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R Knox

Publications and source records attributed to R Knox.

At least 19 recordsLinked to original sources
PubMed2001

Demonstration of the activation of prodrug CB 1954 using human DT-diaphorase mutant Q104Y-transfected MDA-MB-231 cells and mouse xenograft model.

The rat form of DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase; EC 1.6.99.2) is more effective than the human form in activating prodrugs such as CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Our site-directed mutagenesis study has revealed that residue 104 (Tyr in the rat enzyme and Gln in the human enzyme) is an important residue responsible for the catalytic differences between the rat and the human enzymes in the activation of CB 1954 (S. Chen et al., 1997, J. Biol. Chem. 272, 1437-1439). The human mutant Q104Y is capable of reducing CB 1954 at a rate identical to that of the wild-type rat DT-diaphorase. In the present study, we prepared both the wild-type human DT-diaphorase- and the mutant Q104Y-expressing MDA-MB-231 breast cancer cell lines using the cDNA transfection method. The MDA-MB-231 cell line is homozygous for a P187S mutation in the DT-diaphorase gene and has no detectable DT-diaphorase activity. Stable clones for the wild-type transfected cells had the DT-diaphorase activity ranged from 0.1 to 3.8 micromol of DCIP reduced/min/mg of protein and the clones for Q104Y transfected cells had the activity ranged from 0.06 to 1.58 micromol of DCIP reduced/min/mg of protein. Furthermore, in contrast to the cells transfected with only expression vector that were not sensitive to CB 1954 treatment, the wild-type and Q104Y-expressing cells were capable of the reductive activation of CB 1954, resulting in cell eradication. Our data showed that cell killing by CB 1954 followed a dose and incubation-time dependent manner. It was also found that the cell survival upon the treatment of CB 1954 was related to the expressed DT-diaphorase activity in these cells. In the presence of 75 microM CB 1954, a 50% cell killing was achieved in cells containing Q104Y and the wild-type DT-diaphorase with the activity at approximately 0.67 and 3.8 micromol of DCIP reduced/min/mg of protein, respectively. These results agree well with those of the in vitro enzyme assays that show that Q104Y is significantly more active than the wild-type DT-diaphorase in the activation of CB 1954. Finally, the in vivo activation of CB 1954 was demonstrated with a nude mouse model using Q104Y-transfected MDA-MB-231 cells. These studies reveal that DT-diaphorase can activate CB 1954, and human Q104Y mutant enzyme is more active than the wild-type enzyme in the intracellular reductive activation of CB 1954.

Animals↗
PubMed2001

Improvement in asthma symptoms and quality of life in pediatric patients through specialty care delivered via telemedicine.

Asthma is the most common chronic disease affecting children. Studies have demonstrated improvements in asthma control when care is delivered by specialists compared with generalists. We postulated that specialist care delivered by telemedicine would result in similar improvements in control of symptoms and quality of life as compared with face-to-face encounters with specialists. Seventeen patients with persistent asthma, who were cared for by pediatricians in a rural school-based health clinic, were treated over a 6-month period in an asthma specialty program. Patients had face-to-face encounters at week zero, and then telemedicine follow-up visits at weeks 4, 12, and 24. Patients maintained a symptom diary and reliever medication use log. Spirometry and patient and caregiver quality-of-life questionnaires were completed at each visit. Mean number of symptom free days increased 83% from 2.35 days at week 0 to 4.31 days at week 24 (p < 0.05). There was a 44% reduction in mean symptom scores, from 2.32 at week 0 to 1.31 at week 24 (p < 0.001). Nine patients reported having 7 symptom-free days or 7 days of symptom scores of zero in the preceding seven days at week 24 compared with one patient at week 0 (p < 0.002). FEV(1) increased by > or = 12% in seven patients during the study period. Significant improvements in quality of life were reported by patients at week 4 (p < 0.02) and week 24 (p < 0.01), and by caregivers at week 24 (p < 0.002). Specialty asthma care delivered via telemedicine resulted in improvements in asthma symptom control and quality of life similar to improvements reported in face-to-face encounters provided by specialists.

