Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization.

The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.     

Neuroactive and other free amino acids in seed and young plants of Panax ginseng

The seeds and one to three years old plants of Asian ginseng (Panax ginseng C.A. Meyer) were analyzed for their free amino acid contents. The neuro-excitatory beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid), suggested to be the cause of the crippling neurolathyrism, was the major component in the seed extract (70% of the total free amino acids detected) and showed the highest concentration (0.43% by wt) compared to that in the different parts of young plants. beta-ODAP concentration was higher in the shoots as compared to roots and declined in older plants. The amount of beta-ODAP in the roots may be considered as an indirect measure of age and quality. Another neuro-active non-protein amino acid, GABA (gamma-aminobutyric acid), increased dramatically after germination and reached highest concentration in different parts of 3 year-old plants. Glutamine and arginine were the two major free proteinogenic amino acids in the ginseng plants and together they constituted over 50% of all the free amino acids detected in the root.     

Comparison of crystal structures of human androgen receptor ligand-binding domain complexed with various agonists reveals molecular determinants responsible for binding affinity

Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A resolution, respectively. Comparison of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension, presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with the hARLBD. Indeed, THG--which possesses the highest affinity--establishes more van der Waals contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity measured experimentally for DHT over Testo. Moreover, estimation of the ligand-receptor interaction energy through modeling confirms that even minor modifications in ligand structure have a great impact on the strength of these interactions. Our crystallographic data combined with those obtained by modeling will be helpful in the design of novel molecules with stronger affinity for the AR.     

Recycling and adaptation of Klebsormidium flaccidum microalgae for the sustained production of gold nanoparticles

Targeting the development of cell-based bioreactors for the production of metal nanoparticles, the possibility to perform the sustained synthesis of colloidal gold using Klebsormidium flaccidum green algae was studied. A first strategy relying on successive growth/reduction/reseeding recycling steps demonstrated maintained biosynthesis capability of the microalgae but limitation in metal content due to toxic effects. An alternative approach consisting of progressive gold salt addition revealed to be suitable to favor cell adaptation to larger metal concentrations and supported particle release over month periods.     

Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line

Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17β-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [¹⁴C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3β-hydroxysteroid dehydrogenase (3β-HSD1), aromatase and type 1 17β-HSD (17β-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3β-HSD, aromatase and 17β-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3β-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17β-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17β-HSD.     

Agrobacterium tumefaciens-mediated transformation of leek (Allium porrum) and garlic (Allium sativum)

Transgenic leek (Allium porrum) and garlic (Allium sativum) plants have been recovered by the selective culturing of immature leek and garlic embryos via Agrobacterium-mediated transformation using a method similar to that described by Eady et al. (Plant Cell Rep 19:376–381, 2000) for onion transformation. This method involved the use of a binary vector containing the m-gfp-ER reporter gene and nptII selectable marker, and followed the protocol developed previously for the transformation of onions with only minor modifications pertaining to the post-transformation selection procedure which was simplified to have just a single selection regime. Transgenic cultures were selected for their ability to express the m-gfp-ER reporter gene and grown in the presence of geneticin (20 mg/l). The presence of transgenes in the genome of the plants was confirmed using TAIL-PCR and Southern analysis. This is the first report of leek and true seed garlic transformation. It now makes possible the integration of useful agronomic and quality traits into these crops.     

In Chlamydomonas, the loss of ND5 subunit prevents the assembly of whole mitochondrial complex I and leads to the formation of a low abundant 700 kDa subcomplex

In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the beta membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I-complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex.     

Expansion of green alder (Alnus alnobetula [Ehrh] K. Koch) in the northern French Alps: A palaeoecological point of view

Pollen analyses were undertaken on a small peat bog (Ecuelles 06° 49′ 41″E, 45° 58′ 49″N, 1855 m asl), located on the Anterne mountain (Upper-Arve Valley, French north-western Alps). The study highlights the role of green alder (Alnus alnobetula [Ehrh] K. Koch) in Holocene vegetation dynamics of the nowadays treeless subalpine belt. At this place, the onset of human perturbation caused a retreat of fir and arolla-pine stands and an expansion of green alder, which consequently dominated the landscape from 3700 up to 1965 cal. BP. After 1965 cal. BP, the clearings led to the present grasslands with few ligneous species (spruce, larch) on inaccessible cliffs or green alder on the edges of torrents or in avalanche corridors. Picea percentages have increased after 3900 cal. BP, but, due to human activities, spruce has never constituted large stands in the study area. The present general expansion of green alder is due to the decreasing human impact and it constitutes the first step of re-afforestation that should lead to mixed stands of spruce and arolla-pine. The study gives a new evidence of the past diversity of the vegetation cover and do not support the idea that green alder colonization at the subalpine belt constitutes a long-term risk for the vegetation diversity.     

The Use of the Microcomposter to Study the Dynamics of a Mini-Ecosystem

In this activity, students learn about the important topic of invasive species, specifically Zebra Mussels. Students role-play different characters in a real-life situation: the trial of the Zebra Mussel for unlawful disruption of the Great Lakes ecosystem. Students will also learn about jurisprudential inquiry by examining the trial process. This activity will reinforce important knowledge and skills underscored in the Life Science and Science in Personal and Social Perspectives Standards in the National Science Education Standards (National Research Council 1996).     

Unaided dispersal risk of Magallana gigas into and around the UK: combining particle tracking modelling and environmental suitability scoring

Marine non-indigenous species are a significant threat to marine ecosystems with prevention of introduction and early detection considered to be the only effective management strategy. Knowledge of the unaided pathway has received relatively little attention, despite being integral to the implementation of robust monitoring and surveillance. Here, particle tracking modelling is combined with spatial analysis of environmental suitability, to highlight UK coastal areas at risk of introduction and spread of Magallana gigas by the unaided pathway. ‘Introduction into UK’ scenarios were based on spawning from the continental coast, Republic of Ireland, Channel Islands and Isle of Man and ‘spread within UK’ scenarios were based on spawning from known UK wild populations and aquaculture sites. Artificial structures were included as spawning sites in an introduction scenario. The UK coast was scored, based on parameters influencing larval settlement, to reflect environmental suitability. Risk maps were produced to highlight areas of the UK coast at elevated risk of introduction and spread of M. gigas by the unaided pathway. This study highlights that introduction of M. gigas into UK waters via the unaided pathway is possible, with offshore structures increasing the potential geographical extent of introduction. Further, there is potential for substantial secondary spread from aquaculture sites and wild populations in the UK. The results of the study are considered in the context of national M. gigas management, whilst the approach is contextualised more broadly as a tool to further understanding of a little-known, yet significant pathway.