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Biomedical subjects

J Reiser

Publications and source records attributed to J Reiser.

At least 73 records · Page 4Linked to original sources
PubMed1985

Bordetella pertussis filamentous hemagglutinin gene: molecular cloning of a potential coding sequence.

We have constructed an expression library of Bordetella pertussis DNA sequences, cloned in Escherichia coli. Random DNA fragments were inserted into the beta-galactosidase gene of bacteriophage lambda gt11 and plaques were screened with antibodies directed against filamentous hemagglutinin. The antigen-positive clone obtained expressed B. pertussis sequences as polypeptides fused to beta-galactosidase. The fusion polypeptides reacted both with antibodies to beta-galactosidase and with antibodies to filamentous hemagglutinin. The positive clone contains B. pertussis DNA sequences as determined by specific hybridization to Bordetella DNA.

Base Sequence↗
PubMed1983

Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene.

Only one of six point mutations of the sequence around one end of the larger of the introns of the rabbit beta-globin gene seriously affects the normal removal of the intron and splicing of the gene. That mutation converts a GT sequence, invariably found at the 5' end of introns, into an AT, which is no longer recognized as a signal for intron removal. Instead, three normally unused (cryptic) sites are used, leading to aberrant gene transcripts. One of the cryptic sites is an exception to the invariable GT sequence.

Animals↗
PubMed1983

Evidence against a scanning model of RNA splicing.

A cloned rabbit beta-globin gene was modified by introducing a DNA fragment containing the 5' splice region of the large intron upstream or downstream of its natural counterpart. Analogous constructions were carried out with the 3' splice region. The genes were linked to SV40 DNA, transiently expressed in HeLa cells and the transcripts analyzed by S1 mapping. In all cases, the splice site further removed from the intron was utilized to the complete exclusion of its counterpart. This finding argues persuasively against a simple scanning model of RNA splicing, in which the splicing enzyme(s) attaches at the 'donor' spliced region and moves along the intron until it encounters the closest 'acceptor' splice region. A model compatible with the currently known facts is presented.

Base Sequence↗
PubMed1983

Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells.

Cloned rabbit beta-globin genes, modified in vitro by restructuring or site-directed mutagenesis, were introduced into mouse 3T6 cells, and the resulting transcripts were analyzed by nuclease S1 mapping. The first 109 bp preceding the cap site sufficed for maximal beta-globin transcription. This segment contained three functionally important regions of the ATA box region; the CCAAT box region; and the -100 region. The latter consists of an imperfect tandemly repeated sequence of 14 bp and 15 bp, both copies of which are required for optimal promoter function. Each of three regions contains two or more nucleotide positions where the introduction of point mutations reduces transcription by at least a factor of 2.

Animals↗
PubMed1983

Electrophysiologic changes induced by external transthoracic countershock.

The purpose of this study was to characterize the electrophysiological effects of suprathreshold transthoracic shocks. Dogs were anesthetized and two consecutive shocks (10 joules/gk) were applied one minute apart. Ventricular extrasystoles were observed in six of the 11 dogs tested, A-V block in three, ventricular tachycardia in three, and asystole in one. Forty eight hours later the hearts were removed. Conventional electrophysiological techniques revealed the following: 1) injured cells had loss of resting potential, triangularization of the action potential and depressed upstrokes; 2) the functional refractory period was shortened, with an increase in time dependence; 3) action potential alternans; 4) discrete areas of marked depression of conduction; 5) re-excitation with premature stimulation and 6) histological evidence of sub-epicardial injury. These studies suggest that the application of suprathreshold shocks may induce electrophysiological changes potentially inducing tachyarrhythmias.

Animals↗
PubMed1983

Intramyocardial current flow in acute coronary occlusion in the canine heart.

