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F Freund

Publications and source records attributed to F Freund.

15 recordsLinked to original sources
PubMed2001

Initiation of HIV-2 reverse transcription: a secondary structure model of the RNA-tRNA(Lys3) duplex.

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription initiation complex has been thoroughly studied, there is still little information regarding a possible model to describe the secondary structure of the template-primer complex in HIV-2. To determine whether HIV-2 RNA sequences flanking the PBS may specifically interact with the natural primer tRNA, we performed site-directed mutagenesis and enzymatic footprinting. An RNA fragment corresponding to the HIV-2 U5 RNA domain and tRNA(Lys3) were probed either in their free form or in the binary complex. Important reactivity changes to nucleases were obtained upon complex formation. In addition to the canonical contacts between the viral PBS and the 3' end acceptor stem of tRNA(Lys3), we identified two additional interacting domains: (i) the U-rich region of the anticodon loop with the A-rich sequence of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the TPsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA. In view of these experimental data and sequence comparison between different HIV-2 isolates, we propose a model for the secondary structure of the HIV-2 template-primer initiation complex.

Anticodon↗
PubMed2001

Organic protomolecule assembly in igneous minerals.

CH stretching bands, nu(CH), in the infrared spectrum of single crystals of nominally high purity, of laboratory-grown MgO, and of natural upper mantle olivine, provide an "organic" signature that closely resembles the symmetrical and asymmetrical C--H stretching modes of aliphatic -CH(2) units. The nu(CH) bands indicate that H(2)O and CO(2), dissolved in the matrix of these minerals, converted to form H(2) and chemically reduced C, which in turn formed C--H entities, probably through segregation into defects such as dislocations. Heating causes the C--H bonds to pyrolyze and the nu(CH) bands to disappear, but annealing at 70 degrees C causes them to reappear within a few days or weeks. Modeling dislocations in MgO suggests that the segregation of C can lead to C(x) chains, x = 4, with the terminal C atoms anchored to the MgO matrix by bonding to two O(-). Allowing H(2) to react with such C(x) chains leads to [O(2)C(CH(2))(2)CO(2)] or similar precipitates. It is suggested that such C(x)--H(y)--O(z) entities represent protomolecules from which derive the short-chain carboxylic and dicarboxylic and the medium-chain fatty acids that have been solvent-extracted from crushed MgO and olivine single crystals, respectively. Thus, it appears that the hard, dense matrix of igneous minerals represents a medium in which protomolecular units can be assembled. During weathering of rocks, the protomolecular units turn into complex organic molecules. These processes may have provided stereochemically constrained organics to the early Earth that were crucial to the emergence of life.

Journal Article↗
PubMed2001

Inhibition of HIV-1 replication in vitro and in human infected cells by modified antisense oligonucleotides targeting the tRNALys3/RNA initiation complex.

The untranslated 5' leader region of the human immunodeficiency virus type 1 (HIV-1) RNA plays an essential role in retroviral replication. It is the first retrotranscribed RNA region, primed from a cellular tRNALys3 partially annealed to the HIV-1 primer binding site (PBS). The structural and functional features of the HIV-1 reverse transcription initiation complex have been thoroughly studied. In this work, we used chemically modified antisense oligonucleotides (AS-ODN) as competitors of the natural tRNALys3 primer for the PBS region. Modified 2'-O-methyl AS-ODN were able to inhibit in vitro HIV-1 reverse transcription and displace the tRNALys3 previously annealed to the PBS. The destabilization of the initiation complex by 2'-O-methyl ODN was a sequence-specific process. We further demonstrated the importance of an anchor region contiguous to the PBS in the annealing of the antisense molecule, allowing the displacement of tRNALys3. The 20-mer 2'-O-methyl molecules were also able to inhibit viral replication in HIV-1-human infected cells, either by blocking cDNA synthesis during the early phase or by interfering with the annealing of the tRNALys3 primer to the PBS during the late phase of the viral cycle. Thus, the highly conserved retroviral initiation complex was shown to be a promising target when using the antisense strategy.

Base Sequence↗
PubMed2000

Study of HIV-2 primer-template initiation complex using antisense oligonucleotides.

HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1 RNA, no direct evidence of interactions with the U-rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV-1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre-primer binding site (the upstream region contiguous to the HIV-2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A-rich loop of viral RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A-rich region in HIV-2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV-2 reverse transcription.

Base Sequence↗
PubMed1999

Carboxylic and dicarboxylic acids extracted from crushed magnesium oxide single crystals.

