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K Brown

Publications and source records attributed to K Brown.

At least 127 records · Page 7Linked to original sources
PubMed1999

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Animals↗
PubMed1999

Aminoisoquinolines: design and synthesis of an orally active benzamidine isostere for the inhibition of factor XA.

The design, synthesis and SAR of sulfonamidopyrrolidinone fXa inhibitors incorporating a new benzamidine isostere, namely aminoisoquinolines, is described. These inhibitors have higher Caco-2 cell permeability than comparable benzamidines and attain higher levels of exposure upon oral dosing. The most potent member 14b (fXa Ki=6 nM) is selective against other serine proteases of interest (>600 fold).

Administration, Oral↗
PubMed1999

Site-specific tamoxifen-DNA adduct formation: lack of correlation with mutational ability in Escherichia coli.

We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.

Bacteriophage T4↗
PubMed1999

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily.

N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide-activated UDP-GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 A resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long alpha-helical arm: (i) an N-terminal domain which resembles the dinucleotide-binding Rossmann fold; and (ii) a C-terminal domain which adopts a left-handed parallel beta-helix structure (LbetaH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LbetaH domains, the long alpha-helical linkers being seated on top of the arrangement and the N-terminal domains projected away from the 3-fold axis. In addition, the 2.3 A resolution structure of the GlmU-UDP-GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three-dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP-sugar bacterial pyrophosphorylases.

Acetyltransferases↗
PubMed1999

Abnormal microtubule packing in processes of SF9 cells expressing the FTDP-17 V337M tau mutation.

Mutations in the gene for the microtubule associated protein, tau have been identified for fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In vitro data have shown that FTDP-17 mutant tau proteins have a reduced ability to bind microtubules and to promote microtubule assembly. Using the baculovirus system we have examined the effect of the V337M mutation on the organization of the microtubules at the ultrastructural level. Our results show that the organization of the microtubules is disrupted in the presence of V337M tau with greater distances between the microtubules and fewer microtubules per process.

Animals↗
PubMed1999

Expression of a dominant-negative mutant inhibitor-kappaBalpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo.

We demonstrated recently that constitutive expression of proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in head and neck squamous cell carcinoma is correlated with activation of transcription factor nuclear factor (NF)-kappaB/Rel A (p50/p65), which binds the promoter region within each of the genes encoding this repertoire of cytokines. NF-kappaB can be activated after signal-dependent phosphorylation and degradation of inhibitor-kappaBalpha and has been reported to promote cell survival and growth. In the present study, we expressed a phosphorylation site mutant of inhibitor-kappaBalpha (IkappaBalphaM) in head and neck squamous cell carcinoma lines UM-SCC-9, -11B, and -38 to determine the effect of inhibition of NF-kappaB on cytokine expression, cell survival in vitro, and growth in vivo. After transfection with IKBalphaM, only a few UM-SCC-9 clones were obtained that stably expressed the mutant IkappaB, suggesting that expression of a mutant IkappaBalpha may affect survival of the transfected UM-SCC cell lines. After cotransfection of IkappaBalphaM with a Lac-Z reporter, we found that the number of surviving beta-galactosidase-positive cells in the three cell lines was reduced by 70-90% when compared with controls transfected with vector lacking the insert. In UM-SCC-9 cells that stably expressed IkappaBalphaM, inhibition of constitutive and tumor necrosis factor-a induced NF-kappaB activation, and production of all four cytokines was observed. Although UM-SCC-9 IkappaBalphaM-transfected cells proliferated at the same rate as vector-transfected cells in vitro, a significant reduction in growth of tumor xenografts was observed in SCID mice in vivo. The decreased growth of UM-SCC-9 IkappaBalphaM-transfected tumor cells accompanied decreased immunohistochemical detection of the activated form of NF-kappaB in situ. These results provide evidence that NF-KB and IkappaBalpha play an important role in survival, constitutive and inducible expression of proinflammatory cytokines, and growth of squamous cell carcinoma. NF-kappaB could serve as a potential target for therapeutic intervention against cytokine and other immediate-early gene responses that contribute to the survival, growth, and pathogenesis of these cancers.

Animals↗
PubMed1999

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit. The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.

Amino Acid Sequence↗
PubMed1999

IkappaB-alpha enhances transactivation by the HOXB7 homeodomain-containing protein.

Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing protein, which plays a key role in development and differentiation, physically interacted in vitro with IkappaB-alpha, an inhibitor of NF-kappaB activity. This interaction was mediated by the IkappaB-alpha ankyrin repeats and C-terminal domain as well as by the HOXB7 N-terminal domain. In transient transfection experiments, IkappaB-alpha markedly increased HOXB7-dependent transcription from a reporter plasmid containing a homeodomain consensus-binding sequence. This report therefore showed a novel function for IkappaB-alpha, namely a positive regulation of transcriptional activation by homeodomain-containing proteins.

Base Sequence↗
PubMed1999

Studies on the stability of trans-alpha-acetoxytamoxifen in Sprague-Dawley female rat liver slices, homogenate and subcellular fractions including microsomes and mitochondria.

