Daniel G. Peterson

Conjugated Linoleic Acids in the Dairy Cow: Biological Activities and Applications

Daniel G. Peterson — Tue, 26 Apr 2011 11:30:24 PDT

Push Versus Pull: Nutrient Partitioning Toward Immunity

Brooke D. Humphrey et al. — Tue, 26 Apr 2011 11:30:16 PDT

LH increases insl3 mRNA abundance in MA-10 Leydig cells

Mary Elizabeth Strong et al. — Fri, 01 Apr 2011 07:48:17 PDT

Leydig cells produce testosterone in response to luteinizing hormone (LH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A pathway. Additionally, these cells are responsible for producing insulin-like peptide 3 (INSL3), a peptide hormone that is essential for testicular descent. The insl3 promoterin Leydig cells can be activated by cAMP through the transcription factor Nur77. While the mechanism of LH action on testosterone production is well characterized, the effect of LH on insl3 abundance has yet to be shown directly. MA-10 Leydig cells were treated with either 0 and 100 ng/mL LH for 3 and 6 hours in a 2x2 factorial design or 0 and 0.6 mM cAMP for 6 hours. Relative insl3 mRNA abundance was determined byquantitative reverse transcription PCR. While cAMP treatment failed to alter abundance of insl3 mRNA, treatment with 100ng/mL LH for 3 hours induced a significant increase in insl3 mRNA abundance. These data indicate that LH does increase insl3 mRNA abundance in MA-10 cells.

Development of Methods for the Production of Transgenic Quail Expressing an E. Coli Phytase Gene

G. G. Guenther et al. — Fri, 01 Apr 2011 07:48:09 PDT

Excess phosphorus (P) in manure is a major environmental pollutant associated with poultry and swine production and is directly related to eutrophication, which results in the death of aquatic organisms and loss of biodiversity. Phytate accounts for 50–80% of plant phosphorus and this phytate-bound P is unavailable to simple-stomached animals. As a result, this P is not utilized by the animal and is therefore excreted. Phytases are enzymes that cleave inorganic phosphate from phytic acid, thereby improving its bioavailability to simple-stomached animals. Strategies intended to limit levels of P excretion in production animals have included dietary supplementation of phytase, as well as the use of transgenic crops with reduced phytate levels. Additionally, the recent production of transgenic pigs that express recombinant phytase in the digestive tract has been shown to increase phytate-P availability and therefore reduce P excretion. We have developed a phytase transgene construct for production of transgenic quail as a model system for application to commercial poultry. We have isolated and cloned the chicken preprogastrin promoter (PPGP), which is designed to limit the expression of a recombinant phytase to the proventriculus where it will be secreted into the upper GI tract. The PPGP was cloned from genomic DNA isolated from the Cal Poly chicken population, and the phytase gene was cloned from an E. coli isolated from the Cal Poly quail population. The phytase gene sequence is distinct from other AppA phytases previously reported, and codes for a functional phytase enzyme. Expression of this new phytase in a yeast system has yielded a functional protein with enzymatic properties highly suited for use in a recombinant poultry system. We have developed a protocol for the isolation and culture of quail primordial germ cells (PGC) from 5.5-day-old embryos for transfection and repopulation of a developing embryo with PGCs harboring the transgene construct. A green fluorescent protein gene controlled by the T7 promoter was included in the construct to allow for the simple assessment of transgene incorporation in cells, developing embryos, and tissues of the adult animals. 128 The PPGP was cloned from genomic DNA isolated from the Cal Poly chicken population, and the phytase gene was cloned from an E. coli isolated from the Cal Poly quail population. The phytase gene sequence is distinct from other AppA phytases previously reported, and codes for a functional phytase enzyme. Expression of this new phytase in a yeast system has yielded a functional protein with enzymatic properties highly suited for use in a recombinant poultry system. We have developed a protocol for the isolation and culture of quail primordial germ cells (PGC) from 5.5-day-old embryos for transfection and repopulation of a developing embryo with PGCs harboring the transgene construct. A green fluorescent protein gene controlled by the T7 promoter was included in the construct to allow for the simple assessment of transgene incorporation in cells, developing embryos, and tissues of the adult animals.

