The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.     

Transition metal complexes of buparvaquone as potent new antimalarial agents. 1. Synthesis,X-ray crystal-structures,electrochemistry and antimalarial activity against Plasmodium falciparum

New Cu(II), Ni(II), Co(II), Fe(II), and Mn(II) metal complexes of buparvaquone [3-trans(4-tert.-butylcyclohexyl)methyl-2-hydroxy-1,4-naphthoquione] (L1H) have been synthesized and characterized using IR, electron paramagnetic resonance (EPR) spectroscopy, microanalytical methods and single crystal X-ray diffraction methods. The single crystal structures were determined for ligand L1H [space group P-1 with a=6.2072(14) A, b=10.379 (2) A, c=13.840 (3) A, V=878.7(3) A(3), Z=2, D(calcd.)=1.234 mg/m(3)] and copper complex [Cu(L1)(2)(C(2)H(5)OH)(2)] C1 [space group I2/a with a=17.149(14) A, b=9.4492(8) A, c=26.946(3) A, V=4335.3(7)A(3), Z=4, D(calcd.)=1.233 mg/m(3)]. All the metal complexes along with the parent ligand have been studied for their electrochemical properties using cyclic voltammetric techniques. The compounds were tested for their in vitro antimalarial activity against Plasmodium falciparum strains. A correlation between the antimalarial activity and the redox property of these complexes is presented. The copper complex C1 exhibits significantly higher growth inhibitory activity both in vitro and in vivo than the parent ligand.     

Does the size of small objects influence chemical reactivity in living systems?

Previous theoretical works showed that chemical reactions in micro- and nano-droplets, bubbles and solid particles were strongly affected by their confinement. In particular, the smallness of the systems leads to high internal pressure compared to the external pressure, which then significantly modifies the values of chemical equilibrium and kinetic constants. In addition, surface tension or surface stress, reactional dilatation and surface charge play also a major role on the chemical reactivity. As living systems are also made of very complex dispersed subsystems, i.e. organelles, it seemed obvious to illustrate our theory by some biological actual examples encountered in pulmonary alveolae, in vacuolae and in medical applications, such as dissolution of gallstones.     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.     

Physiological adaptations of the gut in the Lake Magadi tilapia, Alcolapia grahami, an alkaline- and saline-adapted teleost fish

We describe the gut physiology of the Lake Magadi tilapia (Alcolapia grahami), specifically those aspects associated with feeding and drinking while living in water of unusually high carbonate alkalinity (titratable base=245 mequiv l(-1)) and pH (9.85). Drinking of this highly alkaline lake water occurs at rates comparable to or higher than those seen in marine teleosts. Eating and drinking take place throughout the day, although drinking predominates during hours of darkness. The intestine directly intersects the esophagus at the anterior end of the stomach forming a 'T', and the pyloric sphincter, which comprises both smooth and striated muscle, is open when the stomach is empty and closed when the stomach is full. This unique configuration (a functional trifurcation) allows imbibed alkaline water to bypass the empty stomach, thereby avoiding a reactive mixing with acidic gastric fluids, and minimizes interference with a full stomach. No titratable base was present in the stomach, where the mean pH was 3.55, but the intestine was progressively more alkaline (foregut 6.96, midgut 7.74, hindgut 8.12, rectum 8.42); base levels in the intestinal fluid were comparable to those in lake water. The gut was highly efficient at absorbing water (76.6%), which accompanied the absorption of Na(+) (78.5%), titratable base (80.8%), and Cl(-) (71.8%). The majority of Na(+), base and water absorption occurred in the foregut by an apparent Na(+) plus base co-transport system. Overall, more than 70% of the intestinal flux occurred via Na(+) plus base co-transport, and less than 30% by Na(+) plus Cl(-) co-transport, a very different situation from the processes in the intestine of a typical marine teleost.     

Canopy transpiration of Jeffrey pine in mesic and xeric microsites: O<Subscript>3</Subscript> uptake and injury response

Canopy transpiration of mature Jeffrey pine was compared in "mesic" and "xeric" microsites differing in topographical position, bole growth, and the level of drought stress experienced. Diurnal and seasonal course of canopy transpiration was monitored with thermal dissipation probes in 1999 and 2000. Mid-canopy measures of diurnal foliar stomatal conductance (gs) were taken in June and August in 1999. In early summer, there was little difference between trees in either microsite with regard to gs (55 mmol H2O m⁻²s⁻¹), canopy transpiration (4.0 l h⁻¹), and total duration of active transpiration (12 h >0.03 l h⁻¹). In late summer, xeric trees had a lower daily maximum gs (by 30%), a greater reduction in whole canopy transpiration relative to the seasonal maximum (66 vs 79%), and stomata were open 2 h less per day than in mesic trees. Based on leaf-level gas exchange measurements, trees in mesic sites had an estimated 46% decrease in O3 uptake from June to August. Xeric trees had an estimated 72% decrease over the same time period. A multivariate analysis of morphological and tissue chemistry attributes in mid-canopy elucidated differences in mesic and xeric tree response. Mesic trees exhibited more O3 injury than xeric trees based on reduced foliar nitrogen content and needle retention in mid-canopy.     

Measurement of dissociation constants of inhibitors binding to Src SH2 domain protein by non-covalent electrospray ionization mass spectrometry

A mass spectrometric protocol for identifying ligands with a wide range of affinities (3-101 microM) and quantitative spectral analysis for non-covalent interactions have been developed using Src SH2 as a target. Dissociation constants of five compounds, three with a phospho moiety, one with a sulphonic acid group and one with carboxylic acid groups only, were determined using one-ligand one-binding-site, two-ligands one-binding site and one-ligand two-binding-sites models. The Kd values determined by ESI-MS of the three compounds containing the phospho moiety (3.2-7.9 microM) were comparable to those obtained from a solution equilibrium fluorescence polarization assay. The compound with a sulphonate group is a much weaker binding ligand (Kd=101 microM by ESI, >300 microM by FP) towards the Src SH2 protein. Two complexes with different stoichiometric ratios 1:1 and 2:1 (ligand-protein) were observed by ESI-MS for the ligand GIXXX630X. Analysis of binding isotherms indicated the presence of two binding sites for the ligand with Kd values of 9.3 and 193 microM. These data confirmed that, for these polar compounds, non-covalent ESI-MS can measure affinity which very closely reflects the affinity measured under true solution equilibrium conditions. ESI-MS has several key advantages over many solution methods: it can identify the existence of and measure the affinity of complexes other than simple 1:1 ligand-enzyme complexes. Moreover, ESI-MS competition experiments can be readily performed to yield data on whether two ligands bind simultaneously or competitively at the same time as measuring the affinity of the ligand.     

Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.Integrons are DNA elements that capture genes, especially antibiotic resistance genes, by a site-specific recombination system (32). The recombination system consists of a DNA integrase (Int) and two types of recombination sites, attI and attC (59-base element). The integrase gene (int) is located in the 5′ conserved segment of the integron structure (Fig. ​(Fig.1)1) and is a member of the tyrosine recombinase family (1, 4, 13, 23, 24). Three types of integrases, sharing around 50% identity among themselves, have been identified; they define integron classes 1, 2, and 3 (30). The 5′ conserved segment found in class 1 integrons also contains a promoter region responsible for the expression of inserted cassettes (11, 21) and the recombination site attI1 (31). The 3′ conserved segment of the class 1 integrons includes an ethidium bromide resistance determinant (qacEΔ1), a sulfonamide resistance gene (sulI), an open reading frame (ORF5) of unknown function, and further sequences that differ from one integron to another (5, 6, 28). The 3′ conserved segment of class 2 integrons includes transposition genes (20) while that of class 3 integrons has not yet been studied (2). The variable region, located between the two conserved segments, usually contains antibiotic resistance genes; In0 contains no inserted genes while In21 possesses eight cassettes with ten genes (or ORFs) in this region (5, 16). These genes are part of mobile cassettes which include a recombination site, attC, that differs from one gene to another (18, 33). Incoming genes must be associated with an attC to be recognized by the integron integrase and are preferentially inserted at the recombination site attI1 (11). Cassettes are excised as circular intermediates and integrated at core sites by the action of the integrase (8–10). The core site, defined as GTTRRRY, makes up the 3′ end of attI1 and attC, with the crossover taking place between the G and the first T (19). Antibiotic selection pressure can reveal cassette rearrangements in which a given resistance is nearest the promoter and thus most strongly expressed (10). Open in a separate windowFIG. 1General structure of class 1 integrons. Cassettes are inserted in the integron variable region by a site-specific recombination mechanism. The attI1 site is shown by a black circle, core sites are represented by ovals, the attC site is indicated by a black rectangle, and promoters are denoted by P. intIl, integrase gene; qacEΔ1, antiseptic resistance gene; sulI, sulfonamide resistance gene; orf5, gene of unknown function.Site-specific recombination, unlike homologous recombination, is characterized by relatively short, specific DNA sequences and requires only limited homology of the recombining partners (12). Site-specific recombination is an entirely conservative process since all DNA strands that are broken (two per exchange site) are rejoined in a process that involves neither ATP nor DNA synthesis. Homology alignments of site-specific recombinases assign them to two families: the resolvase family, named after the TnpR proteins encoded by the transposons γδ and Tn3, and the integrase family. The integrase family includes over 140 members to date, but they are highly diversified proteins (13, 23). Members of this family, which include the well-studied λ integrase, recombine DNA duplexes by executing two consecutive strand breakage and rejoining steps and a topoisomerization of the reactants. The first pair of exchanges form a four-way Holliday junction and the second pair resolve the junction to complete the recombination. The integrase nucleophile is a conserved tyrosine that becomes associated with a phosphate group on DNA. The cleavage sites on each DNA duplex are separated by 6 to 8 bp with a 5′ stagger, and the tyrosine joins to the 3′ phosphate (17).The initial definition of the integrase family was based on comparisons of seven sequences, and three invariant residues were identified: an HXXR cluster and a Y residue (4). Alignment of 28 sequences identified a fourth invariant position, occupied by an arginine residue (1). These four conserved residues are found in two boxes located in the second half of the protein. A recent analysis has shown that the conserved histidine is present in 136 of the 147 members (93%); this residue is then not conserved in all members of the family (13). Another recent analysis has identified three patches of residues located around box I, which seem to be important in the secondary structure of these proteins (23). In this study, we analyzed the properties of several mutants of the conserved residues R146, R280, and Y312 of the integron integrase IntI1 in in vivo recombination and in vitro substrate binding.

Construction of plasmids overexpressing mutant MBP-IntI1 fusion proteins.

