Antisense principle or ribozyme action?

When incorporated into long stretches of antisense RNA, the catalytic domain of the hammerhead type ribozyme significantly increases the extent of antisense-mediated inhibition in living cells. A kinetic model based on the rate constants determined for duplex formation and cleavage of target RNA in vitro is not compatible with the observed biological effects and indicates that further assumptions have to be included to explain the action of ribozymes in vivo.

Crystallization and preliminary X-ray structure analysis of thermally unstable p21(H-ras) guanosine complexes.

p21 is a small guanine nucleotide binding protein that is involved in intracellular signal transduction. Biochemical data suggest that the presence of the beta-phosphate is essential for strong binding of guanine nucleotides to the protein. Guanosine or GMP bind six orders of magnitude more weakly to p21 than GDP or GTP.

Crystallographic studies on p21(H-ras) using the synchrotron Laue method: improvement of crystal quality and monitoring of the GTPase reaction at different time points.

The parameters affecting the crystal quality of complexes between p21(H-ras) and caged GTP have been investigated. The use of pure diastereomers of caged GTP complexed to the more stable p21(G12P)' mutant of p21 and the addition of n-octyl-beta-D-glucopyranoside improved the reproducibility and decreased the mosaicity of the crystals significantly. Furthermore, the crystallization technique was changed from the batch method to the sitting-drop technique.

Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain.

Based on presently available information on the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, peptides have been synthesized which correspond to the sequence of a particular region of the protein involved in formation of the active heterodimeric form of the enzyme. Several peptides that are 15-19 amino acids long and that are derived from the so-called connection domain of the reverse transcriptase are able to inhibit dimerization of the enzyme and thus inhibit development of its enzymatic activities. In particular, a tryptophan-rich 19-mer corresponding to residues 389-407 was relatively efficient, showing an apparent dissociation constant in the micromolar range for one or both of the subunits.

Intrinsic tryptophan fluorescence of bovine liver adenosine kinase, characterization of ligand binding sites and conformational changes.

Bovine liver adenosine kinase is a 45-kDa monomeric protein which exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 284 nm and an emission peak centered at 335 nm. A total of three tryptophan residues/molecule has been estimated by using a fluorescence titration method. Low values of Stern-Volmer quenching constants in the presence of either acrylamide or iodide (4.

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized.

Viral resistance to the thiazolo-iso-indolinones, a new class of nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

Thiazolo-iso-indolinone derivatives with high specificity toward the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) were identified. The most potent compound, BM +51.0836, inhibited HIV-1 RT at a 50% inhibitory concentration of 90 nM in vitro.

Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy.

Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.

Functional nucleotide-binding domain in the F0F1-ATPsynthase alpha subunit from the yeast Schizosaccharomyces pombe.

The segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence.

Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras.

The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21.

Kinetics of interaction of HIV reverse transcriptase with primer/template.

Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1.

Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer.

The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B.

Characterization of the dimerization process of HIV-1 reverse transcriptase heterodimer using intrinsic protein fluorescence.

Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored.

4'-Thio-oligo-beta-D-ribonucleotides: synthesis of beta-4'-thio-oligouridylates, nuclease resistance, base pairing properties, and interaction with HIV-1 reverse transcriptase.

We present the synthesis and the study of properties of a new series of modified oligonucleotides, namely 4'-thio-oligo-beta-D-ribonucleotides (4'-S-RNA). Homo-oligonucleotides of this class (4'-SU6 and 4'-SU12) were prepared from the previously known thionucleosides using the phosphoramidite methodology. The comparison of the substrate properties of 4'-SU6 and its natural analog U6 with respect to four nucleases indicates that the former is much more resistant than the latter.

Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras.

The coordination and binding of the Mg2+ ion in the nucleotide-binding site of p21 have been investigated using site-directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.

Three-dimensional structure and properties of wild-type and mutant H-ras-encoded p21.

Ras (or p21) is the product of the ras proto-oncogene and is believed to be involved in growth-promoting signal transduction. The structure of the guanine nucleotide-binding domain of H-Ras (or p21H-ras) in the triphosphate conformation was determined at very high resolution (1.4 A).

Recovery of planetary images by speckle imaging.

Limits to the recovery of planetary images by speckle imaging were investigated by means of a numerical simulation of the image-forming process. Laboratory measurements established that the numerical model correctly represented the process. With this numerical model we studied the observing conditions required to obtain useful planetary data for Neptune and Io, and we learned which factors are important for the successful recovery of images.

A new high sensitivity 19F probe for labeling cysteine groups of proteins.

A novel iodoacetamide label 4-perfluoro-tert-butyl-phenyliodoacetamide (PFP) containing nine fluorine atoms in equivalent positions has been synthesized. It provides a homogeneous 19F NMR resonance line which can be detected with high sensitivity when coupled to proteins. As an example, the sulfhydryl groups of actin have been labeled with PFP; < 100 nmol of this medium sized protein (corresponding to 2.

Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase.

We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).

Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein.

Studies on the structure and mechanism of H-ras p21.

Current knowledge of the structure of H-ras p21 is reviewed with particular emphasis on the interaction between guanine nucleotides and the active site of the protein. The nature of the conformational change induced by GTP hydrolysis is discussed. The major change is seen in the region known as the effector loop (loop 2), with significant but less well-defined changes occurring in loop 4, which is implicated in the GTPase reaction.

RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms.

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV-1 and HIV-2 reverse transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes.

Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis.

Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay.

Cryo-electron microscopy of vitrified muscle samples.

A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure.

Is there a rate-limiting step before GTP cleavage by H-ras p21?

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J.