Biomedical subjects

X Wu

Publications and source records attributed to X Wu.

At least 73 records · Page 4Linked to original sources

PubMed2003

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser.

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Bacterial Proteins↗

PubMed2003

Bioavailability and pharmacokinetics of florfenicol in broiler chickens.

The bioavailability and pharmacokinetic disposition of florfenicol in broiler chickens were investigated after intravenous (i.v.), intramuscular (i.m.) and oral administrations of 15 and 30 mg/kg body weight (b.w.). Plasma concentrations of florfenicol were determined by a high performance liquid chromatographic method in which plasma samples were spiked with chloramphenicol as internal standard. Plasma concentration-time data after i.v. administration were best described by a two-compartment open model. The elimination half-lives were 168 +/- 43 and 181 +/- 71 min, total body clearance 1.02 +/- 0.17 and 1.02 +/- 0.16 L x kg/h, the volume of distribution at steady-state 4.99 +/- 1.11 and 3.50 +/- 1.01 L/kg after i.v. injections of 15 and 30 mg/kg b.w., respectively. Plasma concentration-time data after i.m. and oral administrations were adequately described by a one-compartment model. The i.m. bioavailability and the oral bioavailability of florfenicol were 95, 98 and 96, 94%, respectively, indicating that florfenicol was almost absorbed completely after i.m. and oral administrations of 15 and 30 mg/kg b.w.

Administration, Oral↗

PubMed2003

Quantitative EEG effects of topiramate.

OBJECTIVE: The study is to invesigate the effect of topiramate (TPM) on EEG by means of quantitative pharmacoelectroencephalography (QPEEG). METHODS: One dose of TPM was administrated to epileptics and healthy adults. The EEG samples were obtained prior to and at regular intervals within the 24 hours following the administration of TPM. The EEG activity was processed with power spectral analysis. RESULTS: The power of slow wave, alpha 1 bands and total power increased after the administration of TPM, the power or slow wave in both occipital areas, and the total power of all scalp areas also increased. The percent of power increased at the theta band and alpha 1 band (healthy adults) or delta band, theta band (patients). CONCLUSION: TPM can change the EEG background activity. These changes are different from other antiepileptic drugs.

Adult↗

PubMed2003

Frequent overexpression of cyclin D1 in sporadic pancreatic endocrine tumours.

Pancreatic endocrine tumours (PETs) occur sporadically or are inherited as part of the multiple endocrine neoplasia type-1 syndrome. Little is known about the molecular events leading to these tumours. Cyclin D1, a key regulator of the G1/S transition of the cell cycle, is overexpressed in a variety of human cancers as well as certain endocrine tumours. We hypothesized that similar to other endocrine tumours, cyclin D1 is overexpressed in human sporadic PETs. Cyclin D1 protein overexpression was found in 20 of 31 PETs (65%) when compared with normal pancreatic tIssue. Furthermore, Northern blot analysis suggests that cyclin D1 up-regulation occurs at the post-transcriptional level in some PETs. Because the key cell growth signalling pathways p42/p44/ERK (extracellular signal-regulated kinase), p38/MAPK (mitogen-activated protein kinase), and Akt/PKB (protein kinase B) can regulate cyclin D1 protein expression in other cell types, pancreatic endocrine tumours were analysed with phospho-specific antibodies against the active forms of these proteins to elucidate a tIssue-specific regulatory mechanism of cyclin D1 in PETs. We found frequent activation of the p38/MAPK and Akt pathways, but down-regulation of the ERK pathway, in cyclin D1 overexpressing PETs. This study demonstrates that cyclin D1 overexpression is associated with human sporadic PET tumorigenesis, and suggests that this up-regulation may occur at the post-transcriptional level. These findings will direct future studies of PETs towards cell cycle dysregulation and the identification of key growth factor pathways involved in the formation of these tumours.

Adenoma, Islet Cell↗

PubMed2002

A framework for integrating the songbird brain.

