaceae Genomics Network (SGN) ("http://sgn.cornell.edu":http://sgn.cornell.edu), we have developed a database to store raw phenotype and genotype data from QTL studies, perform, on the fly, QTL analysis using R/QTL statistical software ("http://www.rqtl.org":http://www.rqtl.org) and visualize QTLs on a genetic map. Users can identify peak, and flanking markers for QTLs of traits of interest. The QTL database is integrated with other SGN databases (eg. Marker, BACs, and Unigenes), and analysis tools such as the Comparative Map Viewer. Using the comparative map viewer, users can compare chromosome with QTL regions to genetic maps of interest from the same or different Solanaceae species. As the tomato genome sequencing advances, users can also identify corresponding BAC sequences or locations on the tomato physical map, which can be suggestive of candidate genes for a trait of interest.Furthermore at SGN, images, quantitative phenotype and genotype data, publications, genetic maps generated by QTL studies are displayed and available for download. Currently, data from three F2 and two backcross population QTL studies on fruit morphology traits (18 – 46 traits per population) is available at the SGN website for viewing at population, accession, and trait levels. Traits are described using ontology terms. Phenotype data is presented in tabular and graphical formats such as frequency distributions with basic descriptive statistics. Mapping data showing location of parental alleles on individual accession genetic maps is also available.SGN is a public database hosted at Boyce Thomson Institute, Cornell University, and funded by USDA CSREES and NSF.

Nitric oxide signaling depends on biotin in Jurkat human lymphoma cells

Biotin affects gene expression through various signaling pathways. Previous studies suggest that cGMP‐dependent signaling also depends on biotin, but the mechanism of cGMP regulation is unknown. Here we tested the hypothesis that effects of biotin in cGMP‐dependent cell signaling are mediated by nitric oxide (NO). Human lymphoid (Jurkat) cells were cultured in media containing deficient, physiological, and pharmacological concentrations of biotin. Both levels of intracellular biotin and NO exhibited a dose‐dependent relationship in regard to biotin concentrations in culture media. Effects of biotin on NO levels were disrupted by both the NO synthase (NOS) inhibitors and NO donors. Biotin‐dependent production of NO was linked with biotin‐dependent expression of endothelial and neuronal NOS, but not the inducible iNOS. Previous studies revealed that NO is an activator of guanylate cyclase. Consistent with these previous observations, biotin‐dependent generation of NO increased the abundance of cGMP in Jurkat cells. Finally, the biotin‐dependent generation of cGMP increased the activity of protein kinase G. Collectively this study is consistent with the hypothesis that biotin‐dependent cGMP signaling in human lymphoid cells is mediated by NO. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Homeostasis of biotin in human lymphoma and liver cells

Biotin is covalently attached to carboxylases and histones in a reaction catalysed by holocarboxylase synthetase (HCS). Here we determined whether biotin concentrations in culture media affect the expression of genes maintaining biotin homeostasis in human lymphoid (Jurkat) cells. As a secondary objective we determined whether mechanisms of biotin homeostasis are distinct in cells with great metabolic activities, i.e., human hepatocarcinoma (HepG2) cells. Jurkat cells were cultured in media containing deficient, physiological, or high concentrations of biotin for 36 d. While the abundances of biotinylated carboxylases responded rapidly (&lt;3 d) to altered biotin supply, changes in histone biotinylation were not apparent until day 36 when biotin supplementation caused increased biotinylation of histones in nuclear extracts. The expression of genes linked with biotin homeostasis depended on biotin in Jurkat cells. The expression of biotin transporters SMVT and MCT1 increased in biotin‐deficient cells, whereas the expression of HCS increased in biotin‐supplemented cells. In ongoing studies we investigate the effects of biotin supply on histone biotinylation at distinct genomic loci by ChIP assays, and we compare biotin homeostasis in Jurkat cells to HepG2 liver cells. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Enrichment of H3K4bio, H3K9bio, H3K18bio, and H4K8bio in distinct genomic loci

Covalent histone modifications play crucial roles in chromatin structure and gene expression. We previously reported a novel modification of histones H3 and H4: biotinylation of lysine (K) residues by holocarboxylase synthetase (HCS). Biotinylation sites include H3K4bio, H3K9bio, H3K18bio, H4K8bio, and H4K12bio. We demonstrated that H4K12bio represses genes and is enriched in repeat elements such as pericentromeric alpha satellite repeats, telomeres, and long terminal repeats. Biological functions of biotinylated histones other than H4K12bio are unknown. Here we tested the hypothesis that H3K4bio, H3K9bio, H3K18bio, and H4K8bio are enriched at distinct genomic loci in humans and other eukaryotes. As a first step towards testing our hypothesis we validated polyclonal antibodies against H3K4bio, H3K9bio, H3K18bio, and H4K8bio. Antibodies against H3K9bio, H3K18bio, and H4K8bio met stringent specificity requirements and are currently being used to map novel histone marks in the human genome by ChIP‐to‐PCR, ChIP‐to‐chip, and ChIP‐to Seq assays. We anticipate these studies providing insights in the biological functions of H3K9bio, H3K18bio, and H4K8bio (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Holocarboxylase synthetase physically interacts with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the covalent binding of biotin to lysine (K) residues in histones H2A, H3, and H4; HCS knockdown disturbs gene regulation and causes distinct phenotypes in eukaryotes. HCS binds to chromosomes despite lacking a strong DNA‐binding domain. Here we tested the hypothesis that binding of HCS to chromosomes is mediated by chromatin proteins. HCS‐associated proteins were precipitated by using an antibody to HCS. Histone H3 was among the most abundant HCS‐bound proteins, and its identity was confirmed by mass spectrometry. Next, we produced recombinant GST‐HCS fusion protein and its substrate p67. The purified HCS fusion protein was bioactive, as evidenced by biotinylation of p67 in vitro. Finally, we synthesized H3‐based peptides containing the biotinylation targets K9 and K18. These peptides were incubated with recombinant HCS and cofactors.HCS catalyzed biotinylation of both K9 and K18 in peptides, as judged by probing with streptavidin, anti‐H3K9bio, and anti‐H3K18bio. In conclusion, these studies provide evidence that HCS physically interacts with histone H3, and that HCS mediates biotinylation of K9 and K18 in histone H3. (Supported by NE ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