Mesenchymal Stem Cells Isolated from a Novel Equine Tissue Exhibit Embryonic Molecular Markers and Adipogenic Differentiation Potential

Jane Marie Isquith et al. — Fri, 01 Apr 2011 07:48:06 PDT

Mesenchymal stem cells (MSC) have been isolated from equine tissues, most notably adipose and bonemarrow. On the basis of ability for self-renewal, molecular marker expression and differentiation potential, we characterized MSCs isolated from a tissue not yet explored in the equine. After isolation, MSCs weremaintained in culture for 25 passages before senescence was observed. Reverse transcription-PCR revealedembryonic transcription factor Oct4 mRNA, as well as mRNA for fucosyltransferase 4 and ST3B-galactosid α 2,3 sialySSEA4, respylxanthine l ltransferase 2, two enzymes responsible for synthesis of embryonic surface markersSSEA1 and ectively. Treatment with insulin, dexamethasone, indomethacin andisobutylmethed to increased oil red O staining (P<0.05) indicating successful adipogenicdifferentiation. These data indicate the stem-like nature of these cells and their successful isolation from this tissue source. However, further characterization is needed to determine their full potential.

Conjugated Linoleic Acids Alter Body Composition Differently According to Physiological Age in Moulard Ducks

Jeffrey Allen Fesler et al. — Fri, 01 Apr 2011 07:48:02 PDT

Recent developments in the field of lipid biology have shown conjugated linoleic acids (CLA) to have remarkable yet inconsistent metabolic effects in mice, rats, hamsters, chickens, cows, and humans. In particular, effects on lipogenesis vary with tissue, physiological state, and specie. In this study, sub-adult (7 wk) and adult (11 wk) moulard ducks were grouped by age and fed a standard corn-soy based diet supplemented with either 5% soybean oil (control) or 5% CLA isomer mixture (30% 9c,11t, 30% 10t,12c; CLA). Animals were harvested after 3 wk or 6 wk for assessment of body composition including liver, adipose, viscera, and empty carcass weight. After 3 wk, the sub-adult CLA group exhibited a 24% decrease in dissectible adipose tissue (P<0.05) while adult animals showed no significant diet effect. After 6 wk, the sub-adult CLA group exhibited a 20% increase in liver mass compared to the control (P<0.05), but no diet effect on adipose tissue, while the adult CLA group showed a 42% decrease in adipose tissue mass compared to the control (P<0.05). These data indicate that CLA have potent effects on lipid metabolism in ducks, but that these effects differ dependent on physiological age.

Isolation and Selective Culture of Quail Primordial Germ Cells and The Development of a Cross Culture System to Harbor Development of Manipulated Embryos

C. M. Kimmell et al. — Fri, 01 Apr 2011 07:47:59 PDT

Primordial germ cells (PGCs) are the embryonic precursors of adult egg and sperm. Isolation, propagation, genetic manipulation and reimplantation of modified PGCs that retain their commitment to the germline have been recently described for the successful generation of transgenic chickens [1]. Here, we report the adaptation and application of these techniques to Japanese quail for the purpose of future applications involving the introduction of a phytase transgene as previously described [2]. Quail PGCs are harvested by aspiration of whole embryonic blood from embryos that have been incubated for 48– 52 h. Blood is aspirated using a mouth pipette fitted with a 37 um i.d. embryo biopsy needle and then cultured in media that has been conditioned on buffalo rat liver feeder cells to selectively propagate PGCs. We are continuing to refine methods for the selective culture and propagation of PGCs for genetic manipulation and reimplantation into developing embryos. Modified PGCs must be reinjected into an embryo of the same developmental stage, and recipient embryos must then be allowed to develop to hatch. This requires the use of a cross culture system involving transfer of the manipulated quail embryo to foster chicken egg shell beds. We have explored various conditions to optimize this system including egg shell bed size, use of antibiotic and fungicidal compounds, incubation environment, and supplementation with thick and thin chicken albumen. For unmanipulated embryos entering the cross culture system, we have experienced hatchability rates ranging between about 10% and 30%. We have also observed that embryos entering this cross culture system require an additional 12–48 h to complete development.

Comparison of the Effects of Growth Hormone on MAC-T Cells and Primary Mammary Cells

Lisa McDonnell et al. — Fri, 01 Apr 2011 07:47:55 PDT

The ability of growth hormone (GH) to increase milk yield through somatomedins is well characterized,however recent studies utilizing mammary epithelial cell (MEC) lines have indicated a possible direct effecton MEC. In the mammary alveolar cell-T (MAC-T) line, relative abundance of mRNA for two milk protein genes, α-lactalbumin and αS1-casein, and GH receptor was increased by GH. MAC-T cells were compared tobovine mammary tissue explants and primary MEC isolated from raw milk through culture in classicallactation medium (dexamethasone, insulin, prolactin) with 0 or 10 ng/mL GH. Explant and primary cellmRNA abundance showed a similar pattern to MAC-T indicating that the effects observed in cell lines maybe relevant in vivo. To begin to understand the nature of the effects of GH in MEC, global protein expressionin MAC-T cells was analyzed by 2-D gel electrophoresis, finding the abundance of 40 proteins altered byinclusion of 10ng/mL GH. Proteins were identified using MALDI-TOF with tandem MS and include enzymesinvolved in glycolysis, lipogenesis, protein processing and transport.

