Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.     

Qualitative analysis and HPLC isolation and identification of procyanidins from Vicia faba

The soluble proanthocyanidins of the coloured seed coats of Vicia faba L. were isolated and separated by solvent partition. The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+)-gallocatechin, (-)-epigallocatechin, (+)-catechin and (-)-epicatechin units. Oligomeric procyanidins were purified by chromatography on Sephadex LH-20 and the accessible compounds were isolated by RP-HPLC using a Licrospher Li 100 Column. The structures of the purified oligomeric procyanidins were elucidated using a procedure involving TLC, UV spectroscopy, ESI-MS and HPLC analysis of the products from the phloroglucinol reaction. The major condensed tannins of Vicia faba comprise six compounds identified as two A-type procyanidin dimers, the procyanidin dimers B1, B2 and B3, and a procyanidin trimer.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.     

The germ line and somatic stem cell gene Cniwi in the jellyfish Podocoryne carnea

In most animal phyla from insects to mammals, there is a clear division of somatic and germ line cells. This is however not the case in plants and some animal phyla including tunicates, flatworms and the basal phylum Cnidaria, where germ stem cells arise de novo from somatic cells. Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. The cnidarian Piwihomolog Cniwiwas cloned from Podocoryne carnea, a hydrozoan with a full life cycle. CniwiRNA is present in all developmental stages with highest levels in the egg and the medusa. In the adult medusa, Cniwi expression is prominent in the gonads where it likely functions as a germ stem cell gene. The gene is also expressed, albeit at low levels, in differentiated somatic cells like the striated muscle of the medusa. Isolated striated muscle cells can be induced to transdifferentiate into smooth muscle cells which proliferate and differentiate into nerve cells. Cniwi expression is upregulated transiently after induction of transdifferentiation and again when the emerging smooth muscle cells proliferate and differentiate. The continuous low-level expression of an inducible stem cell gene in differentiated somatic cells may underlie the ability to form medusa buds from polyp cells and explain the extraordinary transdifferentation and regeneration potential of Podocoryne carnea.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8⁺ T-cells and the use of an in vitro model of naïve CD8⁺ T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8⁺ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8⁺ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8⁺ and CD4⁺ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.     

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.