Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian–dinoflagellate symbiosis

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host–symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol‐trafficking Niemann–Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2‐a protein is mainly expressed in the epidermis, whereas the NPC2‐d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2‐d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2‐d is a cnidarian‐specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann–Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host–symbiont interactions in the anthozoan–dinoflagellate association.     

Predicting the consequences of species loss using size‐structured biodiversity approaches

Understanding the consequences of species loss in complex ecological communities is one of the great challenges in current biodiversity research. For a long time, this topic has been addressed by traditional biodiversity experiments. Most of these approaches treat species as trait‐free, taxonomic units characterizing communities only by species number without accounting for species traits. However, extinctions do not occur at random as there is a clear correlation between extinction risk and species traits. In this review, we assume that large species will be most threatened by extinction and use novel allometric and size‐spectrum concepts that include body mass as a primary species trait at the levels of populations and individuals, respectively, to re‐assess three classic debates on the relationships between biodiversity and (i) food‐web structural complexity, (ii) community dynamic stability, and (iii) ecosystem functioning. Contrasting current expectations, size‐structured approaches suggest that the loss of large species, that typically exploit most resource species, may lead to future food webs that are less interwoven and more structured by chains of interactions and compartments. The disruption of natural body‐mass distributions maintaining food‐web stability may trigger avalanches of secondary extinctions and strong trophic cascades with expected knock‐on effects on the functionality of the ecosystems. Therefore, we argue that it is crucial to take into account body size as a species trait when analysing the consequences of biodiversity loss for natural ecosystems. Applying size‐structured approaches provides an integrative ecological concept that enables a better understanding of each species' unique role across communities and the causes and consequences of biodiversity loss.     

Endogenous nandrolone metabolites in human urine: preliminary results to discriminate between endogenous and exogenous origin.

When administered to human subjects, nandrolone is metabolized into two main products, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Recent studies demonstrated the endogenous production of these compounds in man at concentrations very close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml. Because the possibility of reaching or exceeding this fateful limit is difficult to exclude, a complementary biochemical parameter is necessary for the differentiation of endogenous 19-NA and 19-NE production from residues resulting from nandrolone consumption. We measured the endogenous concentrations of 19-NA and 19-NE in 385 urine samples from professional football players, and we studied the phase II metabolite composition in individuals excreting the highest concentrations. The results showed that around 30% of endogenous 19-norandrosterone was sulfo-conjugated, whereas 100% of 19-norandrosterone was excreted conjugated to a glucuronic acid when nandrolone was administered. This significant qualitative difference appears to be a promising complementary criterion to more definitively conclude about an athlete's culpability, especially when nandrolone metabolites are found in the low ng/ml range.     

Genew: the Human Gene Nomenclature Database

Genew, the Human Gene Nomenclature Database, is the only resource that provides data for all human genes which have approved symbols. It is managed by the HUGO Gene Nomenclature Committee (HGNC) as a confidential database, containing over 16 000 records, 80% of which are represented on the Web by searchable text files. The data in Genew are highly curated by HGNC editors and gene records can be searched on the Web by symbol or name to directly retrieve information on gene symbol, gene name, cytogenetic location, OMIM number and PubMed ID. Data are integrated with other human gene databases, e.g. GDB, LocusLink and SWISS-PROT, and approved gene symbols are carefully co-ordinated with the Mouse Genome Database (MGD). Approved gene symbols are available for querying and browsing at http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl.     

Chemical genomics: massively parallel technologies for rapid lead identification and target validation

Chemical genomics is a new research paradigm with importantapplications in drug discovery. It links genomic targets withsmall-molecule chemistries thereby allowing for efficient targetvalidation and lead compound identification. ACADIA'schemical-genomics platform consists of a large and diverse small-moleculelibrary (800,000), a reference drug library (2,000), druggablegenomic targets (>300) and a cell-based functional assaytechnology (R-SAT^TM; Receptor Selection and AmplificationTechnology) that allows for ultra-high throughput screening(>500,000 data points/week) as well as high throughputpharmacology and profiling over a wide range of targets. Twoexamples are presented that illustrate the success of ourchemical-genomics approach: (i) The validation of inverse agonismat serotonin 5-HT_2A receptors as an antipsychotic mechanismand the subsequent discovery of potent and selectively acting 5-HT_2A inverse agonists, currently in preclinical development,and (ii) the discovery of the first ectopically binding subtype-selective muscarinic m1 agonist.     