Adolescent↗
PubMed2001

The integrin-mediated cyclic strain-induced signaling pathway in vascular endothelial cells.

The irregular distribution of plaque in the vasculature results from the interaction of local hemodynamic forces with the vessel wall. One well-characterized force is cyclic circumferential strain, the repetitive pulsatile pressure distention on the arterial wall. This review summarizes current research, which has aimed to elicit the signal transduction pathway by which cyclic strain elicits functional and structural responses in endothelial cells; specifically, it summarizes the signaling pathway that begins with the reorganization of integrins. One method by which these extracellular matrix receptors affect signal transduction is through their ability to initiate the process of phosphorylation on tyrosine residues of cytoplasmic protein kinases, including focal adhesion kinase. The strain-induced pathway appears to also involve ras and the mitogen-activated protein kinase family of enzymes, and preliminary data suggests a role for src as well. Ultimately, it is the regulation of gene expression through the modulation of transcription factors that allows endothelial cells to respond to changes in local hemodynamics.

Blood Pressure↗
PubMed2000

Structure-function studies of DT-diaphorase (NQO1) and NRH: quinone oxidoreductase (NQO2).

DT-diaphorase, also referred to as NQO1 or NAD(P)H: quinone acceptor oxidoreductase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. NRH (dihydronicotinamide riboside): quinone oxidoreductase, also referred to as NQO2, has a high nucleotide sequence identity to DT-diaphorase and is considered to be an isozyme of DT-diaphorase. These enzymes transfer two electrons to a quinone, resulting in the formation of a hydroquinone product without the accumulation of a dissociated semiquinone. Steady and rapid-reaction kinetic experiments have been performed to determine the reaction mechanism of DT-diaphorase. Furthermore, chimeric and site-directed mutagenesis experiments have been performed to determine the molecular basis of the catalytic differences between the two isozymes and to identify the critical amino acid residues that interact with various inhibitors of the enzymes. In addition, functional studies of a natural occurring mutant Pro-187 to Ser (P187S) have been carried out. Results obtained from these investigations are summarized and discussed.

Amino Acid Sequence↗
PubMed2000

Telehealth in a rural school-based health center.

Telehealth is a new technology for the delivery of health care that allows a provider to deliver health care to a client at a remote setting via full, real-time video and audio interaction (American Nurses' Association, 1996). Telehealth does not change traditional health care standards or practice. School-based health centers are designed specifically for children and offer a continuum of preventive and acute care interventions that few other health care entities can provide (School-Based Adolescent Health Care Program, 1993). Telehealth and a school-based health center were joined together in a pilot project to demonstrate the efficacy of primary care via telehealth and to determine the value of this technology to the school and community. The telehealth capability enhanced the role of the school nurse and increased access to primary care for the students. The students were quick to adapt to the technology. The role of the school nurse was essential to the success of this project.

Child↗
PubMed1999

Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling.

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.

Antineoplastic Agents↗
PubMed1997

Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase.

Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using an Escherichia coli expression method. NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) [also referred to as NAD(P)H:quinone acceptor oxidoreductase] because there is a 49% identity between their amino acid sequences. The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit. Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. It catalyzes a two-electron reduction of quinones and oxidation-reduction dyes. One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2. This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor. The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase. In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954. Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions. NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone. Flavones are inhibitors of NQO2. However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase. The most potent flavone inhibitor tested so far is quercetin (3,5,7,3',4'-. 6pentahydroxyflavone). It has been found that quercetin is a competitive inhibitor with respect to NRH (Ki = 21 nM). NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S. Chen et al., Protein Sci. 3, 51-57, 1994). In order to understand better the basis of catalytic differences between NQO2 and DT-diaphorase, a human NQO2 with 43 amino acids from the carboxyl terminus of human DT-diaphorase (i.e., hNQO2-hDT43) has been prepared. hNQO2-hDT43 still uses NRH as an electron donor. In addition, the chimeric enzyme is inhibited by quercetin but not dicumarol. These results suggest that additional region(s) in these enzymes is involved in differentiating NRH from NAD(P)H.

