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H C Lee

Publications and source records attributed to H C Lee.

At least 163 records · Page 9Linked to original sources
PubMed1998

The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries.

Human recombinant CD38 catalyzes the formation of both cyclic ADP-ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The kcat for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the beta-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre-steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond-breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+.

ADP-ribosyl Cyclase↗
PubMed1998

Fluorescent analogs of NAADP with calcium mobilizing activity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca2+ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N6-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N6-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N6-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3-4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca2+ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 microM and 5 microM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca2+ release with a half-maximal concentration of 60-80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca2+ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca2+ stores that are sensitive to NAADP in live cells.

ADP-ribosyl Cyclase↗
PubMed1998

Calcium signaling by cyclic ADP-ribose, NAADP, and inositol trisphosphate are involved in distinct functions in ascidian oocytes.

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, beta-NAD, into unfertilized oocytes. The changes induced by beta-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte.

ADP-ribosyl Cyclase↗
PubMed1998

Effect of alpha2-adrenoceptor stimulation on isolated canine Purkinje fiber contraction.

We have recently identified the presence of postjunctional alpha2-adrenoceptors in canine Purkinje fibers. In this study, we examined the effects of alpha2-adrenoceptor stimulation on the contraction strength of isolated Purkinje fibers. Exposure to the alpha2-adrenoceptor specific agonist and antagonist, UK 14,304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) and yohimbine (17-hydroxyyohimban-16-carboxylic acid methyl ester hydrochloride) alone at 0.1 microM respectively, did not produce any significant effect on Purkinje contraction strength. Purkinje contraction strength was augmented by isoproterenol (0.1 microM), forskolin (0.1 microM), or 8-bromo-adenosine cyclic 2',3'-monophosphate (8-bromo-cAMP, 10 microM). UK 14,304 significantly reversed the effects of isoproterenol and forskolin but not those of 8-bromo-cAMP on Purkinje contraction strength. After incubation with pertussis toxin, the positive inotropic effect of forskolin on Purkinje contraction strength remained intact, but the forskolin effect could no longer be reversed by UK 14,304. These results suggest that the postjunctional alpha2-adrenoceptors in canine Purkinje fibers are coupled to a pertussis toxin-sensitive G protein, probably Gi. Stimulation of the alpha2-adrenoceptor antagonizes the effect of beta-adrenoceptor stimulation on Purkinje contraction strength in an accentuated antagonism manner.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 microM cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

ADP-ribosyl Cyclase↗
PubMed1998

Independent occurrence of somatic mutations in mitochondrial DNA of human skin from subjects of various ages.

The incidence (frequency of occurrence) and abundance (percentage of mutant out of total mtDNA population) of two different somatic mtDNA mutations in human skin were investigated in 44 subjects ranging from 19 to 87 years of age. Using quantitative allele-specific polymerase chain reaction (AS-PCR) to analyse the A-->G base substitution at nucleotide 3243, 50% of the samples showed detectable levels of that particular mutation, with abundances ranging from 0.01% to 0.12%. In the same set of skin samples, the overall incidence of the 4977 bp "common" deletion was also approximately 50%. Where detected, the abundance of this deletion ranged from 0.0002% to 0.1%. Comparative analyses of the incidence and abundance of these two mutations, collectively and in individual skin samples, led to these two conclusions: (1) there is independent occurrence of these two mtDNA mutations in human skin, and (2) whereas the 4977 bp deletion shows an age-associated accumulation in human skin, no age association is apparent for the 3243 A-->G base substitution. Furthermore, in general, there is a much lower incidence of somatic mutations in mtDNA of human skin as compared to that in postmitotic tissues such as skeletal muscle.

Adult↗
PubMed1998

Functional expression of secreted mouse BST-1 in yeast.

BST-1, a bone marrow stromal cell surface antigen, is a glycosyl phosphotidylinositol-anchored protein that stimulates pre-B-cell growth and has adenosine diphosphate (ADP)-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase activity. The two enzymatic activities are responsible for the synthesis and hydrolysis of cADPR, a novel second messenger of calcium release from intracellular calcium stores. The expression and characterization of human BST-1 in certain mammalian cell lines have been reported. We have expressed the murine BST-1 in yeast as a 6 x His-tagged secreted protein. The recombinant protein has been purified and subjected to structural and functional characterization. It has an apparent molecular mass of 38.5 kDa on SDS-PAGE gel stained with Coomassie blue and is recognized on Western blots by a rabbit polyclonal antibody against BST-1. Deglycosylation of the protein with N-glycanase produces a ladder of bands with molecular sizes ranging from 32 to 39 kDa. The protein possesses the ADP-ribosyl cyclase activity as measured using nicotinamide guanine dinucleotide as substrate.

ADP-ribosyl Cyclase↗
PubMed1998

Calcium signaling by cyclic ADP-ribose and NAADP. A decade of exploration.

Ca2+ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca2+ messenger, inositol trisphosphate (IP3). This article focuses on two new Ca2+ release activators, which mobilize internal Ca2+ stores via mechanisms totally independent of IP3. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.

Adenosine Diphosphate Ribose↗
PubMed1998

Thio-NADP is not an antagonist of NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca2+ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995] J. Biol. Chem. 270, 3216-3223). Its effects in sea-urchin egg homogenates were investigated. At 50 microM, thio-NADP activates partial Ca2+ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca2+ releasing activity of 50 microM thio-NADP and reduces the subsequent inhibition by 73.7 +/- 1.3%. The residual inhibitory effect is no more than that exerted by 50 microM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by 32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific 32P-NAADP binding to egg microsomes with an IC50 of 40 microM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 microM, similar to the extent of inhibition effected by 40 microM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca2+ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca2+ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.

Adenosine Diphosphate Ribose↗
PubMed1998

Prenatal and neonatal intussusception.

Intussusception found in the 1st month of life is rare and usually discussed as one entity, neonatal intussusception, but in fact, includes the intussusceptions occurring both prenatally and neonatally, of which the clinical presentations and results are different. Four full-term babies with prenatal intussusception presenting as intestinal atresia (IA) and three premature babies with neonatal intussusception mimicking necrotizing enterocolitis (NEC) are presented. Prenatal intussusception, as one of the causes of IA produces prominent signs of intestinal obstruction immediately after birth. Preoperative evaluation usually fails to yield a definitive diagnosis, but surgery is usually performed in time and is successful. In neonatal intussusception, full-term infants usually have a pathological lead point and the colon is almost always involved. A barium enema is thus useful in diagnosis. Premature babies, on the other hand, rarely have a colonic component, and the clinical features are insidious and similar to NEC. This results in diagnostic confusion that may lead to a dangerous delay in appropriate surgical correction. A high level of suspicion about this condition in cases diagnosed presumptively with NEC is important. Serial abdominal sonograms may be helpful in the early diagnosis of neonatal intussusception.

Fatal Outcome↗
PubMed1998

Nitric oxide as an autocrine regulator of sodium currents in baroreceptor neurons.

Arterial baroreceptors are mechanosensitive nerve endings in the aortic arch and carotid sinus that play a critical role in acute regulation of arterial blood pressure. A previous study has shown that nitric oxide (NO) or NO-related species suppress action potential discharge of baroreceptors. In the present study, we investigated the effects of NO on Na+ currents of isolated baroreceptor neurons in culture. Exogenous NO donors inhibited both tetrodotoxin (TTX) -sensitive and -insensitive Na+ currents. The inhibition was not mediated by cGMP but by NO interaction with channel thiols. Acute inhibition of NO synthase increased the Na+ currents. NO scavengers (hemoglobin and ferrous diethyldithiocarbamate) increased Na+ currents before but not after inhibition of NO synthase. Furthermore, NO production in the neuronal cultures was detected by chemiluminescence and immunoreactivity to the neuronal isoform of NO synthase was identified in fluorescently identified baroreceptor neurons. These results indicate that NO/NO-related species function as autocrine regulators of Na+ currents in baroreceptor neurons. Modulation of Na+ channels may represent a novel response to NO.

Animals↗
PubMed1998

Forensic applications of DNA typing: part 2: collection and preservation of DNA evidence.

The initial stages of physical evidence examination are pivotal to the successful resolution of criminal investigations. Recent cases clearly reinforce the notion that methods of evidence collection and preservation will continue to be rigorously scrutinized and challenged in court. This article reviews forensic applications of DNA typing, focusing on the collection and preservation of biological evidence. Topics addressed include physical evidence collection at the crime scene, the forensic laboratory, and the autopsy room. Specific concerns pertaining to different sources of DNA evidence are discussed, as are special collection methods associated with various substrates on which the evidence is deposited.

Body Fluids↗
PubMed1998

Electrophysiologic effects of civamide (zucapsaicin) on canine cardiac tissue in vivo and in vitro.

The cardiac electrophysiologic effects of civamide (zucapsaicin), the cis-isomer of the alkyl vanillylamide, capsaicin, were evaluated in intact dogs and isolated Purkinje fibers. In anesthetized dogs, the mechanism of ventricular tachycardia inducible from 1 to 3 h after coronary artery occlusion was determined by activation mapping. Of 16 dogs studied, nine had ventricular tachycardia of focal endocardial origin; four, a reentrant mechanism; and three had no inducible arrhythmia. Civamide (50 microg/kg) was administered to 10 of 13 dogs that were inducible, but three dogs were used as time controls. Transmural activation times were unaltered by civamide, but mean arterial pressure decreased from 76 +/- 10 to 66 +/- 10 mm Hg (p < 0.05), and muscle refractory periods shortened from 138 +/- 3 to 132 +/- 4 ms (p < 0.05). Civamide altered inducibility in five of six dogs with ventricular tachycardia of focal endocardial origin, but those with epicardial reentrant mechanisms were not affected in three of four dogs. With microelectrode techniques in vitro, civamide (10(-5) M) shortened the action-potential duration at 50% repolarization (APD50) from 193 +/- 13 to 177 +/- 12 ms (p < 0.01) and APD90 from 260 +/- 15 to 248 +/- 13 ms (p < 0.01) in isolated Purkinje fibers (n = 10). Nifedipine prevented the effects of civamide in vitro. These results show that civamide may alter inducibility of ventricular tachycardia with focal endocardial origin and shorten APD of Purkinje fibers in vitro. The effects of civamide in vitro are prevented by preexposure of the Purkinje fibers to nifedipine, suggesting that the electrophysiologic effects of civamide may be mediated through blockade of calcium channels.

Action Potentials↗
PubMed1998

Sepsis during total parenteral nutrition: exploration of risk factors and determination of the effectiveness of peripherally inserted central venous catheters.

BACKGROUND: Sepsis is the most frequent serious complication during total parenteral nutrition (TPN), resulting in increased morbidity, mortality and health care costs. Existing reports have not documented the risk factors of sepsis during TPN. The objectives of this study were to determine the rate of sepsis in our practice and to explore the risk factors for sepsis during TPN. We also determined the role and efficacy of using peripherally inserted central venous catheters (PCVC) as insertion catheters to administer TPN. METHODS: From October, 1994, to May, 1996, we administered TPN to 378 pediatric patients hospitalized at Mackay Memorial Hospital. We followed all cases for the occurrences of any complications while administering TPN. We studied all patients who had fever, a clinical presentation of sepsis and a positive blood culture during their course of TPN. RESULTS: During the 20-month period 378 patients received TPN for a total of 6562 days. Fifty-six patients presented with clinical sepsis and positive blood cultures. Significant features in the sepsis group included longer duration of TPN, age < 3 months, usage of central venous catheters, gastrointestinal diseases as indication for TPN, low birth weight and short gestational age in prematurity. Seven patients died despite prompt antimicrobial therapy. One hundred eleven patients received TPN via PCVC for a mean duration of 17.1 days, significantly longer than 10.4 days in the peripheral intravenous catheter group but no difference between the sepsis rates. CONCLUSION: Considering the high incidence of sepsis during TPN, every attempt should be made to minimize the length of TPN therapy and encourage early enteral feeding. We also recommend the use of PCVC in patients requiring prolonged nutritional support.

Catheterization, Central Venous↗
PubMed1998

Analysis-synthesis of the phonocardiogram based on the matching pursuit method.

The matching pursuit method of Mallat and Zhang is applied to the analysis and synthesis of phonocardiograms (PCG's). The method is based on a classical Gabor wavelet or time-frequency atom which is the product of a sinusoid and a Gaussian window function. It decomposes a signal into a series of time-frequency atoms by an iterative process based on selecting the largest inner product of the signal (and the subsequent residues) with atoms from a redundant dictionary. The Gaussian window controls the envelope duration and time position of each atom; and the sinusoid represents the frequency. The method was applied to two sets of PCG's: one with very low-noise level and the other with 10% noise energy. Each data base includes 11 PCG's representing the normal and the pathological conditions of the heart. The normalized root-mean-square error (NRMSE) was computed between the original and the reconstructed signals. The results show that the matching pursuit method is very suitable to the transient and complex properties of the PCG's, as it yielded excellent NRMSE's around 2.2% for the two sets of 11 PCG's tested.

Heart Murmurs↗
PubMed1998

Time-frequency scaling transformation of the phonocardiogram based of the matching pursuit method.

A time-frequency scaling transformation based on the matching pursuit (MP) method is developed for the phonocardiogram (PCG). The MP method decomposes a signal into a series of time-frequency atoms by using an iterative process. The modification of the time scale of the PCG can be performed without perceptible change in its spectral characteristics. It is also possible to modify the frequency scale without changing the temporal properties. The technique has been tested on 11 PCG's containing heart sounds and different murmurs. A scaling/inverse-scaling procedure was used for quantitative evaluation of the scaling performance. Both the spectrogram and a MP-based Wigner distribution were used for visual comparison in the time-frequency domain. The results showed that the technique is suitable and effective for the time-frequency scale transformation of both the transient property of the heart sounds and the more complex random property of the murmurs. It is also shown that the effectiveness of the method is strongly related to the optimization of the parameters used for the decomposition of the signals.

Fourier Analysis↗