Biomedical subjects
Publications and source records attributed to D Ross.
Genetic theories still flounder on the fact that similarity of hand preference is the same in monozygotic (MZ) and dizygotic (DZ) twins. Lateral preference on a well-designed set of 5 activities was obtained from 2,131 male pairs. On item analysis, only "throw" discriminated zygosity, attributable to "excess" nondextral MZ pairs. This item is remarkably free of the intense cultural bias against sinistrality.
We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.
NAD(P)H:quinone oxidoreductase 1 (NQO1) participates in the detoxification of many environmental quinones and related compounds. Recent studies have suggested that individuals with a polymorphism in NQO1 (NQO1*2), which results in a decrease (heterozygous, NQO1*1/*2) or a total loss (homozygous, NQO1*2/*2) of NQO1, may be at increased risk for the development of leukemias. Previous studies have failed to detect NQO1 in freshly aspirated bone marrow including Ficoll-purified mononuclear cells and purified CD34(+) hematopoietic progenitor stem cells. In these studies we examined human bone marrow core biopsies by immunohistochemistry using monoclonal antibodies directed against NQO1. These studies revealed that NQO1 was expressed in human bone marrow but expression of NQO1 was limited to bone marrow endothelium and adipocytes. To confirm the expression of NQO1 in bone marrow endothelial cells we examined an immortalized human bone marrow endothelial cell line (HBMEC-60) for NQO1 protein expression. Immunoblot analysis and an activity assay confirmed the expression of NQO1 in HBMEC-60. These data demonstrate that NQO1 is present in human bone marrow. The increased risk of leukemia associated with a deficit in NQO1 levels due to the NQO1*2 polymorphism may reflect impaired quinone detoxification and an increased susceptibility of endothelial cells in bone marrow to environmental insults.
We study wetting in liquid mixtures of methanol and the n-alkanes. Mixing alkanes of different chain lengths, we can examine the crossover between critical (continuous) and first-order (discontinuous) wetting transitions. Measurements of the film thickness and surface specific heat exponent indicate that for carbon number n between 11 (undecane) and 9 (nonane), there is a crossover from first-order to critical wetting with a tricritical wetting point between an effective alkane carbon number of 9.6 and 10. The observed variation of the specific heat exponent in the tricritical region agrees fairly well with the predictions of a simple mean-field model with only short-range interactions.
BACKGROUND: Given that vagal afferents project to brainstem regions that promote alertness, the authors tested the hypothesis that vagus nerve stimulation (VNS) would improve daytime sleepiness in patients with epilepsy. METHODS: Sixteen subjects with medically refractory seizures underwent polysomnography and multiple sleep latency tests (MSLT) and completed the Epworth Sleepiness Scale (ESS), a measure of subjective daytime sleepiness, before and after 3 months of VNS. Most subjects (>80%) were maintained on constant doses of antiepileptic medications. RESULTS: In the 15 subjects who completed baseline and treatment MSLT, the mean sleep latency (MSL) improved from 6.4 +/- 4.1 minutes to 9.8 +/- 5.8 minutes (+/- SD; p = 0.033), indicating reduced daytime sleepiness. All subjects with stimulus intensities of < or =1.5 mA showed improved MSL. In the 16 subjects who completed baseline and treatment ESS, the mean ESS score decreased from 7.2 +/- 4.4 to 5.6 +/- 4.5 points (p = 0.049). Improvements in MSLT and ESS were not correlated with reduction in seizure frequency. Sleep-onset REM periods occurred more frequently in treatment naps as compared to baseline naps (p < 0.008; Cochran-Mantel-Haenszel test). The amount of REM sleep or other sleep stages recorded on overnight polysomnography did not change with VNS treatment. CONCLUSIONS: Treatment with VNS at low stimulus intensities improves daytime sleepiness, even in subjects without reductions in seizure frequency. Daytime REM sleep is enhanced with VNS. These findings support the role of VNS in activating cholinergic and other brain regions that promote alertness.
