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Biomedical subjects

H C Lee

Publications and source records attributed to H C Lee.

At least 91 records · Page 5Linked to original sources

The Green Revolution: botanical contributions to forensics and drug enforcement.

Forensic botany encompasses many sub-disciplines, including plant anatomy, plant ecology, plant systematics, plant molecular biology, palynology, and limnology. Although the field of forensic botany has been recognized since the mid-1900's, the use of trace plant material as physical evidence in criminal casework is still novel. A review of published forensic casework that used plant evidence is presented here. Cases include the analysis of wood evidence in the Charles Lindbergh baby kidnapping, the use of pollen in establishing the location of a sexual assault, and pollen analysis to determine the time of year for burial in a mass grave. Additional cases discuss the use of plant growth rates to determine the time of a body deposit in a field, the use of diatoms to link individuals to a crime scene, and plant DNA typing to match seedpods to a tree under which a body was discovered. New DNA methods in development for plant species identification and individualization for forensic applications are also discussed. These DNA methods may be useful for linking an individual to a crime scene or physical evidence to a geographic location, or tracking marijuana distribution patterns.

Botany↗

Digital enhancement of sub-quality bitemark photographs.

Digital enhancement software was used to enhance bitemark photographs. This enhancement technique improved the resolution of the bitemark images. Lucis was the software program utilized in this study and case applications. First, this technique was applied on known bitemark images to evaluate the potential effectiveness of this digital enhancement method. Subsequently, Lucis was utilized on two separate unsolved cases involving enhancement of bitemark evidence. One case involved a severely beaten infant with a bitemark on the upper thigh. The second case involves a bitemark observed on the breast of a female sexual assault strangulation victim. In both cases, bitemark images were significantly improved after digital enhancement.

Adult↗

Clostridium bacteremia: emphasis on the poor prognosis in cirrhotic patients.

Bacteremic episodes caused by anaerobes are unusual and the clinical importance of Clostridium bacteremia remains unclear. This retrospective case study examined the risk factors among a group of patients who developed Clostridium bacteremia. Medical records from 73 episodes of clostridial bacteremia in 73 patients treated in a medical center during an 11-year period were reviewed. Of all episodes, 96% were community-acquired. Twelve percent of patients had polymicrobial bacteremia, with Escherichia coli being the most common accompanying bacterium. Diabetes mellitus (26%) and liver cirrhosis (25%) were the most common underlying diseases. The most common etiological organisms were Clostridium perfringens (77%), Clostridium bifermentans (9%), and Clostridium septicum (4%). Only one patient with C. septicum bacteremia had a histocytotoxic infection, which was a fatal gas gangrene. Univariate analysis of data from patients with monomicrobial Clostridium bacteremia revealed that younger age (age < 65 years), underlying liver cirrhosis, and presence of septic shock at initial presentation were associated with fatality; but only the latter two variables were independently associated with fatality in multivariate logistic regression analysis. Appropriate antimicrobial therapy for monomicrobial Clostridium bacteremia did not significantly affect clinical outcomes, which might suggest that Clostridium species in the bloodstream can be regarded as merely contaminants or transient bacteremia. This suggestion was not supported by the finding that seven of 13 cirrhotic patients with monomicrobial Clostridium bacteremia died of sepsis, of whom six had not receive appropriate antimicrobial therapy. Therefore, the clinical importance of Clostridium bacteremia should be interpreted with caution because of its high risk of mortality in susceptible hosts, particularly cirrhotic patients, who do not receive appropriate therapy timely.

Adult↗

Mitochondrial theory of aging matures--roles of mtDNA mutation and oxidative stress in human aging.

