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Biomedical subjects

D Kim

Publications and source records attributed to D Kim.

At least 415 records · Page 23Linked to original sources
PubMed1991

Electroporation of extraneous proteins into CHO cells: increased efficacy by utilizing centrifugal force and microsecond electrical pulses.

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.

Animals↗
PubMed1991

Experimental determination of the linear correlation between in vivo TV fluorescence intensity and vascular and tissue FITC-DX concentrations.

A novel in vivo calibration procedure was developed to determine the microvascular and tissue concentration of fluorescein isothiocyanate-labeled dextrans (FITC-Dx) in the hamster cheek pouch, using intravital fluorescence microscopy with manually controlled TV camera gain and threshold value. Two FITC-dextrans (70,000 and 150,000 MW) were used as tracers. Five minutes after the tracer was administered, selected venules (diameter 20-50 microns) were videotaped, and intravascular gray levels were obtained by digital image processing. Simultaneously, arterial blood samples were taken to measure vascular FITC-Dx concentrations with a spectrofluorometer. The gray levels and the concentrations were used to produce a calibration curve for the vascular FITC-Dx concentration. A similar calibration curve for the interstitial FITC-Dx concentration was obtained by first video recording interstitial space areas saturated with the tracer. After flushing out the tracer in the vessels, the hamster cheek pouch was then cut, weighted, and homogenized. The interstitial FITC-Dx concentration was finally measured with a spectrofluoromet. The gray levels and the concentrations were used to produce a calibration curve for the interstitial FITC-Dx concentration. The gray level was found to vary linearly with both the FITC-Dx vascular concentration (range 0.4-3.0 mg/ml) and the interstitial FITC-Dx concentration (0.12-1.50 mg/ml) in the hamster cheek pouch.

Animals↗
PubMed1991

Two novel cardiac atrial K+ channels, IK.AA and IK.PC.

Two K(+)-selective channels in neonatal rat atrial cells activated by lipophilic compounds have been characterized in detail. The arachidonic acid-stimulated channel (IK.AA) had a slope conductance of 124 +/- 17 pS at +30 mV in symmetrical 140 mM potassium and a mean open time of approximately 1 ms, and was relatively voltage independent. IK.AA activity was reversibly increased by lowering pH to 6.0. Arachidonic acid was most effective in activating this channel, although a number of lipophilic compounds resulted in activation. Surprisingly, choline, a polar molecule, also activated the channel. A second K+ channel was activated by 10 microM phosphatidylcholine applied to the intracellular surface of inside-out atrial patches. This channel (IK.PC) had a slope conductance of 60 +/- 6 pS at +40 mV and a mean open time of approximately 0.6 ms, and was also relatively voltage independent. Fatty acids are probably monomeric in the membrane under the conditions of our recording; thus detergent effects are unlikely. Since a number of compounds including fatty acids and prostaglandins activated these two channels, an indirect, channel-specific mechanism may account for activation of these two cardiac K+ channels.

Animals↗
PubMed1991

Modulation of acetylcholine-activated K+ channel function in rat atrial cells by phosphorylation.

