Biomedical subjects

David C Lee

Publications and source records attributed to David C Lee.

At least 37 records · Page 2Linked to original sources

PubMed2004

Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.

The mechanisms that regulate the transition between the initial priming phase and DNA replication in liver regeneration are poorly understood. To study this transition, we compared events occurring after standard two-thirds partial hepatectomy, which elicits full regeneration, with response to a reduced hepatectomy, one-third partial hepatectomy (1/3PH), which leads to little DNA replication. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between the two procedures, cell cycle progression was significantly blunted in 1/3PH mice. Among the main defects observed in 1/3PH mice were an almost complete deficiency in retinoblastoma phosphorylation and the lack of increase in kinase activity associated with cyclin E. We report that, in two-thirds partial hepatectomy mice, the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) preceded the start of DNA replication and was not detectable in 1/3PH animals. Injection of HB-EGF into 1/3PH mice resulted in a >15-fold increase in DNA replication. Moreover, we show that hepatocyte DNA replication was delayed in HB-EGF knock-out mice. In summary, we show that HB-EGF is a key factor for hepatocyte progression through G(1)/S transition during liver regeneration.

Animals↗

PubMed2004

Cyclin-dependent kinase inhibitor indirubin-3'-oxime selectively inhibits human papillomavirus type 16 E7-induced numerical centrosome anomalies.

Dysregulation of the centrosome duplication cycle has been implicated in tumorigenesis. Our previous work has shown that the human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces aberrant centrosome and centriole duplication in normal human cells. We report here that HPV E7-induced abnormal centriole duplication is specifically abrogated by a small molecule CDK inhibitor, indirubin-3'-oxime (IO), but not a kinase-inactive derivative. Importantly, normal centriole duplication was not markedly affected by IO, and the inhibitory effects were observed at concentrations that did not affect the G1/S transition of the cell division cycle. Depletion of CDK2 by siRNA similarly abrogated HPV E7-induced abnormal centrosome duplication and ectopic expression of CDK2 in combination with cyclin E or cyclin A could rescue the inhibitory effect of IO. IO treatment also reduced the steady-state level of aneuploid cells in HPV-16 E7-expressing cell populations. Our results suggest that cyclin/CDK2 activity is critically involved in abnormal centrosome duplication induced by HPV-16 E7 oncoprotein expression, but may be dispensable for normal centrosome duplication and cell cycle progression.

Base Sequence↗

PubMed2004

Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation.

Parathyroid hormone (PTH) is the major mediator of calcium homeostasis and bone remodeling and is now known to be an effective drug for osteoporosis treatment. Yet the mechanisms responsible for its functions in bone are largely unknown. Here we report that the expression of amphiregulin (AR), a member of the epidermal growth factor (EGF) family, is rapidly and highly up-regulated by PTH in several osteoblastic cell lines and bone tissues. Other osteotropic hormones (1alpha,25-dihydroxyvitamin D3 and prostaglandin E2) also strongly stimulate AR expression. We found all EGF-like ligands and their receptors are expressed in osteoblasts, but AR is the only member that is highly regulated by PTH. Functional studies demonstrated that although AR is a potent growth factor for preosteoblasts, it completely inhibits further differentiation. AR also strongly and quickly stimulated Akt and ERK phosphorylation and c-fos and c-jun expression in an EGF receptor-dependent manner. Moreover, AR null mice displayed significantly less tibial trabecular bone than wild-type mice. Taken together, we have identified a novel growth factor that is PTH-regulated and appears to have an important role in bone metabolism.

Amphiregulin↗

PubMed2004

Transactivation of epidermal growth factor receptor mediates catecholamine-induced growth of vascular smooth muscle.

Stimulation of alpha1-adrenoceptors induces proliferation of vascular smooth muscle cells (SMCs) and contributes to arterial remodeling. Although activation of NAD(P)H oxidase and generation of reactive oxygen species (ROS) are required, little is known about this pathway. In this study, we examined the hypothesis that epidermal growth factor receptor (EGFR) transactivation and extracellular regulated kinases (ERK) are involved in alpha1-adrenoceptor-mediated SMC growth. Phenylephrine increased protein synthesis in association with a rapid (< or =5 minutes) and sustained (> or =60 minutes) doubling of phosphorylation of EGFR and ERK1/2, but not p38 or JNK in the media of rat aorta maintained in organ culture. Antagonists of EGFR phosphotyrosine activity (AG-1478) and ERK phosphorylation (PD-98059, U-0126) abolished phenylephrine-induced protein synthesis, whereas antagonists of p38 or JNK phosphorylation had no specific effect. A competitive antagonist (P22) for heparin binding EGF-like growth factor (HB-EGF) blocked phenylephrine-induced protein synthesis, as did downregulation of pro-HB-EGF (CRM197). Phenylephrine-induced protein synthesis was inhibited by neutralizing antibody to HB-EGF and absent in HB-EGF-/- SMCs. Inhibitors of metalloproteinases (BiPS, KB-R7785) also blocked adrenergic growth. The neutralizing antibody against HB-EGF had no effect on the two-fold increase in ROS generation induced by phenylephrine (DCF fluorescence), suggesting that stimulation of NAD(P)H oxidase by alpha1-adrenoceptor occupation precedes HB-EGF release. Cell culture studies confirmed and extended these findings. These data suggest that alpha1-adrenoceptor-mediated SMC growth requires ROS-dependent shedding of HB-EGF, transactivation of EGFR, and activation of the MEK1/2-dependent MAP kinase pathway. This trophic pathway may link sympathetic activity to arterial wall growth in adaptive remodeling and hypertrophic disease.