Data from numerous experimental infarction studies indicate that rapid myocardial cell depolarization following ischemia causes the flow of injury currents. These currents were measured in the canine myocardium by monitoring voltage gradients across infarct boundaries using silver chloride plunge electrodes, followed by placement of a 100 omega resistor between the electrodes and again measuring the voltage gradients. Current flow was calculated from these measurements with the following results: 1) TQ currents developed within 15 seconds after occlusion and persisted for 120 to 150 minutes, often attaining a magnitude of 1 microA. 2) ST currents also developed within 15 seconds and attained 2 to 3 microA within 15 to 30 minutes, then usually subsided to some degree. 3) T currents were biphasic and attained 2 to 5 microA. Initially, current flowed from normal to ischemic myocardium but usually reversed within 30 minutes after occlusion. 4) The current flow was often disproportionate to the voltage gradient between 120 and 180 minutes after occlusion, possibly indicating electrical uncoupling of the infarcting cells from normal cells. These data indicate that intramyocardial current flow develops early after acute coronary occlusion. These currents may be sufficient to induce reexcitation.

Animals↗
PubMed1983

The effects of insulin and potassium on infarcting canine tissue: an in vitro study.

The electrophysiologic effects of insulin (40 mU/ml) and elevated potassium (4 to 6 mM) on glucose-superfused normal and infarcting tissue from 24 hour coronary artery ligated canine myocardium were studied. With standard intracellular microelectrode techniques, it was observed that insulin infusion for 30 minutes produced an increase in resting membrane potential and a prolongation of action potential duration. In the normal myocardium, the hyperpolarization and the repolarization delay were minimal, but in infarcting tissue with depressed electrophysiologic function, resting membrane potential and action potential duration were significantly improved. This was particularly evident in the presence of an increased potassium concentration (6 mM) when insulin hyperpolarized infarcting cells (n = 8) from 73 +/- 6 to 85 +/- 7 mV (p less than 0.01). In the same studies, action potential amplitudes were increased from 75 +/- 7 to 95 +/- 11 mV (p less than 0.01). In addition, action potential durations at 40 and 80% repolarization were extended from 64 +/- 25 and 141 +/- 46 ms to 132 +/- 34 and 198 +/- 27 ms, respectively (p less than 0.01). Thus, these data are in accord with the reduced ST segment elevation observed in patients treated with glucose-insulin-potassium and support the use of this intervention in the management of acute infarction.

Action Potentials↗
PubMed1981

Oximes: 'enzymatic' slow channel antagonists in canine cardiac purkinje fibers?

In mammalian cardiac muscle voltage-dependent activation of slow channels, e.g., the slow inward current channel, may be possible only when the channels are phosphorylated. We examined the electrophysiological actions of oximes, mile nucleophilic agents which show 'phosphatase-like' activity in isolated enzyme systems, to assess their actions on slow channels in cardiac Purkinje fibers. Diacetyl monoxime (DAM) and pyridine-2-aldoxime (NorPAM) produced a marked, reversible and concentration-dependent reduction in the action potential (AP) plateau duration and abolished spontaneous phase 4 depolarization, but produced only minimal effects on resting potential, dV/dt max, action potential amplitude, duration of phase 3, or membrane resistance. Slow response action potentials evoked in the presence of elevated potassium plus isoproterenol or in Na-free solution were abolished by DAM. The effects of DAM on the AP plateau were antagonized by epinephrine, but an increase in Ca was relatively ineffective. The results suggest that oximes may act as surrogate phosphatases to remove phosphate groups which regulate the availability of slow current channels for voltage-dependent activation.

Action Potentials↗
PubMed1981

Monoclonal antibody with specificity for monocytes and neurons.

We have characterized a monoclonal antibody, called UC45, that reacts with both monocytes and neurons. It was derived from a fusion of the NS-1 plasmacytoma cell line with spleen cells from a mouse immunized with human acute monoblastic leukemia cells. The antibody reacts weakly with viable monocytes in suspension but has specificity for fibrous projections, which are found on monocytes that have adhered to substrate. Other hemopoietically derived cells such as granulocytes and lymphocytes, and many tissue-culture lines, do not react with UC45 by cell-surface immunofluorescence. Similarly, UC45 reacts with the processes of both viable CNS and PNS neurons in tissue culture but with no other neural-tissue-derived cells. The monoclonal antibody has interspecies reactivity, in that it reacts with human, rat and mouse monocytes and neurons. The monocytes and neuronal antigen is present predominantly on a protein of 45 kd. Attempts to identify this protein on monocytes with conventional heteroantisera directed against fibronectin, complement components, fibrinogen, collagen, tubulin and actin have failed. A monoclonal antibody has therefore allowed identification of an antigen, unexpectedly shared by monocytes and neurons. The fact that it is found on cell processes of both cell types suggests that it may be performing some similar function for these cells, whose other activities differ substantially.