Biological systems by default involve complex components with complex relationships. To decipher how biological systems work, we assume that one needs to integrate information over multiple levels of complexity. The songbird vocal communication system is ideal for such integration due to many years of ethological investigation and a discreet dedicated brain network. Here we announce the beginnings of a songbird brain integrative project that involves high-throughput, molecular, anatomical, electrophysiological and behavioral levels of analysis. We first formed a rationale for inclusion of specific biological levels of analysis, then developed high-throughput molecular technologies on songbird brains, developed technologies for combined analysis of electrophysiological activity and gene regulation in awake behaving animals, and developed bioinformatic tools that predict causal interactions within and between biological levels of organization. This integrative brain project is fitting for the interdisciplinary approaches taken in the current songbird issue of the Journal of Comparative Physiology A and is expected to be conducive to deciphering how brains generate and perceive complex behaviors.

Animals↗

PubMed2002

Dynamic and static interactions between bitumen droplets in water.

In the commercial bitumen extraction operation, dynamic and static interaction forces between bitumen drops in water determine the likelihood of desirable bitumen coalescence at different process stages. These dynamic and static forces were measured using colloidal particle scattering and hydrodynamic force balance techniques, respectively. In the former technique, dynamic interactions are studied through droplet-droplet collision trajectory measurement. In the latter technique, the static attractive forces between droplets are determined when a doublet is separated with a known and adjustable hydrodynamic force. The dynamic force measurement implies the presence of rigid chains on bitumen surfaces. The mean chain lengths for deasphalted bitumen at pH 7, whole bitumen at pH 7, and whole bitumen at pH 8.5 are 50, 78, and 41 nm, respectively. However, the static force measurement indicates much shorter mean chain lengths (<9 nm) in these three bitumen systems. Shorter chain length indicates weaker repulsive force. This finding of a much weaker repulsion between bitumen droplets under static conditions has important implications on the commercial bitumen extraction operation.

Journal Article↗

PubMed2002

A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.

Animals↗

PubMed2002

[Comparison between line immunoassay (LIA) and enzyme-linked immunosorbent assay (ELISA) for the determination of antibodies to extractable nuclear antigenes (ENA) with reference to other laboratory results and clinical features].

INTRODUCTION: The line immunoassay (LIA) for the determination of antibodies to individual extractable antinuclear antigens (ENA) is a development of the enzyme-linked immunosorbent assay (ELISA) in which the antigens to be tested are adsorbed onto a nylon test strip. In addition, the antigen subspecificities B/B' and D in the case of Sm, the 70 kD, A and C components in the case of U1-snRNP and the Ro52 and Ro60 components in the case of SSA/Ro are present separately on the strips. The aim of the study was to determine whether the line immunoassay is suitable for routine laboratory use by means of a comparison with the ELISA. METHODS: Sera from 92 patients stored in our serum bank with known ENA profile as determined by ELISA and with at least one antibody to ENA were tested again with LIA. The clinical features and other available laboratory data taken from the patient records were used to classify the disease according to the relevant diagnostic criteria. In discrepant cases, antibodies to ENA were also determined by Western blot. These data were used to determine which of the two methods gave the more plausible results and to calculate the sensitivity and specificity for each autoantibody. RESULTS: There was good correlation between the two methods, especially for anti-CENP-B (centromere protein B) and anti-Jo1 (histidyl-tRNA transferase) antibodies. For anti-Sm, there was a trend toward higher specificity with the LIA in patients with systemic lupus erythematosus (SLE) if antibodies to Sm D were detected. The LIA was significantly more specific for the detection of antibodies to ribonucleoprotein (RNP) in mixed connective tissue disease and, if antibodies to the 70 kD component were present, also in SLE, although the sensitivity was significantly lower in this case. For anti-SSA/Ro, the specificity of the LIA was significantly higher than ELISA if anti-Ro60 was detected. In the case of anti-SS-B/La, anti-Scl70 (topoisomerase I), anti-CENP-B (centromere protein B) und anti-Jo-1 (histidyl-tRNA transferase), the sensitivity and specificity of the two methods were not significantly different. The LIA was significantly more specific but less sensitive for the detection of anti histone antibodies. CONCLUSION: The suitability of the LIA for routine laboratory determinations was confirmed.

