Publications by authors named "Stanislaw Kahl"

19 Publications

  • Page 1 of 1

The effect of delayed feeding post-hatch on caeca development in broiler chickens.

Br Poult Sci 2021 Apr 9. Epub 2021 Apr 9.

United States Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705.

Broiler chicks are frequently deprived of food up to 72 h due to uneven hatching rates, management procedures and transportation to farms. Little is known about the effect of delayed feeding due to extended hatching times on the early neonatal development of the caeca. Therefore, the objective of this study was to investigate the developmental changes and effects of a 48-h delay in feed access immediately post-hatch (PH) on the caeca.After hatch, birds (Ross 708) were randomly divided into two treatment groups (n=6 battery pen/treatment). One group (early fed; EF) received feed and water immediately after hatch, while the second group (late fed; LF) had access to water but had delayed access to feed for 48 h. Contents averaging across all regions of the caeca were collected for mRNA expression as well as for histological analysis at -48, 0, 4 h PH and then at 1, 2, 3, 4, 6, 8, 10, 12 and 14 days PH.Expression of MCT-1 (a nutrient transporter), Cox7A2 (related to mitochondrial function) IgA, pIgR, and ChIL-8 (immune function) genes was affected by delayed access to feed that was dependent by the time PH. Expression of immune and gut barrier function related genes (LEAP2 and MUC2, respectively) was increased in LF group. There was no effect of feed delay on expression of genes related to mitochondrial functions in the caeca, although developmental changes were observed (ATP5F1B, Cox4|1). Caecal mucus and muscle thickness were affected by delayed access to feed during caeca development.The data suggested a limited effect of delayed feed access PH on the developmental changes in caecal functions. However, the caeca seemed to be relatively resistant to delayed access to feed early PH, with only a few genes affected.
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http://dx.doi.org/10.1080/00071668.2021.1912291DOI Listing
April 2021

The effects of tributyrin supplementation on weight gain and intestinal gene expression in broiler chickens during Eimeria maxima-induced coccidiosis.

Poult Sci 2021 Apr 18;100(4):100984. Epub 2021 Jan 18.

Animal Biosciences and Biotechnology Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD 20705, USA. Electronic address:

Butyrate is a feed additive that has been shown to have antibacterial properties and improve gut health in broilers. Here, we examined the performance and gene expression changes in the ileum of tributyrin-supplemented broilers infected with coccidia. Ninety-six, Ross 708 broilers were fed either a control corn-soybean-based diet (-BE) or a diet supplemented with 0.25% (w/w) tributyrin (+BE). Birds were further divided into groups that were inoculated with Eimeria maxima oocysts (EM) or sham-inoculated (C) on day 21 posthatch. At 7 d postinfection (7 d PI), the peak of pathology in E. maxima infection, tributyrin-supplemented birds had significantly improved feed conversion ratios (FCR, P < 0.05) and body weight gain (BWG, P < 0.05) compared with -BE-infected birds, despite both groups having similar feed intake (FI, P > 0.05). However, at 10 d post-infection (10 d PI) no significant effects of feed type or infection were observed. Gene expression in the ileum was examined for insights into possible effects of infection and tributyrin supplementation on genes encoding proteins related to immunity, digestion, and gut barrier integrity. Among immune-related genes examined, IL-1B and LEAP2 were only significantly affected at 7 d PI. Transcription of genes related to digestion (APN, MCT1, FABP2, and MUC2) were primarily influenced by infection at 7 d PI and tributyrin supplementation (FABP2 and MUC2) at 10 d PI. With exception of ZO1, tight junction genes were affected by either infection or feed type at 7 d PI. At 10 d PI, only CLDN1 was not affected by either infection or feed type. Overall tributyrin shows promise as a supplement to improve performance during coccidiosis in broiler chickens; however, its effect on gene expression and mode of action requires further research.
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http://dx.doi.org/10.1016/j.psj.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921011PMC
April 2021

Research Note: Effect of butyric acid glycerol esters on ileal and cecal mucosal and luminal microbiota in chickens challenged with Eimeria maxima.

Poult Sci 2020 Oct 3;99(10):5143-5148. Epub 2020 Jul 3.

United States Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705, U.S.A.