Adrenergic alpha-1 Receptor Agonists↗

PubMed2004

Mass antibiotic treatment and community protection in trachoma control programs.

Azithromycin is highly effective against trachoma, but the practical difficulties of community-wide distribution often leave many individuals untreated. We demonstrate, after mass azithromycin treatment of a population in Ethiopia, an indirect protective effect that occurred among untreated children who resided in villages in which most individuals had been treated. Similarities with the indirect protection within a treated community (i.e., "herd protection") that has been observed in vaccination programs are discussed.

Anti-Bacterial Agents↗

PubMed2004

Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency.

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.

ADAM Proteins↗

PubMed2004

Bilateral nursemaid's elbow.

Explore the source record for details and available documents.

Female↗

PubMed2004

The Meixner test in the detection of alpha-amanitin and false-positive reactions caused by psilocin and 5-substituted tryptamines.

STUDY OBJECTIVE: The Meixner test has been suggested to identify the presence of alpha-amanitin, one of the toxic compounds in Amanita mushrooms. We attempted to determine the detection limit of the Meixner test for alpha-amanitin and to determine the percentage of positive sample interpretation compared with other mushroom indole compounds. METHODS: This was a 2-part in vitro experiment. In part 1, we applied the Meixner test to a series of dilutions of alpha-amanitin (0 microg, 0.8 microg, 1.0 microg, 2.0 microg, and 4.0 microg) on telephone book paper, which were then presented to 5 blinded emergency physicians. We sought to determine the lowest amount of alpha-amanitin that was universally recognized as positive by the physicians (the detection limit). In the second part, 5 emergency physicians were presented simultaneously with 10 Meixner processed samples, including the mushroom indole compounds alpha-amanitin (2 microg and 10 microg), psilocin (20 microL and 60 microL of mushroom extract), 5-hydroxytryptamine (100 microg and 200 microg), and 5-methoxy-N,N-dimethyltryptamine (100 microg and 200 microg), as well as 2 negative controls (20 microL of water and methanol). We determined how likely these other indoles are to be mistaken for a positive alpha-amanitin Meixner reaction result by determining the percentage of positive sample interpretation for each compound and comparing them with the rate for alpha-amanitin. Fisher's exact test was used to determine any significant difference (P<.05) between the samples. RESULTS: The minimum amount of alpha-amanitin that was identified with 100% agreement by testers was 2 microg. For the second part, there was 100% agreement that psilocin gives a positive Meixner test (100% false positive) and a 35% recognition rate of the 5-substituted tryptamine compounds as a positive Meixner test. There was no statistical difference between the interpretation of alpha-amanitin and psilocin, suggesting the test is unable to differentiate between them. CONCLUSION: Although the Meixner test has a good detection limit for toxic amounts of alpha-amanitin, a positive Meixner reaction does not adequately distinguish between alpha-amanitin and other mushroom indoles.

Agaricales↗

PubMed2004

Reading and understanding employee benefit plan financial statements.

If your employee benefit plan has more than 100 participants, chances are you've had to work your way through the audited financial statements you're required to include with your Form 5500 filing. These statements contain a wealth of information about the financial health of your plan, and understanding them is an important fiduciary responsibility. To strengthen your grasp of financial statements, this article gives an overview that will make a plan's financial statements more informative, explains their basic structure and provides information on some of the more arcane aspects (such as actuarial tables). While this article focuses on Taft-Hartley (multiemployer) plans, much of it applies to other types of employee benefit plans.

Accounting↗

PubMed2003

Form and function of developing heart valves: coordination by extracellular matrix and growth factor signaling.

It is becoming clear that converging pathways coordinate early heart valve development and remodeling into functional valve leaflets. The integration of these pathways begins with macro and molecular interactions outside the cell in the extracellular matrix separating the myocardial and endocardial tissue components of the rudimentary heart. Such interactions regulate events at the cell surface through receptors, proteases, and other membrane molecules which in turn transduce signals into the cell. These signals trigger intracellular cascades that transduce cellular responses through both transcription factor and cofactor activation mediating gene induction or suppression. Chamber septation and valve formation occur from these coordinated molecular events within the endocardial cushions to sustain unidirectional blood flow and embryo viability. This review discusses the emerging connection between extracellular matrix and growth factor receptor signaling during endocardial cushion morphogenesis by highlighting the extracellular component, hyaluronan, and erbB receptor functions during early valve development.

Animals↗

PubMed2003

Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.

Heparin-binding epidermal growth factor (HB-EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB-EGF and BTC alleles by homologous recombination. Most HB-EGF(-/-) mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC(-/-) mice were viable and fertile and displayed no overt defects, the lifespan of double null HB-EGF(-/-)/BTC(-/-) mice was further reduced, apparently due to accelerated heart failure. HB-EGF(-/-) newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB-EGF transcripts were localized to endocardial cells lining the margins of wild-type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor-alpha converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE-derived soluble HB-EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context.

ADAM Proteins↗

PubMed2003

Beta-blocker use in the emergency department in patients with acute myocardial infarction undergoing primary angioplasty.

Our objectives were to evaluate the frequency of beta-blocker administration in the setting of acute myocardial infarction (AMI) where angioplasty is the primary treatment, and to investigate emergency physician's (EPs) attitudes toward beta-blockers. We performed a retrospective chart review of all patients who presented with symptoms and electrocardiogram (EKG) criteria consistent with AMI in the defined study period. Charts were reviewed for beta-blocker administration and other treatments. A survey was subsequently distributed to all EPs to determine self-reported reasons for withholding beta-blockers. There were 91 patients identified. Of those who did not have contraindications, 99% (89/90) received aspirin, 97% (88/91) received heparin, 94% (84/89) received nitrates, but only 28% (19/68) received beta-blockers. Ninety-six percent of beta-blocker-eligible patients received them as inpatients. Eighty-six percent (44/52) of EPs completed the survey. Physicians felt strongly about avoiding beta-blockers in patients with asthma exacerbation, severe congestive heart failure, and high degree AV block. Bradycardia was the most frequent reason for withholding beta-blockers. In this series of patients presenting with AMI, beta-blockers were greatly underutilized. The self-reported reasons of EPs for withholding beta-blocker therapy did not explain why 72% (49/68) of patients without contraindications did not receive beta-blockers.

Adrenergic beta-Antagonists↗

PubMed2003

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads.

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.

Amino Acid Sequence↗

PubMed2003

Substrate specificity and inducibility of TACE (tumour necrosis factor alpha-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity.

Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

ADAM Proteins↗

PubMed2003

Magnesium-aluminum hydroxide suspension for the treatment of dermal capsaicin exposures.

OBJECTIVES: To determine whether magnesium-aluminum-hydroxide-simethicone suspension (MgAl) is an effective treatment for dermal capsaicin exposures. METHODS: The authors performed a double-blind, randomized, controlled, pilot study comparing the effect of MgAl with that of saline in the treatment of dermal capsaicin exposures. Ten volunteers were sprayed with a commercial defensive spray containing 10% capsaicin on the flexor surface of both forearms. A dressing embedded with MgAl (Maalox) suspension was randomly applied to one arm and a saline-embedded dressing was applied to the other arm. Pain was assessed on a 10-cm visual analog scale at 0, 10, 20, 30, 60, 90, and 120 minutes. RESULTS: Mean pain scores were significantly lower in the MgAl group as compared with the saline (S) group at 10, 20, and 30 minutes. Differences in pain scores were not statistically significant at times 60, 90, and 120 minutes. CONCLUSIONS: During the initial 30 minutes of treatment, there was a statistically significant decrease in pain scores with MgAl as compared with saline treatments. Although the difference in means may have questionable clinical significance, MgAl is cheap and readily available, and has minimal side effects. Thus, MgAl may be an appropriate treatment for dermal capsaicin exposure.

Administration, Cutaneous↗

PubMed2003

TACE/ADAM17 processing of EGFR ligands indicates a role as a physiological convertase.

EGF family growth factors, including transforming growth factor-alpha (TGFalpha), amphiregulin (AR), and heparin-binding EGF (HB-EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble growth factor. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFalpha from its transmembrane precursor, proTGFalpha. Here, we review our findings that primary keratinocytes from Tace(deltaZn/deltaZn) mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFalpha compared to their normal counterparts, even though TGFalpha mRNA and cell-associated protein levels were similar in the two cell populations. Restoration of TACE activity in Tace(deltaZn/deltaZn) cells increased shedding of TGFalpha species, including the mature, 6-kDa protein. Further, exogenous TACE enzyme accurately cleaved the N-terminal processing site of proTGFalpha in cell lysates, as well as both physiologic sites of a soluble proTGFalpha ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB-EGF proteins from Tace(deltaZn/deltaZn) cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open-eye defect of Egfr(wa-2/wa-2) newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

ADAM Proteins↗

PubMed2002

Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identified the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

Antibody Specificity↗

PubMed2002

Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo.

A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.

Amino Acid Sequence↗