Growth Hormone Alters the Proteome of Differentiated MAC-T Cells

Tasha Lynn Johnson et al. — Fri, 01 Apr 2011 07:47:53 PDT

Growth Hormone (GH) improves growth and lactation in many species. Mammary epithelium is responsive to GH though the mechanism of these effects has not yet been fully characterized. Bovine Mammary Alveolar Cell-T (MAC-T) cells uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin (DIP) and are a widely used ruminant mammary cell model. We have found significant changes in triacyglycerol, cholesterol and phospholipid distribution in MAC -T cells stimulated with 10 ng/ml GH. To characterize these direct effects of GH on a proteomic level, total protein was isolated from MAC-T cells after differentiation for seven days with DIP and either 0 or 10 ng/ml GH. Proteins were separated by isoelectric point and molecular weight using two-dimensional gel electrophoresis. We have observed a change in the abundance of multiple proteins when MAC-T cells are stimulated with GH and the identification of these proteins is being pursued using MALDI-TOF mass spectrometry.

Effects of a Marginal Zinc Diet on Intestinal Health and Immune Function

Daniel G. Peterson et al. — Fri, 01 Apr 2011 07:47:50 PDT

Zinc (Zn) is an essential nutrient for overall health and proper immune function, especially within the digestive system, although the specific mechanisms by which it exerts these effects are not well understood. In this study we examined the effects of short term exposure to a marginal Zn diet on the intestinal health and immune function of lipopolysaccharide (LPS) challenged mice through plasma cytokine profiling and histologic evaluation of intestinal tissue sections. Adult male mice were fed a Zn-adequate (30 ppm) or Zn-marginal (3 ppm) diet for 4 wk and then a bacterial challenge was simulated with an intraperitoneal injection of LPS (10 ug/g BW) or saline (control). Plasma and tissues were collected at 0, 6, or 24 h post-challenge for analysis. Ileal, jejunal and cecal samples were thinly sectioned, mounted on slides and stained with hematoxylin-eosin for histological evaluation of villi length, villi width, lamina propria width, crypt depth, intraepithelial leukocyte number, lamina propria leukocyte number and presence or absence of Peyer’s patches. Plasma was analyzed for IL-1 beta, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, IFN gamma, and TNF alpha. The marginal Zn diet led to shorter and wider villi in the ileum and jejunum (P<0.05), greater lymphocyte infiltration in the lamina propria after LPS challenge (P<0.05), and higher plasma IL-6 levels at 24 h post-LPS (P<0.01). Results indicate that Zn status substantially impacts the intestinal response to LPS through modulation of the cytokine response and leukocyte recruitment, and this impact is evident even with short-term (4 wk) Zn depletion.

Growth Hormone, Prolactin, and Sodium Acetate Have Complex and Non-Uniform Effects on the Lipid Distribution of a Differentiated Bovine Mammary Epithelial Cell Line

Brent Fujimoto et al. — Fri, 01 Apr 2011 07:47:45 PDT

Non-polar lipids (NPL) accumulate within the mammary epithelial cell during lactation; however there are no reported systematic studies that utilize NPL composition as a sign of differentiation. Additionally, there are no reported systematic studies on the response of a bovine mammary alveolar cell line (MAC-T), to a range of lactogenic hormone concentrations, or different lipogenic substrates. In a 3 x 3 factorial arrangement, the effect of growth hormone (GH; 0, 10, 100 ng/ml), and prolactin (PRL; 0, 5, 10 µg/ml) on the lipid composition of MAC-T was examined using thin layer chromatography. Using the optimal hormone treatment (10 ng/ml GH, 5 µg/ml PRL) for NPL synthesis, inclusion of 10 mM sodium acetate (NaOAc) was assessed to determine the effect of lipogenic substrate on lipid composition. GH had a significant effect on NPL, cholesterol, phosphatidylcholine, phosphatidylinostitol, and sphingomyelin (SM), while PRL only affected SM. NaOAc had a significant effect on NPL, phosphatidylethanolamine and SM. The proportion of NPL peaked with 10 ng/ml GH, but dropped to near-control levels with 100 ng/ml GH. This study underscores the importance of testing a range of hormone concentrations when assessing lipogenic profile of MAC-T cells.