Coccidiosis is one of the most prevalent diseases seen in the poultry industry leading to excessive economic losses. The aim of this study was to investigate the effect of butyric acid glycerol esters (BE) on the ileal and cecal microbiota in birds challenged with Eimeria maxima (EM). Ross 708 male broilers were fed a diet supplemented with 0 (control) or 0.25% BE from day 1. On day 21, half of the birds were infected with 10 EM oocysts. For determing microbiota, ileal and cecal contents and epithelial scrapings were collected at 7 and 10 D postinfection (PI). Alpha diversity of bacterial communities was mostly affected (P < 0.05) by time PI and EM infection. The richness of luminal bacterial populations in the ileum and ceca was affected (P < 0.05) by addition of BE and by time PI × EM × BE interaction, respectively. In the ileal and cecal luminal and mucosal bacterial communities, permutational multivariate analysis of variance (PERMANOVA, unweighted UniFrac) showed significant (P < 0.05) differences because of time PI and interaction between time PI, EM, and BE. Significant (P < 0.05) differences in taxonomic composition at the family level were observed in microbiota of luminal and mucosal populations of the ileum and ceca owing to time PI, EM, BE, and their interactions. The bacterial community present in the cecal lumen was characterized by the lowest number of differential bacteria, whereas the cecal mucosal community was characterized by the highest number of differentially abundant bacteria. In conclusion, our results show that EM infection and time PI has the biggest impact on microbial diversity in the chicken gut. The presence of BE in the diet had a limited effect on gut microbiota.
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http://dx.doi.org/10.1016/j.psj.2020.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598111PMC
October 2020

Effect of delayed feeding post-hatch on expression of tight junction- and gut barrier-related genes in the small intestine of broiler chickens during neonatal development.

Poult Sci 2020 Oct 3;99(10):4714-4729. Epub 2020 Jul 3.

Animal Biosciences and Biotechnology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.

The gut not only plays a key role in digestion and absorption of nutrients but also forms a physical barrier and first line of defense between the host and the luminal environment. A functional gut barrier (mucus and epithelial cells with tight junctions [TJ]) is essential for optimal health and efficient production in poultry. In current broiler system, chicks are deprived of food and water up to 72 h due to uneven hatching, hatchery procedures, and transportation. Post-hatch feed delay results in lower BW, higher FCR and mortality, and delayed post-hatch gut development. Little is known about the effects of early neonatal development and delayed feeding immediately post-hatch on gut barrier function in chickens. Therefore, the aim of the present study was to characterize the expression pattern of gut barrier-related and TJ-related genes in the small intestine of broiler chickens during early development and delay in access to feed. Newly hatched chicks received feed and water immediately after hatch or were subjected to 48 h delayed access to feed to mimic commercial hatchery setting and operations. Birds were sampled (n = 6) at -48, 0, 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h post-hatch. Jejunum and ileum were collected, cleaned of digesta, and snap-frozen in liquid nitrogen or fixed in paraformaldehyde. The relative mRNA levels of gut barrier- and TJ-related protein genes were measured by quantitative PCR and analyzed by 2-way ANOVA. In both tissues, changes (P < 0.05) in gene expression pattern of gut barrier-related and TJ-related genes were detected due to delayed access to feed post-hatch and/or development. In general, expression of TJ-related genes was downregulated while mRNA levels of gut barrier-related genes were upregulated during development. Histological differences and changes in mucin staining due to age and treatment were observed. These results suggest that delayed access to feed post-hatch may affect TJ structure and/or function and therefore gut barrier function and overall health of the chicken small intestine.
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http://dx.doi.org/10.1016/j.psj.2020.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598124PMC
October 2020

Effect of bovine genotype on innate immune response of heifers to repeated lipopolysaccharide (LPS) administration.

Vet Immunol Immunopathol 2019 Sep 31;215:109914. Epub 2019 Jul 31.

