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Biomedical subjects

D C Lee

Publications and source records attributed to D C Lee.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1998

Expression of transforming growth factor alpha and epidermal growth factor receptor in adult polycystic kidney disease.

Adult polycystic kidney disease (APKD) is a common genetic disease with a frequency of 1:1000. Evidence suggests that transforming growth factor alpha (TGF alpha) signaling may contribute to the hyperproliferation of the cystic epithelia in APKD. TGF alpha and epidermal growth factor (EGF) are well known mitogens expressed in the kidney and both exert their biological activities through binding to the same EGF receptor. A transgenic mouse that over-expressed TGF alpha developed renal cysts; raised levels of TGF alpha and EGF receptor mRNA were found in kidneys from two autosomal dominant APKD patients. To study the role of TGF alpha in cyst formation, we analyzed nine anatomically diagnosed adult polycystic kidneys and four normal kidneys using immunohistochemistry. We also traced the possible origins of the cysts by staining with the proximal convoluted tubule (PCT) marker, gp330, and the distal convoluted tubule (DCT) and collecting tubule (CT) marker, peanut agglutinin (PNA). In normal kidneys, TGF alpha protein was concentrated in the DCT and CT and EGF receptor protein in all three tubule types. In the early cysts of APKD, the cystic epithelia showed strong positive staining with TGF alpha, EGF receptor and gp330 but negative with PNA. Strong TGF alpha and EGF receptor staining was also found in the mixture of advanced cysts in the end-stage cystic kidneys although the cysts are likely to be derived from different segment of the renal tubules. This increased TGF alpha and EGF receptor expression in all cases and all types of cysts suggests that autocrine/paracrine stimulation by TGF alpha may be a common mechanism in cyst development in APKD.

Adult↗
PubMed1998

Hypothermia from realistic fluid resuscitation in a model of hemorrhagic shock.

STUDY OBJECTIVE: To correlate changes in core body temperature with changes in mean arterial pressure (MAP) and cardiac output (CO) and with the administration of room-temperature intravenous fluids in a clinically relevant large-animal model of uncontrolled hemorrhage. METHODS: Ten swine were subjected to uncontrolled hemorrhage through a flow-monitored shunt placed between the femoral artery and the peritoneal cavity. Animals were randomly assigned to a treatment or a control group. The control group (n=5) received no intravenous fluids. The treatment group (n=5) received 80 mL/kg (3:1 crystalloid/blood loss) ambient-temperature lactated Ringer's solution over a 10-minute resuscitation phase initiated 10 minutes after injury. CO and core body temperature, measured with the use of a pulmonary artery catheter, and MAP were the primary outcomes. We analyzed differences between groups with the use of repeated-measures ANOVA. Change of temperature was analyzed against the change in CO, and against fluid infusion for each interval, by means of regression analysis. RESULTS: The unresuscitated control animals had no change in core temperature despite profound hemorrhagic shock and hypotension. The animals treated with fluids had a mean 2.6 degrees C decrease in core temperature during fluid resuscitation (95% confidence interval [CI], 1.8 to 3.5). A 1.5 degrees C decrease in core temperature (95% CI, .1 to 2.0) persisted at the end of 60 minutes (40 minutes after fluid resuscitation was discontinued). Core temperatures in control animals were 2.8 degrees C lower than those in treated animals after fluid resuscitation (95% CI, .8 to 4.8). Decreases in core temperature correlated with fluid infusion (beta=-35.2 mL/kg x degrees C, R2=.75) and increases in CO (beta=-1.46 L/min x degrees C, R2=.69). CONCLUSION: Ambient-temperature crystalloid resuscitation in a clinically relevant large-animal model of hemorrhagic shock causes small decreases in core body temperature. Resuscitation rather than shock is the main cause of decreased body temperature in this model.

Analysis of Variance↗
PubMed1998

Intraoperative flexion against gravity as an indication of ultimate range of motion in individual cases after total knee arthroplasty.

To assess a method of predicting the final postoperative flexion in individual cases after total knee arthroplasty, 364 primary posterior cruciate-retaining total knee arthroplasties were reviewed retrospectively. The knees were subdivided into three preoperative flexion groups--I: poor motion (0 degrees to 85 degrees), II: intermediate motion (90 degrees to 110 degrees), and III: good motion (115 degrees to 140 degrees). There were 302 cases of osteoarthritis and 62 rheumatoid knees (12 juvenile rheumatoid). Correlation was made between preoperative; intraoperative, and postoperative (minimum 2-year follow-up) passive knee flexion for individuals. Intraoperative flexion against gravity was measured after capsular closure by passively flexing the patient's hip 90 degrees and allowing the weight of the lower leg to flex the knee joint. The overall mean value of postoperative flexion for all three groups was similar to preoperative and intraoperative flexion in both osteoarthritis and rheumatoid arthritis. In the poor motion group (I), postoperative flexion (103 degrees) was increased over preoperative flexion (84 degrees) but similar to intraoperative flexion (104 degrees). In the intermediate group (II), postoperative flexion (110 degrees) was similar to both the preoperative flexion (108 degrees) and intraoperative flexion (110 degrees). In the good group (III), postoperative flexion (119 degrees) tended to be less than preoperative flexion (123 degrees) and more than intraoperative flexion (116 degrees), but the differences were not statistically significant. When comparing preoperative and intraoperative flexion to postoperative flexion for individual cases, 55% of knees had postoperative flexion +/-10 degrees of their preoperative value, while 97% of knees had postoperative flexion +/-10 degrees of their intraoperative value. This study indicates that the final postoperative mean flexion for a group of patients with poor preoperative flexion (<85 degrees) and for individual cases (regardless of their preoperative mobility) can best be predicted by intraoperative flexion against gravity rather than by a preoperative value.

Arthritis, Juvenile↗
PubMed1998

Role of integrins in mouse eyelid development: studies in normal embryos and embryos in which there is a failure of eyelid fusion.

Eyelid fusion normally occurs between E15.5 and E16.5 of mouse embryonic development and results from the migration of a population of periderm-derived epithelial cells over the corneal surface. Cell migration is known to depend on extracellular matrix receptors of the integrin family and to be regulated by growth factors. We were therefore interested that a failure of eyelid fusion has been reported in mice that are homozygous null for the transforming growth factor alpha (TGF-alpha) gene and in mice (invalpha5beta1) in which a transgenic alpha5beta1 integrin under the control of the involucrin promoter is misexpressed in differentiating keratinocytes. We examined expression of the alpha2beta1, alpha3beta1, alpha5beta1 and alpha6beta4 integrins during eyelid fusion in wild-type embryos and found selective upregulation of the alpha5beta1 integrin and its ligand, fibronectin, in the migrating eyelid tip cells. In TGF-alpha null embryos, the failure of eyelid fusion was correlated with a failure to upregulate the alpha5beta1 integrin and fibronectin in the tip cells. Using beta-galactosidase as a reporter gene in transgenic mice, we observed specific activity of the involucrin promoter in the eyelid tip cells. In invalpha5beta1 mice the transgenic human integrin was overexpressed not only in the tip cells but throughout the eyelid epidermis. In contrast, the endogenous, murine, alpha5beta1 integrin was only weakly expressed in the tip cells. We speculate that selective and coordinated expression of the alpha5beta1 integrin and fibronectin in eyelid tip cells is required for eyelid fusion and may be under the control of growth factors that include TGF-alpha.

Animals↗
PubMed1998

The use of pamidronate for hypercalcemia secondary to acute vitamin D intoxication.

INTRODUCTION: Pamidronate disodium, a bone resorption inhibitor through osteoclast mediation, has been used to treat chronic hypercalcemia secondary to malignancy and chronic renal failure. We report the use of pamidronate for acute, severe hypercalcemia secondary to iatrogenic vitamin D poisoning. CASE REPORT: A 77-year-old female nursing home resident was inadvertently administered 50,000 units of oral vitamin D daily for 6 days. The patient presented with lethargy, abdominal pain, and vomiting. The patient's initial serum calcium concentration was 5.25 mmol/L (21 mg/dL). The patient was initially treated with hydration and furosemide but developed congestive heart failure. Pamidronate was used and calcium concentrations normalized by 24 hours after treatment. CONCLUSION: We report a case of the use of pamidronate for significant hypercalcemia secondary to acute vitamin D poisoning. Although evidence of congestive heart failure was evident, dialysis was avoided without significant sequelae. Pamidronate therapy should be considered in patients with hypercalcemia secondary to acute vitamin D poisoning.

Aged↗
PubMed1998

Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland.

The complex system of ERBB receptors and ligands is implicated in growth and differentiation of the mammary gland. However, it has not been comprehensively examined in this dynamic tissue. Combined RNA and protein analyses of glands in different stages from virgin to involution revealed differential expression of the four ERBB receptors, as well as distinctive patterns of ERBB ligand expression that suggested specialized function. ERBB localization was linked to mammary gland function. Thus, in the virgin gland, ERBB1 and ERBB2 were colocalized to all major cell types during ductal morphogenesis but differentially localized in the mature gland. All four ERBB receptors were restricted to epithelia in the differentiated gland. Analyses of ERBB tyrosine phosphorylation provided strong evidence of interaction between the four receptors in this physiological context. Thus, exogenous EGF induced stage-dependent transphosphorylation of ERBB2-4 as well as ERBB1, whereas endogenous phosphorylation of all four receptors peaked in late pregnancy and lactation.

Animals↗
PubMed1997

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene.

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.

Animals↗