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J D Harding

Publications and source records attributed to J D Harding.

At least 19 recordsLinked to original sources
PubMed2001

Electrophysiological alterations after mechanical circulatory support in patients with advanced cardiac failure.

BACKGROUND: Recognizing that mechanical circulatory support with a left ventricular assist device (LVAD) induces changes in myocardial structure and contractile function, we examined whether there are changes in ventricular conduction and/or repolarization among failing human hearts after LVAD implantation. METHODS AND RESULTS: We examined 12-lead electrocardiograms before surgery, immediately after LVAD placement, and at a delayed (>1 week) postoperative time point in 23 patients who were receiving LVAD support for refractory heart failure. The immediate effects of hemodynamic unloading via LVAD placement included a decrease in QRS duration from 117+/-6 to 103+/-6 ms (P<0.01), an increase in absolute QT duration from 359+/-6 to 378+/-8 ms (P<0.05), and an increase in the heart rate-corrected QT interval (QTc) from 379+/-10 to 504+/-11 ms (P<0.01). None of these immediate changes were observed among 22 patients undergoing routine coronary artery bypass grafting. With sustained cardiac unloading via LVAD support, there was a marked decrease in the QTc from 504+/-11 to 445+/-9 ms (P<0.001). Studies in isolated cardiac myocytes, obtained at the time of transplantation, confirmed that delayed decreases in heart rate-adjusted QTc were the result of decreases in action potential duration after LVAD support. CONCLUSIONS: Acute electrocardiogram responses to LVAD placement demonstrate the dependence of QRS and QT duration on load in the failing human heart. Delayed decreases in QTc and action potential duration reflect reversal of electrophysiologic remodeling in the failing heart. Shortening of the action potential duration likely contributes to the improved cellular contractile performance observed after sustained LVAD support.

Action Potentials↗
PubMed1989

Detection of DNA targets with biotinylated and fluoresceinated RNA probes. Effects of the extent of derivitization on detection sensitivity.

The substituted nucleotide aminohexyl-ATP (AH-ATP) was used for synthesis of RNA probes from a plasmid template using the T7 phage promoter. Following synthesis, RNA probes were modified by reaction with N-hydroxysuccinimide (NHS) esters of biotin or fluorescein. Nearest-neighbor analysis was used to quantitate both the incorporation of the substituted nucleotide into RNA and the subsequent modification of the incorporated nucleotide by the NHS esters. The results indicate that AH-ATP is efficiently incorporated into RNA and that modification of the amine group is also efficient. The T7 polymerase shows a bias for ATP over AH-ATP and truncated transcripts are produced if 100% AH-ATP is used for synthesis. However, the use of 50% AH-ATP in the synthesis reaction yields full-length RNA probes that contain on average one amine-labeled nucleotide every 12 bases. This RNA is readily modified by the respective NHS esters to obtain one biotin group per 15-18 total RNA bases or one fluorescein group per 25-35 bases. Probes modified with biotin or fluorescein were used to detect picogram levels of target DNA in a dot blot hybridization format.

Adenosine Monophosphate↗
PubMed1989

Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.

Bacteriophages↗
PubMed1988

Transcriptional activity and factor binding are stimulated by separate and distinct sequences in the 5' flanking region of a mouse tRNAAsp gene.

The transcriptional properties of two cloned mouse tRNAAsp genes were examined in vitro. The tRNA(2Asp) gene displays a five fold greater transcriptional activity than the tRNA(1Asp) gene and a greater ability to form stable complexes with transcription factors. Transcription of a hybrid gene with swapped 5' flanking sequences and of 5' flanking region deletion mutants demonstrates that the differential transcription of the genes results from stimulatory sequences in the 5' flanking region of the tRNA(2Asp) gene. Distal sequences including those between positions -53 and -31 stimulate transcription but do not affect factor binding. Proximal sequences between positions -9 and -1 enhance factor binding. Thus, binding of transcription factors and later steps required for transcription can be modulated by separate and distinct 5' flanking sequence motifs in eukaryotic tRNA genes.

