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Biomedical subjects

D C Lee

Publications and source records attributed to D C Lee.

At least 37 records · Page 2Linked to original sources
PubMed2000

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

Amino Acid Sequence↗
PubMed2000

Fatal nifedipine ingestions in children.

Nifedipine is a prototypical dihydropyridine calcium channel "blocker" that can cause hypotension and cardiac conduction abnormalities. When compared to other calcium channel antagonists, overdoses have been reported to be relatively benign with treatment consisting mainly of supportive care. We report two pediatric cases of death secondary to accidental ingestion of long acting nifedipine (Adalat). Both cases did not respond to aggressive supportive care that included calcium, atropine, epinephrine, glucagon, sodium bicarbonate, and transthoracic pacing.

Accidents, Home↗
PubMed2000

Reconstruction of pulp defect using a free finger-pulp flap from an excised extra digit.

We present a case of a post-traumatic finger-pulp reconstruction in which we used a free finger-pulp flap elevated from the excised extra digit in a patient with coincidental polydactyly of the thumb that had not been previously corrected. It resulted in satisfactory reconstruction of the defect with a well-padded, sensitive fingertip (static two-point discrimination of 3 mm) and excellent cosmetic appearance with the simultaneous correction of the polydactyly.

Adult↗
PubMed2000

Low plasma thiamine levels in elder patients admitted through the emergency department.

OBJECTIVES: To determine the prevalence of thiamine deficiency in a high-risk group of elder emergency department (ED) patients who reside in nursing homes and need admission to the hospital, and to determine the effect of patients' diets on this prevalence. METHODS: This was an observational pilot study of 75 consecutive ED patients aged 65 years or older who lived in a nursing home and were admitted to the hospital. Plasma thiamine levels were measured by high-pressure liquid chromatography on serum samples collected within 24 hours of hospital admission. Nursing home records were reviewed to determine whether patients received nutritional supplementation or enteral tube feedings. RESULTS: Seventy patients participated and had a mean plasma thiamine level of 27.3 microg/dL (95% CI = 20.2 to 34.4). Fourteen percent (n = 10, 95% CI = 8% to 24%) were thiamine-deficient (<10 microg/dL). Patients not receiving dietary supplements or tube feedings (n = 26) had lower mean thiamine levels (20.3 microg/dL, 95% CI = 12.7 to 27.9) and were thiamine-deficient more often (27%) than patients receiving dietary support (n = 44, 31.5 microg/dL, 95% CI = 24.7 to 38.3, 7% thiamine-deficient). CONCLUSIONS: Elder nursing home patients seen in the ED and admitted to the hospital are frequently thiamine-deficient. Empiric thiamine supplementation is often used in the ED for other high-risk patients, such as alcoholic individuals, and may be appropriate for high-risk elder patients. Further research is needed to determine whether thiamine supplementation in these patients can improve their clinical outcomes.

Age Distribution↗
PubMed1999

Kap104p-mediated nuclear import. Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna.

Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p. We demonstrate direct binding of Kap104p to each of these substrates. We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo. The nuclear localization signals map to similar arginine/glycine-rich RNA-binding domains in both proteins and are thus termed rg-nuclear localization signals to distinguish them from classical nuclear localization signals. We also demonstrate that Kap104p, like other known beta-karyopherins (or importins), interacts directly with the small GTPase Ran/Gsp1. However, unlike other known import factors, Ran binding is not sufficient to mediate release of substrates from Kap104p; efficient Ran-GTP-mediated substrate release requires RNA. Also, addition of Kap104p to Nab2p and Nab4p/Hrp1p prebound to single-stranded DNA-cellulose stimulated release of both proteins from the resin. We suggest a simple cycle in which Nab2p and Nab4p/Hrp1p, upon import, are released in the nucleus at sites of transcription by the concerted action of Ran-GTP and binding to newly synthesized mRNA. The resulting ribonucleoprotein complexes are exported to the cytoplasm, where Kap104p rebinds to Nab2p and Nab4p/Hrp1p, contributing to their release from mRNA.