Department of Animal Science, University of Minnesota, Saint Paul, MN, 55108, USA. Electronic address:

This pilot study provides a preliminary assessment of the impact of genotype on acute innate immune pro-inflammatory, metabolic and endocrine responses to repeated lipopolysaccharide (LPS) administered to growing heifers. Heifers (n = 4/genotype) were from unselected (stable milk yield since 1964, UH) or contemporary (CH) Holstein cows that differed in milk yield (6200 vs 11,100 kg milk/305 d) or from contemporary Black Angus (CA) cows bred to contemporary Red Angus bulls. Heifers were challenged with iv administration of 0.5 μg LPS/kg body weight on day 1 (Challenge 1) and d 5 (Challenge 2) of study to assess endotoxin tolerance. Plasma was collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to each LPS administration. Rectal body temperature (BT) was measured before each blood sampling and at 5 and 7 h. Data were analyzed by repeated measures with sampling time as the repeated effect. Each genotype had at least one pro-inflammatory response that indicated it might have a more robust response than the other genotypes. The CH heifers had a greater TNF-α response, UH heifers had greater IL-6 and XO responses and CA heifers had greater BT and SAA response to LPS than the other genotypes. There was a genotype by time by interaction as cortisol peaked earlier in CH and UH than in CA heifers. Glucose response was less in CA and insulin response was greater in CH heifers. Endotoxin tolerance to LPS was evident as pro-inflammatory, cortisol, glucose and insulin responses were less during Challenge 2 than during Challenge 1. Differences among genotypes during Challenge 1 were eliminated during Challenge 2 except for the greater SAA response in CA heifers and indicate the potential for differential impacts of genotype on the development of endotoxin tolerance. Specific reasons for these effects of genotype are not clear from these data but the results support the hypothesis for differential innate immune signaling among these bovine genotypes.
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http://dx.doi.org/10.1016/j.vetimm.2019.109914DOI Listing
September 2019

Effect of early neonatal development and delayed feeding post-hatch on jejunal and ileal calcium and phosphorus transporter genes expression in broiler chickens.

Poult Sci 2019 04;98(4):1861-1871

United States Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705, USA.

Calcium (Ca) and phosphorus (P) are essential minerals involved in many biological processes including bone development and mineralization. Plasma concentration of both minerals is tightly regulated, and Ca and P homeostasis is maintained via intestinal absorption, bone storage and exchange, and renal reabsorption. In the current broiler production systems, chicks are deprived of food and water for up to 72 h due to uneven hatching, hatchery procedures, and transportation time to farms. Post-hatch (PH) feed delay results in lower body and organ weight, higher feed conversion ratio and mortality, and delayed PH growth and GIT development. Little is known about the effects of early neonatal development and delayed or immediate feeding PH on Ca and P transporters. Therefore, the aim of the present study was to characterize expression patterns of Ca and P transporter genes in small intestine during the first 2 wk PH in chickens fed immediately after hatch (FED) or subjected to 48 h delayed feeding (NOTFED). Expression of all Ca and P transporters in jejunum and ileum was significantly (P < 0.05) affected by age. Among Ca transporter genes, only mRNA expression of Calbidin D28k in jejunum and Ca sensing receptor (CaSR) in ileum were significantly (P < 0.05) affected by delay in feed access. For P transporter genes' expression, only P transporter type III (PIT1) mRNA was significantly affected by age, delay in feed access, and their interaction (P < 0.05). In summary, we have shown, for the first time, early developmental changes of Ca and P transporter genes in broiler chickens. Results suggest that an increase in gene expression of some of the transporters corresponds with the switch from yolk to high starch diet. Overall, our results can be helpful in better understanding of Ca and P homeostasis in broilers.
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http://dx.doi.org/10.3382/ps/pey546DOI Listing
April 2019

Temporal pattern changes in duodenal protein tyrosine nitration events in response to Eimeria acervulina infection in chickens.

J Anim Sci 2018 Jun;96(6):2125-2138

Department of Oncology, Center for Biomedical Research of La Rioja (CIBIR), Spain.

Intracellular generation of nitric oxide (NO) and superoxide anion (SOA) can result in the formation of 3'-nitrotyrosine proteins (NTp). Nitrated proteins usually are associated with significant perturbation in protein function, apoptosis, autophagy, and cell death. We undertook the present study to establish the temporal dynamics of NTp generation in cytokeratin-18-positive epithelial cells (ETCs) of broiler chickens in response to infection with Eimeria acervulina. Duodenal tissue was harvested from noninfected (NOI) and infected (INF) broilers on days (d) 1, 3, 6, 7, and 10 postinfection (PI) and fixed, embedded, and sectioned for quantitative image analysis, immunohistochemistry with antibodies specific to NTp and the SOA-generating enzyme xanthine oxidase (XO). The pixel density characteristics for NTp and XO representative of ETCs demonstrated that NTp and XO increased in intestinal villi as early as d1 PI (P < 0.05 vs. NOI). Progressive increases in NTp were evident in ETCs through d6 PI. For XO, increases in cell content increased only through d3. On d6 and d7 PI, high levels of NTp were present in immune infiltrating cells (IIC) where no XO was detected. The increases in ETC NTp occurred in a defined pattern, significant by villus-to-crypt location for day of infection, initiating in the distal villus and progressing down into the crypts. Two NTp patterns were observed for ETCs: a high level associated with ETCs harboring parasites and a low-level increase in ETCs not containing Eimeria but in proximity to such. The data suggest that NTp and XO responses may mediate some of the processes through which ETCs respond to Eimeria to limit the extent of infection by this pathogen.
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http://dx.doi.org/10.1093/jas/sky140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095395PMC
June 2018