The benzene metabolite hydroquinone (HQ) is postulated to exert its myelotoxicity by bioactivation to reactive quinone derivatives in myeloperoxidase (MPO)-containing cells. In this study, the role of caspases in hydroquinone-induced apoptosis in MPO-rich HL-60 promyelocytic leukemia and MPO-deficient Jurkat T-lymphoblastic leukemia cells was investigated. HQ-induced apoptosis in both cell types was accompanied by phosphatidylserine (PS) exposure, caspases-3/-7 activation, PARP cleavage, DNA fragmentation, and ultrastructural changes as assessed by electron microscopy. In HL-60 cells, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked activation of caspases-3/-7, cleavage of PARP, and DNA, but PS externalization and cytoplasmic changes were not significantly affected. In marked contrast, all features of apoptosis were completely inhibited by Z-VAD.FMK in HQ-treated Jurkat cells. These data provide evidence for Z-VAD.FMK-insensitive and caspases-3/-7-independent pathway(s) in the externalization of PS and cytoplasmic changes during HQ-induced apoptosis in HL-60 cells. In contrast, in Jurkat cells, all of these changes required caspase activation. The ability of HQ to induce equivalent apoptosis in both MPO-deficient Jurkat cells and MPO-rich HL-60 cells demonstrates that MPO-catalyzed bioactivation of HQ is not a prerequisite for toxicity. The differential mechanisms of apoptosis in HL-60 and Jurkat T cells may reflect the MPO activity of these cells and, as a result, the amount of reactive BQ and other metabolites that are generated.
A technique is described for the measurement of fluid temperatures in microfluidic systems based on temperature-dependent fluorescence. The technique is easy to implement with a standard fluorescence microscope and CCD camera. In addition, the method can be used to measure fluid temperatures with micrometer spatial resolution and millisecond time resolution. The efficacy of the method is demonstrated by measuring temperature distributions resulting from Joule heating in a variety of microfluidic circuits that are electrokinetically pumped. With the equipment used for these measurements, fluid temperatures ranging from room temperature to 90 degrees C were measured with a precision ranging from 0.03 to 3.5 degrees C-dependent on the amount of signal averaging done. The spatial and temporal resolutions achieved were 1 microm and 33 ms, respectively.
A pulsed UV excimer laser (KrF, 248 nm) was used to modify the surface charge on the side wall of hot-embossed microchannels fabricated in a poly(methyl methacrylate) substrate. Subablation level fluences, less than 2,385 mJ/cm2, were used to prevent any changes in the physical morphology of the surface. It is shown that the electroosmotic mobility, induced by an electric field applied along the length of the channel, increases by an average of 4% in the regions that have been exposed to UV laser pulses compared to nonexposed regions. Furthermore, application of UV modification to electroosmotic flow around a 90 degrees turn results in a decrease in band broadening, as measured by the average decrease in the plate height of 40% compared to flow around a nonmodified turn. The ability to modify the surface charge on specific surfaces within a preformed plastic microchannel allows for fine control, adjustment, and modulation of the electroosmotic flow without using wall coatings or changing the geometry of the channel to achieve the desired flow profile.
BACKGROUND: Reperfusion injury (RI) is a major cause of mortality and morbidity among lung transplant recipients. We sought to determine if prophylactic administration of inhaled nitric oxide (NO) to lung transplant recipients at reperfusion would prevent RI. We also hypothesized that if prophylactic NO proves ineffective in preventing RI then it may improve pulmonary hemodynamics and gas exchange in the subset of patients who develop RI. METHODS: After informed consent, 28 consecutive, adult lung transplant recipients received NO at 20 ppm at reperfusion. NO was withdrawn for 15 min at 6 and 12 hr after reperfusion, and gas exchange and hemodynamics were measured. RESULTS: Five of the 28 lung transplant recipients (18%) developed RI. Withdrawal of NO for 15 min in this subset of patients resulted in a significant rise in mean pulmonary artery pressure and a reduction in oxygenation index. All five patients with RI were kept on inhaled NO until full functional recovery of the allograft and were then weaned from mechanical ventilation. None required extracorporeal membrane oxygenation support; the early mortality in this group was 20% (1/5). The remaining 23 patients without RI had uneventful early postoperative course and were weaned from NO and mechanical ventilation within 36 hr of transplantation. CONCLUSIONS: Prophylactic-inhaled NO does not prevent RI in human lung transplantation. However, inhaled NO, started at reperfusion, improves gas exchange and reduces pulmonary artery pressure in those patients who develop RI.
We observed simultaneous focusing in both space and time for light pulses propagating in a planar waveguide. In particular, 60 fs pulses with a width of 170 microm were injected into a planar glass waveguide in the anomalous dispersion regime. Output pulses as short as 30 fs and as narrow as 20 microm were measured. The results suggest that multiphoton absorption and intrapulse stimulated Raman scattering arrest the spatiotemporal contraction. The results were compared to the pulse evolution in zero and normal dispersion regimes and were shown to be significantly different. All of the experimental results were reproduced by a numerical model.
