Estradiol regulation of the human retinoic acid receptor alpha gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs.

Estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines express significantly higher levels of retinoic acid receptor alpha (RAR alpha) (isoform 1) mRNA than ER-negative HBCs. Estradiol enhances RAR alpha mRNA expression in different ER-positive HBCs by 2-3-fold, which in turn results in increased sensitivity of ER-positive HBCs to the growth inhibitory effects of retinoic acid. To investigate the regulatory mechanisms of estradiol-mediated enhancement of RAR alpha mRNA expression, the functional promoter for the human RAR alpha isoform 1 was cloned and used to assess estradiol-mediated promoter-dependent enhancement of firefly luciferase reporter gene activity in transiently transfected ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231) HBCs. Deletional promoter constructs were obtained to further delineate the promoter region responsible for estradiol-mediated enhancement of promoter activity. Here, we present evidence that approximately 130 bp of the promoter fragment preceding the transcriptional start site are responsible for estradiol-mediated enhancement of hRAR alpha gene expression. The estradiol-mediated enhancement is dependent on ER binding. Further deletional analysis showed that a promoter sequence of 42 base pairs, located approximately 100 bases upstream of the transcriptional start site, contains elements for estradiol-mediated enhancement. Specific deletion of either the Sp1 motif or mutations in the imperfect half-palindromic estrogen response element motif of this fragment abolish its estradiol responsiveness in transient transfections.

Regulation of the human retinoic acid receptor alpha gene in the estrogen receptor negative human breast carcinoma cell lines SKBR-3 and MDA-MB-435.

Estradiol-mediated enhancement of retinoic acid receptor alpha (RARalpha) expression in the estrogen receptor (ER)-positive human breast carcinoma (HBC) cells results in their sensitivity to RA-mediated growth inhibition (A. K. Rishi et al., Cancer Res., 55: 4999-5006, 1995). Most ER-negative HBCs are known to express lower levels of RARalpha and are resistant to RA-mediated inhibition of growth. We show that ER-negative SKBR-3 and MDA-MB-435 HBCs express approximately 2-fold higher levels of RARalpha isoform 1 mRNA when compared to the ER-negative MDA-MB-231 and MDA-MB-468 HBCs. SKBR-3 cells are sensitive to growth inhibition by RA, and by using RARalpha-selective synthetic retinoids, we demonstrate that the antiproliferative effects of RA in the SKBR-3 cell line are accomplished, in part, via activation of RARalpha. Both MDA-MB-231 and MDA-MB-468 HBCs are not growth inhibited by RA or any of the retinoids tested. Transient transfection experiments using a 5.0-kb RARalpha promoter fragment fused to the luciferase reporter gene showed 2-3-fold higher transcriptional activation in SKBR-3 cells when compared to MDA-MB-468 cells. We report identification of a 72-bp fragment of RARalpha promoter that contains unique cis elements responsible for mediating an estradiol-independent 2.5-fold enhancement of RARalpha gene expression in SKBR-3 and MDA-MB-435 cells.

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.

Influence of abomasal carbohydrates on subcutaneous, omental, and mesenteric adipose lipogenic and lipolytic rates in growing beef steers.

To determine the response to alteration in site and form of carbohydrate delivery to the digestive tract, in vitro rates of lipogenesis and lipolysis in mesenteric (MESA), omental (OMA), and subcutaneous (SQA) adipose depots were compared. Forty crossbred beef steers (243 +/- 2 kg of BW) were fed 161 (LI) or 214 (HI) kcal of ME/(kg of BW(0.75) x d) or they were fed LI and infused for 35 d into the rumen (R) or abomasum (A) with starch hydrolysate (SH) or into the abomasum with glucose (G). Jugular blood samples were collected, steers were slaughtered, and adipose depots were sampled and prepared for assessment of lipogenesis and lipolysis in vitro. Blood concentrations of glucagon were increased (P = 0.04) in HI-H2O compared with LI-H2O steers, whereas A-SH tended to increase (P = 0.08) circulating IGF-I relative to R-SH, and A-G tended to have elevated (P = 0.09) T3 compared with A-SH. Lipolysis, as assessed by NEFA release, was unaffected by treatment. Glycerol release by the MESA and SQA was increased or tended to be increased (P < or = 0.08) in HI-H2O compared with LI-H2O steers. In A-G compared with A-SH steers, glycerol release from OMA increased (P = 0.008) and from SQA tended to be increased (P = 0.08). Acetate incorporation into total neutral lipids (TNL) increased or tended to increase with ME intake and SH infusion (P < or = 0.09) across all depots. Rates of acetate incorporation into fatty acids (FA) also increased or tended to be increased (P < or = 0.1) by SH infusion across all depots, but only that of SQA was increased with ME intake (HI-H2O vs. LI-H2O; P = 0.02). Rates of acetate incorporation into FA and TNL in MESA were increased (P < or = 0.03) by A-SH compared with R-SH, but site of SH infusion did not affect the rates in SQA or OMA. Glucose incorporation into TNL for MESA and SQA increased or tended to be increased (P < or = 0.1) by dietary and infused energy, whereas for OMA they tended to be increased (P = 0.1) only by SH infusion. In contrast, glucose incorporation into FA was unaffected by energy supply but tended to be increased (P = 0.07) by SH in MESA and tended to be greater (P = 0.08) for A-G than A-SH in OMA. The general across-depot pattern of acetate incorporation rate into FA and TNL was SQA > OMA > MESA, whereas, for glucose incorporation, rates across depots were equivalent. These data provide evidence that the postruminal supply of energy, specifically carbohydrate, stimulates lipogenesis from acetate and glucose and is more pronounced in abdominal depots relative to the subcutaneous depot.

