Acoustic tokamak with strongly coupled toroidal bubbles.

Gas bubbles stabilized in toroidal 3D-printed cages are good acoustic resonators with an unusual topology. We arrange them in a circular array to obtain what we call an "acoustic tokamak" because of the torus shape of the whole array. We demonstrate experimentally and theoretically that the system features several acoustic modes resulting from the acoustic interaction between tori. The fundamental acoustic mode has a much lower frequency than that of the individual bubbles. The acoustic field along the circle inside the acoustic tokamak is remarkably homogeneous, as shown by our 3D simulations.

Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase.

HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D64, D116 and E152 of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V151E152S153 region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3'-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V151E152S153 region involvement in the integration reaction is more important than for the 3'-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.

Functional interactions of human immunodeficiency virus type 1 integrase with human and yeast HSP60.

Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.

Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis.

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells.

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins.

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.

Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.

The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.

Identification of a plasma membrane-associated guanine nucleotide exchange factor for ARF6 in chromaffin cells. Possible role in the regulated exocytotic pathway.

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.

Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae.

The integration of proviral DNA into the genome of the host cell is an essential step in the replication of retroviruses. This reaction is catalyzed by a viral-encoded enzyme, the integrase (IN). We have previously shown that human immunodeficiency virus type 1 (HIV-1) IN causes a lethal effect when expressed in yeast cells. This system, called yeast lethal assay, was used as a tool to study IN activity in a cellular context. The yeast lethal assay allowed the selection and characterization of mutations affecting both the lethal phenotype and the in vitro IN activities. IN mutants were produced by random PCR mutagenesis in an IN gene bearing the inactivating D116A mutation in the catalytic site. The corresponding D116A substituted IN does not lead to lethality in yeast. Subsequent selection of mutants able to restore the lethal effect of IN was carried out using the yeast lethal assay. We isolated three mutants presenting a restored phenotype. The mutated IN genes were sequenced and the corresponding proteins were purified to characterize their in vitro activities. The three mutants presented restoration of the in vitro strand transfer activity, while 3' processing was only partially restored.The three mutants differ from D116A IN by at least one amino acid substitution located in the N-terminal domain of the protein, outside of the active site. These new mutated HIV-1 INs may therefore allow a better understanding of the N-terminal domain function in the integration reaction. In addition, these results support our hypothesis that explains the lethal effect as a consequence of the nuclear damage caused by wild-type IN in yeast cells. These data also indicate that the yeast lethal assay can be used as a tool to study the retroviral integration mechanism in a cellular context and to select specific inhibitors.

Inhibition of HIV-1 integrase-catalysed reaction by new DNA minor groove ligands: the oligo-1,3-thiazolecarboxamide derivatives.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus, responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecarboxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1-4 Tz rings and different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated. Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units. The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this complex should provide a rationale for the design of drugs targeting the integration step.

High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.

Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D.

The ADP-ribosylation factor (ARF) GTP-binding proteins have been implicated in a wide range of vesicle transport and fusion steps along the secretory pathway. In chromaffin cells, ARF6 is specifically associated with the membrane of secretory chromaffin granules. Since ARF6 is an established regulator of phospholipase D (PLD), we have examined the intracellular distribution of ARF6 and PLD activity in resting and stimulated chromaffin cells. We found that stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. To probe the existence of an ARF6-dependent PLD in chromaffin cells, we measured the PLD activity in purified plasma membranes. PLD could be activated by a nonhydrolyzable analogue of GTP and by recombinant myristoylated ARF6 and inhibited by specific anti-ARF6 antibodies. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 inhibited both PLD activity and catecholamine secretion in calcium-stimulated chromaffin cells. The possibility that ARF6 participates in the exocytotic reaction by controlling a plasma membrane-bound PLD and thereby generating fusogenic lipids at the exocytotic sites is discussed.

Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening.

The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage. To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression. However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase. Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro. We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo.