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Biomedical subjects

L Ng

Publications and source records attributed to L Ng.

At least 55 records · Page 3Linked to original sources
PubMed1991

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.

Amino Acid Sequence↗
PubMed1991

Enzymologic mechanism of calf thymus DNA polymerase delta.

The catalytic core protomer of calf thymus DNA polymerase delta (pol delta) was purified to apparent homogeneity by a modified procedure, and its enzymologic mechanism was investigated using a combination of steady-state kinetics and semiquantitative sedimentation binding analyses. Like DNA polymerase alpha (pol alpha), in the absence of a primer, pol delta was able to bind single-stranded but not double-stranded DNA. This, in conjunction with the observation of induced substrate (dNTP) inhibition of pol delta in the presence of a correctly base-paired 2',3'-dideoxyribonucleotide-terminated primer, suggests that pol delta follows an ordered sequential ter-reactant mechanism of substrate recognition and binding similar to that elucidated for pol alpha. Pol delta binds template first followed by primer and then template-directed dNTP. With suitable substrates, addition to incubations of proliferating cell nuclear antigen, the pol delta auxiliary factor, leads to a reduction in Km and increase in Vmax. This suggests that proliferating cell nuclear antigen enhances the processivity of pol delta by increasing both the residence time of pol delta on the DNA template-primer and the rate at which individual nucleotides are incorporated.

Animals↗
PubMed1990

One amino acid change produces a high affinity cGMP-binding site in cAMP-dependent protein kinase.

Discrimination between cAMP and cGMP is a critical feature of cAMP- and cGMP-dependent protein kinases. An alanine/threonine difference in the cyclic nucleotide-binding sites has been proposed to provide a structural basis for this functional distinction. Site-directed mutagenesis of this alanine to a threonine in a cAMP-binding site of cAMP kinase produced a mutant with markedly increased cGMP affinity as determined by cGMP binding and protein kinase activation assays. Studies of other mutants at this position support the role of the threonine hydroxyl group as the component that enhances cGMP binding affinity.

Alanine↗
PubMed1990

Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart.

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrated with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacted with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis revealed that the putative Drosophila PCNA was highly homologous to human PCNA. Of the first 22 amino acids, 16 were identical, and 4 of the remaining 6 were changed conservatively. Results of total amino acid analysis were also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrated that Drosophila PCNA could substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-reacted with human PCNA and were able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ. These antibodies thus promise to be useful probes for the study of cell proliferation in this rapidly developing organism.

Amino Acid Sequence↗
PubMed1989

Recognition and binding of template-primers containing defined abasic sites by Drosophila DNA polymerase alpha holoenzyme.

Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points.

Animals↗
PubMed1989

Correlation of nonlinear mathematical transformation of the normal electrocardiogram with the severity of coronary artery disease.

Signal processing was applied to normal ECGs to study whether the results could predict coronary artery disease (CAD) on angiography. ECGs determined to be normal by conventional criteria for 116 white males and 84 white females were subjected to signal processing. The technique applied nonlinear mathematical transformation to a segment of seven ECG leads which were photographically imaged, automatically digitized, normalized for time, and had topographical coordinate transformation using an IBM-XT microcomputer. The resulting curvilinear display on the computer monitor was termed a biopotential coordinate transformation (BCT). Successive superimposition of BCTs from normal ECGs of patients with normal angiograms developed boundaries of a normal template for each lead. Study BCTs were then compared to the normal templates to predict the presence or absence of CAD. Correlation of the BCT results with the severity of CAD for the males resulted in average sensitivities of 80%, 84.4%, and 91.7% for single, double, and triple-vessel CAD, respectively. Similarly, for the females average sensitivities were 59.1%, 73.9%, and 88.9%, respectively. In past studies on the same population, the BCT process detected CAD with average sensitivity of 85.1% and specificity of 81.8% for white males and average sensitivity of 73% and specificity 81% for white females. As such, computerized signal processing of ECGs may provide a noninvasive method for detection of CAD in patients with seemingly normal resting ECGs. The results of this study indicate that the sensitivity of the process increases in direct proportion to the severity of CAD. Further investigation is warranted for process refinement and verification.