Antibodies, Antinuclear↗

PubMed2002

Caspase cleavage of the nuclear autoantigen LEDGF/p75 abrogates its pro-survival function: implications for autoimmunity in atopic disorders.

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear autoantigen in atopic disorders implicated in cellular protection against stress-induced apoptosis. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD generated by caspases-3 and -7 cleaving at three sites: DEVPD30/G, DAQD486/G and WEID85/N. Sequence analysis revealed that the DEVPD30/G and WEID85/N sites lie within the highly conserved HATH (homologous to amino terminus of hepatoma-derived growth factor) region, also known as PWWP domain. Alignment of proteins containing this domain failed to reveal conservation of the DEVPD30/G and WEID85/N sites, suggesting that the HATH/PWWP domain of LEDGF/p75 may be specifically targeted by caspases. Overexpression of LEDGF/p75 protected HepG2 cells from serum starvation-induced cell death, whereas expression of the 65 kD fragment failed to protect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abrogating its pro-survival function and enhancing its immunogenicity.

Apoptosis↗

PubMed2001

Tolerance to nickel: oral nickel administration induces a high frequency of anergic T cells with persistent suppressor activity.

We adapted our mouse model of allergic contact hypersensitivity to nickel for the study of tolerance. Sensitization in this model is achieved by the administration of nickel ions with H(2)O(2); nickel ions alone are unable to prime naive T cells, but can restimulate primed ones. A 4-wk course of oral or i.p. administration of 10 mM NiCl(2) to naive mice induced tolerance, preventing the induction of hypersensitivity for at least 20 wk; long term desensitization of nickel-sensitized mice, however, required continuous NiCl(2) administration. When splenic T cells of orally tolerized donors, even after a treatment-free interval of 20 wk, were transferred to naive recipients, as with lymph node cells (LNC), they specifically prevented sensitization of the recipients. The LNC of such donors were anergic, because upon in vivo sensitization with NiCl(2) in H(2)O(2) and in vitro restimulation with NiCl(2), they failed to show the enhanced proliferation and IL-2 production as seen with LNC of mice not tolerized before sensitization. As few as 10(2) bulk T cells, consisting of both CD4(+) and CD8(+) cells, were able to specifically transfer tolerance to nickel. A hypothesis is provided to account for this extraordinarily high frequency of nickel-reactive, suppressive T cells; it takes into account that nickel ions fail to act as classical haptens, but form versatile, unstable metal-protein and metal-peptide complexes. Furthermore, a powerful amplification mechanism, such as infectious tolerance, must operate which allows but a few donor T cells to tolerize the recipient.

Adjuvants, Immunologic↗

PubMed2001

Linkage studies between attention-deficit hyperactivity disorder and the monoamine oxidase genes.

Attention-deficit hyperactivity disorder (ADHD) is a prevalent behavioral disorder in children and the etiology of this disorder is not clear. Molecular genetic and pharmacological studies suggest the involvement of dopaminergic and noradrenergic neurotransmitter systems in ADHD, e.g., several reports have found association between ADHD and the dopamine receptor gene DRD-4, the dopamine transporter gene DAT1, and the catecholamine clearance enzyme catechol-O-methyltransferase. Monoamine oxidase (MAO) A and B genes encode enzymes that participate in the metabolism of neurotransmitters of the dopaminergic and noradrenergic systems. MAO inhibitors have been shown to be effective in the treatment of ADHD. Our previous studies showed an association between ADHD and the DXS7 locus, which is located in close vicinity to the MAO genes on chromosome X. These findings suggest that there might be linkage between ADHD and MAO genes. To test this hypothesis, we used the transmission/disequilibrium test (TDT) to test for linkage between a VNTR polymorphism at the MAOA(CA)(n) or MAOB(GT)(n) locus and DSM-III-R-diagnosed ADHD in 82 nuclear families of the Chinese population. The TDT analysis revealed linkage between ADHD and the MAOA(CA)(n) locus (chi-square = 15.25, df = 7, P < 0.05), but not the MAOB(GT)(n) locus (chi-square = 11.18, df = 7, P > 0.05). The data showed that ADHD was in linkage with the MAOA gene and suggested that MAOA might be a susceptibility factor for ADHD.

