Biomedical subjects
Publications and source records attributed to M Lee.
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The coupling between conduction charges and the vibrational modes of the molecular lattice plays a defining role in the transport characteristics of organic semiconductors. Using electron tunneling spectroscopy, we obtain the electron--optical-phonon coupling spectrum in photodoped pentacene crystals at energies <30 meV. Comparison of the tunneling spectrum to infrared absorption data on the optical phonon density of states yields the energy dependence of the electron-phonon scattering matrix element. The integrated spectral weight of the electron-phonon coupling shows that superconductivity in pentacene is likely of electron-phonon origin.
In this study, we identified AT-rich element located at positions -504 to -516 in the rat p53 promoter by DNase I foot printing assay. This region was previously identified as a positive regulatory element in the murine p53 promoter and designated as PBF1 (p53 binding factor 1) binding site. However, the proteins binding to this AT-rich element have not been identified yet. Therefore, we characterized the binding protein by various biochemical methods. First, we confirmed that by the oligonucleotide competition assay, nuclear factors bound to the AT-rich element in a sequence-specific manner. Two binding proteins were identified in southwestern blotting analysis and the molecular masses of the proteins were 60 and 40 kDa, respectively. The proteins were stable to denaturants or ionic strength. Treatment of chelators showed that the binding proteins did not require divalent cation for DNA-binding activity. In addition, the binding proteins were labile to protease treatment. This study showed that 60 and 40 kDa proteins bound to AT-rich element and the physico-chemical properties provided new insights into the binding proteins.
Progesterone has been used as an ingredient of anticancer drug for patients with ovarian carcinoma. However, the mechanism of anticancer effects by progesterone has not been understood. In this study, the effects of progesterone on ovarian cancer cells, SNU-840, were investigated. After the incubation with progesterone, the viability of the cells was evaluated by MTT assay. As a result, 45% of the cells were viable after 48 h of incubation with 100 microM progesterone. In addition, [(3)H]thymidine incorporation assay showed that the proliferation of the cells was completely inhibited by progesterone after 48 h of incubation at 100 microM concentration. Colorimetric TUNEL assay revealed the fragmentation of the chromosomal DNA, suggesting that the process of the cell death was apoptosis. The level of the p53 mRNA was determined by northern blotting assay, since many apoptosis processes are mediated by up-regulation of the p53 expression. The level of the p53 mRNA reached its maximum at 12 h and decreased after 24 h of incubation with progesterone. In conclusion, progesterone inhibits the proliferation and elicits apoptosis of SNU-840 cells. Also, it up-regulates the p53 mRNA transiently.
To assess the extent to which the results reported by Warner and Bradley (1991) can be generalized beyond the population of undergraduate psychology students, 132 adults in the metropolitan Harrisburg area were asked to evaluate the competency and traits of clinical psychologists, licensed professional counselors, and psychiatrists. Results indicated that the general public believed counselors to be more caring than psychologists and psychiatrists. All three professional groups were perceived to be comparable in their ability to treat the least severe disorders. Licensed counselors were perceived to be most competent to treat disorders rated by the subjects as moderately severe, comparable in competence to clinical psychologists for adjustment disorders, and comparable to psychiatrists for marital problems. Psychiatrists and psychologists were considered the most competent to treat the most severe disorder, major depression. The results indicate that while clinical psychologists often were viewed as competent, they were not viewed as the single practitioner of choice for any diagnostic classification, nor were they noted to have positive character traits.
Inversions are structural chromosome abnormalities that may be associated with infertility, multiple miscarriage and chromosomally unbalanced offspring. Preimplantation genetic diagnosis (PGD) with subtelomeric probes was used to select for transfer only those embryos that were normal or balanced for three pericentric inversions. In contrast to previous protocols the present procedure allows the detection of unbalanced embryos that might arise from U-recombination in the inverted region. Additionally, aneuploidy screening was carried out in two cases by a second round of fluorescent in situ hybridization (FISH) with centromeric probes. Of the three couples that underwent the procedure one became pregnant twice. The first pregnancy delivered a healthy and chromosomally normal baby and the second pregnancy is ongoing with triplets.
We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.
