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N J Koszewski

Publications and source records attributed to N J Koszewski.

28 records · Page 2Linked to original sources
PubMed1993

Estrogen-responsive elements contain non-B DNA.

The estrogen receptor, a hormone-regulated transcription factor, regulates gene expression by interacting with a specific nucleotide sequence called the estrogen-responsive element (ERE). In this report we demonstrate by potassium permanganate, osmium tetroxide and diethylpyrocarbonate reactivity and S1 nuclease sensitivity that the nucleotides either within or in the immediate region of imperfect and perfect EREs are in a non-B DNA conformation. The presence of nucleotides in a non-B DNA conformation in the ERE is an intrinsic property of the DNA and is independent of whether the ERE is in linear or supercoiled DNA. S1 nuclease sensitivity was peculiar to the ERE as it was not detected in the thyroid hormone-responsive element. Our results suggest that the nucleotides comprising the ERE are structurally labile. We propose that this intrinsic lability of the ERE could be constrained in vivo such that a unique DNA tertiary structure is formed which may facilitate recognition of the ERE by the estrogen receptor.

Animals↗
PubMed1992

Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding.

We have shown that the 32P-phosphorylation of the nuclear estrogen receptor from human MCF-7 cells or the calf uterus is estrogen-dependent. Within 2 min of estradiol treatment the phosphorylation of the estrogen receptor from MCF-7 cells doubled, and increased 4-fold within 20-40 min of estradiol treatment. Progesterone was ineffective in stimulating the phosphorylation of the estrogen receptor. Phosphoamino acid analysis indicated that the estrogen-stimulated phosphorylation of the human or calf estrogen receptor occurred only on serine residue(s). Phosphotryptic peptide analysis of the human estrogen receptor by two-dimensional peptide mapping or reverse-phase high pressure liquid chromatography revealed that only a single tryptic peptide (site) was phosphorylated. Treatment of the estrogen receptor with potato acid phosphatase resulted in the dephosphorylation of the 32P-labeled estrogen receptor and a decrease of the receptor's affinity for specific DNA sequences. These data suggest that transcriptional activation by the estrogen receptor involves an estrogen-dependent phosphorylation of the receptor resulting in its increased affinity for specific DNA sequences.

Adenocarcinoma↗
PubMed1991

Phosphate-sensitive binding of the estrogen receptor to its response elements.

Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference. Specific ER-DNA complexes were evident in electrophoretic gel mobility shift assays using DNA fragments containing either the perfect ERE (-625 relative to gene start site; 5'-GGTCAGCGTGACC) or the imperfect ERE (-353; 5'-GGTCAACATAACC). The phosphate ethylation interference footprint identified a 2-fold, symmetrical exclusion of phosphate residues essential for specific binding to the perfect ERE with a 5' stagger, indicating that each monomer of the ER dimer is bound in the major groove of the DNA. The interference footprint of the imperfect ERE did not detect interactions between the receptor and the phosphate residues in the 3' half of the response element on the noncoding strand. In contrast, the corresponding footprint of the perfect ERE displayed strong interactions between the ER and the phosphate backbone of the DNA. Consequently, the absence of these electrostatic contacts very likely accounts for the reduced binding affinity of the ER for the imperfect ERE. These results indicate that specific contacts between the ER and the sugar-phosphate backbone of its cognate response elements are an important aspect of DNA sequence recognition and high affinity binding.

Base Sequence↗
PubMed1990

Developmental changes in rat kidney 1,25-dihydroxyvitamin D receptor.

Kidney 1,25-dihydroxyvitamin D receptor (VDR) was examined in both young and aged male Fischer 344 rats. Cytosols prepared by direct homogenization of the kidney indicated no significant difference in the amount of unoccupied VDR in young (149 +/- 8 fmol/mg) and aged (155 +/- 8 fmol/mg) rats. Binding of kidney VDR to DNA-cellulose, however, was significantly different for the two groups. The assay indicated that about 44% and 24% of the VDR prepared from young and aged rats, respectively, were bound to calf thymus DNA. Elution profiles from DNA-cellulose chromatography displayed the presence of two peaks from young kidneys, while a single broad peak was evident from aged rats. Immunoblot analysis confirmed the existence of two receptor bands at 52K and 50K. The presence of the 50K band was greatly diminished or absent in aged samples. The 50K receptor form was observed to elute from DNA-cellulose at a higher salt concentration than the 52K-form. Similarly, prepared receptor extracts from intestinal tissue produced only a single band at 52K. These results demonstrate for the first time that the rat kidney possesses two forms of the receptor which have different affinities for DNA.

Aging↗
PubMed1990

1 alpha-hydroxylation of 24-hydroxyvitamin D2 represents a minor physiological pathway for the activation of vitamin D2 in mammals.

C24-Hydroxylation was evaluated as a possible activation pathway for vitamin D2 and vitamin D3. Routine assays showed that 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 could be detected in rats receiving physiological doses (100 IU/day) of vitamin D2; however, 24-hydroxyvitamin D3 could not be detected in rats receiving similar doses of vitamin D3. In rats, 24-hydroxyvitamin D2 was very similar to 25-hydroxyvitamin D2 at stimulating intestinal calcium transport and bone calcium resorption. The biological activity of 24-hydroxyvitamin D2 was eliminated by nephrectomy, suggesting that 24-hydroxyvitamin D2 must undergo 1 alpha-hydroxylation to be active at physiological doses. In vivo experiments suggested that when given individually to vitamin D deficient rats, 24-hydroxyvitamin D2, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 were 1 alpha-hydroxylated with the same efficiency. However, when presented simultaneously, 24-hydroxyvitamin D2 was less efficiently 1 alpha-hydroxylated than either 25-hydroxyvitamin D3 or 25-hydroxyvitamin D2. 1,24-Dihydroxyvitamin D2 was also approximately 2-fold less competitive than either 1,25-dihydroxyvitamin D2 or 1,25-dihydroxyvitamin D3 for binding sites on the bovine thymus 1,25-dihydroxyvitamin D receptor. These results demonstrate that 24-hydroxylation followed by 1 alpha-hydroxylation of vitamin D2 represents a minor activation pathway for vitamin D2 but not vitamin D3.

