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We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.

Tuberous sclerosis (TSC) is a genetic disorder that results in the development of hamartomatous lesions in a variety of organ systems. Both the prevalence of the disease and the often devastating consequences of these tumors pose a serious health and medical care problem. The disease has been mapped to two distinct genetic loci in humans, and although the genes (TSC1 and TSC2) for both loci have recently been cloned, their function remains an enigma. Data presented here demonstrates that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes. These data place TSC2 into a growing list of nuclear receptor coregulators and strengthen the expanding body of evidence that these coregulators may play critical roles in cellular differentiation. Tuberous sclerosis (TSC)1 is an autosomal dominant neurocutaneous disorder associated with the development of hamartomatous lesions in a wide variety of tissues, including skin, kidney, brain, eye, lungs, and heart (1). The birth frequency has been estimated at approximately 1 in 6000 (2). Pathologically, TSC is classified as a disorder of cellular migration, proliferation, and differentiation. The tumors associated with TSC are diagnostically distinct, very seldom progress to malignancy, but often have very devastating consequences. The most classical are those that occur in the brain, where they frequently cause epilepsy, mental retardation, autism, and/or attention deficit disorders (1, 3). The relatively high prevalence and serious consequences of TSC make it an important health concern both here in the United States and throughout the world.TSC has been genetically linked to two loci in humans (4). About 60% of TSC cases are sporadic, but in families it has autodominant inheritance with high penetrance. TSC was first linked to chromosome 9q34 in 1987 (5), and this locus was termed TSC1. Later studies identified a second locus on chromosome 16p13 (6) that was called TSC2. The focal nature of TSC tumors suggests that TSC1 and TSC2 may function as tumor suppressor genes, and this is further supported by evidence for inactivating TSC2 germ line mutations and loss of heterozygosity at the TSC2 locus in TSC-associated tumors.The genes for both TSC1 and TSC2 have recently been cloned. Both were cloned by consortiums of investigators sequencing the specific regions of chromosome 9 (7) and chromosome 16 (8) to which TSC1 and TSC2 had been mapped, respectively. TSC1, or hemartin as it was named by the authors, encodes a cDNA of 8599 base pairs containing a 1164-amino acid open reading frame. TSC2, or tuberin, also encodes a rather large protein of 198 kilodaltons as specified by a cDNA of 5474 base pairs containing a 1784-amino acid open reading frame. The lack of any obvious structural similarities between TSC1 and TSC2 would suggest their relationship to tuberous sclerosis is either through some functional homology or through some converging function.Very little is known about the function of TSC1. The large TSC2...

This experiment was conducted to evaluate the effects of feeding dietary fiber on cecal short-chain fatty acid (SCFA) concentration and cecal microbiota of broiler and laying-hen chicks. The lower fiber diet was based on corn-soybean meal (SBM) and the higher fiber diet was formulated using corn-SBM-dried distillers grains with solubles (DDGS) and wheat bran to contain 60.0 g/kg of both DDGS and wheat bran from 1 to 12 d and 80.0 g/kg of both DDGS and wheat bran from 13 to 21 d. Diets were formulated to meet or exceed NRC nutrient requirements. Broiler and laying-hen chicks were randomly assigned to the high and low fiber diets with 11 replicates of 8 chicks for each of the 4 treatments. One cecum from 3 chicks was collected from each replicate: one cecum underwent SCFA concentration analysis, one underwent bacterial DNA isolation for terminal restriction fragment length polymorphism (TRFLP), and the third cecum was used for metagenomics analyses. There were interactions between bird line and dietary fiber for acetic acid (P = 0.04) and total SCFA (P = 0.04) concentration. There was higher concentration of acetic acid (P = 0.02) and propionic acid (P < 0.01) in broiler chicks compared to laying-hen chicks. TRFLP analysis showed that cecal microbiota varied due to diet (P = 0.02) and chicken line (P = 0.03). Metagenomics analyses identified differences in the relative abundance of Helicobacter pullorum and Megamonas hypermegale and the genera Enterobacteriaceae, Campylobacter, Faecalibacterium, and Bacteroides in different treatment groups. These results provide insights into the effect of dietary fiber on SCFA concentration and modulation of cecal microbiota in broiler and laying-hen chicks.

Circulating parathyroid hormone (PTH) is a mixture of PTH-1-84 and carboxy-terminal (C-PTH) fragments. Recently, the "intact" PTH assay was reported to detect not only PTH-(1-84) but also a C-PTH fragment, presumably PTH-(7-84). The purpose of this study was to determine whether PTH-(7-84) antagonizes the PTH-(1-84) effects on bone. Forty-eight rats were thyroparathyroidectomized (TPTX), eight were used as controls and the remaining TPTX rats (10/group) were nephrectomized (Nx) and subsequently given PTH-(1-84), PTH-(7-84), PTH-(1-84) and PTH-(7-84) or no PTH peptide. Another ten rats were sham-operated and served as controls. Administration of PTH-(1-84) brought serum calcium and osteoblast activity i.e., bone turnover, to normal levels. Concomitant administration of PTH-(7-84) abrogated these effects. Administration of PTH-(7-84) alone did not further suppress the levels of serum calcium and bone turnover in these TPTX-Nx rats. Moreover, radioligand binding experiments demonstrate that PTH-(7-84) binds specifically to SaOS cells and is equally displaced by both PTH-(1-84) and (7-84), but only partially displaced by PTH-(1-34). These data indicate that PTH-(7-84) antagonizes PTH-(1-84) effect not only on serum calcium but also on osteoblasts to affect bone turnover.