Minor diterpenoid glycosides from the leaves of Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-β-d-xylopyranosyl-β-d-glucopyranosyl) ester (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (2), and 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data.     

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.     

Proteomic analysis using protein chips to detect biomarkers in cervical and amniotic fluid in women with intra-amniotic inflammation

Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1-3, calgranulin A and B.     

Novel pathways of bile acid metabolism involving CYP3A4

The hepatic predominating cytochrome P450, CYP3A4, plays an essential role in the detoxification of bile acids and is important in pathological conditions such as cholestasis where CYP3A4 is adaptively up-regulated. However, the mechanism that triggers the up-regulation of CYP3A4 is still not clear. In this study, using recombinant CYP3A4 and human liver microsomes, we demonstrate that CYP3A4 can metabolise lithocholic acid into 3-dehydrolithocholic acid, a potent activator of the nuclear receptors, pregnane X receptor and 1,25-dihydroxy vitamin D3 receptor, which are known to regulate the expression of CYP3A4. This process thus provides a feed-forward metabolism of toxic bile acid that may be of importance in maintaining bile acid homeostasis. We also provide evidence for a novel CYP3A4-mediated metabolic pathway of the secondary bile acid deoxycholic acid. Patients treated with the antiepileptic drug carbamazepine, a CYP3A4 inducer, had markedly elevated urinary excretion of 1beta-hydroxydeoxycholic acid compared to healthy controls. The importance of CYP3A4 in this process was verified by incubations with recombinant CYP3A4 and human liver microsomes, both of which efficiently converted deoxycholic acid into 1beta-hydroxydeoxycholic acid. Interestingly, CYP3A4 was also found to be active against the secondary bile acid ursodeoxycholic acid.     

ADAM12-mediated focal adhesion formation is differently regulated by beta1 and beta3 integrins

ADAM12, adisintegrin and metalloprotease, has been demonstrated to be upregulated in human malignant tumors and to accelerate the malignant phenotype in a mouse model for breast cancer. ADAM12 is a substrate for beta1 integrins and may affect tumor and stromal cell behavior through its binding to beta1 integrins. Here, we report that cells deficient in beta1 integrin or overexpressing beta3 integrin can bind to recombinant full-length human ADAM12 via beta3 integrin. Furthermore, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions, which are not formed upon beta1 integrin-mediated cell attachment. We also show that RhoA is involved in beta3 integrin-mediated focal adhesion formation.     

Crystal structure of the biologically active form of class Ib ribonucleotide reductase small subunit from Mycobacterium tuberculosis

Two nrdF genes of Mycobacterium tuberculosis code for different R2 subunits of the class Ib ribonucleotide reductase (RNR). The proteins are denoted R2F-1 and R2F-2 having 71% sequence identity. The R2F-2 subunit forms the biologically active RNR complex with the catalytic R1E-subunit. We present the structure of the reduced R2F-2 subunit to 2.2 A resolution. Comparison of the R2F-2 structure with a model of R2F-1 suggests that the important differences are located at the C-terminus. We found that within class Ib, the E-helix close to the iron diiron centre has two preferred conformations, which cannot be explained by the redox-state of the diiron centre. In the R2F-2 structure, we also could see a mobility of alphaE in between the two conformations.