Prognosis and treatment of solid tumors are directly dependent on the stage of disease. For any type of cancer, tumor characteristics such as size, multiplicity, and metastatic potential are highly heterogeneous among patients. Our understanding of the genetic determinants of tumor burden is rudimentary. Here, rats carrying a germline mutation of the gene Tsc2 were found to develop variable size and number of renal tumors. We hypothesize that "modifier" genes unlinked to Tsc2 affect its expressivity. Using a backcross (BC) analysis between the two strains that showed the greatest difference in tumor size (Fischer344 and Brown Norway), we mapped a quantitative trait locus based on tumor volume to rat chromosome 3q, lying in the interval between D3Mit3 and D3Rat17, with a maximum lod score of 4.4. This locus, Mot1 (modifier of Tsc2 1), accounts for approximately 35% of the genetic variation in tumor size between the two strains. No significant difference in tumor multiplicity was noted between Brown Norway and Fischer344 rats. This suggests that Mot1 modulates the rate of disease progression and not tumor initiation. Candidate genes on rat chromosome 3 included Tsc1, whose product interacts biochemically with the TSC2 protein, but it was excluded on the basis of linkage analysis (LOD=0.01). Comparative genomics suggest that the Mot1 region is represented by human chromosomes 15q and 20pq. Our results provide the first evidence of a modifier gene affecting the Tsc2 pathway in the progression of renal tumorigenesis.
Our objective was to design a water-soluble lipopolymer (WSLP) and an interleukin-12 (IL-12) expression plasmid for enhanced delivery of the IL-12 gene. We synthesized WSLP using branched polyethylenimine (PEI) of 1800 Da and cholesteryl chloroformate, and constructed p2CMVmIL-12, encoding the IL-12 subunits p35 and p40, each under the transcriptional control of a separate cytomegalovirus (CMV) promoter. The percentage of cholesterol conjugated to PEI was about 47% and the average molecular weight of WSLP was approximately 2000 Da. The mean particle size of WSLP/p2CMVmIL-12 complexes formulated in 5% glucose was 26 to 62 nm and xi potential was 8 to 60 mV. The WSLP/p2CMVmIL-12 complexes were nontoxic to CT-26 colon carcinoma cells at the N/P ratio (nitrogen atoms of WSLP/phosphate of plasmid DNA) of 20 and below; PEI25000/pDNA complexes were highly toxic. WSLP/p2CMVmIL-12 complexes demonstrated higher transfection in CT-26 cells compared with the DNA formulations prepared using PEI of molecular weights 1800, 10,000 and 25,000 Da. Transfection efficiency increased with an increase in N/P ratios from 5 to 15, then there was no significant increase in transfection up to the N/P ratio of 30/1. There was an increase in the level of IL-12 when free or complexed p2CMVmIL-12 was compared with free or complexed pIRESmIL-12 in which the p35 and p40 subunits were linked to the internal ribosome entry sites (IRES). At 48 hours post-injection of WSLP/p2CMVmIL-12 complexes into BALB/c mice bearing CT-26 subcutaneous tumors, the levels of IL-12, IFN-gamma, and nitric oxide (NO) in the supernatant of the cultured tumors were higher for the WSLP/p2CMVmIL-12 complexes than for the naked p2CMVmIL-12, WSLP, and 5% glucose injected groups. There was a significant improvement in the survival rate and the inhibition of tumor growth after a single injection of WSLP/p2CMVmIL-12 complexes. We have designed an effective, nontoxic WSLP and an IL-12 expression plasmid with two CMV promoters.
It was previously reported that silencing of the expression of glutamic acid decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet beta-cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can prevent autoimmune destruction of pancreatic beta-cells. To repress GAD expression in islet beta-cells, we delivered an antisense GAD mRNA expression plasmid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g-PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid: PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in beta-cells and expression was dependent on glucose level. The repression of GAD after transfection of pRIP-AS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression.
Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet beta-cell destruction belong to the Th1 subset (producing IL-2 and IFN-gamma), whereas regulatory T cells are Th2 type (producing IL-4 and IL-10). Here, we evaluate the effect of combined delivery of plasmid DNA encoding IL-4 and IL-10 using a degradable, cationic polymeric carrier, poly[gamma-(4-aminobutyl)-L-glycolic acid] (PAGA), in nonobese diabetic (NOD) mice. In the liver of NOD mice, we detected mouse Il4 and Il10 mRNA 5 days after intravenous injection of both PAGA-Il4 and PAGA-Il10 plasmid complexes. We found that 6 weeks after injection, 75% of observed islets were intact compared with less than 3% in the control group. Furthermore, in the treatment group, only 5% of the islets were severely infiltrated by the lymphocytes compared with over 30% in the control group. We measured glucose levels weekly up to the age of 32 weeks, revealing that co-injection of PAGA-Il4 and PAGA-Il10 plasmids prevented the development of diabetes in 75% of the treated animals. Thus, combined administration of mouse Il4 and Il10 plasmids prevents the development of autoimmune diabetes in NOD mice.