Animals↗
PubMed1981

Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper.

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.

Animals↗
PubMed1981

Preferential sensitivity of the left canine purkinje system to cardiac glycosides.

Previous studies have shown that the toxic effects of cardiac glycosides are not manifested uniformly throughout the myocardium. The purpose of our study was to determine whether cardiac glycosides exert different effects on the right vs. left peripheral Purkinje systems and to ascertain mechanisms involved. Control in vitro measurements of paired right and left canine Purkinje fibers showed higher spontaneous rates in left (24.2 +/- 1.75 beats/min) than in right (11.6 +/- 1.55 beats/min, P less than 0.01, n = 81) Purkinje fiber bundles. Following overdrive stimulation, left Purkinje fiber bundles also showed earlier escape beats. After ouabain exposure (2 X 10-7 M), left Purkinje fiber bundles showed earlier signs of toxicity in 20 of 28 experiments, as determined by changes in the maximum diastolic potential, the degree of diastolic depolarization, spontaneous escape intervals, and the magnitude of delayed after-depolarizations. The enhanced sensitivity of left Purkinje fiber bundles was independent of the extracellular potassium concentration and glycoside polarity, and was also observed in situ. We conclude that distal Purkinje fibers are functionally dissimilar and that the left Purkinje system shows greater sensitivity to cardiac glycosides than the right Purkinje system. These data also support the observation that digitalis-induced dysrhythmias arise in the left ventricle.

Action Potentials↗
PubMed1980

Sensitive immunological detection of translation products in SV40 plaques.

A simple and sensitive in situ radioimmunoassay, using simian virus 40 (SV40) proteins as a model, has been developed for the detection of specific translation products of foreign genes cloned in viral vectors. This assay is based on the coupling of all proteins in viral plaques to diazophenylthioether (DPT)-paper. Specific proteins bound to the filter are detected by autoradiography after sequential incubation with (i) unfractionated and unlabeled specific antiserum and (ii) 125I-labeled protein A from Staphylococcus aureus. This assay detects SV40-specific proteins in individual plaques as early as 42 h after infection and its sensitivity limit is below 5 x 10(8) molecules of the major SV40 capsid protein, VP1.

Antigens, Neoplasm↗
PubMed1980

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin.

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.

Animals↗
PubMed1980

Differences in automaticity between Purkinje strands from right and left dog ventricle.

Previous in situ studies have implicated differences in the pacemaker rates between the left and right ventricular conduction system. Our studies were conducted to characterize automaticity in peripheral right and left canine Purkinje fibers in vitro. Standard microelectrode techniques were utilized. In paired right (RPF) and left (LPF) canine Purkinje fibers, control intrinsic rate (IR) was found to be higher in LPF (24.8 +/- 1.7 beats/min) than in RPF (11.5 +/- 1.5 beats/min, P less than 0.01). Following overdrive stimulation at various cycle lengths, LPF consistently showed earlier escape beats. In low K0+ (1.35 mM), IR increased comparably in both RPF and LPF. However, high K0+ (5.4 mM) resulted in a greater reducton of the IR in RPF (-78%) than in LPF (-58%). After epinephrine (10(-6) M) the IR of LPF > RPF although the chronotropic response was more pronounced in RPF. Adrenergic stimulation preceded by beta-adrenergic blockade did not produce significant differences in the intrinsic rate between RPF and LPF. These findings indicate that automaticity in right and left Purkinje strands is functionally dissimilar.

Action Potentials↗