This work examined the stability of trans alpha-acetoxytamoxifen in Krebs-Henseleit buffer (pH 7.4), in the presence of Sprague-Dawley female rat liver slices, rat liver homogenate and hepatic subcellular fractions, including microsomes and mitochondria, at pH 7.4 and at 37 degrees C over 300 min. The rate of hydrolysis was determined using high-performance liquid chromatography, and degradation profiles were obtained from which the rate and order of degradation were both evaluated. By applying zero-, first-, second- and third-order models of drug disappearance and the generation of by-products, first- and second-order appeared to produce the best fit. trans-alpha-Acetoxytamoxifen degraded rapidly in buffer and more slowly in the biological systems, probably due to the fact that the agent partitions into the hydrophobic component of the biological tissue and hence degrades at a much slower rate. The principal degradation products were trans-alpha-hydroxytamoxifen and, to a lesser extent, cis-alpha-hydroxytamoxifen. Another peak could not be identified. The production of trans-alpha-hydroxytamoxifen was enhanced in the presence of biological enzymes, whereas the concentration of the cis isomer remains relatively constant in buffer only (pH 7.4) and in the presence of biological enzymes. Therefore, the formation of identical adducts with DNA is consistent, because it has been shown that alpha-acetoxytamoxifen breaks down to form alpha-hydroxytamoxifen in vitro. The percentage of trans-alpha-acetoxytamoxifen remaining after 300 min was 40% in mitochondria and 32% in homogenate. The half-life (t1/2) was calculated for each condition by applying zero-, first- and second-order rate kinetics.

Animals↗
PubMed1999

Fatal invasive aspergillosis in an adolescent with cystic fibrosis.

We report on a 13-year-old girl with cystic fibrosis (CF) who developed refractory airflow obstruction despite high-dose steroids. She developed invasive aspergillosis and died despite oral and intravenous antifungal therapy. We speculate that the increasing use of immunosuppressive strategies and aggressive antipseudomonal therapy in CF may lead to an increase in aspergillus lung disease, including invasive aspergillosis in the future.

Adolescent↗
PubMed1999

Do memory clinics improve the quality of life of carers? A randomized pilot trial.

OBJECTIVE: To determine the effects of attendance at a memory clinic on the psychosocial health of carers. DESIGN: Randomized control trial. PARTICIPANTS: Fifty community dwelling subjects with mild to moderate dementia and their carers. MAIN OUTCOME MEASURES: Carer burden, psychological morbidity and psychosocial health related quality of life at 6 and 12 months post intervention. MAIN RESULTS: There was significant improvement in psychosocial health related quality of life of carers as measured by the psychosocial domain of the Functional Limitation Profile (FLP) at 6 months (p < 0.01), including improvement in the subgroups of alertness behaviour (p < 0.05) and social interaction (p < 0.01), after adjustment for age of subject and baseline scores. The improvement in social interaction was maintained at 12 months (p < 0.05). There was no significant difference in carer psychological morbidity (General Health Questionnaire), carer burden (Zarit family interview) or carer knowledge of dementia (Dementia Knowledge Test) at 6 or 12 months, between groups. CONCLUSIONS: These results demonstrate improved psychosocial health related quality of life for carers of those with mild to moderate cognitive impairment attending a memory clinic. Further research in this area is indicated, comparing memory clinic intervention with alternative support.

Aged↗
PubMed1999

Heterogeneity of in vitro radiosensitivity in human bladder cancer cells.

Human bladder cancer is often heterogeneous containing biologically different populations. Radiotherapy plus chemotherapy is the most common treatment for invasive disease. However few studies have investigated the role of heterogeneity in determining radiosensitivity. The radiation sensitivities of a parent human bladder cancer cell line (UCRU-BL-17CL) and nine cloned cell lines derived from it were determined. These cloned cell lines were previously shown to exhibit different biological characteristics when grown in nude mice. Radiation sensitivity was determined using both MTT and clonogenic assays. The radiobiological parameters, alpha,beta, and surviving fractions at 2 Gy and 8 Gy from the linear-quadratic model, were used to assess radiation sensitivity in the statistical analyses. The nine clones differed in radiosensitivity by both assays. By MTT, but not by the clonogenic assay, their radiation sensitivities were relatively consistent within each of the three biological groups (non-tumorigenic, tumorigenic, invasive); invasive clones were more sensitive than those of the non-tumorigenic and the tumorigenic groups for all the three-test criteria. The heterogeneity exhibited by this cell line may explain some of the variations in the clinical responses seen in the radiation treatment of invasive bladder cancer.

Animals↗
PubMed1999

Etiology and incidence of pressure ulcers in surgical patients.

This experimental study was designed to identify the etiology of pressure ulcers in a surgical sample and to evaluate a special OR mattress overlay in preventing pressure ulcer development. Surgical patients (N = 413) were randomized to receive "usual perioperative care" or the new mattress overlay. Over six postoperative days, 89 patients (21.5%) developed pressure ulcers, primarily stage I. Only 2% developed stage II or IV ulcers. Patients with ulcers were statistically older, had diabetes, were smaller in body mass, had lower Braden Scale scores on admission, and used the new mattress overlay (P < .02). Pressure ulcers that presented as "burns" or ecchymosis did not deteriorate to stage III or IV ulcers during the study. The mattress overlay was not effective in preventing pressure ulcer development.