Effects of heat stress on carbohydrate and lipid metabolism in growing pigs.

Physiol Rep 2015 Feb 25;3(2). Epub 2015 Feb 25.

Department of Animal Science, Iowa State University, Ames, Iowa, USA.

Heat stress (HS) jeopardizes human and animal health and reduces animal agriculture productivity; however, its pathophysiology is not well understood. Study objectives were to evaluate the direct effects of HS on carbohydrate and lipid metabolism. Female pigs (57 ± 5 kg body weight) were subjected to two experimental periods. During period 1, all pigs remained in thermoneutral conditions (TN; 20°C) and were ad libitum fed. During period 2, pigs were exposed to: (1) constant HS conditions (32°C) and fed ad libitum (n = 7), or (2) TN conditions and pair-fed (PFTN; n = 10) to minimize the confounding effects of dissimilar feed intake. All pigs received an intravenous glucose tolerance test (GTT) and an epinephrine challenge (EC) in period 1, and during the early and late phases of period 2. After 8 days of environmental exposure, all pigs were killed and tissue samples were collected. Despite a similar reduction in feed intake (39%), HS pigs tended to have decreased circulating nonesterified fatty acids (NEFA; 20%) and a blunted NEFA response (71%) to the EC compared to PFTN pigs. During early exposure, HS increased basal circulating C-peptide (55%) and decreased the insulinogenic index (45%) in response to the GTT. Heat-stressed pigs had a reduced T3 to T4 ratio (56%) and hepatic 5'-deiodinase activity (58%). After 8 days, HS decreased or tended to decrease the expression of genes involved in oxidative phosphorylation in liver and skeletal muscle, and ATGL in adipose tissue. In summary, HS markedly alters both lipid and carbohydrate metabolism independently of nutrient intake.
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http://dx.doi.org/10.14814/phy2.12315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393217PMC
February 2015

Alpha-Tocopherol Alters Transcription Activities that Modulates Tumor Necrosis Factor Alpha (TNF-α) Induced Inflammatory Response in Bovine Cells.

Gene Regul Syst Bio 2012 5;6:1-14. Epub 2011 Dec 5.

Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, ARS, USDA, 10300 Baltimore Ave, Beltsville, MD 20705.

To further investigate the potential role of α-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-α as an immuno-stimulant to simulate inflammation response in cells with or without α-tocopherol pre-treatment. Using microarray global-profiling and IPA (Ingenuity Pathways Analysis, Ingenuity(®) Systems, http://www.ingenuity.com) data analysis on TNF-α-induced gene perturbation in those cells, we focused on determining whether α-tocopherol treatment of normal bovine cells in a standard cell culture condition can modify cell's immune response induced by TNF-α challenge. When three datasets were filtered and compared using IPA, there were a total of 1750 genes in all three datasets for comparison, 97 genes were common in all three sets; 615 genes were common in at least two datasets; there were 261 genes unique in TNF-α challenge, 399 genes were unique in α-tocopherol treatment, and 378 genes were unique in the α-tocopherol plus TNF-α treatment. TNF-α challenge induced significant change in gene expression. Many of those genes induced by TNF-α are related to the cells immune and inflammatory responses. The results of IPA data analysis showed that α-tocopherol-pretreatment of cells modulated cell's response to TNF-α challenge. In most of the canonical pathways, α-tocopherol pretreatment showed the antagonistic effect against the TNF-α-induced pro-inflammatory responses. We concluded that α-tocopherol pre-treatment has a significant antagonistic effect that modulates the cell's response to the TNF-α challenge by altering the gene expression activities of some important signaling molecules.
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http://dx.doi.org/10.4137/GRSB.S8303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256998PMC
August 2012

Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells.