Mitochondrial theory of aging, a variant of free radical theory of aging, proposes that accumulation of damage to mitochondria and mitochondrial DNA (mtDNA) leads to aging of humans and animals. It has been supported by the observation that mitochondrial function declines and mtDNA mutation increases in tissue cells in an age-dependent manner. Age-related impairment in the respiratory enzymes not only decreases ATP synthesis but also enhances production of reactive oxygen species (ROS) through increased electron leakage in the respiratory chain. Human mtDNA, which is not protected by histones and yet is exposed to high levels of ROS and free radicals in the matrix of mitochondria, is susceptible to oxidative damage and mutation in tissue cells. In the past decade, more than one hundred mtDNA mutations have been found in patients with mitochondrial disease, and some of them also occur in aging human tissues. The incidence and abundance of these mutant mtDNAs are increased with age, particularly in tissues with great demand for energy. On the other hand, recent studies have revealed that the ability of the human cell to cope with oxidative stress is compromised in aging. Comparative analysis of gene expression by microarray technology has shown that a number of genes related to oxidative stress response are altered in aging animals. We discovered that the transcripts of early growth response protein-1, growth arrest and DNA damage-inducible proteins and glutathione S-transferase genes are increased in response to oxidative stress in human skin fibroblasts. Moreover, the activities of Cu,Zn-SOD, catalase and glutathione peroxidase decrease with age, whereas Mn-SOD activity increases with age up to 65 years and slightly declines thereafter in skin fibroblasts. Such an imbalance in the function of antioxidant enzymes may result in excess production of damaging ROS in the cell. This notion is supported by the observation that intracellular levels of H2O2 and oxidative damage to DNA and lipids are significantly increased with age of the fibroblast donor. Furthermore, the mitochondrial pool of reduced glutathione declines and DNA damage is enhanced in aging tissues. Taken together, these observations and our previous findings that mtDNA mutations and oxidative damage are increased in aging human tissues suggest that mitochondrial theory of aging is mature.

Aging↗

Klebsiella pneumoniae psoas abscess: predominance in diabetic patients and grave prognosis in gas-forming cases.

Seven cases of psoas abscess caused by Klebsiella pneumoniae were observed at the National Cheng Kung University Hospital within a period of 4.5 years. These cases constituted 25% of a total 28 episodes of non-tuberculous psoas abscess, ranking second to those caused by Staphylococcus aureus (8 cases). Eight cases of psoas abscess caused by K. pneumoniae were identified from Medline, and 5 of which were reported from Taiwan. Of these 8 cases, 1 neonatal case was excluded, and the remaining 7 adult cases were combined with the 7 cases in this series for analysis. The mean age was 53.8 years, and diabetes mellitus was the most common underlying disease. Fever and pain on the flank and back area were the common findings. The interval between the onset of symptoms and diagnosis ranged from 1 to 60 days. The most common sites of concurrent infection were the urinary tract (6 cases; 43%) and bone (3 cases; 21%). All patients received percutaneous or surgical drainage in addition to antibiotic treatment. Gas formation was present in 5 of the 12 patients recorded, and 4 of them died during hospitalization. Only 1 patient had a metastatic infection with osteomyelitis of the left radius and right humerus; he had experienced 5 episodes of recurrent K. pneumoniae infections in different sites. We concluded that K. pneumoniae should be considered as an important endemic pathogen of psoas abscess in diabetics in Taiwan. The high mortality rate in the gas-forming cases should also be highlighted. Early recognition, empiric antimicrobial coverage for K. pneumoniae, and aggressive drainage or debridement are indicated in these patients.

Adolescent↗

Minimal change nephrotic syndrome associated with malignant thymoma: case report and literature review.

We report on a patient with nephrotic syndrome with myasthenia gravis and malignant thymoma status post thymectomy and radiotherapy. This 44-year-old woman underwent a thymectomy and localized radiotherapy for invasive thymoma in 1991. She also took azathioprine and pyridostigmine regularly for the control of her symptoms of myasthenia gravis following thymectomy. Neither evidence of recurrence of myasthenia gravis, nor enlargement of residual thymoma was noted following treatment in 1991. Unfortunately, this patient developed a pronounced nephrotic syndrome in 1999, a renal biopsy revealing a minimal-change glomerulonephritis as being present. The patient entered remission subsequent to steroid and cyclosporin therapy, whereas many previously-reported cases of a similar nature either died or remained with a persistent proteinuria. Herein, we review the literature pertaining to examples of nephrotic syndrome for those patients diagnosed with malignant thymoma, and discuss the possible mechanisms for this association, and emphasize the need for the aggressive treatment of the condition, recommending the early use of steroids and cyclosporins.

Adult↗

Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue.

A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of beta cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic beta cells by autoimmune responses specific to beta cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic beta cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

Animals↗

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni.

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.

Chromatography, Gel↗

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

ADP-ribosyl Cyclase↗

Heat shock-induced, caspase-3-independent rapid breakdown of Akt and consequent alteration of its total phosphorylation/activity level.