Carbohydrate Response Element Binding-Protein is Activated by Elevated Glucose Levels in the Normal Murine Liver Cell Line NMuLi

Jackson Adams et al. — Fri, 01 Apr 2011 07:47:38 PDT

Carbohydrate response element (ChRE) binding protein (ChREBP) is a transcription factor that is regulated by intracellular glucose levels and activates several lipogenic genes including acetyl CoA carboxylase (ACC). Previous studies of the ChREBP system have used both in vivo models as well as isolated primary hepatocytes. Due to the difficulties in isolation and genetic variability of primary cell culture, we propose the use of a normal murine liver cell line (NMuLi) as a convenient alternative to primary isolated hepatocytes. We show that high (25 mM) but not low (0.5 or 5 mM) glucose activates a luciferase reporter driven by 4 repeated ChREs, as well as the murine ACC promoter segment from –220 to +21. Furthermore, using small interfering RNA to knock-down ChREBP, we show that ChREBP is required for this glucose-induced luciferase reporter activation. Finally, using chromatin immunoprecipitation we show that ChREBP directly binds to the ChRE within the promoter region of the ACC gene in the presence of high but not low glucose. Together, these results suggest that NMuLi cells may be used as an alternative to primary isolated hepatocytes for studies of the ChREBP system.

Update on Conjugated Linoleic Acids (CLA)

D. E. Bauman et al. — Mon, 14 Mar 2011 10:59:01 PDT

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

Andrew K. Benson et al. — Mon, 14 Mar 2011 10:59:00 PDT

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genomewide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

Moderate Zinc Restriction Affects Intestinal Health and Immune Function in Lipopolysaccharide-Challenged Mice

Daniel G. Peterson et al. — Mon, 14 Mar 2011 10:58:58 PDT

Zinc (Zn) is an essential nutrient that affects immune function, especially within the digestive system, although the underlying mechanisms are not well understood. This study examined the effects of short-term moderate Zn restriction on intestinal health and immune function in lipopolysaccharide (LPS)-challenged mice through plasma cytokine profiling and histological evaluation of intestinal tissue sections. Adult male mice were fed with a Zn-adequate (40 ppm) or a Zn-marginal (4 ppm) diet for 4 weeks, and then a bacterial challenge was simulated by intraperitoneal injection of LPS (10 μg/g body weight [BW]) or saline (control). BW was recorded weekly, and feed intake was recorded daily over the last week. Voluntary locomotor activity was assessed 6 and 24 h after the challenge. Plasma and tissues were collected 0, 6 or 24 h after the challenge for analysis. Histological analysis of intestinal samples included evaluation of villi length and width, lamina propria (LP) width, crypt depth and intraepithelial as well as LP leukocyte numbers. Plasma was analyzed for IL-1β, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, interferon gamma and tumor necrosis factor α. Diet did not affect BW and feed intake. The LPS challenge led to decreased voluntary locomotor activity (P<.05). Moderate Zn restriction led to greater leukocyte infiltration in the LP after the LPS challenge (P<.05) and higher plasma IL-6 and IL-10 levels 24 h after the LPS challenge (P<.01). Results indicate that Zn status impacts intestinal responses to LPS through modulation of the cytokine response and leukocyte recruitment, and this impact is evident even with short-term (4weeks) moderate Zn restriction.

Growth Hormone Alters Lipid Composition and Increases the Abundance of casein and lactalbumin mRNA in the MAC-T Cell Line

Tasha L. Johnson et al. — Mon, 14 Mar 2011 10:58:57 PDT

The MAC-T cell line has been used extensively to investigate bovine mammary epithelial cell function. A lactogenic phenotype is generally induced in this cell line by a combination of dexamethasone, insulin and prolactin and has typically been assessed by milk protein production. Few studies have focused on identifying other factors that may affect milk protein synthesis in the MAC-T cell line, and none have considered the lipid class distribution of MAC-T cells as a component of the lactogenic phenotype. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and has been shown to alter the lipogenic profile of mammary explant models. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations to the lipid class distribution as well as to milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium significantly induced production of alpha-s1 casein and alpha-lactalbumin mRNA. GH also significantly affected the proportion of triacylglycerol and sphingomyelin. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and support the possibility of a direct effect of GH on milk synthesis in vivo.