K12‐biotinylated histone H4 is enriched in human telomeric repeats

Covalent modifications of histones play a role in regulating telomere attrition and cellular senescence. Biotinylation of lysine (K) residues in histones, mediated by holocarboxylase synthetase (HCS), is a novel diet‐dependent mechanism to regulate chromatin structure and gene expression. We have previously shown that biotinylation of K12 in histone H4 (H4K12bio) is a marker for heterochromatin and is enriched in pericentromeric alpha satellite repeats. Here we hypothesized that H4K12bio is also enriched in telomere repeats. We used human IMR‐90 lung fibroblasts and immortalized IMR‐90 cells overexpressing human telomerase (hTERT) to quantify H4K12bio in telomeres from young and old cells. About 30% of the histone H4 molecules in telomeres were biotinylated in position K12. The abundance of H4K12bio in telomeres decreased by 42% during telomere attrition in senescent IMR‐90 cells; overexpression of telomerase prevented the loss of H4K12bio. Possible confounders such as decreased expression of HCS and biotin transporters were formally excluded. These preliminary data suggest that H4K12bio might play a role in telomere structures. (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)

Cloning and characterization of 3‐hydroxyacyl‐CoA dehydrogenase from Micrococcus luteus WIUJH20

Soapstock is a by‐product of edible oil refining process with low cash value. It is rich in oleic and linoleic acids which can be converted to value‐added compounds by certain microorganisms. Nocardia cholesterolicum NRRL 5767 (N. cholesterolicum NRRL 5767) is a well‐known microbe that can transform oleic acid to 10‐hydroxyoctadecanoic acid (10‐HOOD, 85%) and 10‐oxo‐octadecanoic (10‐OXOD, 10%). The 10‐OXOD contaminant complicates downstream purification of 10‐OHOD. The conversion reactions are catalyzed by oleate dehydrogenase and secondary alcohol dehydrogenase. The long‐term goal is strain improvement of N. cholesterolicum NRRL 5767 by blocking 2‐ADH to create an industrially useful microbe for the production of 10‐OHOD from oleic acid. M. luteus was used for 2‐ADH purification due to its lack of oleate hydratase which hampered the purification of 2‐ADH from N. cholesterolicum NRRL 5767.The 2‐ADH from M. luteus WIUJH20 has been purified to homogeneity and its N‐terminal sequence determined. Using BLAST search tool, the 2‐ADH gene has been identified as belonging to the 3‐hydroxyacyl CoA dehydrogenase superfamily. The gene has been amplified by PCR and cloned into pGEX2T. The recombinant 2‐ADH protein has been overexpressed in E. coli, purified, and its Km and Vmax determined.Supported in part by grants from USDA CSREES 04‐35504‐14712 and 02‐015470.

Differences in variety of vegetable consumption in low income women

MyPyramid includes recommendations for quantity and variety of vegetable intake. In previous studies, increased variety of vegetable intake was inversely related to BMI. We examined this association in low‐income women in California. Participants (n=112) completed survey instruments to assess dietary intake, behaviors and attitudes related to food intake. Three groups were formed based on variety of vegetables eaten weekly: low variety (LV) ≤ 5 different vegetables/wk, moderate (MV) 6‐9 vegetables/wk, high (HV) ≥ 10 vegetables/wk. BMI did not differ between groups, but descriptors of diet quality were different. Daily consumption of all vegetables averaged 1.0 cups in LV, and 2.8 cups in HV (p&lt;0.001). The Healthy Eating Index score was lower and energy density of the diet was higher in LV compared to MV and HV (p&lt;0.001). Top 5 vegetables most commonly consumed by LV included 3 starchy vegetables of high energy density, but only nutrient dense vegetables were in the list of top 5 for HV. More women in HV thought it was important that foods they ate were healthy (p&lt;0.03). Although variety of vegetables was not related to BMI in this low‐income sample, greater vegetable variety was linked to better diet quality and higher quantities of vegetables consumed. Interventions designed to encourage and support variety in vegetable intake for this population are needed. Supported by USDA CSREES NRI grant #2004‐35215‐14441.