1. In voltage-clamped whole cells dialysed with GTP, extracellular application of ACh elicits an inwardly rectifying K+ current which subsequently decreases to a steady-state level well below the maximally induced current (desensitization). The mechanism of desensitization of the acetylcholine (ACh)-activated K+ channel current was studied in rat neonatal atrial cells at the single-channel level using the patch-clamp technique. 2. In cell-attached patches with ACh in the pipette, a similar pattern of K+ channel current desensitization was present. Single-channel analyses revealed that the initial rapid decrease in channel activity was associated with progressive shortening of the mean open time (tau o) and prolongation of the mean closed time (tau c) of the K+ channel. 3. In excised, inside-out patches with ACh in the pipette, GTP activated K+ channels with a tau o of approximately 1.0 ms. Addition of ATP to the cytosolic surface resulted in progressive increases in tau o (from 1 to 5 ms) and channel activity. These changes are similar but opposite in direction to those observed during the early phase of ACh-induced channel desensitization in cell-attached patches. 4. The effect of ATP on the channel kinetics was abolished in Mg(2+)-free solution AMP-PNP (adenylyl-imidodiphosphate, a non-hydrolysable analogue of ATP), ADP, CTP (cytidine triphosphate), ITP (inosine triphosphate) or UTP (uridine triphosphate) did not alter the channel kinetics, suggesting that the ATP effect on channel gating probably occurs via phosphorylation by a membrane-bound kinase. H-8 (an isoquinolinesulphonamide derivative which inhibits protein kinases A and C) failed to prevent the action of ATP on the channel. 5. The increases in tau o and channel activity produced by ATP could be completely reversed by an elevation of cytosolic [Ca2+] to 3 x 10(-5) M or above. 6. The effect of Ca2+ on the ATP-induced changes in channel kinetics was blocked by sodium vanadate, a general phosphatase inhibitor. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, did not block the Ca2+ effect. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), trifluoroperazine, and calmidazolium, partially blocked the effect of Ca2+. 7. Alkaline phosphatase (20 units/ml) reversed the ATP-induced increases in tau o and channel activity. These results suggest that the ACh-activated K+ channel can be modulated by phosphorylation and dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1991

Fast selective black blood MR imaging.

To overcome the problems associated with gradient-echo (GRE) magnetic resonance (MR) angiography ("bright blood" imaging) and "black blood" imaging with presaturated spin-echo (SE) pulse sequences, the authors devised a new approach for black blood imaging. Their method, selective preinversion fast imaging with steady precession (turboFISP), uses a segmented GRE sequence for fast data acquisition. Nulling of vascular signal results, and stationary tissue appears bright. The method was compared with flow-compensated GRE imaging in a phantom and with GRE imaging and presaturated SE imaging in seven healthy volunteers and nine patients with various cardiac diseases. With phantoms, the selective preinversion turboFISP sequence produced better flow contrast than did GRE sequences. Selective preinversion turboFISP was often superior to SE imaging for depicting vessel lumina, particularly in patients with slowly flowing blood. Arteries appeared dark in selective black blood angiograms, but veins did not. Selective preinversion turboFISP can be used with bright blood GRE imaging to depict vessel lumina, and its capability for image acquisition within a breath hold and with cardiac gating minimizes artifacts from respiration and motion of the vessel wall.

Adult↗
PubMed1991

Endothelin activation of an inwardly rectifying K+ current in atrial cells.

Various tissues including heart express specific binding sites for endothelin. Endothelins have been reported to increase the force of contraction of cardiac muscle, presumably via specific receptors. Specific binding of endothelin to atrial tissue is particularly high. In spontaneously contracting rat atrial cells used in this study, all three isoforms of endothelin (endothelin-1, endothelin-2, and endothelin-3) decreased the rate of beating and caused an increase in inwardly rectifying K+ current in voltage-clamped whole cells. Endothelin-3 was the most potent isoform, and its effects on beating rate and K+ current were present at a concentration as low as 100 pM (Kd, approximately 1 nM). the atrial cells did not have the hyperpolarization-activated current (the pacemaker current), If. In excised inside-out patches, all three isoforms of endothelin activated a population of K+ channels with kinetic properties identical to those of acetylcholine (muscarinic)-activated K+ channels, and this was GTP dependent. Endothelin failed to decrease the beating rate or to elicit the K+ current in pertussis toxin-treated cells. These results indicate that endothelin has a potent negatively chronotropic effect by activation of the inwardly rectifying, muscarinic K+ channel and therefore could be an important regulator of heart function.

Acetylcholine↗
PubMed1991

Renal artery imaging: a prospective comparison of intra-arterial digital subtraction angiography with conventional angiography.