In Vitro Cell Dev Biol Anim 2012 Jan 17;48(1):1-11. Epub 2011 Dec 17.

USDA, ARS, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705, USA.

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.
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http://dx.doi.org/10.1007/s11626-011-9467-7DOI Listing
January 2012

Enhanced mitochondrial complex gene function and reduced liver size may mediate improved feed efficiency of beef cattle during compensatory growth.

Funct Integr Genomics 2010 Mar 24;10(1):39-51. Epub 2009 Sep 24.

Beltsville Agricultural Research Center, ARS, USDA, MD 20705, USA.

Growing ruminants under extended dietary restriction exhibit compensatory growth upon ad libitum feeding, which is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify mechanisms contributing to these physiological changes, 8-month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n = 6) for a period of 12 weeks. All steers were fed ad libitum for the remaining 8 weeks of experimentation (realimentation). Liver was biopsied at days -14, +1, and +14 relative to realimentation for gene expression analysis by microarray hybridization. During early realimentation, feed-restricted steers exhibited greater rates of gain and feed efficiency than controls and an increase in expression of genes functioning in cellular metabolism, cholesterol biosynthesis, oxidative phosphorylation, glycolysis, and gluconeogenesis. Gene expression changes during feed restriction were similar to those reported in mice, indicating similar effects of caloric restriction across species. Based on expression of genes involved in cell division and growth and upregulation of genes encoding mitochondrial complex proteins in early realimentation, it was concluded that reduced hepatic size and increased mitochondrial function may contribute to improved feed efficiency observed during compensatory growth.
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http://dx.doi.org/10.1007/s10142-009-0138-7DOI Listing
March 2010

AKT/eNOS signaling module functions as a potential feedback loop in the growth hormone signaling pathway.

J Mol Signal 2009 Mar 25;4. Epub 2009 Mar 25.

Bovine Functional Genomics laboratory, Animal and Natural Resources Institute, Agricultural Research Service, US Department of Agriculture, 10300 Baltimore Ave,, BARC EAST, Building 200, Room 209, Beltsville, MD 20705, USA.

Background: While evidence suggested that the activity states of Protein kinase B (AKT/PKB) and endothelial nitric oxide synthase (eNOS) play an important role in the progression of the Growth Hormone (GH) signal cascade, the implication of the activation of AKT/PKB and eNOS in terms of their function in the signaling pathway was not clear.

Results: Using a specific AKT/PKB inhibitor and a functional proteomic approach, we were able to detect the activities of multiple signal transduction pathway elements, the downstream targets of the AKT/PKB pathway and the modification of those responses by treatment with GH. Inhibiting the AKT/PKB activity reduced or eliminated the activation (phosphorylation) of eNOS. We demonstrated that the progression of the GH signal cascade is influenced by the activity status of AKT and eNOS, wherein the suppression of AKT activity appears to augment the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2) and to antagonize the deactivation (phosphorylation) of cyclin-dependent kinase 2 (CDC2/Cdk1) induced by GH. Phosphorylation of GSK3a/b (glycogen synthase kinase 3), the downstream target of AKT/PKB, was inhibited by the AKT/PKB inhibitor. GH did not increase phosphorylation of ribosomal S6 kinase 1 (RSK1) in normal cells but increases phosphorylation of RSK1 in cells pre-treated with the AKT and eNOS inhibitors.

Conclusion: The MAP kinase and CDC2 kinase-dependent intracellular mechanisms are involved in or are the targets of the GH's action processes, and these activities are probably directly or indirectly modulated by AKT/PKB pathways. We propose that the AKT/PKB-eNOS module likely functions as a negative feedback mediator of GH actions.
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http://dx.doi.org/10.1186/1750-2187-4-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666727PMC
March 2009

Lipopolysaccharide-induced early response genes in bovine peripheral blood mononuclear cells implicate GLG1/E-selectin as a key ligand-receptor interaction.

Funct Integr Genomics 2009 Aug 5;9(3):335-49. Epub 2009 Mar 5.

Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, BARC East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA.