Carboxylic and dicarboxylic acids (glycolic, oxalic, malonic and succinic) have been extracted with tetrahydrofuran (THF) and H2O from large synthetic MgO crystals, crushed to a medium fine powder. The extracts were characterized by infrared spectroscopy and 1H-NMR. The THF extracts were derivatized with tert-butyldimethylsilyl (t-BDMS) for GC-MS analysis. A single crystal separated from the extract was used for an x-ray structure analysis, giving the monoclinic unit cell, space group P21/c with ao = 5.543 A, bo = 8.845 A, co = 5.086 A, and beta = 91.9 degrees, consistent with beta-succinic acid, HOOC(CH2)COOH. The amount of extracted acids is estimated to be of the order of 0.1 to 0.5 mg g-1 MgO. The MgO crystals from which these organic acids were extracted grew from the 2860 degrees C hot melt, saturated with CO/CO2 and H2O, thereby incorporating small amounts of the gaseous components to form a solid solution (ss) with MgO. Upon cooling, the ss becomes supersaturated, causing solute carbon and other solute species to segregate not only to the surface but also internally, to dislocations and subgrain boundaries. The organic acids extracted from the MgO crystals after crushing appear to derive from these segregated solutes that formed C-C, C-H and C-O bonds along dislocations and other defects in the MgO structure, leading to entities that can generically be described as (HxCyOz)n-. The processes underlying the formation of these precursors are fundamental in nature and expected to be operational in any minerals, preferentially those with dense structures, that crystallized in H2O-CO2-laden environments. This opens the possibility that common magmatic and metamorphic rocks when weathering at the surface of a tectonically active planet like Earth may be an important source of abiogenically formed complex organic compounds.

Carboxylic Acids↗
PubMed1998

Modified (PNA, 2'-O-methyl and phosphoramidate) anti-TAR antisense oligonucleotides as strong and specific inhibitors of in vitro HIV-1 reverse transcription.

Natural beta-phosphodiester 16mer and 15mer antisense oligonucleotides targeted against the HIV-1 and HIV-2 TAR RNAs respectively were previously described as sequence-specific inhibitors of in vitro retroviral reverse transcription. In this work, we tested chemically modified oligonucleotide analogues: alpha-phosphodiester, phosphorothioate, methylphosphonate, peptide nucleic acid or PNA, 2'- o -methyl and (N3'-P5') phosphoramidate versions of the 16mer anti-TAR oligonucleotide. PNA, 2'- O -methyl and (N3'-P5') phosphoramidate oligomers showed a strong inhibitory effect compared with the unmodified 16mer, with reverse transcription inhibition (IC50) values in the nanomolar range. The inhibition was sequence-specific, as scrambled and mismatched control oligonucleotides were not able to inhibit cDNA synthesis. No direct binding of the 2'- O -methyl, PNA or (N3'-P5') phosphoramidate anti-TAR oligonucleotides to the HIV-1 reverse transcriptase was observed. The higher T m obtained with 2'- O -methyl, (N3'-P5') phosphoramidate and PNA molecules concerning the annealing with the stem-loop structure of the TAR RNA, in comparison with the beta-phosphodiester oligonucleotides, is correlated with their high inhibitory effect on reverse transcription.

Anti-HIV Agents↗
PubMed1998

Initiation of in vitro reverse transcription from tRNA(Lys3) on HIV-1 or HIV-2 RNAs by both type 1 and 2 reverse transcriptases.

HIV reverse transcription is initiated from a cellular tRNA partially associated with the retroviral genome. Here we studied homologous HIV-2 cDNA synthesis using natural or synthetic primers. With natural tRNA(Lys3), synthesis of early products comprising nucleotides +5 to +7 preceded the elongation step leading to synthesis of (-) strong-stop cDNA. In the presence of a poly(A) x oligo(dT) trap, no full-length product was observed while early products were still present, showing a transition between initiation and elongation. With DNA primers only an unspecific elongation was found. Our data show a similar mechanism of reverse transcription initiation by HIV-1 and HIV-2 reverse transcriptases. Furthermore, using a heterologous system we found that HIV-1 RNA, in contrast to data reported in the literature, was an excellent template for HIV-2 reverse transcriptase.

Base Sequence↗
PubMed1997

Antibodies directed against a variable and neutralizable region of the HTLV-I envelope surface glycoprotein.