The immediate effect of a 15-min heat shock was examined on the level and the activity of Akt. Following heat shock, the Akt level decreased by 15-70% in a temperature-dependent and phosphorylation status-independent manner. This decrease of Akt level was not prevented by caspase inhibitors. At 48 degrees C, the extent of the breakdown was so immense that the total phosphorylation/activity level of Akt was not increased over the control level, implying that the total cellular activity of Akt governed by the level and the molar activity does not necessarily undergo the ensuing change.

Animals↗

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in hepatocyte nucleus.

CD38 is a type II transmembrane glycoprotein found on both hematopoietic and non-hematopoietic cells. It is known for its involvement in the metabolism of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. It is generally believed that CD38 is an integral protein with ectoenzymatic activities found mainly on the plasma membrane. Here we show that enzymatically active CD38 is present intracellularly on the nuclear envelope of rat hepatocytes. CD38 isolated from rat liver nuclei possessed both ADP-ribosyl cyclase and NADase activity. Immunofluorescence studies on rat liver cryosections and isolated nuclei localized CD38 to the nuclear envelope of hepatocytes. Subcellular localization via immunoelectron microscopy showed that CD38 is located on the inner nuclear envelope. The isolated nuclei sequestered calcium in an ATP-dependent manner. cADPR elicited a rapid calcium release from the loaded nuclei, which was independent of inositol trisphosphate and was inhibited by 8-amino-cADPR, a specific antagonist of cADPR, and ryanodine. However, nicotinic acid adenine dinucleotide phosphate failed to elicit any calcium release from the nuclear calcium stores. The nuclear localization of CD38 shown in this study suggests a novel role of CD38 in intracellular calcium signaling for non-hematopoietic cells.

ADP-ribosyl Cyclase↗

Identification of the enzymatic active site of CD38 by site-directed mutagenesis.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.

ADP-ribosyl Cyclase↗

Multiple calcium stores: separate but interacting.

Multiple mechanisms exist for increasing the concentration of intracellular calcium. This Perspective by Lee is one in a series on intracellular calcium release mechanisms and focuses on the calcium store operated by nicotinic acid adenine dinucleotide phosphate (NAADP). The characterization of the NAADP-operated calcium store as separate from the inositol trisphosphate (IP(3))-operated and cyclic ADP-ribose (cADPR)-operated calcium stores is discussed. Lee also addresses the role of NAADP in regulating intracellular calcium fluctuations during fertilization and hormonal activation of pancreatic acinar cells.

Adenosine Diphosphate Ribose↗

Functional analysis of heme regulatory elements of the transcriptional activator Hap1.

Heme regulation of the activity of diverse proteins was thought to be mediated by heme-responsive motifs (HRMs). The yeast transcriptional activator Hap1 contains seven HRMs: HRM1-7. Three copies of a 17-amino-acid repeat are also located in the region encompassing HRM1 to -6. We examined the effects of these HRMs and repeats on heme regulation of Hap1 activity by deletion analysis and by Ala substitutions of key residues. We found that the effect of mutation or deletion of one HRM or 17-amino-acid repeat on Hap1 heme responsiveness is different from the effect of mutation or deletion of another HRM or repeat. Our data suggest that HRM7 plays a dominant role in mediating heme activation of Hap1 in heme-sufficient cells while HRM1-6 may scavenge heme and cause a low level of Hap1 activation in heme-deficient cells. These results may help in understanding the roles of HRMs in other hemoproteins.

Amino Acid Motifs↗

Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells.

Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H(2)O(2) at concentrations of 90-360 microM. After the treatment for 24-72 h, we found that cells were arrested at G(0) and G(1) phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H(2)O(2) treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.

Cell Cycle↗

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy.

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.

Binding Sites↗

Origin of the native driving force for protein folding.

We derive an expression with four adjustable parameters that reproduces well the 20x20 Miyazawa-Jernigan potential matrix extracted from known protein structures. The numerical values of the parameters can be approximately computed from the surface tension of water, water-screened dipole interactions between residues and water and among residues, and average exposures of residues in folded proteins.

Electrochemistry↗

Mean-field HP model, designability and alpha-helices in protein structures.

Analysis of the geometric properties of a mean-field HP model on a square lattice for protein structure shows that structures with a large number of switchbacks between surface and core sites are chosen favorably by peptides as unique ground states. Global comparison of model (binary) peptide sequences with concatenated (binary) protein sequences listed in the Protein Data Bank and the Dali Domain Dictionary indicates that the highest correlation occurs between model peptides choosing the favored structures and those portions of protein sequences containing alpha helices.

Computer Simulation↗