This study uses integrated global gene expression information and knowledge of the regulatory events in cells to identify transcription networks controlling peripheral blood mononuclear cells' (PBMCs) immune response to lipopolysaccharide (LPS) and to identify the molecular and cellular pathways' responses to LPS. We identified that 464 genes, including at least 17 transcription factors, are significantly induced by 2-h LPS stimulation using a high-density bovine microarray platform at a very stringent false discovery rate = 0%. The networks show that, in the LPS-stimulated PBMCs, altered gene expression was transcriptionally regulated via those transcription factors through potential interaction within the pathway networks. Functional analyses revealed that LPS induces unique pathways, molecular functions, biological processes, and gene networks. In particular, gene expression data identified Golgi complex-localized glycoprotein 1/endothelial-selectin as a key ligand-receptor interaction in the early response of cells.
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http://dx.doi.org/10.1007/s10142-009-0116-0DOI Listing
August 2009

Growth hormone (GH)-associated nitration of Janus kinase-2 at the 1007Y-1008Y epitope impedes phosphorylation at this site: mechanism for and impact of a GH, AKT, and nitric oxide synthase axis on GH signal transduction.

Endocrinology 2007 Aug 17;148(8):3792-802. Epub 2007 May 17.

US Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, Beltsville, Maryland 20705, USA.

A generalized increase in liver protein tyrosine nitration (3'-nitrotyrosine, 3'-NT) occurs after GH injection in a time frame consistent with observed acute GH hyporesponsiveness. Here we investigated whether the GH-associated nitration process might be targeted to the (1007)Y-(1008)Y-phosphorylation epitope of Janus kinase (JAK)-2 because of its homology to a defined peptide nitration motif. Using antibodies we developed to the 3'NT-substituted peptide analog of the (1007)Y-(1008)Y-JAK2 site (nitro-JAK2), we demonstrated a rapid increase in membrane-associated nitro-JAK2 after GH. In vivo (bovine liver) and in vitro (porcine hepatocytes), GH-induced cellular levels of nitro-(1007)Y-(1008)Y-JAK2 persisted significantly longer after a stimulatory GH pulse than did levels of phospho-JAK2. Treatment of cultured cells with inhibitors of AKT or endothelial nitric oxide synthase prior to GH challenge attenuated the increases in nitro-JAK2 predominantly in the membrane subcellular fraction. In instances in which GH effected orthophosphorylation of (694)Y-signal transducer and activator of transcription (STAT)-5b, the addition of AKT and endothelial nitric oxide synthase inhibitors prior to GH significantly increased the levels of phospho-(694)Y-STAT5b and phospho-(1007)Y-JAK2 over those arising from GH alone. Nuclear magnetic resonance molecular modeling of natural and 3'-NT- and orthophosphate-substituted peptide analogs of the (1007)Y-(1008)Y site demonstrated significant effects of 3'-nitration on the planar orientation and intramolecular stabilizing points of the affected tyrosines. When these peptides were used as substrates for in vitro tyrosine kinase phosphorylation reactions, 3'-NT in the (1007)Y and/or (1008)Y positions blocked the generation of (1007)Y-phosphotyrosine. The data suggest that the nitration of JAK2 may act as an inhibitory counterpart to phosphorylation activation, reflecting a very localized break on the progression of GH signal transduction processes spanning JAK-STAT-AKT interactions.
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http://dx.doi.org/10.1210/en.2006-1736DOI Listing
August 2007

Caveolae nitration of Janus kinase-2 at the 1007Y-1008Y site: coordinating inflammatory response and metabolic hormone readjustment within the somatotropic axis.

Endocrinology 2007 Aug 17;148(8):3803-13. Epub 2007 May 17.

Growth Biology Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, USA.