The Crepis palaestina cDNA Cpal2 encodes a delta 12-epoxygenase that can catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of the napin seed-specific promoter in Arabidopsis thaliana (L.) Heynh., the seed lipids accumulated only low levels of 18:1E and also 12,13-epoxy-cis-9,15-octadec-2-enoic acid (18:2E). Despite the fact that the levels of these epoxy fatty acids comprised only up to 6.2% of the total fatty acids, there was a very marked increase in oleic acid (18:1) and decrease in linoleic (18:2) and alpha-linolenic (18:3) acids in these plants, indicating that endogenous delta 12-desaturation was greatly reduced in these plants. Significant between-line differences in the levels of Cpal2 mRNA were observed during seed development, but were not associated with any major variation in mRNA levels for the endogenous Arabidopsis delta 12-desaturase (Fad2). This suggests that if an unfavourable interaction occurs between the transgenic delta 12-epoxygenase and the endogenous delta 12-desaturase, which decreases the level of desaturation, it occurs at either the translational or post-translational level. We further show that the co-expression of a delta 12-desaturase gene from C. palaestina in Cpal2 transgenic Arabidopsis returns the relative proportions of the C18 seed fatty acids to normal levels and results in an almost twofold increase in total epoxy fatty acids.
Little research has been carried out on human performance in optimization problems, such as the Traveling Salesman problem (TSP). Studies by Polivanova (1974, Voprosy Psikhologii, 4, 41-51) and by MacGregor and Ormerod (1996, Perception & Psychophysics, 58, 527-539) suggest that: (1) the complexity of solutions to visually presented TSPs depends on the number of points on the convex hull; and (2) the perception of optimal structure is an innate tendency of the visual system, not subject to individual differences. Results are reported from two experiments. In the first, measures of the total length and completion speed of pathways, and a measure of path uncertainty were compared with optimal solutions produced by an elastic net algorithm and by several heuristic methods. Performance was also compared under instructions to draw the shortest or the most attractive pathway. In the second, various measures of performance were compared with scores on Raven's advanced progressive matrices (APM). The number of points on the convex hull did not determine the relative optimality of solutions, although both this factor and the total number of points influenced solution speed and path uncertainty. Subjects' solutions showed appreciable individual differences, which had a strong correlation with APM scores. The relation between perceptual organization and the process of solving visually presented TSPs is briefly discussed, as is the potential of optimization for providing a conceptual framework for the study of intelligence.
Degranulated mast cells are present in the human arterial intima. After degranulation of rat serosal mast cells, the secreted neutral serine protease chymase remains bound to the heparin proteoglycan matrix of the exocytosed granules, forming granule remnants. Addition of granule remnants to human aortic intimal fluid results in proteolysis of the apoAI present in the intimal fluid, which contains physiological inhibitors of chymase. To study the physiological mechanism of this protection of granule remnant-bound chymase against its inhibitors, we performed experiments using HDL3 as substrate. Chymase, when bound to the heparin proteoglycans of granule remnants, but not when released from them, resisted inhibition by the mammalian protease inhibitors alpha1-antitrypsin, alpha2-antichymotrypsin, alpha2-macroglobulin, and eglin C. Importantly, the heparin proteoglycan-bound chymase, but not unbound chymase, degraded its inhibitor (alpha1-antitrypsin) in the presence of its substrate (HDL3). Finally, binding to heparin proteoglycans of a physiological inhibitor of chymase (mucus protease inhibitor (MPI)) or of another substrate of chymase (LDL) did not inhibit the degradation of HDL3 by granule remnant-bound chymase. This study demonstrates that binding of chymase to the heparin proteoglycan chains of the exocytosed mast cell granules allows the protease to remain active and degrade HDL3 in the presence of its physiological inhibitors and in the presence of high concentrations of LDL, such as are found in the interstitial fluid of the arterial intima.