The majority of neutralizing antibodies of HTLV-I are directed against linear epitopes of the envelope surface glycoprotein (gp46) in the immunodominant region 175-199. Although gp46 presents a remarkable degree of conservation, the substitution of the proline at position 192 by a serine is described for 10 isolates among the 54 sequenced ones. This amino acid substitution is known to induce an important change in the orientation of the exposed residues of this region and has drastic consequences on the immunogenicity of the neutralizable epitopes located in this region. We developed monoclonal antibodies directed against epitopes located in this region containing a proline or a serine at position 192. The six monoclonal antibodies obtained recognize the gp46 at the surface of living HTLV-I producing cells, two of them are specific of a 190-197 epitope with a serine at position 192. This demonstrates that the antigenicity of this epitope differs depending on the presence of a proline or a serine at position 192. Altogether, these results demonstrate that the immunodominant neutralizable region 175-199 is antigenically variable.

Amino Acid Sequence↗
PubMed1991

Clathrate hydrate formation in amorphous cometary ice analogs in vacuo.

The presence of clathrate hydrates in cometary ice has been suggested to account for anomalous gas release at large radial distances from the sun as well as the retention of volatiles in comets to elevated temperatures. However, how clathrate hydrates can form in low-pressure environments, such as in cold interstellar molecular clouds, in the outer reaches of the early solar nebula, or in cometary ices, has been poorly understood. Experiments performed with the use of a modified electron microscope demonstrate that during the warming of vapor-deposited amorphous ices in vacuo, clathrate hydrates can form by rearrangements in the solid state. Phase separations and microporous textures that are the result of these rearrangements may account for a variety of anomalous cometary phenomena.

Chemical Phenomena↗
PubMed1991

Positive holes in magnesium oxide. Correlation between magnetic, electric, and dielectric anomalies.

Magnetic susceptibility measurements of high purity MgO single crystals (<50-wt. ppm transition metals) by means of a vibrating-sample magnetometer shows an anomaly at 800 K. At the same temperature the electric conductivity increases anomalously, the static dielectric constant epsilon increases from 9 to approximately 150, a pronounced positive surface charge appears, and Fe2+ in the MgO matrix oxidizes to Fe3+. The data are consistent with O2(2-) (peroxy) defects, representing self-trapped, spin-paired positive holes at Mg2+ vacancy sites. Diamagnetic at low temperatures, the holes start to decouple their spins > 600 K, probably forming at first V0 centers (two O- at an Mg2+ vacancy), then V- centers (single O- at an Mg2+ vacancy), and releasing mobile O- states. These O- represent itinerant charge carriers on acceptor levels near the O 2p-dominated valence band and conduct by O- /O2- valency fluctuations. The O- concentration is of the order of 8 X 10(19) cm-3.

Crystallization↗
PubMed1991

Highly mobile oxygen hole-type charge carriers in fused silica.

Some peculiar positive charge carriers are thermally generated in fused silica above 500 degrees C. These charge carriers appear to be positive holes, chemically O- states, probably arising from dissociation of peroxy defects. The charge carriers give rise to a pronounced positive surface charge which disappears upon cooling but can be quenched by rapid quenching from approximately 800 degrees C. Reheating to approximately 200 degrees C remobilizes these charge carriers and causes them to anneal below 400 degrees C. The generation of positive holes charge carriers may be important to understand failure mechanisms of SiO2 insulators.

Electric Conductivity↗
PubMed1988

The nature and origin of interstellar diamond.

Microscopic diamond was recently discovered in oxidized acid residues from several carbonaceous chondrite meteorites (for example, the C delta component of the Allende meteorite). Some of the reported properties of C delta seem in conflict with those expected of diamond. Here we present high spatial resolution analytical data which may help to explain such results. The C delta diamond is an extremely fine-grained (0.5-10 nm) single-phase material, but surface and interfacial carbon atoms, which may comprise as much as 25% of the total, impart an 'amorphous' character to some spectral data. These data support the proposed high-pressure conversion of amorphous carbon and graphite into diamonds due to grain-grain collisions in the interstellar medium although a low-pressure mechanism of formation cannot be ruled out.

Carbon↗
PubMedDate not supplied

The emission of atoms and molecules accompanying fracture of single-crystal MgO.

We have been investigating the emission of particles due to deformation and fracture of materials. We observe the emission of electrons (exoelectron emission), ions, neutral species, photons (triboluminescence), as well as long wavelength electromagnetic radiation; collectively we refer to these emissions as fractoemission. In this paper we describe measurements of the neutral emission accompanying the fracture of single-crystal MgO. Masses detected are tentatively assigned to the emission of H2, CH4, H2O, CO, O2, CO2, and atomic Mg. Other hydrocarbons are also observed. The time dependencies of some of these emissions relative to fracture are presented for two different loading conditions.

Crystallization↗