Animals↗
PubMed1989

Metabolism of vitamin D2 in pig liver homogenates: evidence for a free radical reaction.

In vitro hydroxylation of vitamin D2 at carbon-24 (C-24) was demonstrated with pig liver homogenate. The putative 24-hydroxyvitamin D2 (24-OHD2) comigrated with standard 24-OHD2 on a Zorbax Sil column developed in hexane/isopropanol (98/2). Rechromatography in methylene chloride/methanol (99.8/0.2) resolved the putative 24-OHD2 into two components. The identity of these compounds was determined to be 24(R)-OHD2 and 24(S)-OHD2 (epimers) by low resolution mass spectroscopy and proton NMR spectroscopy. The fact that epimers of 24-OHD2 were produced from vitamin D2 in the absence of pig liver homogenate in vitro was strong evidence for the participation of free radicals in the reaction. Further support for free radical involvement was provided by the following observations: (a) hydroxyl free radical scavengers such as alpha-tocopherol, catalase, and ethanol reduced the amount of 24-OHD2 produced by 18-64%; (b) use of autoclaved homogenate in the incubation mixture had little or no effect on the amount of 24-OHD2 produced; and (c) the failure of the enzyme-substrate saturation curve to level off as expected with high levels of vitamin D2 (100-2000 micrograms = 50-1009 microM). Maximum production of 24-OHD2 was obtained at pH 4.75 and represented a sevenfold increase relative to the amount produced at pH 7.4. The omission of citrate or the addition of electron transport inhibitors, cyanide or antimycin, had little or no effect on the reaction. These data suggested that C-24 hydroxylation of vitamin D2 in vitro was a free radical-mediated process not involving the electron transport system. In vitro hydroxylation at C-24 appeared to be driven by free radicals, and the dominance of this reaction made it difficult to determine whether there was an enzyme involved in the reaction.

Animals↗
PubMed1988

24,26-Dihydroxyvitamin D2: a unique physiological metabolite of vitamin D2.

A new vitamin D2 metabolite, 24,26-dihydroxyvitamin D2, has been detected in the plasma of rats fed physiologic amounts of vitamin D2. The identity of the new metabolite (isolated from cow plasma) was established by ultraviolet absorbance, mass spectroscopy, chemical reactivity, and NMR spectroscopy. Among these, the mass spectrum was unique for the presence of a peak at M-48 that was attributed to an intramolecular rearrangement involving both the C-24 and C-26 hydroxyl groups. A 300-MHz 1H NMR spectrum of 40 micrograms of metabolite indicated a downfield shift of the C-28 methyl group signal to delta 1.30 and a multiplet at delta 3.66 corresponding to the hydroxylated C-26 methyl group. We determined that the formation of 24,26-dihydroxyvitamin D2 represented a major pathway for further metabolism of 24-hydroxyvitamin D2 in rats, exceeding the formation of 24,25-dihydroxyvitamin D2. Standard bioassays revealed that 24,26-dihydroxyvitamin D2 possessed very little biological activity and most likely represents a deactivation pathway for 24-hydroxyvitamin D2.

Animals↗
PubMed1987

Use of Fourier transform 1H NMR in the identification of vitamin D2 metabolites.

The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.

Ergocalciferols↗
PubMed1986

24-Hydroxylation of 1,25-dihydroxyergocalciferol. An unambiguous deactivation process.

1,24,25-Trihydroxyergocalciferol was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyergocalciferol and from chick kidney homogenates incubated with 24,25-dihydroxyergocalciferol. The identity was established by ultraviolet absorbance, sensitivity to periodate, nuclear magnetic resonance, and mass spectrometry. The new metabolite had an affinity equal to 1,24,25-trihydroxycholecalciferol for the bovine-thymus and chick-intestinal 1,25-dihydroxyvitamin D receptor and had an affinity twice that of 1,24,25-trihydroxycholecalciferol for the rat-intestinal receptor. It was 3- and 6-fold less competitive than either 1,25-dihydroxycholecalciferol or 1,24,25-trihydroxycholecalciferol, respectively, for the rat plasma vitamin D transport protein. 1,24,25-Trihydroxyergocalciferol was at least 10-fold less active than 1,25-dihydroxycholecalciferol, 1,25-dihydroxyergocalciferol, and 1,24,25-trihydroxycholecalciferol at stimulating intestinal-calcium transport and was also relatively ineffective at stimulating bone-calcium resorption in rats. Moreover, in rats, [3H]1,24,25-trihydroxyergocalciferol was cleared from plasma approximately 40% faster than [3H]1,24,25-trihydroxycholecalciferol. These data suggest that C-24 hydroxylation of 1,25-dihydroxyergocalciferol represents a significant in vivo deactivation step, whereas equivalent deactivation of 1,25-dihydroxycholecalciferol seems to involve metabolic steps subsequent to C-24 hydroxylation (C-24 ketonization). C-24 ketonization of 1,25-trihydroxyergocalciferol would not be anticipated due to the presence of the 24(S)-methyl group. These results reveal further dissimilarities between ergocalciferol and cholecalciferol metabolism in mammals and suggest a mechanism for the lesser tendency of ergocalciferol to cause hypercalcemia relative to cholecalciferol.

Animals↗