Hyperlipoproteinemia phenotypes (HLP), one of genetic disorders with an estimated prevalence of 0.5-2% in the general population, is responsible for 10% of premature CHD. After first screening with the high cholesterol (>6.47 mM/l) and triglyceride (TG) (>2.6 mM/l) levels without medication, subjects were typed for HLP classification. Differential metabolic effects of HLP types on plasma lipid profiles and the reverse cholesterol transport system (RCT) were studied in 196 HLP types (91.2%) and 19 non-HLP (8.8%). A total of 45% of subjects had primary HLP and the others had NIDDM (10.7%), hypertension (9.3%) and other chronic diseases. Type IV HLP (58.6%) was most predominant and Types IIa, IIb, III and V comprised 16.7, 12.1, 2.3 and 1.4% of the HLP. Type I was not found. Plasma lipids excluding apo A-I and Lp(a) were significantly different among HLP compared to non-HLP (P<0.001). Since Type V and III impact the clearance of TG-riched lipoproteins, TG and VLDL-C levels were higher in V and III. TG and LDL-C were higher in Type II than those in the others because of defect of LDL receptors. LCAT activity, lower in Type III and Type IV and highest in Type V, was highly associated with plasma free cholesterol levels and the ratio of apoB/apoA and LDL/HDL. CETP activity was highest in Type V due to high VLDL-C and TG and low HDL-C. The ratio of LCAT/CETP was not different among HLP types but was significantly lower in HLP than in non-HLP. CETP increased 2-3 times as well as LCAT decreased among HLP patients compared to non-HLP. We conclude non-HLP subjects with high cholesterol and TG levels do not always mean high risk of CHD and the intervention effects of HLP types would lead to impose the risk of CHD by the impact of RCT.
BACKGROUND: Trinucleotide repetition combined with variable penetrance of expression could be responsible for the complex transmission pattern observed in bipolar affective disorder (BPAD). The purpose of this study was to investigate the association of excess longer allele of KCNN3 and CTG18.1 in the patients with BPAD. METHODS: CAG/CTG repeat distribution in KCNN3, CTG 18.1 and ERDA1 was examined and the copy number of ligation product in repeat expansion detection (RED) was measured in Korean bipolar patients in comparison to ethnically matched healthy controls. RESULTS: No significant difference was found in the allele distribution of those repeats between bipolar patients and controls. Ligation product size in RED was not increased in bipolar patients. However, the copy number of ligation product in RED was highly correlated with CAG/CTG copies of ERDA1 (P=0.0001), partly with CTG 18.1 (P=0.04), but not with KCNN3. CONCLUSIONS: A longer CAG repeat alleles of KCNN3 or CTG 18.1 may not be a risk factor for BPAD in Korean population and the copy number of ligation product in RED in the patients with BPAD is influenced by the longer allele of CAG/CTG of ERDA1 or CTG 18.1.
The effect of endogenous dopamine (DA) on measurement of neostriatal DA D(2) receptor binding potential (D(2)RBP) in vivo was evaluated with positron emission tomography (PET) and the radiotracer [11C]raclopride by comparing the D(2)RBP before and after acute DA depletion. DA depletion was achieved by per-oral administration of 4.5 g alpha-methyl-para-tyrosine (AMPT) given in 25 h. Six healthy subjects completed the protocol. The AMPT treatment increased D(2)RBP significantly from 3.11 +/- 0.25 to 3.68 +/- 0.23 and decreased plasma levels of the DA metabolite homovanillic acid by 71 +/- 11% and levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenethyleneglycol by 53 +/- 7%. Increase in D(2)RBP correlated with decrease in attentiveness and with increase in errors of commission from Conners' Continuous Performance Test. On AMPT, a significant decrease in subjective happiness scores was observed. The results imply that a noninvasive [11C]raclopride PET protocol coupled with relatively brief administration of a rather low total dose of AMPT resulted in measurable acute DA depletion that might provide estimates of synaptic neostriatal DA concentration.
BACKGROUND: An intervention to improve the testing and treatment of Helicobacter pylori (HP) in patients receiving chronic acid suppression (AS) therapy was developed at Harvard Pilgrim Health Care (HPHC), a mixed-model not-for-profit health maintenance organization. METHODS: Ten full-time primary care physicians (4 staff model and 6 group practice) were interviewed in 1999 about their knowledge, attitudes, and practice regarding dyspepsia, the use of chronic AS drugs, and approaches to HP infection, as well as about the feasibility and acceptability of various potential interventions that might be used in a quality improvement program. RESULTS: Self-reported practice regarding dyspepsia and HP infection were relatively uniform, and physicians were generally aware of current recommendations. Three common misperceptions acted as barriers to optimal HP management: Untreated HP was not considered an important problem; patients who used drugs for chronic AS rarely had HP infection; and chronic use of AS drugs was considered effective and without adverse consequence. All physicians wanted brief educational materials with explicit guidelines, preferably locally adapted and endorsed by local experts. All informants agreed that the main barrier to successful QI interventions was the requirement for any extra time or effort, particularly when directed at populations of patients who do not have symptomatic complaints. DISCUSSION: The interviews revealed the many barriers to improving the management of HP infection and to targeting educational messages and tailoring different methods for facilitating practice change across different managed care settings. Evidence-based components of the intervention program include physician education, a notification/reminder system, and practice-based tools to facilitate change and minimize workload.