Serum pneumoproteins and biomarkers of exposure to urban air pollution: a cross-sectional comparison of policemen and foresters

Very few biomarkers are available for the non-invasive detection of effects of urban air pollution on the respiratory tract. The objective was to evaluate whether Clara cell protein (CC16) and surfactant-associated protein-A (SP-A), two pulmonary secretory proteins, were useful in the detection of effects of urban air pollutants on the pulmonary epithelium. These proteins were determined in the serum of 53 policemen working in Brussels, Belgium, and a control group of 59 foresters working in the countryside. Except for ozone (O₃), annual concentrations of the main air pollutants (PM₁₀, NO₂, CO, SO₂ and benzene) were significantly higher in Brussels than in the country. The proportion of smokers was lower in urban policemen compared with foresters, but they smoked on average a similar number of cigarettes per day as confirmed by their urinary excretion of cotinine. Muconic acid, a marker of benzene exposure, was significantly higher in urban policemen than in foresters, in both smokers and non-smokers. Multiple regression analysis showed that the type of work, smoking habits and time spent outdoors and in a car were significant determinants of benzene uptake. Tobacco smoking impaired lung function to a similar extent in urban policemen and foresters. The serum levels of SP-A were significantly increased in smokers but were not different between policemen and foresters. Serum CC16 was significantly reduced by tobacco smoking and slightly decreased in policemen compared with foresters. Interestingly, the reduction of serum CC16 was more pronounced in the subgroup of traffic compared with survey policemen, the latter being also less exposed to benzene. The results suggest that serum pneumoproteins and especially serum CC16 could be useful in the detection of chronic effects of urban air pollutants on the respiratory epithelium of populations particularly at risk.     

Effect of a Dietary Herbal Supplement Containing Caffeine and Ephedra on Weight,Metabolic Rate,and Body Composition*

Objective: To evaluate the effect of a dietary supplement containing herbal caffeine (70 mg/dose) and ephedra (24 mg/dose; C&E) on metabolic rate, weight loss, body composition, and safety parameters. Research Methods and Procedures: In phase I, 12 healthy subjects with a BMI of 25 to 35 kg/m² had resting metabolic rate (RMR) measured for 2 hours after ingesting C&E or a placebo on two occasions 1 week apart, followed by a 1‐week washout before phase II. In phase II, these 12 and 28 additional subjects were randomized to a 12‐week, double‐blind trial comparing C&E (3 times/day) to placebo. In phase III, the C&E group was given open‐label C&E for 3 months, and the placebo group was given C&E for 6 months. Results: In phase I, C&E gave an average 8 ± 0.1% (SE) rise in RMR over 2 hours compared with placebo (p < 0.01). In phase II, weight loss at 12 weeks was 3.5 ± 0.6 kg with C&E compared with 0.8 ± 0.5 kg with placebo (p < 0.02). The percentage fat lost, shown by DXA, was 7.9 ± 2.9% with C&E and 1.9 ± 1.1% with placebo (p < 0.05). Pulse decreased more in the placebo group that in the C&E group (p < 0.03). There were no differences in lipid levels or blood pressure. In phase III, there was a 6‐month loss of 7.3% and 7.8% of initial body weight for the groups on placebo and C&E during phase II, respectively. There were no serious adverse events. Discussion: C&E increased RMR significantly by 8% compared with placebo, promoted more weight and fat loss than placebo, and was well tolerated.     

Lumbricus terrestris Hemoglobin—The Architecture of Linker Chains and Structural Variation of the Central Toroid

The extracellular giant hemoglobin from the earthworm Lumbricus terrestris was reconstructed at 14.9-Å resolution from cryo-electron microscope images, using a new procedure for estimating parameters of the contrast transfer (CTF) function. In this approach, two important CTF parameters, defocus and amplitude contrast ratio, can be refined iteratively within the framework of 3D projection alignment procedure, using minimization of sign disagreement between theoretical CTF and cross-resolution curves. The 3D cryo-EM map is in overall good agreement with the recent X-ray crystallography map of Royer et al. (2000, Proc. Natl. Acad. Sci. USA 97, 7107–7111), and it reveals the local threefold arrangement of the three linker chains present within each 1/12 of the complex. The 144 globin chains and 36 linker chains within the complex are clearly visible, and the interdigitation of the 12 coiled-coil helical spokes forming the central toroidal piece is confirmed. Based on these findings, two mechanisms of the dodecameric unit assembly are proposed and termed “zigzag” and “pairwise” polymerizations. However, the detection by cryo-EM of 12 additional rod-like bodies within the toroid raises the possibility that the architecture of the toroid is more complex than previously thought or that yet unknown ligands or allosteric effectors for this oxygen carrier are present.     

Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase.

Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants. Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E. coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa. Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP. Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1). The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of [(18)O]AMP produced from (18)O transfer using [carboxyl-(18)O]pantoate. Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction. These data also suggest that both half-reactions are partially rate-limiting. Reverse isotope exchange of [(14)C]-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products.     

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in syrian hamsters

Summary This article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms. The cell line described in this article has been deposited with the European Collection of Cell Cultures with the accession number 98061003.