Life-threatening proinflammatory response (PR) induces severe GH resistance. Although low-level PR is much more commonly encountered clinically, relatively few studies have investigated the accompanying change in GH signal transduction progression and, in particular, the impact of low-level PR on Janus kinase (JAK)-2. Using a low-level, in vivo endotoxin [lipopolysaccharide (LPS)] challenge protocol, we demonstrated that the liver tissue content of JAK2 declined 24 h (62%, P < 0.02) after LPS and that tyrosine-nitrated JAK2 could be immunoprecipitated from post-LPS liver biopsy homogenates. With antibodies developed to probe specifically for nitration at the (1007)Y-(1008)Y phosphorylation epitope of JAK2, we demonstrated that the nitrated (1007)Y-(1008)Y-JAK-2 (nitro-JAK2) coimmunoprecipitated with caveolin-1 and (1177)phospho-SER-endothelial nitric oxide synthase when post-LPS liver homogenates were treated with anticaveolin-1 and protein A/G. The magnitude of increase in nitro-JAK2 was attenuated in animals treated with vitamin E prior to LPS. The increase in nitro-JAK2 after LPS was greater in a line of experimental animals with a genetic propensity for higher PR at the given LPS dose than responses measured in their normal counterparts. The development and remission of nitro-JAK2 was temporally concordant with changes in plasma concentrations of IGF-I; hepatocellular IGF-I mRNA content was inversely proportional to nitro-JAK2 content. Localized changes in the state of nitration of regulatory phosphorylation domains of JAK2 in caveolar microenvironments and tissue content of JAK2 during PR suggest a unique mechanism through which discrete signal transduction switching might occur in the liver to fine tune cellular responses to the endocrine-immune signals that develop during low-level, transient proinflammatory stress.
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http://dx.doi.org/10.1210/en.2006-1737DOI Listing
August 2007

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection.

Domest Anim Endocrinol 2007 Feb 13;32(2):138-54. Epub 2006 Mar 13.

USDA-ARS, Growth Biology Laboratory, Beltsville, MD 20705, US.

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.
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http://dx.doi.org/10.1016/j.domaniend.2006.02.004DOI Listing
February 2007

Temporal response of liver signal transduction elements during in vivo endotoxin challenge in cattle: effects of growth hormone treatment.

Domest Anim Endocrinol 2007 Feb 24;32(2):79-92. Epub 2006 Jan 24.

Growth Biology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Building 200, Room 209, Beltsville, MD 20705, USA.

We quantified the changes in abundance of inducible nitric oxide synthase (iNOS) and associated tissue signal transduction pathway elements (STPEs) in the bovine liver in response to lipopolysaccharide (LPS) challenge and further assessed the impact on the LPS-driven variable responses as affected by daily treatment with recombinant growth hormone (GH) prior to LPS challenge. Twenty-four cross-bred beef steers were divided into GH-treated (recombinant bovine GH, Monsanto Inc., St. Louis, MO; 0.1mg/kg BW, i.m., daily for 12 days) and non-GH-treatment (control) groups (n=12/group). Liver biopsy samples were obtained from all animals at 0, 3, 6, and 24h after LPS challenge (E. coli 055:B5, 2.5 microg/kg BW, i.v. bolus) for Western blot analyses of iNOS and STPEs. In response to LPS, tissue levels of iNOS increased significantly (P<0.001) in the first 3h and persisted at levels greater than those at time 0 until 24h. GH further augmented levels of iNOS at 0, 3, and 6h resulting in an overall significant increase in the iNOS protein level (P<0.01). AKT/protein kinase B (AKT/PKB) phosphorylation levels at time 0 were not different between GH-treated and control animals; LPS increased the phosphorylation of AKT/PKB with GH treatment stimulating a four-fold further increase of AKT/PKB phosphorylation. Effects similar to those on AKT/PKB were also observed on signal transducer and activator of transcription 5b (STAT5b). The family of mitogen-activated protein kinase (MAPK) showed different pattern of response. ERK1/2 phosphorylation increased 3h after LPS challenge but only in GH-treated group (P<0.01). Compared to 0 h, SAPK/JUN phosphorylation increased in both experimental groups 3, 6h (P<0.01), and 24h (P<0.05) after LPS. However, at 3h the increase was greater (P<0.01) in GH-treated than in control animals. No effect of LPS challenge or GH treatment on p38(MAPK) was observed. These results suggest that GH treatment has a significant impact on the differential activation of STPEs in the clinical response to LPS.
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http://dx.doi.org/10.1016/j.domaniend.2006.01.001DOI Listing
February 2007

Mechanisms underlying growth hormone effects in augmenting nitric oxide production and protein tyrosine nitration during endotoxin challenge.

Endocrinology 2004 Jul 24;145(7):3413-23. Epub 2004 Mar 24.

United States Department of Agriculture, Agricultural Research Service, Growth Biology Laboratory, B-200, Room 201, Beltsville Agricultural Research Center-East, Beltsville, Maryland 20705, USA.