Coronary Angiography↗
PubMed1989

Effects of hypothermia and hemodilution on oxygen metabolism and hemodynamics in patients recovering from coronary artery bypass operations.

The coexistence of hypothermia and hemodilution in patients in the intensive care unit immediately postoperatively after coronary artery bypass graft operations presents concerns regarding the adequacy of hemodynamics and oxygen metabolism. We evaluated the hemodynamic status and oxygen metabolism during the postoperative recovery period in six patients with moderate hemodilution (hematocrit value 34% +/- 3%) and in eight patients with marked hemodilution (hematocrit value 23% +/- 2%). All patients were well sedated and paralyzed with pancuronium bromide during the study period, during which their body temperature was slowly returning toward normal. In both groups, cardiac index at 34 degrees C was about 40% lower than at 37 degrees C. This was associated with 50% higher systemic vascular resistance and 30% lower oxygen availability to tissue. Oxygen consumption, however, was proportionally lower (45%) and coronary perfusion pressure was higher (28%) at 34 degrees C than at 37 degrees C; thus neither mixed venous nor coronary sinus blood oxygen saturation was compromised under hypothermic conditions. Although the trends in hemodynamic changes were similar in both groups, cardiac indices in patients with marked hemodilution were higher than cardiac indices in those with moderate hemodilution at all temperatures. This observation indicates that the hemodilution-induced rise in cardiac index remains intact even under hypothermic conditions. Under the conditions we studied, hypothermia with or without hemodilution had no significant adverse effects on hemodynamics and oxygen metabolisms of the whole body or of the heart.

Aged↗
PubMed1988

Plasma acetate levels in response to intravenous fat or glucose/insulin infusions in diabetic and non-diabetic subjects.

We measured plasma levels of acetate, glucose, insulin, fatty acids and 'ketone bodies' (KB), during fat infusion and continuous simultaneous infusion of insulin and glucose according to a computerized algorithm to maintain fasting euglycaemia and derive indices of tissue insulin sensitivity (hyperinsulinaemic euglycaemic clamping HEC). (i) Plasma acetate levels (mmol/l) approximately doubled (0.14 +/- (SEM) 0.02 to 0.25 +/- 0.02, p less than 0.01) during INTRALIPID infusion in 7 non-diabetic individuals while total 'ketone bodies' and non-esterified fatty acids (NEFAs) increased 10-fold. (ii) Early in the HEC, plasma acetate levels decreased as did NEFAs in 13 non-diabetic (0.17 +/- 0.01 to 0.12 +/- 0.01, p less than 0.001) and 9 diabetic (0.22 +/- 0.02 to 0.15 +/- 0.01, p less than 0.005) individuals. However while acetate levels later rose to fasting values in the non-diabetics, they remained low in the diabetics. NEFA levels were low throughout the clamp but glucose flux was increased as judged from the glucose infusion even with maintained euglycaemia. The change in acetate values during the second hour of the clamp correlated with neither BMI nor two indices of insulin sensitivity (glucose metabolic clearance rate and steady state glucose infusion rate). These results accord with acetate production from glucose and fat oxidation, via acetyl CoA. The differing metabolism of acetate in the second hour of clamping between diabetics and non-diabetics may reflect altered post-receptor glucose metabolism with the onset of diabetes.

Acetates↗
PubMed1988

Detection of coronary artery disease from the normal resting ECG using nonlinear mathematical transformation.

In order to enhance subtle changes not apparent in resting ECGs by conventional criteria, a computerized signal processing technique, biopotential coordinate transformation (BCT), has been developed to increase the physician's perceptibility of CAD in seemingly "normal" resting ECGs. ECGs initially read as normal then can be tested with this process to predict the presence or absence of CAD. A blinded, retrospective study of 93 Caucasian patients with normal ECGs was performed using coronary angiography as the test standard. The BCT process identified the presence or absence of CAD with an 84.3% sensitivity and 81.8% specificity in 62 men. Similarly, a 76.2% sensitivity and 80% specificity was obtained for 31 women. These results suggest that the BCT process may provide the emergency physician with a noninvasive screening test for the detection of CAD in patients with apparently normal resting ECGs. Further studies are necessary for process refinement and verification.