Amino Acid Sequence↗
PubMed1999

Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding.

The extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) comprises four subdomains (I-IV) and mediates binding of several different polypeptide ligands, including EGF, transforming growth factor-alpha, and heparin-binding EGF. Previous studies have predominantly implicated subdomain III in ligand binding. To investigate a possible role for sequences in subdomain IV, we constructed several mutant EGFRs in which clusters of charged or aromatic amino acids were replaced with alanine. Analysis of stably transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of the wild-type receptor. Although tyrosine phosphorylation of most mutants was markedly induced by EGF, a cluster mutation (mt25) containing four alanine substitutions in the span of residues 521-527 failed to respond. EGF-induced tyrosine phosphorylation of an alternative mutant (DeltaEN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whereas the response to transforming growth factor-alpha was undetectable. These results suggest that mt25 might be defective with respect to either ligand binding or receptor dimerization. Quantitative analyses showed that binding of (125)I-EGF to mt25 and DeltaEN was reduced to near background levels, whereas binding of EGF to other cluster mutants was reduced 60-70% compared with wild-type levels. Among the mutants, only mt25 and DeltaEN failed to form homodimers or to transphosphorylate HER2/Neu in response to EGF treatment. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF family ligands. Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequently used for structure/function studies of the EGFR extracellular region.

Animals↗
PubMed1999

Modeling, simulation, and kinetic analysis of a heterogeneous reaction system for the enzymatic conversion of poorly soluble substrate.

We developed a kinetic model that describes a heterogeneous reaction system consisting of a solid substrate suspension for the production of D-amino acid using D-hydantoinase. As a biocatalyst, mass-produced free and whole cell enzymes were used. The heterogeneous reaction system involves dissolution of a solid substrate, enzymatic conversion of the dissolved D-form substrate, spontaneous racemization of an L-form substrate to D-form, and deactivation of the enzyme. In the case of using whole cell enzymes, transfer of the dissolved substrate and product through the cell membrane was considered. The kinetic parameters were determined from experiments, literature data, and by using Marquardt's method of nonlinear regression analysis. The model was simulated using the kinetic parameters and compared with experimental data, and a good agreement was observed between the experimental results and the simulation ones. Factors affecting the kinetics of the heterogeneous reaction system were analyzed on the basis of the kinetic model, and the efficiency of the reaction systems using free and whole cell enzymes was also compared.

Enzymes↗
PubMed1999

Dextromethorphan modulates the AP-1 DNA-binding activity induced by kainic acid.

We have recently reported that dextromethorphan attenuates the neurotoxicity induced by kainic acid in a dose-related fashion. Pretreatments with dextromethorphan (50 mg/kg, p.o. x2) significantly reduced the activator protein-1 DNA-binding activity and the Fos-related antigen-immunoreactive protein induced by kainic acid (10 mg/kg, i.p.) in the CA1, but not the CA3 or the dentate gyrus sector of the rat hippocampus. Paradoxically, dextromethorphan itself caused an elevated activator protein-1 DNA-binding activity and Fos-related antigen-immunoreactive protein in the CA1 region which lasted for at least 4 days. The results suggest that the CA1 area is the critical site for mediating the putative neuroprotective effect induced by dextromethorphan.

Animals↗
PubMed1999

Blood lead levels in emergency department patients with retained lead bullets and shrapnel.

OBJECTIVE: To determine whether patients with retained lead shrapnel who present to the ED have elevated whole blood lead levels (BLLs) compared with matched control patients. METHODS: Test subjects were patients with x-ray evidence of retained lead shrapnel who presented to any of three urban EDs, and consented to inclusion in the study. BLLs were obtained from 15 ED patients, and data regarding time since injury and symptoms of plumbism were collected. Control subjects consisted of 15 ED patients, without similar lead exposure, matched for age, sex, race, and place of dwelling as determined by zip code, who also presented to any of the same three EDs. RESULTS: The mean (+/-SD) BLL in the test subjects was 17+/-9.78 microg/dL (range 7-50 microg/dL). Time since impalement ranged from 1 to 45 years. The mean BLL in the control subjects was 7+/-3.77 microg/dL (0-16 microg/dL). This difference was statistically significant (two-tailed t-test p = 0.002). CONCLUSIONS: In this preliminary study, the patients with retained lead shrapnel who presented to the ED had significantly elevated BLLs, compared with the matched control subjects. Chronic plumbism may be considered in patients with retained lead shrapnel, and a history should be taken to assess the presence of symptoms referable to plumbism. These patients may require long-term follow-up to assess the development of elevated blood lead level and lead toxicity.

Adult↗
PubMed1999

Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development.

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.

Amphiregulin↗
PubMed1999

Purification and characterization of thermostable D-hydantoinase from Bacillus thermocatenulatus GH-2.

A thermostable D-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography. The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric. Isoelectric pH of the enzyme was found to be approx 4.3. The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate. The optimal pH and temperature for catalytic activity were about 7.5 and 65 degrees C, respectively. The half-life of the enzyme was estimated to be about 45 min at 80 degrees C.

Journal Article↗
PubMed1998

Optimization of a heterogeneous reaction system for the production of optically active D-amino acids using thermostable D-hydantoinase.

A thermostable D-hydantoinase from Bacillus stearothermophilus SD-1 was previously mass-produced by batch cultivation of the recombinant E. coli harboring the gene encoding the enzyme (Lee et al., 1997). In this work, we attempted to optimize the process for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is readily hydrolyzed to D-p-hydroxyphenylglycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using the mass-produced D-hydantoinase. In an effort to overcome the low solubility of the substrate, enzyme reaction was carried out in a heterogeneous system consisting of a high substrate concentration up to 300 g/L. In this reaction system, most of substrate is present in suspended particles. Optimal temperature and pH were determined to be 45 degrees C and 8.5, respectively, by taking into account the reaction rate and conversion yield. When the free enzyme was employed as a biocatalyst, enzyme loading higher than 300 unit/g-substrate was required to achieve maximum conversion. Use of whole cell enzyme resulted in maximum conversion even at lower enzyme loadings than the free enzyme, showing 96% conversion yield at 300 g/L substrate. The heterogeneous reaction system used in this work might be applied to the enzymatic production of other valuable compounds from a rarely water-soluble substrate.

Amidohydrolases↗
PubMed1998

An essential role for ectodomain shedding in mammalian development.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

ADAM Proteins↗
PubMed1998

Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1.

The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a 'pore' (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an alpha-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an alpha-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.

Amino Acid Sequence↗
PubMed1998

Sex difference in immunostaining of RET in the adult mouse kidney.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which is important for the development of the kidney and the enteric nervous system. During nephrogenesis, c-ret is expressed in the ureteric bud epithelium and later in its derivative, the collecting duct. This takes place during 11-17.5 days post-coitum (d.p.c.) in the mouse and our immunohistochemical study showed that the RET protein co-localized with the transcript. At 18.5 d.p.c. the kidney is fully differentiated. At 18.5 d.p.c., 1 week and 10 weeks old, RET was found in the proximal convoluted tubules, which is formed from the condensed mesenchyme. This suggests that c-ret may also play a role in kidney function. For the 10 weeks old kidney, RET immunostaining in male was concentrated on the basolateral side while female had a stronger staining in the whole cell. Furthermore, cytoplasmic staining was observed in male whereas both cytoplasmic and nuclear staining was found in female. c-ret transcript was detected by RT-PCR, and in situ hybridization showed its expression throughout the kidney. The reason for the sex-specific staining and the role of RET in kidney function remain to be determined.

Animals↗