Alleles↗

PubMed2001

Discovery of small-molecule inhibitors of Bcl-2 through structure-based computer screening.

Bcl-2 belongs to a growing family of proteins which regulates programmed cell death (apoptosis). Overexpression of Bcl-2 has been observed in 70% of breast cancer, 30-60% of prostate cancer, 80% of B-cell lymphomas, 90% of colorectal adenocarcinomas, and many other forms of cancer. Thereby, Bcl-2 is an attractive new anti-cancer target. Herein, we describe the discovery of novel classes of small-molecule inhibitors targeted at the BH3 binding pocket in Bcl-2. The three-dimensional (3D) structure of Bcl-2 has been modeled on the basis of a high-resolution NMR solution structure of Bcl-X(L), which shares a high sequence homology with Bcl-2. A structure-based computer screening approach has been employed to search the National Cancer Institute 3D database of 206 876 organic compounds to identify potential Bcl-2 small-molecule inhibitors that bind to the BH3 binding site of Bcl-2. These potential Bcl-2 small-molecule inhibitors were first tested in an in vitro binding assay for their potency in inhibition of the binding of a Bak BH3 peptide to Bcl-2. Thirty-five potential inhibitors were tested in this binding assay, and seven of them were found to have a binding affinity (IC(50) value) from 1.6 to 14.0 microM. The anti-proliferative activity of these seven active compounds has been tested using a human myeloid leukemia cell line, HL-60, which expresses the highest level of Bcl-2 protein among all the cancer cell lines examined. Compound 6 was the most potent compound and had an IC(50) value of 4 microM in inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compounds had moderate activity in inhibition of cell growth. Compound 6 was further evaluated for its ability to induce apoptosis in cancer cells. It was found that 6 induces apoptosis in cancer cells with high Bcl-2 expression and its potency correlates with the Bcl-2 expression level in cancer cells. Furthermore, using NMR methods, we conclusively demonstrated that 6 binds to the BH3 binding site in Bcl-X(L). Our results showed that small-molecule inhibitors of Bcl-2 such as 6 modulate the biological function of Bcl-2, and induce apoptosis in cancer cells with high Bcl-2 expression, while they have little effect on cancer cells with low or undetectable levels of Bcl-2 expression. Therefore, compound 6 can be used as a valuable pharmacological tool to elucidate the function of Bcl-2 and also serves as a novel lead compound for further design and optimization. Our results suggest that the structure-based computer screening strategy employed in the study is effective for identifying novel, structurally diverse, nonpeptide small-molecule inhibitors that target the BH3 binding site of Bcl-2.

Amino Acid Sequence↗

PubMed2001

The allatostatin gene of the cricket Gryllus bimaculatus (Ensifera, Gryllidae).

The gene encoding allatostatins (AST) of the FGLamide family from the cricket Gryllus bimaculatus is expressed in the brain. The mRNA, which contains four polyadenylation signals, encodes a hormone precursor that is split into at least 14 putative hormones. Five of them have been previously found in the cricket, six to seven others, or their close homologues, are known from other insects. Hormone AST 2 contains an internal cleavage site and may exist in a shorter version 2b. The hormones AST 3 and 4 are identical. The cDNA sequence revealed that a single point mutation and a single deletion eliminated an additional hormone between AST 12 and 13. The deduced hormone precursor is very similar to that in cockroaches, but is different from a shorter precursor in locusts, indicating that the gene evolved very fast in the latter. Regions conserved between cockroaches and crickets include parts of the acidic spacers that separate clusters of hormones, suggesting that these spacers may have additional functions.

