Differential gene expression during terminal erythroid differentiation.

Terminal erythroid differentiation in mammals is the process whereby nucleated precursor cells accumulate erythroid-specific proteins such as hemoglobin, undergo extensive cellular and nuclear remodeling, and ultimately shed their nuclei to form reticulocytes, which then become mature erythrocytes in the circulation. Little is known about the mechanisms that enable erythroblasts to undergo such a transformation. We hypothesized that genes involved in these mechanisms were likely expressed at restricted times during the differentiation process and used differential display reverse transcriptase polymerase chain reaction as a first step in identifying such genes. We identified three differentially expressed cDNAs that we termed late erythroblast (LEB) 1-3. None of these cDNAs were previously identified as being expressed in erythroblasts and their patterns of expression indicated they are likely to be involved in the differentiation process. LEB-1 cDNA was derived from the gene A330102K04Rik (approved gene symbol Apoll1), and shares homology with members of the apolipoprotein L family in humans. LEB-3 cDNA was derived from the novel gene D930015E06Rik, that has no known function. LEB-2 cDNA was derived from the gene ranBP16 (approved gene symbol Xpo7), a nuclear exportin. D930015E06Rik mRNA is also strongly expressed in the testis and was localized to a region of the seminiferous tubule where secondary spermatocytes and early spermatids are found, suggesting a role for D930015E06Rik in spermatogenesis as well as terminal erythroid differentiation. We have thus identified three genes not previously described as being expressed in erythroblasts that could be relevant in elucidating mechanisms involved in terminal erythroid differentiation.

Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

Endometriosis is a debilitating disease found in 10-15% of reproductive-age women and is characterized by the presence of endometrial tissue outside of the uterus. The present study characterizes the expression of AhR and ARNT mRNA in a human endometrial explant culture model in the absence and presence of TCDD exposure. In a parallel, companion study using this model, TCDD exposure was shown to induce CYP1A1 mRNA, CYP1B1 mRNA, EROD (7-ethoxyresorufin-O-deethylase) activity, and CYP1B1 protein in human endometrial explants. Explants were prepared from specimens obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. These specimens were a subset of the specimens used in the parallel study. The explants were cultured in medium containing 10 nM estradiol (E(2)) or 1 nM estradiol plus 500 nM progesterone (E(2) + P(4)) with or without TCDD (first 24 h). After culture, AhR and ARNT mRNA expression were quantified by RT-PCR. TCDD treatment significantly increased the expression of AhR mRNA, but not ARNT mRNA. The expression of both genes was similar for all individual explants and the ratio of AhR:ARNT mRNA expression across all samples was 1.7 to 1.8. Constitutive AhR mRNA expression was donor age dependent (increasing with age), while ARNT mRNA expression was donor age and tissue phase dependent (increased in older and proliferative phase specimens). Similar to results in the parallel study on expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein, the presence of endometriosis did not affect the expression of AhR or ARNT mRNA, either constitutively or following TCDD exposure. However, the detection of disease-specific change was limited by small sample size and variability in tissue cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase, hormonal exposure, and donor age.

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.

A salt-free isocratic high-pressure liquid chromatographic method for the quantitation of benzodiazepines in serum.

A simple, rapid high-pressure liquid chromatography (HPLC) method was developed for detecting and quantifying benzodiazepines in serum. Seven major benzodiazepines were extracted from spiked serum samples using solid-phase extraction with prazepam as the internal standard. The eluted drugs were then resolved isocratically by HPLC within 11 min using a reversed-phase C8 column with a mobile phase consisting of acetonitrile, methanol, water, and perchloric acid. All drugs gave responses that varied linearly with concentration over the ranges studied. Within-day imprecision (CV) varied from 3.9 to 14.9%, day-to-day CV from 4.8 to 17.0%, absolute recoveries from 67% to 114%, and detection limits from 10 to 110 ng/mL. Tricyclic antidepressants did not interfere, and clinical results were in good agreement with those obtained by a gas chromatographic method. The advantage of this method is that it uses a salt-free isocratic mobile phase that can be easily manipulated to effect difficult benzodiazepine separations.

Cyclic adenosine 3',5'-monophosphate-dependent phosphorylation of HMG 14 inhibits its interactions with nucleosomes.

The high mobility group protein HMG 14, which is preferentially associated with nucleosomes containing active gene sequences, is phosphorylated on different sites according to the tissue and stimulus being studied. In the thyroid, HMG 14 displays TSH-dependent phosphorylation that is mediated by cAMP-dependent protein kinase (A-kinase). We have, therefore, studied how phosphorylation of HMG 14 on its major and minor A-kinase sites (Ser-6 and -24) affects its interactions with nucleosomes and various forms of DNA, since this could reflect a means of regulating its function of binding to active chromatin. Approximately twice as much Ser-6 phospho- and 4 times as much Ser-6,24 diphospho-HMG 14 were required to produce the same degree of nucleosome band displacement as that caused by native unphosphorylated HMG 14. Phosphorylation also reduced the ability of HMG 14 to protect the ends of nucleosomal DNA from thermal denaturation. When the electrophoretic mobility of naked DNA was examined, the Ser-6 phospho-HMG 14 was about half as effective as native HMG 14 in retarding the various forms of double stranded DNA, and Ser-6,24 diphospho-HMG 14 was even less effective. Our data demonstrate that electrostatic interactions between DNA and basic amino acids in two highly conserved regions (residues 1-5 and 16-27) can be modulated by phosphorylation at Ser-6 and Ser-24. The ability of mammalian HMG 14, but not HMG 17, to display hormone-dependent phosphorylation may indicate a route for differentially modulating their binding to transcriptionally active chromatin.

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components.

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices.

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).