Coronary Angiography↗
PubMed1988

Effects of CGS-13080, a thromboxane inhibitor, on pulmonary vascular resistance in patients after mitral valve replacement surgery.

Pulmonary hypertension with an elevated pulmonary vascular resistance was observed during the immediate recovery period in patients who underwent mitral valve surgery. In eight such patients, intravenous infusion of CGS-13080, imidazo(1,5-a)pyridine-5-hexanoic acid (a thromboxane synthetase inhibitor), at a dose range of 0.08-0.1 mg/kg/hr, effectively reduced pulmonary hypertension (from a mean pulmonary arterial pressure of 36 +/- 2 to 31 +/- 2 torr) and pulmonary vascular resistance (from 339 +/- 38 to 238 +/- 37 dynes.sec.cm-5) within 30 minutes and remained reduced for the entire infusion period (48 hours in five patients and 18 hours in three patients). Mean arterial pressure or systemic vascular resistance were not significantly affected by the drug infusion. Serum thromboxane B2 levels (a stable metabolic product of thromboxane A2) were significantly reduced after administration of the compound, with the maximum effect of greater than 90% reduction. All patients tolerated the drug infusion without significant side effects.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Nonlinear transformation of the resting electrocardiogram in the diagnosis of coronary artery disease.

Despite the common use of the standard 12-lead ECG, its reliability as an indicator of coronary artery disease (CAD) is poor. The normal ECG is falsely negative in more than 50% of angiographically proven CAD. The waveform of the standard ECG, however, can be transformed mathematically by nonlinear signal transformation to enhance its interpretation by computer. Using such a process it is possible that abnormalities can be identified in "normal" ECGs that can be correlated with CAD, thus identifying high-risk patients. A computer template that represents grouped data of normal ECGs for patients who also have normal coronary angiography was developed. Unblinded, preliminary testing of the template on normal ECGs of 107 white patients who had normal or abnormal coronary angiograms was performed. The process identified presence or absence of CAD with 82% specificity and 71% sensitivity for 53 women, and with 82% specificity and 86% sensitivity for 54 men. These preliminary results are promising, but further refinement of the templates is required and blinded studies with larger numbers and varieties of patients are needed.

Angiography↗
PubMed1984

Construct validity of heroin abuse estimators.

The construct validity of two methodologically unrelated procedures for estimating the prevalence of heroin abuse was studied using a covariance structure model-fitting approach. A factor analytic estimation procedure and a log-linear-based capture-recapture method of estimation were simultaneously applied to 24 major metropolitan areas of the nation. A test of the construct validity of these procedures for estimating the prevalence of heroin abuse was carried out in the context of structural equation models where the results of the two methods were compared directly.

Data Collection↗
PubMed1981

Alterations in rat central nervous system endorphins following transauricular electroacupuncture.

Auricular electro-stimulation (electroacupuncture) was found to produce naloxone-reversible analgesia in the rat. These behavioral effects were accompanied by significant increases in cerebrospinal fluid (CSF) levels of endorphins with concomitant decreases in the basomedial hypothalamus and medial thalamus of beta-endorphin-like immunoreactivity as well as endorphin-like radioreceptor activity. In addition, the radioreceptor assay also revealed a decrease in endorphin-like radioreceptor activity in the periaqueductal gray (PAG) matter. These results are interpreted to imply that electroacupuncture in the rat produces at least part of its analgesic action by activating central nervous system endorphinergic circuitry which results in a release and depletion of endorphins in certain brain loci and a concomitant elevation in the CSF. Hypophyseal endorphins do not appear to be involved in mediating acupuncture-induced analgesia in the rat since plasma levels of endorphins were not altered by this manipulation.

Acupuncture Therapy↗