Origin of deoxycorticosterone sulfate (DOC-SO4) in plasma of pregnant women: Pregnenolone-3,21-disulfate is a placental precursor of DOC-SO4

The plasma levels of deoxycorticosterone sulfate (DOC-SO₄) in near-term pregnant women are 100 times those in plasma of men or non-pregnant women. Yet, neither the tissue site of synthesis nor the precursor of DOC-SO₄ that enters maternal plasma is known. Several potential sources have been excluded: plasma DOC-SO₄ is not derived from plasma DOC; and the secretion of C₂₁-steroids (other than aldosterone) from the maternal adrenals during human pregnancy is not increased. Similarly, the transfer of DOC-SO₄ from fetal plasma cannot account for the high level of DOC-SO₄ in the maternal compartment, and a reduced clearance of plasma DOC-SO₄ during pregnancy cannot account for the high levels of DOC-SO₄. Indeed, the rate of clearance of DOC-SO₄ from plasma is 10–100 times that of most other steroid sulfates. To address this question further, we evaluated the possibility that fetal plasma pregnenolone-3,21-disulfate serves as a precursor for DOC-SO₄ formation in the placenta. The preferential hydrolysis of the 3β-sulfate of pregnenolone-3,21-disulfate in placenta would give rise to pregnenolone-21-monosulfate, which, if acted upon by placental 3β-hydroxysteroid dehydrogenase/Δ^{5 → 4} isomerase, could give DOC-SO₄. [³H]Pregnenolone-3,21-disulfate was incubated with minces of human placental tissue for 5, 20, 60 and 120 min. Radiolabelled DOC-SO₄, DOC, and pregnenolone-21-monosulfate were isolated from the incubation media and quantified. After a 5 min incubation, 7.5% of substrate was converted to DOC-SO₄; and after 20, 60 and 120 min 30% of the [³H]pregnenolone-3,21-disulfate was recovered from the media of these incubations as [³H]DOC-SO₄. [³H]DOC was also present in the incubation media and the concentrations of this product increased as a function of incubation time. Therefore, pregnenolone-3,21-disulfate, which is present in very high concentrations in fetal plasma (1000 ng/ml), is metabolized in the placenta to DOC-SO₄. Because of the fetal and maternal vascular arrangements of the hemochorioendothelial placenta of human pregnancy, steroids produced in syncytiotrophoblasts preferentially enter the intervillous space; thus, fetal plasma pregnenolone-3,21-disulfate may serve as a placental precursor of maternal plasma DOC-SO₄.     

Active proton uptake by chromaffin granules: observation by amine distribution and phosphorus-31 nuclear magnetic resonance techniques.

The hydrogen ion activity within isolated chromaffin granules can be estimated from the distribution of the weak base methylamine and from phosphorus-31 nuclear magnetic resonance spectra of ATP contained in the granules. Following the addition of ATP to the external medium, the internal pH drops by 0.2 to 0.5 unit. This change occurs only in medium containing a permeant anion such as chloride and is abolished by an uncoupler of oxidative phosphorylation. These results indicate that the chromaffin granule membrane possess an electrogenic proton pump directed inward.     

Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA.

RNA editing plays a central role in the life cycle of hepatitis D virus (HDV), a subviral human pathogen. Previous studies (J.L. Casey, K.F. Bergmann, T.L. Brown, and J.L. Gerin, Proc. Natl. Acad. Sci USA 89:7149-7153, 1992; H. Zheng, T.-B. Fu, D. Lazinski, and J. Taylor, J. Virol. 66:4693-4697, 1992) had concluded that the genomic RNA of HDV was the target for RNA editing and that the editing reaction was a conversion of U to C. However, we show here that the antigenomic RNA of HDV is in fact the target for HDV RNA editing, which is therefore a conversion of A to G. This result is verified by using an assay specific for editing on the antigenomic RNA and by analyzing the editing of site-directed mutant RNAs in transfected cells and in cell extracts. Because editing occurs in the absence of viral antigens and the specificity for the HDV editing target site is present even in extracts from Drosophila cells, it is likely that HDV RNA is edited by one or more cellular factors that are conserved among higher eukaryotes. These results raise the likelihood that double-stranded RNA adenosine deaminase specifically edits HDV antigenomic RNA.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

Aortic ascorbic acid, trace elements, and superoxide dismutase activity in human aneurysmal and occlusive disease

Altered trace elements and ascorbic acid metabolism have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, their role in the disease process, or the effect of atherosclerosis on their tissue levels within plaque, is poorly understood. The present study analyzes the concentrations of Fe, Cu, Zn, and Mn, and ascorbic acid and superoxide dismutase (SOD) activity in tissue samples from 29 patients with abdominal aortic aneurysms (AAA) and 14 patients with atherosclerotic occlusive disease (AOD). It was observed that the Fe and Mn concentrations in AAA and AOD tissue were higher than the levels in nondiseased control aorta, whereas Cu and Zn levels in AAA and AOD tissue were similar to the levels in controls. The Zn:Cu ratio was significantly lower in the AAA tissue in comparison to both AOD and control tissue. In addition, AAA and AOD tissue had low ascorbic acid levels and low Cu,Zn-SOD activity with Cu,Zn-SOD:Mn-SOD ratios of 0.27 and 0.19, respectively, compared to a ratio of 3.20 in control aorta. These data indicate that aorta affected by aneurysms and occlusive disease have altered trace element and ascorbic acid concentrations, as well as low Cu,Zn-SOD activity. Although these observations do not directly support the hypothesis that AAA is associated with aortic Cu deficiency they do suggest a role for oxygen radicals or increased lipid peroxidation in occlusive and aneurysmal disease of the aorta.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Androgen receptor activation by G(s) signaling in prostate cancer cells

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.