Amino Acid Sequence↗
PubMed1997

Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse.

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.

Amino Acid Sequence↗
PubMed1997

Selective cell ablation in transgenic mice expression E. coli nitroreductase.

The gene encoding E. coli nitroreductase (NTR) was expressed in the luminal cells of the mammary gland of transgenic mice using the ovine beta-lactoglobulin promoter. Treatment of NTR expressing animals with the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) resulted in a rapid and selective killing of this population of cells whereas the closely associated myoepithelial cells were unaffected. NTR-mediated inducible cell ablation offers a number of advantages over the use of HSV1-tk for the selective killing of cells in vivo.

Animals↗
PubMed1995

A five-year experience with severe injuries in elderly patients.

Recent reports of injury in elderly patients document that aggressive care is justified. Our experience indicates that geriatric injury differs from that of other trauma patients. All patients entered into a large urban trauma center registry over a five-year period were analyzed. Variables reviewed included demographics, trauma indices, mechanism of injury, mean number of hospital days, and morbidity and mortality for patients under 60 years old and for geriatric patients defined as those aged 60 and above. There were 3,064 patients in the reviewed group, of whom 243 qualified for geriatric analysis. Blunt injuries in males were the most typical scenario. Failure to use safety belts and alcohol intoxication persist into the geriatric age group (83% and 13%, respectively). Trauma indices including Injury Severity Score (ISS) were slightly greater in the geriatric group (27 versus 23) as compared to younger patients; plus geriatric patients tolerated head injury less well (GCS in those who died 6.7 versus 4.6, respectively; p < 0.001). Mortality in the geriatric group was 31% while being 17.1% in the younger group (p < 0.005). Days in the hospital were 20 for the geriatric compared to 13 for the younger group (p < 0.025). Infections and chest complications were twice as common in the elderly and dysrhythmias were five times more frequent. Elderly patients constitute 8% of the trauma population and suffer a magnitude of injury at least comparable to the general population. Their mortality is approximately 50% above the population as a whole and morbidity twice as common, accounting for the prolonged hospital stay.

Accidents, Traffic↗
PubMed1995

Catalytic properties of NAD(P)H:quinone acceptor oxidoreductase: study involving mouse, rat, human, and mouse-rat chimeric enzymes.

NAD(P):quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes. In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and EO9 [5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione]. By using Escherichia coli-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous finding showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities. Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage. To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated. MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxy-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase. In addition, MR-P and RM-P were found to be inhibited by flavones with Ki values similar to those for rat and mouse quinone reductases, respectively. Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones.

Amino Acid Sequence↗
PubMed1993

Fibrinogen and the albumin-globulin ratio in recurrent stroke.

