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Biomedical subjects

A Hampel

Publications and source records attributed to A Hampel.

At least 37 records · Page 2Linked to original sources
PubMed1995

Rapid desilylation of oligoribonucleotides at elevated temperatures: cleavage activity in ribozyme-substrate assays.

Treatment of 2'-O-silyl-oligoribonucleotides with triethylamine trihydrofluoride in DMF at 55 degrees C for 1 h effected complete desilylation. The product was isolated by a single addition of 1-butanol to the reaction mixture. The resulting RNA was found to be identical with that obtained by traditional desilylation methods as analyzed by HPLC, enzyme digest and ribozyme-substrate assays.

Base Sequence↗
PubMed1994

Mutagenesis of the hairpin ribozyme.

Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.

Base Composition↗
PubMed1994

Alteration of hairpin ribozyme specificity utilizing PCR.

We have developed a method by which a researcher can quickly alter the specificity of a trans hairpin ribozyme. Utilizing this PCR method, two oligonucleotides, and any target vector, new ribozyme template sequences can be generated without the synthesis of longer oligonucleotides. We have produced templates with altered specificity for both standard and modified (larger) ribozymes. After transcription, these ribozymes show specific cleavage activity with the new substrate beta-glucuronidase (GUS), and no activity against the original substrate (HIV-1, 5' leader sequence). Utilizing this technique, it is also possible to produce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical system for the assessment of trans ribozyme activity.

Base Sequence↗
PubMed1993

A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1.

Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the beta-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

Antiviral Agents↗
PubMed1993

New technique for the sterile introduction of flexible nasopharyngolaryngoscopes.

Since the inception of flexible fiberoptic endoscopes, disinfection of these instruments has been a problem. Soaking in glutaraldehyde does not always achieve sterilization, and often damages the scopes. Ethylene oxide can sterilize endoscopes; however, it is economically impractical because of a required downtime of 24 hours. Thus, it is obvious, especially with respect to human immunodeficiency virus, hepatitis B virus, and Mycobacterium, that a new technique to attain sterility is necessary. This paper discusses a new method of sterile introduction of the flexible nasopharyngolaryngoscope. The technique employs disposable sterile sheaths that are prepackaged and made from a thermoplastic elastomer with a clear optical end. The sheaths can be applied in seconds and tightly adhere to the flexible insertion portion of the scope. Results to date indicate that the performance of the endoscope is unhindered by using the sheaths. Furthermore, there has been no break in the integrity of the sheaths or damage to instruments. It is our opinion that these devices will greatly improve the level of sterility while at the same time reducing costs and downtime.

Cross Infection↗
PubMed1992

A method for generating transcripts with defined 5' and 3' termini by autolytic processing.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

Autolysis↗
PubMed1992

Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Base Sequence↗
PubMed1992

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.

Base Sequence↗
PubMed1990

'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA.

We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.

Base Sequence↗
PubMed1989

RNA catalytic properties of the minimum (-)sTRSV sequence.

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

Base Sequence↗
PubMed1988

Leucine-tRNA ligase complexes.

The methodologies described in this chapter allow the reproducible preparation of native high-molecular-weight synthetase complexes of leuRL. These complexes have the ability to preferentially utilize extracellular leucine immediately upon transport and are likely the forms of the enzyme most important in the utilization of leucine for protein synthesis.

Amino Acyl-tRNA Synthetases↗
PubMed1986

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.

Base Sequence↗
PubMed1985

External auditory canal dysplasia: CT evaluation.

Dysplasias of the external auditory canal can be evaluated with high resolution CT using proper technique. Images are studied to determine the type of atresia (membranous or bony) and the thickness of the associated atretic plate (if present). Other findings sought include the degree of pneumatization, the status of the fenestrae, the size of the tympanic cavity, and the location of both the mandibular condyle and the facial nerve canal.

Bone Diseases, Developmental↗
PubMed1984

A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex.

The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts025Cl with a defect in leucyl-tRNA synthetase ( LeuRS ) does not have an inherently more thermolabile LeuRS , but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same Km for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS .

Amino Acyl-tRNA Synthetases↗
PubMed1984

Cell culture mutants as aminoacyl-tRNA synthetase complex probes.

A number of Chinese hamster ovary cell culture mutants have been characterized by us for their high-molecular-weight aminoacyl-tRNA synthetase (aaRS) complexes. The results all support a model of aaRS complex function wherein the high-molecular-weight complexes form aminoacyl-tRNA by utilizing extracellular amino acids immediately on their transport and before they have equilibrated with the internal pool. The low-molecular-weight aaRS forms exclusively utilize amino acids from only the intracellular pool.

Amino Acids↗
PubMed1981

Reformation of leucyl-tRNA synthetase complexes in revertants from CHO mutant tsH1.

A direct correlation was found to exist between increased thermolability of leucyl-tRNA synthetase and loss of the high-molecular-weight enzyme complexes in the CHO cell mutant tsH1 and its revertants. This was shown to occur apart from a differential thermostability between the complexes themselves and is supported by Michaelis constant determinations.

Amino Acyl-tRNA Synthetases↗