The plasmids encoding various mutants of MBP-IntI1 were constructed by PCR using pLQ369 (50 ng) as a template (15). Two primer pairs, designed with the OLIGO software package (version 4.1; National Biosciences, Plymouth, Minn.), were used to construct each set of mutants. The R146 mutants were constructed with an XcmI-BamHI primer pair [IntI1(R146)-XcmI, 5′-TTCACCAGCTTCTGTATGGAACGGGCATG(A/G)(A/T)AATCAG-3′; IntI1(R146)-BamHI, 5′-CCGGATCCCTACCTCTCACT-3′], the R280 mutants were constructed with an NruI-XmnI primer pair [IntI1(R280)-NruI, 5′-AGCCGTCGCGAACGAGTGC(C/T)(C/T)GAGGG-3′; IntI1(R280)-XmnI, 5′-ACCCCTAATGAAGTGGTTCGTATCC-3′], and the Y312 mutants were constructed with a AatII-ScaI primer pair [IntI1(Y312)-AatII, 5′-ATTCCGACGTCTCTACTACGATGATTT(C/T)CACGC-3′; pLQ369-ScaI, 5′-ATGCTTTTCTGTGACTGGTG-3′] (restriction sites within primer sequences are underlined). PCR conditions were 10 min at 94°C, three cycles consisting of 45 s at 94°C, 45 s at 47°C, and 90 s at 72°C, 30 cycles consisting of 45 s at 94°C, 45 s at 60°C (50°C for Y312 mutants), and 90 s at 72°C, and a final elongation step of 10 min at 72°C. The XcmI, NruI, and AatII primers were degenerate in one or two positions, so that a single primer could give all mutants. Mutant PCR fragments were digested and cloned directly into pLQ369 digested with the same enzymes, except for the R146 mutant fragments that were subcloned into pLQ364 at first. New mutant PCR fragments were then amplified on these subclones, using IntI1(R146)-BamHI and IntI1(R280)-XmnI primers. These mutant PCR fragments were cleaved with BamHI and XmnI, and the resulting fragments were cloned into pLQ369. This avoids the necessity of partial digestion of pLQ369 with XcmI. Mutant clones were digested with restriction endonucleases and sequenced to determine the mutation.

In vivo recombination.

Mutant MBP-IntI1 clones were introduced into Escherichia coli TB1 {F′ araΔ(lac-proAB) rpsL (Str^r) [φ80dlacΔ(lacZ)M15] hsdR(r_K⁻m_K⁻)} containing pLQ428 by transformation (Fig. ​(Fig.22 and Table ​Table1).1). E. coli TB1 cells containing pLQ428 and one of the MBP-IntI1 mutants were grown at 37°C for 3 h in Luria-Bertani medium. Excision of the aacA1-ORFG and/or ORFH cassettes was induced by the overexpression of the malE-intI1 gene by using 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma Chemical Co.) and by incubation at 37°C for another 3 h. Cell culture was done in the presence of 50 μg of ampicillin per ml, 15 μg of amikacin per ml, and 50 μg of chloramphenicol per ml. Plasmid DNA was then prepared from 5-ml cultures with the Perfect Prep DNA extraction kit (Mandel Corporation). In order to determine the capacity of mutant MBP-IntI1 proteins to excise aacA1-ORFG and/or ORFH cassettes of In21, we used PCR primers pACYC184-5′ (5′-TGTAGCACCTGAAGTCAGCC-3′) and pACYC184-3′ (5′-ATACCCACGCCGAAACAAG-3′) (Fig. ​(Fig.2,2, primers 1 and 2) to detect the reduction of pLQ428 length. PCR conditions were 10 min at 94°C, 30 cycles consisting of 1 min at 94°C, 1 min at 60°C, and 5 min at 72°C, and a final elongation step of 10 min at 72°C. A major PCR fragment can be seen in each lane containing a DNA preparation from a mutant clone (Fig. ​(Fig.3,3, lanes 2 to 9). This band is 2,499 bp long and, as determined by restriction enzyme digestions, represents the pLQ428 clone without any cassette excision (data not shown). This band is also observed in the negative control, which is the pMAL-c2 vector without any gene fused to malE (Fig. ​(Fig.3,3, lane 12). Open in a separate windowFIG. 2Representation of plasmids used in this study. The positions of the three invariant residues of the integrase family are indicated, along with restriction sites used to construct mutant proteins. Core sites are represented by black circles, and attCs are shown by white boxes. The numbered arrows represent the PCR primers used to detect excision events, pACYC184-5′ (1) and pACYC184-3′ (2). bla, gene encoding β-lactamase; cat, gene encoding chloramphenicol acetyltransferase; intIl, gene encoding the integron integrase (IntI1); malE, gene encoding the maltose binding protein (MBP); ori, origin of replication; P_tac, tac promoter; P_tet, tetracycline promoter. Only relevant restriction sites are indicated.

TABLE 1

Plasmids used in this study

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m² solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m² artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different. This revised version was published online in June 2006 with corrections to the Cover Date.