Influence of anode pretreatment on its microbial colonization.

AIMS: To assess the influence of chemical treatment of the anode of a marine sediment biofuel cell (MSBFC) on the microbial diversity of the anode biofilm. METHODS AND RESULTS: A MSBFC was equipped with two graphite plate anodes, one pretreated by electrochemical oxidation in sulfuric acid and the other untreated. After 6 weeks of operation, 16S rRNA clone libraries were constructed from each anode biofilm. The pretreated anode exhibited a fourfold depletion in gamma-proteobacteria, a fourfold enrichment in delta-proteobacteria, a sixfold increase in sulfate reducers, a fivefold enrichment in unclassified micro-organisms, and 6% of the colonies were sulfur oxidizers while none were detected on the untreated anode. CONCLUSION: Anode pretreatment significantly affects the anode-colonized microbial communities of MSBFCs. SIGNIFICANCE AND IMPACT OF THE STUDY: The MSBFC is one of a new class of microbial fuel cells in which the anode is spontaneously colonized by a subset of micro-organisms indigenous to a complex anaerobic mixture (such as sewage and food processing effluents). These micro-organisms utilize the anode as an oxidant, catalysing power generation by oxidizing fuel in the mixture and reducing the anode. This study reveals that pretreatment of the anode can greatly affect the composition of the microbial colony of such fuel cells.

Influence of ruminal and postruminal carbohydrate infusion on visceral organ mass and adipose tissue accretion in growing beef steers.

Forty crossbred beef steers (243 +/- 2 kg of BW) with ruminal and abomasal infusion catheters were used to test 2 hypotheses: 1) visceral mass is responsive to energy input and site of carbohydrate (CHO) infusion and 2) rate and site of adipose accretion are dependent on site of CHO infusion and complexity. Treatments included a pelleted, forage-based, basal diet fed at 161 (LI) or 214 (HI) kcal of ME/(kg of BW(0.75) x d), LI plus ruminal (R-SH) or abomasal (A-SH) infusion of a partial starch hydrolysate (SH), and LI plus abomasal infusion of glucose (A-G). The basal diet was fed in 12 equal portions daily at 2-h intervals, with starch and glucose infused over a 22-h period at rates of 12.6 and 14.4 g/(kg of BW(0.75) x d). After 35 d of infusion, steers were slaughtered; and visceral organ and adipose mass, subcutaneous adipose thickness over the 5th and 12th rib, and LM intramuscular fat concentration were determined. Total intake energy (IE) increased (P = 0.0001) with ME intake. Dietary IE was similar between LI and CHO treatments, but total IE increased (P < 0.001) with CHO infusion. Greater dietary ME intake and CHO infusion increased or tended (P < or = 0.09) to increase final BW and HCW. As a percentage of empty BW, total stomach complex, rumen, omasum, liver, pancreas, and kidney weights were greater (P < or = 0.05) for HI vs. LI. Stomach complex, rumen, pancreas, and kidney weights as a percentage of empty BW were greater (P < or = 0.05) for R-SH vs. A-SH. Compared with ASH, A-G increased (P < or = 0.02) total and mucosal weights from the 10-cm sections of the ileum. Increases in rumen mass were associated with no change or an increase in rumen total and mucosal DNA concentrations. Greater dietary ME tended (P = 0.06) to increase subcutaneous fat thickness at the 5th rib but did not affect alimentary adipose accretion on an empty BW basis. Omental and total alimentary adipose weights were increased (P < or = 0.04) by A-G compared with A-SH. Although SH infusion did not alter adiposity, there was a consistent numerical pattern in total alimentary and subcutaneous fat depots with CHO infusion (A-G > ASH > R-SH). Our findings demonstrate that increasing ruminal CHO supply results in a disproportionate increase in rumen mass, whereas increasing small intestinal CHO supply does not alter gastrointestinal organ mass. Small intestinal energy in the form of glucose resulted in greater adipose accretion, particularly the omental depot.

Characterization of a normalized cDNA library from bovine intestinal muscle and epithelial tissues.

Tissue-specific cDNA library sequences (expressed sequence tags, or EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e., microarrays) for gene expression profiling. The objective of this study was to develop and characterize an intestine-specific cDNA library to examine the transcriptome of the bovine gut and identify expressed genes that influence ruminant nutrition and health. We describe BARC-8BOV, a normalized cDNA library developed from mRNA isolated from four distinct intestinal locations (duodenal, jejunal and ileal small intestine, colon) of Holstein dairy cattle resulting in 19,110 5'-EST deposited into the NCBI GenBank EST database. Assembly and clustering of these 19,110 clone sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal species, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not previously represented as EST in nucleotide or protein databases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. Ultimately, these data will also contribute in efforts to annotate the bovine genome.