Animals↗
PubMed1986

Processing of mammalian tRNA transcripts in vitro: different pre-tRNAs are processed along alternative pathways that contain a common rate-limiting step.

We have analyzed the pathways and kinetics of processing of mouse tRNA gene transcripts in vitro. Different transcripts are processed along two alternative pathways. The 3' trailer sequence of the tRNA His primary transcript is excised before the 5' leader sequence. In contrast, for the tRNA Gly primary transcript, the 5' leader sequence is excised before the 3' trailer sequence, as has been found for other monomeric eukaryotic tRNA gene transcripts. Computerized analysis of the kinetics of processing indicates that tRNA Asp, tRNA Gly, tRNA Glu and tRNA His transcripts are processed in a substrate concentration-dependent manner and also reveals the existence of a common rate-limiting step, the rate constant of which is equivalent for three of the four transcripts tested. The processing of one pre-tRNA transcript can be competitively inhibited by addition of another pre-tRNA transcript to the processing reaction. The common rate-limiting step is associated with the conversion of the primary transcript to an intermediate and is independent of sequence and the particular processing pathway of the transcript.

Animals↗
PubMed1986

Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes.

We determined the nucleotide sequences of three mouse tRNAHis genes and a tRNAGly gene present in two different lambda clones. One lambda clone contained two tRNAHis genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHis and a tRNAGly gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHis if posttranscriptional modifications are not considered. Notably, the three tRNAHis genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.

Animals↗
PubMed1986

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.

Animals↗
PubMed1984

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands.

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.

Animals↗
PubMed1984

Structure and evolution of mammalian tRNA genes: sequence of a mouse tRNAiMet gene, the 5'-flanking region of which is homologous to a human gene.

From a recombinant lambda phage, we have determined a 317-bp sequence containing a mouse tRNAiMet gene. The coding region is precisely homologous to mammalian tRNAiMet if post-transcriptional modifications (including addition of the 3'-terminal CCA) are not considered. The gene does not contain introns and has a typical RNA polymerase III termination site in the 3'-flanking region. It is transcribed by RNA polymerase III in the HeLa cell S-100 system in vitro. Notably, the 5'-flanking region of the mouse tRNAiMet gene shares a "patchwork" pattern of homology with one of the human tRNAiMet genes of Santos and Zasloff [Cell 23 (1981) 699-710]. The 5'-flanking regions of the two genes contain strings of nucleotides, 6 to 32 bp in length, the homology of which is 76-100%. These are separated by short strings of unrelated nucleotides. This is one of the first examples of tRNA genes containing homologous 5'-flanking regions isolated from distantly related mammals. We also report a novel method for constructing deletion mutants of sequences cloned in M13 vectors.

Animals↗
PubMed1983

Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp.

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.

Animals↗
PubMed1983

Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis--nucleotide sequence of a mouse lysine tRNA gene.

From a recombinant lambda phage, we have determined a 387 bp sequence containing a mouse lysine tRNA gene. The putative lys tRNA (anticodon UUU) differs from rabbit liver lys tRNA at five positions. The flanking regions of the mouse gene are not generally homologous to published human and Drosophila lys tRNA genes. However, the mouse gene contains a 14 bp region comprising 13 A-T base pairs, 30-44 bp from the 5' end of the coding region. Cognate A-T rich regions are present in human and Drosophila genes. The coding region is flanked by two 11 bp direct repeats, similar to those associated with alu family sequences. The sequence was determined by a "walking" protocol that employs, as a novel feature, iodinated single-stranded M13 probes to identify M13 subclones which contain sequences partially overlapping and contiguous to an initially determined sequence. The probes can also be used to screen lambda phage and in Southern and dot blot experiments.

Animals↗
PubMed1982

Isolation and nucleotide sequence of a mouse histidine tRNA gene.

We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T residues. 523 residues after the 3' end of the his tRNA coding region, the mouse DNA contains a sequence 72% homologous to part of the consensus sequence of the B1 (alu) family.

Animals↗