Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Hormonal effects of zinc on growth in children

Zinc deficiency impairs the metabolism of thyroid hormones, androgens, and above all growth hormones. In view of their important role in growth, it is not surprising to find growth disorders associated with zinc deficiency. Stunted growth linked to zinc deficiency is found during gestation, and also in the newborn and children up to adolescence. Depending on the country, 5–30% of children suffer from moderate zinc deficiency, responsible for small-for-age height. Zinc supplementation has proven effective in many studies, mainly in children where zinc deficiency has first been found. Finally, zinc supplementation makes it possible in certain cases to overcome resistance to growth hormone treatment.     

Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors,silver and cobalt

The effects of CoCl₂, AgNO₃ and ethylene released by exogenous 2-chloroethylphosphonic acid (Ethephon), were studied on shoot regeneration from cotyledons of Helianthus annuus cv. E8206R, a poorly regenerative cultivar. Inhibition of ethylene biosynthesis by CoCl₂, at concentrations of 20 K, provoked a substantial enhancement of shoot regeneration (30 %): the control was poorly regenerative. However, CoCl₂ had no effect when Ethephon was supplied. Inhibition of ethylene action by AgNO₃, at concentrations of 10–25 M, caused a significant increase in plant regeneration: 25 % instead of 1.2 % in the control. Furthermore, addition of Ethephon to AgNO₃-treated tissues failed to reduce the stimulation of shoot regeneration caused by AgNO₃. On the basis of these findings, it is suggested that ethylene inhibits the regeneration process from cotyledons of sunflower.Abbreviations NAA 1-naphthalene acetic acid - BAP 6-benzylamino-purine - GA₃ gibberellic acid - Ethephon 2-chloroethylphosphonic acid - MS Murashige and Skoog medium - AVG aminoethoxyvinylglycine     

IF-3 crosslinking to Escherichia coli ribosomal 30 S subunits by three different light-dependent procedures: Identification of 30 S proteins crosslinked to IF-3—Utilization of a new two-stage crosslinking reagent, p-nitrobenzylmaleimide

We here report the results of using three light-dependent procedures for crosslinking IF-3 to 30 S proteins within an IF-3·30 S complex. In the first procedure, employing FMN as a photosensitizer, protein S12 is found to be the only major crosslinked protein. In the second procedure, IF-3 is first reacted with the new two-stage crosslinking reagent, p-nitrobenzylmaleimide (PNBM), and the PNBM—IF-3·30 S complex is irradiated. The major crosslinked proteins are S3 > S2, S12, S18. Small amounts of crosslinked S11 and S21 are also found. In the third procedure, the IF-3·30 S complex is reacted with PNBM and then irradiated. The major crosslinked proteins are S12 > S3 > S11 and small amounts of crosslinked S1, S13, and S21 are also found. These results are compared with results obtained by others using different crosslinking procedures and are used to discuss the Lake and Kahan model (J. A. Lake and L. Kahan, 1975, J. Mol. Biol., 99, 631–644, and J. A. Lake, 1978, in Advanced Techniques in Biological Electron Microscopy II, Koehler, J. K., ed., pp. 173–211, Springer-Verlag, Berlin) for IF-3 binding to 30 S subunits.     

Sur le mode de croissance de Nuia,algue incertae sedis

The authors recall some data concerning the distribution, the ecology and the taxonomy of Nuia and describe in detail its mode of growth, distinguished by 6 different growth-forms.     

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase from Zea mays: General properties and regulation by tryptophan

In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn⁺⁺ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn⁺⁺and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )     

Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

The association and dissociation mechanisms of lectin membrane receptor microclustering on HeLa cells have been studied by measuring resonance energy transfer between fluoresceinated and rhodaminated lentil lectin. Compounds known to affect membrane receptor mobility, such as Ca²⁺ ions, methylamine, cytochalasin D and nocodazole, did not modify the association kinetics nor the maximal energy transfer values at 4 and 37 °C. Dissociation of the membrane receptor microclusters was followed by measuring the temporal decrease in energy transfer values at 4 °C after preincubation for different time intervals at 37 °C. The rate of dissociation of the lectin receptors decreased in the presence of Ca²⁺ ions (10⁻³ M) and after cross-linking with anti-lectin antibodies. An increase was observed in the presence of cytochalasin D (10⁻⁶ M) and, to a lesser extent, of methylamine (10⁻² M). When cytochalasin D and methylamine were combined at subliminal concentrations, a partial synergistic effect was observed. Nocodazole (10⁻⁶ M) had no effect. The results suggest that the association of lectin membrane receptors in microclusters is mediated only by physicochemical parameters. Ca²⁺ ions, cytochalasin D (microfilaments) and methylamine (transglutaminase)-sensitive components appear, however, to play an important role in the stabilization of the receptor microclusters.     

Author index for volume 49

Using mouse thymocytes, mitogen-induced [³H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G₀ and G₁ phase of thymocytes. PHA induces a transient but considerable G₀-G₁ shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G₀-G₁ shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [³H]thymidine uptake in thymocytes and a shift from G₁ to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G₁ cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G₀-G₁ phases of the cell cycle.