Animals↗

PubMed2001

Photoreaction of platinum(II) beta-diketonate complexes with olefins.

Pt(hfac)(2) [hfac = 1,1,1,5,5,5-hexafluoro-2,4-pentanedionato] reacts with ethylene when irradiated at 350 nm to yield a single photoproduct which we have isolated for the first time. X-ray diffraction reveals this to be Pt(hfac)(2)(eta(2)-C(2)H(4)), a five coordinated complex, with two bidentate hfac ligands remaining intact. When Pt(hfac)(2) and ethylene were irradiated at 300 nm, a second product that could not be isolated was detected by (1)H NMR and (19)F NMR. Pt(hfac)(2)(eta(2)-C(2)H(4)) was also shown not to be stable at shorter wavelengths. The corresponding photoproducts of Pt(hfac)(2) with propene and of Pt(tfac)(2) [tfac = 1,1,1-trifluoro-2,4-pentanedionato] with ethylene were detected by (1)H NMR spectroscopy.

Journal Article↗

PubMed2001

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Gamma-radiation-induced G2 delay, apoptosis, and p53 response as potential susceptibility markers for lung cancer.

Gamma-radiation results in cell cycle arrest and apoptosis in a wide variety of cells. Cell cycle arrest provides time for the cell to repair damaged DNA before entering the next phase of the cycle. If the damage is severe and cannot be repaired, the cells undergo apoptosis. However, if the damaged cells continue to grow without repair or apoptosis, then carcinogenic transformation may occur. We hypothesized that individuals with inherited disruption in cell cycle control and/or apoptosis and/or DNA repair may be susceptible to lung cancer development. The cells from susceptible individuals would have a shorter G2 period and less apoptosis compared with cells from normal individuals upon exposure to gamma-radiation. To test this hypothesis, the following methods were used: (a) fluorescence-activated cell sorting method was used to measure apoptosis and G2 cell cycle delay; (b) the ELISA method was used to measure p53 protein expression levels in these cell lines; and (c) gamma-radiation-induced chromatid breaks were counted as a marker for DNA damage or repair. Next, gamma-radiation-induced G2 delay and apoptosis were tested in three lymphoblastoid cell lines to determine the dose response effect and optimal time points of gamma-radiation. Finally, these assays were tested in lymphoblastoid cell lines from 30 lung cancer patients and 22 healthy controls. We found a dose-response relationship for gamma-radiation-induced G2 delay and apoptosis. The optimal time points to detect differential G2 delay and apoptotic index were 10 h and 48 h after gamma-radiation, respectively. The mean G2 delay was 22.5% +/- 10.5% for the control cell lines and 14.71% +/- 8.8% for case cell lines (P < 0.01). The mean apoptotic index was 20.4% +/- 11.7% for the controls and 14.3% +/- 7.8% for the cases (P < 0.05). The controls had a significantly higher p53 response ratio and fewer chromatid breaks than the cases. We also found that a p53 increasing ratio was strongly related to cell cycle G2 delay (gamma = 0.413; P = 0.002) and chromatid breaks (gamma =0.384; P = 0.028). Therefore, we concluded that gamma-radiation-induced G2 delay, apoptosis, p53 increasing ratio, and chromatid breaks might potentially be used as susceptibility markers for lung cancer risk. A large epidemiology study is in progress to confirm these findings.

Adult↗

PubMed2001

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances lung cancer susceptibility.

