Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.     

Iron-sulfur cluster biogenesis in chloroplasts. Involvement of the scaffold protein CpIscA

The chloroplast contains many iron (Fe)-sulfur (S) proteins for the processes of photosynthesis and nitrogen and S assimilation. Although isolated chloroplasts are known to be able to synthesize their own Fe-S clusters, the machinery involved is largely unknown. Recently, a cysteine desulfurase was reported in Arabidopsis (Arabidopsis thaliana; AtCpNifS) that likely provides the S for Fe-S clusters. Here, we describe an additional putative component of the plastid Fe-S cluster assembly machinery in Arabidopsis: CpIscA, which has homology to bacterial IscA and SufA proteins that have a scaffold function during Fe-S cluster formation. CpIscA mRNA was shown to be expressed in all tissues tested, with higher expression level in green, photosynthetic tissues. The plastid localization of CpIscA was confirmed by green fluorescent protein fusions, in vitro import, and immunoblotting experiments. CpIscA was cloned and purified after expression in Escherichia coli. Addition of CpIscA significantly enhanced CpNifS-mediated in vitro reconstitution of the 2Fe-2S cluster in apo-ferredoxin. During incubation with CpNifS in a reconstitution mix, CpIscA was shown to acquire a transient Fe-S cluster. The Fe-S cluster could subsequently be transferred by CpIscA to apo-ferredoxin. We propose that the CpIscA protein serves as a scaffold in chloroplast Fe-S cluster assembly.     

Expression of a mouse selenocysteine lyase in Brassica juncea chloroplasts affects selenium tolerance and accumulation

Selenium is an essential nutrient for many organisms, as part of certain selenoproteins. However, selenium is toxic at high levels, which is thought to be due to non-specific replacement of cysteine by selenocysteine leading to disruption of protein function. In an attempt to prevent non-specific incorporation of selenocysteine into proteins and to possibly enhance plant selenium tolerance and accumulation, a mouse selenocysteine lyase was expressed in Brassica juncea (Indian mustard) chloroplasts, the site of selenocysteine synthesis. This selenocysteine lyase specifically breaks down selenocysteine into elemental selenium and alanine. The transgenic cpSL plants showed normal growth under standard conditions. Selenocysteine lyase activity in the cpSL transgenics was up to 6-fold higher than in wild-type plants. The cpSL transgenics contained up to 40% less selenium in protein compared to wild-type plants, indicating that Se flow in the plant was successfully redirected. Surprisingly, the selenium tolerance of the transgenic cpSL plants was reduced, perhaps due to interference of produced elemental selenium with chloroplastic sulphur metabolism. Shoot selenium levels were enhanced up to 50% in the cpSL transgenics, but only during the seedling stage.     

The multiple flavors of GoU pairs in RNA

Wobble GU pairs (or GoU) occur frequently within double‐stranded RNA helices interspersed within the standard G═C and A─U Watson‐Crick pairs. However, other types of GoU pairs interacting on their Watson‐Crick edges have been observed. The structural and functional roles of such alternative GoU pairs are surprisingly diverse and reflect the various pairings G and U can form by exploiting all the subtleties of their electronic configurations. Here, the structural characteristics of the GoU pairs are updated following the recent crystallographic structures of functional ribosomal complexes and the development in our understanding of ribosomal translation.     

Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN⁺]-based experimental system to explore the nature of transmission barriers. [PIN⁺], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1''s prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN⁺] prion state in vivo. Subsequent analysis of [PIN⁺] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold.     

The chloroplast NifS-like protein of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> is required for iron–sulfur cluster formation in ferredoxin

Plastids are known to be able to synthesize their own iron–sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron–sulfur (Fe–S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe–S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe–S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe–S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.