Age Factors↗
PubMed1999

Comparing physician-specific two-year patient outcomes after coronary angiography: methodologic issues and results.

OBJECTIVES: We sought to evaluate methodologies to compare physician-related long-term patient outcomes appropriately. BACKGROUND: Evaluation of physicians on the basis of short-term patient outcome is becoming widely practiced. These analyses fail to consider the importance of long-term outcome, and methods appropriate to such an analysis are poorly defined. METHODS: All patients undergoing coronary angiography between 1992 and 1994 who received all of their cardiac care at our institution were followed for 27+/-13 months (mean+/-SD). Patients (n = 754) were cared for by one or more of 17 staff physicians. Risk-adjusted models were developed for four candidate clinical end points and cost. Physicians were then evaluated for each outcome measure. RESULTS: Of the clinical end points, death could be modeled most accurately (c-statistic = 0.83). The c-statistics for other end points ranged from 0.63 to 0.70. Physicians with outcomes statistically different (p < 0.05) from other physicians were identified more commonly than would be expected from the play of chance (p = 0.005). However, improvement in the c-statistics by the addition of physician identifiers was very modest. Physician's evaluations by the four measures of clinical outcome were variably correlated (r = .00 to .85). Graphic display of clinical and cost results for each physician did identify certain physicians who might be judged to provide more cost-effective care than others. CONCLUSIONS: Although comparisons of groups of physicians on the basis of long-term patient outcomes may have merit, individual physician-to-physician comparisons will be more difficult, owing to 1) multiple physicians contributing care to individual patients; 2) the poor predictive capacity of models other than that for survival; and 3) the modest apparent impact of differences in physician providers on long-term patient outcome. With these caveats in mind, modeling to compare patient outcomes of individual physicians with homogeneous patient populations or to identify gross outliers (good or bad) may be practicable in some patient-care systems, but may be inappropriate in others.

Aged↗
PubMed1999

Attribution of responsibility for injury and long-term outcome of patients with paralytic spinal cord trauma.

STUDY DESIGN: Survey. OBJECTIVE: To assess the association between patients' perceptions of responsibility for the injury and long term outcomes. SETTING: Duke University Medical Center Spinal Injury Service. METHODS: One hundred and forty patients, who suffered spinal cord injuries between 1985 and 1990, were selected to participate in the study, and 26 (19%), including eight with quadriplegia, completed a telephone interview. The mean age at interview was 39+/-12 years and 12% were women. The mean time from injury to completion of the study was 8.7+/-1.5 years. Attribution of cause of injury, the Craig Handicap Assessment Reporting Technique, and the SF36 mental health depression/vitality surveyed instruments attribution of responsibility for the injury, functional capacity, mental health depression, and vitality. RESULTS: The majority (18/26) of patients either totally blamed themselves or others for their injuries. Overall, there was no statistically significant association between attribution of injury and long-term outcomes. Outcomes were similar in patients who either totally blamed themselves or others for their predicament. CONCLUSION: In this study, long-term outcomes were not influenced by whether patients perceived themselves as being responsible for their injuries.

Activities of Daily Living↗
PubMed1999

The incidence of detrusor instability before and after colposuspension: a study using conventional and ambulatory urodynamic monitoring.

OBJECTIVE: To determine the incidence of detrusor instability (DI) detected by conventional and ambulatory bladder pressure monitoring, any link between urodynamic findings before and after colposuspension, and between patients' symptoms and the urodynamic findings. PATIENTS AND METHODS: Sixty-five patients, scheduled to undergo colposuspension on the basis of pure genuine stress incontinence on conventional cystometrography (CMG), also underwent ambulatory monitoring (AM) before surgery. All were requested to return 3 months afterward for repeat CMG and AM in random order. In all, 56 patients completed all assessments before and after surgery. In addition to the urodynamic assessment, the patients' symptoms before and after surgery were compared using a detailed questionnaire. RESULTS: On AM before surgery, half the patients showed some DI; afterward, the incidence on AM was 70% and on CMG was 27%. However, on AM, 25% of patients who had DI before surgery showed no evidence of it afterward. Of the 19 women who complained of urgency preoperatively, 16 showed DI on preoperative AM. However, of the 37 women denying urgency preoperatively, 12 (32%) also showed evidence of DI on preoperative AM. CONCLUSIONS: There is a significant incidence of DI detected by CMG after colposuspension in patients who are stable before surgery. The incidence of DI on AM was significantly higher than on CMG both before and after surgery. However, the preoperative urodynamic finding of increased DI on AM does not exclusively predict its presence postoperatively on AM or CMG. In addition, a patient history of urgency does not correlate well with the urodynamic findings before or after surgery. Therefore, from this study it appears that the postoperative symptomatic or urodynamic state cannot be predicted reliably from those before surgery.

Adult↗