Extracellular matrix-degrading matrix metalloproteinase-1 (MMP-1) is one of the interstitial collagenases likely to be involved in tumor invasion and metastasis. MMP-1 may also contribute to tumor initiation and development by altering the cellular microenvironment that facilitates tumor formation. Recent studies have found that overexpression of MMP-1 is associated with the initial stages of cancer development in addition to promoting cellular invasion; however, preexisting oncogenic mutations or chemical carcinogens are required to initiate tumorigenesis as well. There is a single nucleotide polymorphism located in the promoter region of MMP-1 that partially regulates gene expression. The 2G/2G genotype enhances transcriptional activity and may be associated with an increased lung cancer risk. Using a case-control study, we tested the hypotheses that (a) individuals with the 2G/2G genotype may be at an increased risk for lung cancer; and (b) the risk should be greatly elevated in smoking individuals. PCR-RFLP was used to determine the MMP-1 genotypes in 456 lung-cancer cases and 451 frequency-matched controls of Caucasian ethnicity. Overall, there was a significant association between the 2G/2G genotype and lung cancer risk [odds ratio (OR), 1.76; 95% confidence interval (CI), 1.29-2.39]. In current smokers, the lung cancer risk associated with the 2G/2G genotype was significantly elevated (OR, 3.16; 95% CI, 1.87-5.35). However, this association was less evident in former smokers (OR, 1.23; 95% CI, 0.81-1.87) and absent in never smokers (OR, 1.09; 95% CI, 0.31-3.91). Similarly, this risk was more evident in heavy smokers (OR, 2.55; 95% CI, 1.61-4.03) than in light smokers (OR, 1.40; 95% CI, 0.84-2.32). Interestingly, men were observed to have a 2.15-fold increased lung cancer risk (OR, 2.15; 95% CI, 1.42-3.26) compared with women (OR, 1.34; 95% CI, 0.84-2.15). Furthermore, subjects with 2G/2G genotype developed lung cancer earlier (60.94 +/- 0.64 years old) than patients with 1G/1G and 1G/2G genotypes (62.91 +/- 0.59 years old; P = 0.024). Our data demonstrate that the 2G/2G genotype enhances lung cancer susceptibility especially in current smokers. To our knowledge, these results report the first molecular epidemiological evidence of the MMP-1 promoter polymorphism associated with the development of lung cancer in the presence of continuing carcinogenic exposure.

Age Factors↗

PubMed2001

Kinetic studies, mechanism, and substrate specificity of amadoriase I from Aspergillus sp.

Amadoriase is a flavoenzyme that catalyzes the oxidative deglycation of Amadori products (fructosyl amino acids or aliphatic amines) to yield free amine, glucosone, and hydrogen peroxide. The mechanism of action of amadoriase I from Aspergillus sp. has been investigated by stopped-flow kinetic studies using fructosyl propylamine and O(2) as substrates in 10 mM Tris HCl, pH 7.9, 4 degrees C. Using both substrate analogues and fast kinetic techniques, the active configuration of the substrate was found to be the beta-pyranose form. Stopped-flow studies showed that the reductive half-reaction is triphasic and generates intermediates that absorb at long wavelengths and is consistent either with (i) the reaction of the substrate with the flavin followed by iminium deprotonation or hydrolysis and then product release or with (ii) the formation of flavin reduction intermediates (carbanion equivalents or adducts), followed by product release. The rate of product release after flavin reduction is lower than the aerobic turnover rate, 14.4 s(-1), suggesting that it is not involved in the catalytic cycle and that reoxidation of the reduced enzyme occurs in the E(red)-product complex. In the oxidative half-reaction, the reduced flavin is oxidized by O(2) in a single phase. The observed rate constant has a linear dependence on oxygen concentration, giving a bimolecular rate constant of 4.9 x 10(4) M(-1) s(-1) in the absence of product, and 3.6 x 10(4) M(-1) s(-1) when the product is bound. The redox potentials of amadoriase have been measured at pH 7.0, 25 degrees, giving values of +48 and -52 mV for the oxidized enzyme/anionic semiquinone and anionic semiquinone/reduced enzyme couples, respectively.

Amino Acid Oxidoreductases↗