BACKGROUND AND PURPOSE: In following patients initially recruited for a cross-sectional study of blood viscosity in ischemic cerebrovascular disease, it was noted that those having a low albumin-globulin ratio appeared to experience the majority of subsequent vascular events. Accordingly, a prospective study in which subjects were assigned to a high or low albumin-globulin cohort was undertaken to examine the relation between a low albumin-globulin ratio, the presence of clinical risk factors for stroke, and the occurrence of subsequent stroke, myocardial infarction, or vascular death. METHODS: Three groups of subjects were followed for an average of 1.5 +/- 0.8 years to ascertain vascular end points. Group 1 consisted of 126 patients with acute ischemic stroke; group 2 included 109 subjects matched with group 1 for age, medications, and recognized clinical risk factors for stroke; and group 3 was composed of 84 healthy volunteers, matched for age with groups 1 and 2. The median albumin-globulin ratio for group 1 at enrollment, 1.45, was used to dichotomize patients into two cohorts: all subjects with an albumin-globulin ratio of 1.45 or less were assigned to the "low" albumin-globulin cohort; those whose ratio was greater than 1.45 were assigned to the "high" albumin-globulin cohort. The occurrence of vascular end points was verified during subsequent hospitalizations and outpatient clinic visits and by telephone interviews of patients and providers. RESULTS: A total of 51 vascular events occurred, including 39 in group 1, 8 in group 2, and 4 in group 3. Subjects in either group 1 or 2 who were in the low albumin-globulin cohort had at least double the risk for a subsequent vascular event compared with their counterparts in the high albumin-globulin cohort (P < .01 and P < .03, respectively). In comparison with the high albumin-globulin cohort, significantly more patients in the low albumin-globulin cohort in group 1 had a history of prior stroke (P < .03). When groups 1 and 2 were combined, both a low albumin-globulin ratio and diabetes had a significant independent association with increased risk for subsequent vascular events in a Cox proportional-hazards model (P < .01 and P < .03, respectively). CONCLUSIONS: The results of this study indicate that significantly increased risk for subsequent vascular events in stroke patients and in subjects with clinical risk factors for stroke is associated with a shift in the concentrations of blood proteins to a prothrombotic environment characterized by lower levels of albumin and an increased concentration of globulins and fibrinogen.

Aged↗
PubMed1993

Placement of hydroxyapatite-coated endosseous implants in fresh extraction sites: a case report.

This case illustrates the use of the principles of guided tissue regeneration to achieve osseointegration of an endosseous dental implant. One implant was placed immediately, with hydroxyapatite and a barrier membrane, in an extraction site, while another was placed in a healed site. Eighteen months postinsertion, the implant placed in the extraction socket exhibited better maintenance of crestal height than did the implant inserted in the healed site.

Adult↗
PubMed1992

Steroid use is associated with pneumonia in pediatric chest trauma.

A review of pediatric trauma focused on pediatric chest injuries was performed at a trauma center specializing in neurologic trauma. Eighty of 342 (23%) pediatric trauma patients admitted to the center had chest injuries. Age, gender, mechanism of injury, magnitude of injury, incidence of pulmonary infection, chest tube usage, endotracheal intubation, steroid or antibiotic usage, morbidity, and mortality data were reviewed. Sixteen of 78 children (20%) with chest injuries developed pulmonary infections and were compared with the noninfected group. Patients with pneumonia had a higher morbidity with significantly longer mean hospital stay (43.0 vs. 12.7 days; p = 0.001), duration of intubation (8.4 vs. 1.5 days; p = 0.001), and total days with chest tubes, (2.2 vs. 1.4 days; p = 0.02). Pneumonia was significantly associated with longer mean duration of steroid usage (6.4 vs. 0.8 days; p = 0.0001). Duration of steroid administration for the treatment of concomitant brain injury was a significant independent risk factor for the occurrence of pneumonia.

Adolescent↗
PubMed1991

Effect of fat pre-feeding on bile flow and composition in the rat.

Studies were conducted in rats to determine if the increase in lymph triacylglycerol output on pre-feeding a 20% glyceryltrioleate diet (Mansbach, C.M., II and Arnold, A. (1986) Am. J. Physiol. 251, G263-269) was due to an increase in phosphatidylcholine output into bile. Rats who were fed chow or pre-fed the 20% fat diet were equipped with biliary and duodenal cannulas and infused with glucose-saline while bile was collected hourly. The next day a taurocholate-glyceryltrioleate infusion was given and bile collected for 5 h. Bile flow, bile acid, phosphatidylcholine and cholesterol output were greater in the chow fed group than controls during the 6 h of the glucose saline period. Outputs were low overnight. During the taurocholate-glyceryltrioleate infusion, bile flow, bile acid, phosphatidylcholine and cholesterol output were all greater in the fat pre-fed group than the chow fed controls. We conclude that fat pre-feeding profoundly influences biliary composition and flow. The 2-fold increase in biliary phosphatidylcholine output during duodenal lipid infusion offers a potential explanation for the increased delivery of triacylglycerol into the lymph in rats on a similar fat pre-feeding program.

Animals↗