This study describes a systematic comparison of intra-arterial digital subtraction angiography (DSA) of the main renal arteries with conventional angiography (CA), the currently accepted "gold standard" for the diagnosis of renal artery stenosis. Twenty-five patients scheduled for abdominal aortography for various indications underwent first DSA then CA. The DSA and CA images were evaluated for number of renal arteries, presence and grade of renal artery stenosis, presence of post-stenotic dilation or fibromuscular changes, and diagnostic and pictorial adequacy of the images. DSA was found to be diagnostically adequate in 92% of cases, compared with 96% for CA. In evaluating significant main renal artery stenosis, which the authors assumed to be any stenosis greater than 50%, there were 10 such stenoses seen by CA. DSA also detected 10 cases, but there was 1 false positive and 1 false negative, yielding a sensitivity of 90% and a specificity of 98%. There was also close correlation of DSA and CA for the few cases of post-stenotic dilatation and fibromuscular dysplasia encountered. The authors conclude that DSA is an acceptable substitute for CA in the evaluation of patients for main renal artery stenosis.

Angiography↗
PubMed1991

[Imaging of thoracic and abdominal aortic aneurysms using MR angiography].

Five patients with suspected dissecting aneurysm of the thoracic aorta and nine patients with fusiform aneurysm of the abdominal aorta underwent time-of-flight (TOF) MR angiography. MR angiography revealed dissecting aneurysms in four patients and a dilated ascending aorta without evidence of dissection in one patient. These findings correlated well with angiography, computed tomography and surgical findings. Proximal and distal extension as well as the relationship to vessels originating from the aortic arch was assessed correctly in all cases. There was a good correlation between MR angiography, CT, and angiography in the patients with abdominal aortic aneurysms. The status of the renal arteries was shown in all patients with MR angiography, but the inferior mesenteric artery was not regularly demonstrated. MR angiography is a promising method for non-invasive assessment of thoracic and abdominal aortic aneurysms.

Aortic Dissection↗
PubMed1991

Magnetic resonance imaging: a reliable test for the evaluation of proximal atherosclerotic renal arterial stenosis.

Symptomatic renal artery stenosis is a significant and treatable clinical problem. A reliable and accurate noninvasive method of screening for renal artery stenosis has not yet been found. We used magnetic resonance imaging to study 37 patients who had undergone recent renal angiography. Fourteen patients had normal renal arteries by angiography. In 23 patients either unilateral or bilateral stenosis or occlusion was present. The disease process in all patients appeared to be atherosclerosis. The average age of the 37 patients was 68 years. The magnetic resonance scans and angiograms were read independently by two different radiologists, each of whom was blinded to the clinical history and the results of the other study. Renal arterial stenoses found on angiogram and magnetic resonance scans were graded as absent (0% to 24%), mild (25% to 49%), moderate (50% to 74%), or severe (75% to 99%). The magnetic resonance imaging results concurred with the angiographic findings in 70 of 77 arteries (91%). Magnetic resonance imaging predicted the presence of a greater than 50% stenosis of the renal artery with a sensitivity of 100% and a specificity of 94%. Magnetic resonance imaging may prove to be the best noninvasive screening test for proximal atherosclerotic renal arterial stenosis.

Adult↗
PubMed1991

[MR angiography of the abdominal veins].

On the basis of the time-of-flight effect in 18 normal volunteers and 119 patients with different diseases of the abdominal veins (inferior vena cava, porto-splenic system, renal/hepatic/iliac veins) magnetic resonance (MR) angiograms were compared with the DSA, CT and US results. The MR technique included a series of 2D gradient-echo (Flash) images in which the patients held their breath and projection angiograms (PA) (MIP algorithm). PA of the inferior vena cava and renal veins had a sensitivity of 90% and a specificity of 88.8%. The results demonstrate that in all cases diseases of the large veins could be detected using all the MR information available. It is suggested that this method is so far not satisfactory in the evaluation of small vessels and slow intravascular flow conditions.

Abdomen↗
PubMed1990

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells.

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.

Adenosine Triphosphate↗
PubMed1990

Yeast tRNATrp genes with anticodons corresponding to UAA and UGA nonsense codons.

Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.

Anticodon↗
PubMed1990

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing.

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.

Base Sequence↗