Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth,Plutella xylostella

Calreticulin (CRT) plays pivotal roles in Ca²⁺ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full-length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly-A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.     

Gut microbiota composition is associated with environmental landscape in honey bees

There is growing recognition that the gut microbial community regulates a wide variety of important functions in its animal hosts, including host health. However, the complex interactions between gut microbes and environment are still unclear. Honey bees are ecologically and economically important pollinators that host a core gut microbial community that is thought to be constant across populations. Here, we examined whether the composition of the gut microbial community of honey bees is affected by the environmental landscape the bees are exposed to. We placed honey bee colonies reared under identical conditions in two main landscape types for 6 weeks: either oilseed rape farmland or agricultural farmland distant to fields of flowering oilseed rape. The gut bacterial communities of adult bees from the colonies were then characterized and compared based on amplicon sequencing of the 16S rRNA gene. While previous studies have delineated a characteristic core set of bacteria inhabiting the honey bee gut, our results suggest that the broad environment that bees are exposed to has some influence on the relative abundance of some members of that microbial community. This includes known dominant taxa thought to have functions in nutrition and health. Our results provide evidence for an influence of landscape exposure on honey bee microbial community and highlight the potential effect of exposure to different environmental parameters, such as forage type and neonicotinoid pesticides, on key honey bee gut bacteria. This work emphasizes the complexity of the relationship between the host, its gut bacteria, and the environment and identifies target microbial taxa for functional analyses.     

Catalytic differences between porcine blastocyst and placental aromatase isozymes.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

Physics of Root Growth

THE elongation of a plant cell involves the yielding of the cell wall under the action of tensile stresses in the wall¹. The rate of elongation, R, can be expressed simply as R=mW, where m is the extensibility of thé cell wall material and W is the wall pressure. Changes in either m or W have been used to explain the effect of biochemical factors on the growth rate of plant cells^2,3. Cell growth is also affected by the physical environment and this becomes particularly important in the case of plant roots where soil water potential and the mechanical resistance of the soil to deformation can become rate controlling. Working with 3-5 day old radicles of Pisum sativum, growing in soil cores, we have obtained values of wall pressure in terms of these two properties and we find that the rate of root elongation can be described by a simple extensibility equation.     

Structure and diversity of the Mesozoic wood genus Xenoxylon in Far East Asia: implications for terrestrial palaeoclimates

Although the faunal elements of Far East Asian Mesozoic terrestrial biota have attracted much attention in recent years, their palaeoecology remains poorly known. In particular, features of the palaeoclimate are highly controversial. To address this point we used the Mesozoic fossil wood Xenoxylon , a genus recognized as an indicator of wet temperate biotopes and which is common in the area during the Carnian–Maastrichtian interval. We re-appraised bibliographic data and gathered new data for Xenoxylon in the Mesozoic of Far East Asia. This demonstrated that previous taxonomic approaches to the genus have been so far idiosyncratic. We examined the anatomical diversity of morphogenus Xenoxylon in Far East Asia and compared it to that of samples from Europe. This indicates that in an area centred on north-eastern China, Xenoxylon reached a level of anatomical diversity unmatched elsewhere in the world. We hypothesize that this diversity witnesses the persistence of palaeoecological conditions particularly suitable for Xenoxylon and that a wet temperate climate prevailed over most of the area throughout the Carnian–Maastrichtian interval. It is in this setting that the famous Jehol Biota probably evolved.     

Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.