To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-diaphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adenocarcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphism. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined by the thiazolyl blue (MTT) assay] to known antitumor quinones and newer clinical candidates. Inhibition of NQO1 by pretreatment with an irreversible inhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone]. RH1 was evaluated further by clonogenic assay for cytotoxic response and was more cytotoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhibition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-links than did BE cells in response to RH1 treatment. DNA cross-linking in BE-NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirming that NQO1 activates RH1 to a potent cross-linking species. Further studies using streptonigrin, ES921, and RH1 were undertaken to analyze the relationship between NQO1 activity and quinone toxicity. Toxicity of these compounds was measured in a panel of BE-NQ cells expressing a range of NQO1 activity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induced toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (>77 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antitumor quinones in this system and suggest that a threshold level of NQO1 activity is required to initiate toxic events.
We have characterized electroosmotic flow in plastic microchannels using video imaging of caged fluorescent dye after it has been uncaged with a laser pulse. We studied flow in microchannels composed of a single material, poly(methyl methacrylate) (acrylic) or poly(dimethylsiloxane) (PDMS), as well as in hybrid microchannels composed of both materials. Plastic microchannels used in this study were fabricated by imprinting or molding using a micromachined silicon template as the stamping tool. We examined the dispersion of the uncaged dye in the plastic microchannels and compared it with results obtained in a fused-silica capillary. For PDMS microchannels, it was possible to achieve dispersion similar to that found in fused silica. For the acrylic and hybrid microchannels, we found increased dispersion due to the nonuniformity of surface charge density at the walls of the channels. In all cases, however, electroosmotic flow resulted in significantly less sample dispersion than pressure-driven flow at a similar velocity.
Neutrinos are elementary particles that carry no electric charge and have little mass. As they interact only weakly with other particles, they can penetrate enormous amounts of matter, and therefore have the potential to directly convey astrophysical information from the edge of the Universe and from deep inside the most cataclysmic high-energy regions. The neutrino's great penetrating power, however, also makes this particle difficult to detect. Underground detectors have observed low-energy neutrinos from the Sun and a nearby supernova, as well as neutrinos generated in the Earth's atmosphere. But the very low fluxes of high-energy neutrinos from cosmic sources can be observed only by much larger, expandable detectors in, for example, deep water or ice. Here we report the detection of upwardly propagating atmospheric neutrinos by the ice-based Antarctic muon and neutrino detector array (AMANDA). These results establish a technology with which to build a kilometre-scale neutrino observatory necessary for astrophysical observations.
BACKGROUND: NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1's ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. RESULTS: Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. CONCLUSIONS: The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.
PURPOSE: Mitomycin C has been used in the treatment of primary acquired melanosis and melanomas of the conjunctiva. Because there is increasing evidence that NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, NQO1) or DT-diaphorase plays an important role in the bioactivation of mitomycin C, we examined pathologic specimens of these tumors for NQO1 by immunohistochemistry. METHODS: Formalin-fixed, paraffin-embedded sections with histologic diagnoses of primary acquired melanosis or conjunctival melanomas were obtained from the Eye Pathology Laboratory, University of Colorado Health Sciences Center. Detection of NQO1 in tissues was performed using standard immunohistochemical techniques with monoclonal antibodies against NQO1 and immunoperoxidase staining. Samples were examined by two independent reviewers and NQO1 staining was graded from 0 (no staining) to 3+ (intense staining). RESULTS: Eleven of 11 melanomas (95% confidence interval, 72% to 100%) and three of three lesions with primary acquired melanosis with atypia stained positively for NQO1. In the melanomas, staining was relatively uniform, while in primary acquired melanosis there was cell-to-cell variability in the staining. CONCLUSIONS: NQO1 was detected by immunohistochemistry in every examined section of primary acquired melanosis and melanoma of the conjunctiva, suggesting that NQO1 may play a role in the bioactivation of mitomycin C in these tumors.
This study sought to: a) ascertain the effect on rates of violence by varying its operational definition and b) compare characteristics of violent and nonviolent patients. Aggressive behavior was recorded daily for every patient (N = 78) during a 2-year period. Standardized rating scales were used to rate psychopathology and functioning. Almost two thirds of patients were aggressive to others, and 26% violently assaulted another person. Official incident reports underestimated rates of violence to others, self- harm, and property damage. Multivariate predictive models that greatly improved accuracy over base rates showed that violent patients tended to be female, schizophrenic (nonparanoid type), and abusive of alcohol before admission. Violence is more common in treatment resistant psychotic inpatients than suggested by incident reports. Standardized definitions of violence are urged in order to accurately study its prevalence and correlates. Models combining both historical/demographic and clinical data may enhance prediction of violence.
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The NAD(P)H:quinone oxidoreductase 1 (NQO1)*2 polymorphism is characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.