The present study defined the effects of GH administration on components of the nitric oxide (NO)-generating cascade to account for observed increases in NO production and protein nitration after an immune challenge. Calves were assigned to groups with or without GH treatment (100 microg GH/kg body weight or placebo im, daily for 12 d) and with or without low-level endotoxin [lipopolysaccharide (LPS), 2.5 microg/kg, or placebo, iv]. Plasma was obtained for estimation of NO changes as [NO(2)(-) + NO(3)(-)] (NO(x)). Transcutaneous liver biopsies were collected for measurement of protein tyrosine nitration, cationic amino acid transporter (CAT)-2 mRNA transporter, and constitutive NO synthase (cNOS), inducible NOS (iNOS), and arginase activity. Liver protein nitration increased more than 10-fold 24 h after LPS and an additional 2-fold in animals treated with GH before LPS. GH increased plasma NO(x) after LPS to levels 27% greater than those measured in non-GH-treated calves. LPS increased CAT-2 mRNA after LPS; GH was associated with a 24% reduction in CAT-2 mRNA content at the peak time response. cNOS activity was 3-fold greater than iNOS after LPS. NOS activities were increased 140% (cNOS) at 3 h and 169% (iNOS) at 6 h, respectively, after LPS; GH treatment increased cNOS activity and the phosphorylation of endothelial NOS after LPS more than 2-fold over that measured in non-GH-treated calves. The data suggest that an increased production of nitrated protein develops in the liver during low-level, proinflammatory stress, and nitration is increased by GH administration through a direct effect on the competing activities of NOS and arginase, modulatable critical control points in the proinflammatory cascade.
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http://dx.doi.org/10.1210/en.2004-0063DOI Listing
July 2004

Effect of progressive cachectic parasitism and growth hormone treatment on hepatic 5'-deiodinase activity in calves.

Domest Anim Endocrinol 2002 Jun;22(4):211-21

Growth Biology Laboratory, US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Building 200, Room 211E, Beltsville, MD 20705, USA.

Thyroid status is compromised in a variety of acute and chronic infections. Conversion of thyroxine (T(4)) into the metabolically active hormone, triiodothyronine (T(3)), is catalyzed by 5'-deiodinase (5'D) mainly in extrathyroidal tissues. The objective of this study was to examine the effect of protozoan parasitic infection (Sarcocystis cruzi) on hepatic 5'D (type I) activity and plasma concentrations of T(3) and T(4) in placebo- or bovine GH (bGH)-injected calves. Holstein bull calves (127.5+/-2.0 kg BW) were assigned to control (C, ad libitum fed), infected (I, 250,000 S. cruzi sporocysts per os, ad libitum fed), and pair-fed (PF, non-infected, fed to intake of I treatment) groups placebo-injected, and three similar groups injected daily with pituitary-derived bGH (USDA-B-1, 0.1mg/kg, i.m.) designated as C(GH), I(GH) and PF(GH). GH injections were initiated on day 20 post-infection (PI), 3-4 days prior to the onset of clinical signs of the acute phase response (APR), and were continued to day 56 PI at which time calves were euthanized for liver collection. Blood samples were collected on day 0, 28, and 55 PI. Alterations in nutritional intake did not affect type I 5'D in liver. Treatment with bGH increased (P<0.05) 5'D activity in C (24.6%) and PF (25.5%) but not in I calves. Compared to PF calves, infection with S. cruzi reduced 5'D activity 25% (P<0.05) and 47.8% (P<0.01) in placebo- and bGH-injected calves, respectively. Neither nutrition nor bGH treatment significantly affected plasma concentrations of T(4) and T(3) on day 28 and 55 PI. However, plasma thyroid hormones were reduced by infection. On day 28 PI, the average plasma concentrations of T(3) and T(4) were reduced in infected calves (I and I(GH)) 36.4% (P<0.01) and 29.4% (P<0.05), respectively, compared to pair-fed calves (PF and PF(GH)). On day 55 PI, plasma T(3) still remained lower (23.7%, P<0.01 versus PF) in infected calves while plasma T(4) returned to control values. The data suggest that parasitic infection in growing calves inhibits both thyroidal secretion and extrathyroidal T(4) to T(3) conversion during the APR. After recovery from the APR, thyroidal secretion returns to normal but basal and bGH-stimulated generation of T(3) in liver remains impaired.
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http://dx.doi.org/10.1016/s0739-7240(02)00127-3DOI Listing
June 2002